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Sample records for lef-3 knockout bacmid

  1. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    International Nuclear Information System (INIS)

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F.

    2007-01-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes

  2. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  3. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  4. The Knockout Mouse Project

    Science.gov (United States)

    Austin, Christopher P; Battey, James F; Bradley, Allan; Bucan, Maja; Capecchi, Mario; Collins, Francis S; Dove, William F; Duyk, Geoffrey; Dymecki, Susan; Eppig, Janan T; Grieder, Franziska B; Heintz, Nathaniel; Hicks, Geoff; Insel, Thomas R; Joyner, Alexandra; Koller, Beverly H; Lloyd, K C Kent; Magnuson, Terry; Moore, Mark W; Nagy, Andras; Pollock, Jonathan D; Roses, Allen D; Sands, Arthur T; Seed, Brian; Skarnes, William C; Snoddy, Jay; Soriano, Philippe; Stewart, David J; Stewart, Francis; Stillman, Bruce; Varmus, Harold; Varticovski, Lyuba; Verma, Inder M; Vogt, Thomas F; von Melchner, Harald; Witkowski, Jan; Woychik, Richard P; Wurst, Wolfgang; Yancopoulos, George D; Young, Stephen G; Zambrowicz, Brian

    2009-01-01

    Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain. PMID:15340423

  5. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... advancements in the area of (α, 2α) reactions and heavy cluster knockout reactions are discussed. Importance of the finite-range vertex and the final-state interactions are brought out. Keywords. Cluster knockout reactions; FR-DWIA calculations; t-matrix effective interaction. PACS Nos 14.20.Pt; 24.10.

  6. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... Cluster knockout reactions are expected to reveal the amount of clustering (such as that of , d and even of heavier clusters such as 12C, 16O etc.) in the target nucleus. In simple terms, incident medium high-energy nuclear projectile interacts strongly with the cluster (present in the target nucleus) as if it ...

  7. KnockoutJS blueprints

    CERN Document Server

    Russo, Carlo

    2015-01-01

    If you are a JavaScript developer and already know the basics of KnockoutJS and you want to get the most out of it, then this book is for you. This book will help in your transition from a small site to a large web application that is easily maintainable.

  8. Heterologous expression, purification and characterization of human β-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

    Science.gov (United States)

    Miyazaki, Takatsugu; Kato, Tatsuya; Park, Enoch Y

    2018-02-03

    β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. KnockoutJS essentials

    CERN Document Server

    Ferrando, Jorge

    2015-01-01

    If you are a JavaScript developer who has been using DOM manipulation libraries such as Mootools or Scriptaculous, and you want go further in modern JavaScript development with a simple and well-documented library, then this book is for you. Learning how to use Knockout will be perfect as your next step towards building JavaScript applications that respond to user interaction.

  10. Cluster knockout reactions

    Indian Academy of Sciences (India)

    2014-04-07

    Apr 7, 2014 ... (figure 2), the corresponding α–α t-matrix effective interactions are seen to be widely different (figure 3). 2. Formalism. The finite-range transition matrix element T n,l. BA in the triple differential cross-section [13], d3σ d 1d 2dE1. = Fkin × Sl × |T n,l. BA (kf 1, kf 2, ki)|2 for the (α, 2α) cluster knockout reactions is ...

  11. Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

    Directory of Open Access Journals (Sweden)

    Ogata Makoto

    2009-06-01

    Full Text Available Abstract Background Sialic acid is a deoxy uronic acid with a skeleton of nine carbons which is mostly found on cell surface in animals. This sialic acid on cell surface performs various biological functions by acting as a receptor for microorganisms, viruses, toxins, and hormones; by masking receptors; and by regulating the immune system. In order to synthesize an artificial sialoglycoprotein, we developed a large-scale production of rat α2,6-sialyltransferase (ST6Gal1. The ST6Gal1 was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP--Chi- bacmid. The expressed ST6Gal1 was purified, characterized and used for sialylation of asialoglycopolypeptide. We tested the inhibitory effect of the synthesized α2,6-sialoglycopolypeptide on hemagglutination by Sambucus nigra (SNA lectin. Results FLAG-tagged recombinant ST6Gal1 was expressed efficiently and purified by precipitation with ammonium sulphate followed by affinity chromatography on an anti-FLAG M2 column, generating 2.2 mg purified fusion protein from only 11 silkworm larvae, with a recovery yield of 64%. The purified ST6Gal1 was characterized and its N-glycan patterns were found to be approximately paucimannosidic type by HPLC mapping method. Fluorescently-labelled N-acetyllactosamine (LacNAc glycoside containing dansyl group was synthesized chemo-enzymatically as high-sensitivity acceptor substrate for ST6Gal1. The acceptor substrate specificity of the enzyme was similar to that of rat liver ST6Gal1. The fluorescent glycoside is useful as a substrate for a highly sensitive picomole assay of ST6Gal1. Asialoglycopolypeptide was regioselectively and quantitatively sialylated by catalytic reaction at the terminal Gal residue to obtain α2,6-sialoglycopolypeptide using ST6Gal1. The α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra (SNA lectin

  12. KnockoutJS web development

    CERN Document Server

    Farrar, John

    2015-01-01

    This book is for web developers and designers who work with HTML and JavaScript to help them manage data and interactivity with data using KnockoutJS. Knowledge about jQuery will be useful but is not necessary.

  13. The serotonin transporter knockout rat : A review

    NARCIS (Netherlands)

    Olivier, Jocelien; Cools, Alexander; Ellenbroek, Bart A.; Cuppen, E.; Homberg, Judith; Kalueff, Allan V.; LaPorte, Justin L.

    2010-01-01

    This chapter dicusses the most recent data on the serotonin transporter knock-out rat, a unique rat model that has been generated by target-selected N-ethyl-N-nitrosourea (ENU) driven mutagenesis. The knock-out rat is the result of a premature stopcodon in the serotonin transporter gene, and the

  14. Nucleon knockout: off-shell effects

    International Nuclear Information System (INIS)

    Stephenson, G.J. Jr.

    1977-01-01

    The effect of the off-energy-shell extrapolation of the proton-proton scattering amplitude on the analysis of (p,2p) reactions is discussed. In particular, the range of expected variations in this extrapolation is explored and the possibility of using knock-out reactions to limit models of the p-p amplitude is studied

  15. Universal statistics of the knockout tournament

    Science.gov (United States)

    Baek, Seung Ki; Yi, Il Gu; Park, Hye Jin; Kim, Beom Jun

    2013-11-01

    We study statistics of the knockout tournament, where only the winner of a fixture progresses to the next. We assign a real number called competitiveness to each contestant and find that the resulting distribution of prize money follows a power law with an exponent close to unity if the competitiveness is a stable quantity and a decisive factor to win a match. Otherwise, the distribution is found narrow. The existing observation of power law distributions in various kinds of real sports tournaments therefore suggests that the rules of those games are constructed in such a way that it is possible to understand the games in terms of the contestants' inherent characteristics of competitiveness.

  16. High-throughput knockout screen in fission yeast

    OpenAIRE

    Gregan, Juraj; Rabitsch, Peter K; Rumpf, Cornelia; Novatchkova, Maria; Schleiffer, Alexander; Nasmyth, Kim

    2006-01-01

    We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all pred...

  17. Pion-induced knock-out reactions

    International Nuclear Information System (INIS)

    Jain, B.K.; Phatak, S.C.

    1977-01-01

    A strong absorption model for pion-induced Knock-out reactions is proposed. The distortion of the in-coming and out-going pions has been included by (1) computing pion wave number in nuclear medium (dispersive effect) and (2) excluding the central region of the nucleus where the real pion-absorption is dominant (absorption effect). In order to study the dependence of the (π + π + p) reaction on the off-shell pion-nucleon t-matrix, different off-shell extrapolations are used. The magnitude of the cross-sections seems to be sensitive to the type of off-shell extrapolation; their shapes, however, are similar. The theoretical results are compared with experimental data. The agreement between the theoretical results for separable off-shell extrapolation and the data is good. (author)

  18. Interrater agreement of an observational tool to code knockouts and technical knockouts in mixed martial arts.

    Science.gov (United States)

    Lawrence, David W; Hutchison, Michael G; Cusimano, Michael D; Singh, Tanveer; Li, Luke

    2014-09-01

    Interrater agreement evaluation of a tool to document and code the situational factors and mechanisms of knockouts (KOs) and technical knockouts (TKOs) in mixed martial arts (MMA). Retrospective case series. Professional MMA matches from the Ultimate Fighting Championship-2006-2012. Two nonmedically trained independent raters. The MMA Knockout Tool (MMA-KT) consists of 20 factors and captures and codes information on match characteristics, situational context preceding KOs and TKOs, as well as describing competitor states during these outcomes. The MMA-KT also evaluates the mechanism of action and subsequent events surrounding a KO. The 2 raters coded 125 unique events for a total of 250 events. The 8 factors of Part A had an average κ of 0.87 (SD = 0.10; range = 0.65-0.98); 7 were considered "substantial" agreement and 1 "moderate." Part B consists of 12 factors with an average κ of 0.84 (SD = 0.16; range = 0.59-1.0); 7 classified as "substantial" agreement, 4 "moderate," and 1 "fair." The majority of the factors in the MMA-KT demonstrated substantial interrater agreement, with an average κ of 0.86 (SD = 0.13; range = 0.59-1.0). The MMA-KT is a reliable tool to extract and code relevant information to investigate the situational factors and mechanism of KOs and TKOs in MMA competitions.

  19. Sleep in Kcna2 knockout mice

    Directory of Open Access Journals (Sweden)

    Messing Albee

    2007-10-01

    Full Text Available Abstract Background Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3–4 h/day instead of 8–14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system. Results Continuous (24 h electroencephalograph (EEG, electromyogram (EMG, and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ and wild-type (WT pups (P17 and HZ and WT adult mice (P67. Sleep stages were scored visually based on 4-s epochs. EEG power spectra (0–20 Hz were calculated on consecutive 4-s epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups ( Conclusion Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep.

  20. High-throughput knockout screen in fission yeast.

    Science.gov (United States)

    Gregan, Juraj; Rabitsch, Peter K; Rumpf, Cornelia; Novatchkova, Maria; Schleiffer, Alexander; Nasmyth, Kim

    2006-01-01

    We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.

  1. Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

    Science.gov (United States)

    Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

  2. Akt2/LDLr double knockout mice display impaired glucose tolerance and develop more complex atherosclerotic plaques than LDLr knockout mice

    NARCIS (Netherlands)

    Rensing, Katrijn L.; de Jager, Saskia C. A.; Stroes, Erik S.; Vos, Mariska; Twickler, Marcel Th B.; Dallinga-Thie, Geesje M.; de Vries, Carlie J. M.; Kuiper, Johan; Bot, Ilze; von der Thüsen, Jan H.

    2014-01-01

    To characterize the phenotype of Akt2/low-density-lipoprotein receptor double knockout (dKO) (Akt2/LDLr dKO) mice with respect to insulin resistance and features of atherosclerotic plaque progression. Metabolic profile and atherosclerotic plaque progression were compared between LDLr KO mice and

  3. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2014-01-01

    Full Text Available Glycosaminoglycans (GAGs are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs.

  4. Rapid curation of gene disruption collections using Knockout Sudoku.

    Science.gov (United States)

    Anzai, Isao A; Shaket, Lev; Adesina, Oluwakemi; Baym, Michael; Barstow, Buz

    2017-10-01

    Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is ∼100× faster and 30× lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial pooling and next-generation sequencing. This method facilitates the rapid, algorithmically guided condensation and curation of the progenitor collection into a high-quality, nonredundant collection that is suitable for rapid genetic screening and gene discovery.

  5. Pauli blocking and medium effects in nucleon knockout reactions

    International Nuclear Information System (INIS)

    Bertulani, C. A.; De Conti, C.

    2010-01-01

    We study medium modifications of the nucleon-nucleon (NN) cross sections and their influence on the nucleon knockout reactions. Using the eikonal approximation, we compare the results obtained with free NN cross sections with those obtained with a purely geometrical treatment of Pauli blocking and with NN obtained with more elaborated Dirac-Bruecker methods. The medium effects are parametrized in terms of the baryon density. We focus on symmetric nuclear matter, although the geometrical Pauli blocking also allows for the treatment of asymmetric nuclear matter. It is shown that medium effects can change the nucleon knockout cross sections and momentum distributions up to 10% in the energy range E lab =50-300 MeV/nucleon. The effect is more evident in reactions involving halo nuclei.

  6. Physiological roles of CNS muscarinic receptors gained from knockout mice

    DEFF Research Database (Denmark)

    Thomsen, Morgane; Sørensen, Gunnar; Dencker, Ditte

    2017-01-01

    , knockout mice are likely to continue to provide valuable insights into brain physiology and pathophysiology, and advance the development of new medications for a range of conditions such as Alzheimer's disease, Parkinson's disease, schizophrenia, and addictions, as well as non-opioid analgesics...... receptors modulating neuronal activity and neurotransmitter release in many brain regions, shaping neuronal plasticity, and affecting functions ranging from motor and sensory function to cognitive processes. As gene targeting technology evolves including the use of conditional, cell type specific strains...

  7. One-neutron knockout from 51-55Sc

    International Nuclear Information System (INIS)

    Schwertel, S.; Maierbeck, P.; Gernhaeuser, R.; Bildstein, V.; Boehmer, M.; Eppinger, K.; Faestermann, T.; Friese, J.; Fabbietti, L.; Maier, L.; Winkler, S.; Kruecken, R.; Kroell, T.; Alvarez-Pol, H.; Benjamim, E.A.; Benlliure, J.; Caamano, M.; Cortina-Gil, D.; Gascon, M.; Kurtukian, T.; Perez, D.; Rodriguez-Tajes, C.; Aksouh, F.; Aumann, T.; Behr, K.; Boretzky, K.; Bruenle, A.; Chatillon, A.; Chulkov, L.V.; Geissel, H.; Gerl, J.; Gorska, M.; Kojouharov, I.; Klimkiewicz, A.; Kurz, N.; Nociforo, C.; Schaffner, H.; Simon, H.; Stanoiu, M.; Suemmerer, K.; Weick, H.; Borge, M.J.G.; Pascual-Izarra, C.; Perea, A.; Tengblad, O.; Buerger, A.; Casarejos, E.; Brown, B.A.; Enders, J.; Schrieder, G.; Hansen, P.G.; Jonson, B.; Nyman, G.; Kanungo, R.; Kiselev, O.; Larsson, K.; Le Bleis, T.; Mahata, K.; Nilsson, T.; Prochazka, A.; Rossi, D.; Sitar, B.; Otsuka, T.; Tostevin, J.A.; Rae, W.D.M.

    2012-01-01

    Results are presented from a one-neutron knockout experiment at relativistic energies of ∼ 420 A MeV on 51-55 Sc using the GSI Fragment Separator as a two-stage magnetic spectrometer and the MINIBALL array for gamma-ray detection. Inclusive longitudinal momentum distributions and cross-sections were measured enabling the determination of the contributions corresponding to knockout from the νp 1/2 , νp 3/2 , (L = 1) and νf 7/2 , νf 5/2 (L = 3) neutron orbitals. The observed L = 1 and L = 3 contributions are compared with theoretical cross-sections using eikonal knockout theory and spectroscopic factors from shell model calculations using the GXPF1A interaction. The measured inclusive knockout cross-sections generally follow the trends expected theoretically and given by the spectroscopic strength predicted from the shell model calculations. However, the deduced L = 1 cross-sections are generally 30-40% higher while the L = 3 contributions are about a factor of two smaller than predicted. This points to a promotion of neutrons from the νf 7/2 to the νp 3/2 orbital indicating a weakening of the N = 28 shell gap in these nuclei. While this is not predicted for the phenomenological GXPF1A interaction such a weakening is predicted by recent calculations using realistic low-momentum interactions V low k obtained by evolving a chiral N3LO nucleon-nucleon potential. (orig.)

  8. Single proton knock-out from 24F

    International Nuclear Information System (INIS)

    Thoennessen, M.; Baumann, T.; Brown, B.A.; Enders, J.; Frank, N.H.; Hansen, P.G.; Heckman, P.; Luther, B.A.; Seitz, J.P.; Stolz, A.; Tryggestad, E.

    2004-01-01

    The measurement of the single proton knock-out reaction from 24 F on a 12 C target at 46.7 MeV/nucleon yielded a 23 O ground state population of (6.6+/-1.0) mb. The data were compared to calculations based on the many-body shell model and the eikonal theory. The results are consistent with a [0d5/26]-bar 1s1/2 configuration of 23 O

  9. Robust and sensitive analysis of mouse knockout phenotypes.

    Directory of Open Access Journals (Sweden)

    Natasha A Karp

    Full Text Available A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student's t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene's function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained.

  10. Pre-Equilibrium Cluster Emission with Pickup and Knockout

    International Nuclear Information System (INIS)

    Betak, E.

    2005-01-01

    We present a generalization of the Iwamoto-Harada-Bisplinghoff pre-equilibrium model of light cluster formation and emission, which is enhanced by allowing for possible admixtures of knockout for strongly coupled ejectiles, like α's. The model is able to attain the Weisskopf-Ewing formula for compound-nucleus decay at long-time limit; it keeps the philosophy of pre-equilibrium decay during the equilibration stage and it describes the initial phase of a reaction as direct process(es) expressed using the language of the exciton model

  11. A STAT-1 knockout mouse model for Machupo virus pathogenesis

    Directory of Open Access Journals (Sweden)

    Shurtleff Amy C

    2011-06-01

    Full Text Available Abstract Background Machupo virus (MACV, a member of the Arenaviridae, causes Bolivian hemorrhagic fever, with ~20% lethality in humans. The pathogenesis of MACV infection is poorly understood, and there are no clinically proven treatments for disease. This is due, in part, to a paucity of small animal models for MACV infection in which to discover and explore candidate therapeutics. Methods Mice lacking signal transducer and activator of transcription 1 (STAT-1 were infected with MACV. Lethality, viral replication, metabolic changes, hematology, histopathology, and systemic cytokine expression were analyzed throughout the course of infection. Results We report here that STAT-1 knockout mice succumbed to MACV infection within 7-8 days, and presented some relevant clinical and histopathological manifestations of disease. Furthermore, the model was used to validate the efficacy of ribavirin in protection against infection. Conclusions The STAT-1 knockout mouse model can be a useful small animal model for drug testing and preliminary immunological analysis of lethal MACV infection.

  12. Efficient CRISPR/Cas9-based gene knockout in watermelon.

    Science.gov (United States)

    Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

    2017-03-01

    CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

  13. Proton-induced knockout reactions with polarized and unpolarized beams

    Science.gov (United States)

    Wakasa, T.; Ogata, K.; Noro, T.

    2017-09-01

    Proton-induced knockout reactions provide a direct means of studying the single particle or cluster structures of target nuclei. In addition, these knockout reactions are expected to play a unique role in investigations of the effects of the nuclear medium on nucleon-nucleon interactions as well as the properties of nucleons and mesons. However, due to the nature of hadron probes, these reactions can suffer significant disturbances from the nuclear surroundings and the quantitative theoretical treatment of such processes can also be challenging. In this article, we review the experimental and theoretical progress in this field, particularly focusing on the use of these reactions as a spectroscopic tool and as a way to examine the medium modification of nucleon-nucleon interactions. With regard to the former aspect, the review presents a semi-quantitative evaluation of these reactions based on existing experimental data. In terms of the latter point, we introduce a significant body of evidence that suggests, although does not conclusively prove, the existence of medium effects. In addition, this paper also provides information and comments on other related subjects.

  14. Bone phenotypes of P2 receptor knockout mice

    DEFF Research Database (Denmark)

    Orriss, Isabel; Syberg, Susanne; Wang, Ning

    2011-01-01

    The action of extracellular nucleotides is mediated by ionotropic P2X receptors and G-protein coupled P2Y receptors. The human genome contains 7 P2X and 8 P2Y receptor genes. Knockout mice strains are available for most of them. As their phenotypic analysis is progressing, bone abnormalities have...... been observed in an impressive number of these mice: distinct abnormalities in P2X7-/- mice, depending on the gene targeting construct and the genetic background, decreased bone mass in P2Y1-/- mice, increased bone mass in P2Y2-/- mice, decreased bone resorption in P2Y6-/- mice, decreased bone...... formation and bone resorption in P2Y13-/- mice. These findings demonstrate the unexpected importance of extracellular nucleotide signalling in the regulation of bone metabolism via multiple P2 receptors and distinct mechanisms involving both osteoblasts and osteoclasts....

  15. Antiatherogenic effects of oleanolic acid in apolipoprotein E knockout mice

    DEFF Research Database (Denmark)

    Buus, Niels Henrik; Hansson, Nicolaj Christopher; Rodriguez-Rodriguez, Rosalia

    2011-01-01

    Oleanolic acid (OA) is a plant triterpenoid steroid with potentially antiatherogenic properties. We investigated whether OA affected atherosclerosis development and vascular function in apolipoprotein E knockout (ApoE(-/-)) mice. ApoE(-/-) mice were fed a high cholesterol Western-type diet...... in combination with OA (100 mg/kg/day), fluvastatin (5 mg/kg/day) or vehicle, with wild type (WT) mice serving as controls. After 8 weeks of treatment atherosclerotic plaque areas in the aortic arch and plasma lipid concentrations were determined. Vasoconstriction and relaxation of the proximal part of aorta...... were investigated in vitro. Inducible nitric oxide synthase (iNOS) was visualized using immunoblotting. As opposed to WT and fluvastatin- and vehicle-treated mice, OA-fed ApoE(-/-) mice gained no weight during the treatment period. Plasma concentrations of total-cholesterol and triglyceride were...

  16. Patched Knockout Mouse Models of Basal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Frauke Nitzki

    2012-01-01

    Full Text Available Basal cell carcinoma (BCC is the most common human tumor. Mutations in the hedgehog (HH receptor Patched (PTCH are the main cause of BCC. Due to their high and increasing incidence, BCC are becoming all the more important for the health care system. Adequate animal models are required for the improvement of current treatment strategies. A good model should reflect the situation in humans (i.e., BCC initiation due to Ptch mutations on an immunocompetent background and should allow for (i BCC induction at a defined time point, (ii analysis of defined BCC stages, and (iii induction of BCC in 100% of animals. In addition, it should be easy to handle. Here, we compare several currently existing conventional and conditional Ptch knockout mouse models for BCC and their potential use in preclinical research. In addition, we provide new data using conditional Ptchflox/flox mice and the K5-Cre-ERT+/− driver.

  17. Drop tests of the Three Mile Island knockout canister

    International Nuclear Information System (INIS)

    Box, W.D.; Aaron, W.S.; Shappert, L.B.; Childress, P.C.; Quinn, G.J.; Smith, J.V.

    1987-01-01

    A type of Three Mile Island Unit 2 (TMI-2) defueling canister, called a ''knockout'' canister, was subjected to a series of drop tests at the Oak Ridge National Laboratory's Drop Test Facility. These tests confirmed the structural integrity of internal fixed neutron poisons in support of a request for NRC licensing of this type of canister for the shipment of TMI-2 reactor fuel debris to the Idaho National Engineering Laboratory (INEL) for the Core Examination R and D Program. This report presents the data generated and the results obtained from a series of four drop tests that included two drops with the test assembly in the vertical position and two drops with the assembly in the horizontal position

  18. Medium effects on spin observables of proton knockout reactions

    Energy Technology Data Exchange (ETDEWEB)

    Krein, G. [Instituto de Fisica Teorica (IFT), Sao Paulo, SP (Brazil); Maris, T.A.J.; Rodrigues, B.B.; Veit, E.A. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Inst. de Fisica

    1994-07-01

    Medium modifications of the properties of bound nucleons and mesons are investigated by means of medium energy quasi free proton knockout reactions with polarized incident protons. The sensitivity of the spin observables of these reactions to modifications of the nucleon and meson properties is studied using the Bonn one-boson exchange model of the nucleon-nucleon interaction. A method proposed to extract the pp analysing power in medium from the (p, 2 p) asymmetries indicates a reduction of this quantity compared to its free space value. This reduction is linked to modifications of masses and coupling constants of the nucleons and mesons in the nucleus. The implications of these modifications for another spin observable to be measured in the future are discussed. (author). 39 refs, 9 figs.

  19. Piroxicam treatment augments bone abnormalities in interleukin-10 knockout mice

    DEFF Research Database (Denmark)

    Holgersen, Kristine; Dobie, Ross; Farquharson, Colin

    2015-01-01

    BACKGROUND: Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut...... inflammation in an experimental colitis model. METHODS: Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone...... microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. RESULTS: PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia...

  20. Preaxial Polydactyly in Sost/Sostdc1 Double Knockouts

    Energy Technology Data Exchange (ETDEWEB)

    Yee, C M; Collette, N M; Loots, G G

    2011-07-29

    In the United States, {approx}5% are born with congenital birth defects due to abnormal function of cellular processes and interactions. Sclerosteosis, a rare autosomal recessive disease, causes hyperostosis of the axial and appendicular skeleton, and patients present radial deviation, digit syndactyly, nail dysplasia, and overall high bone mineral density. Sclerosteosis is due to a loss of function of sclerostin (Sost). Sost is a Wnt (abbrev.) antagonist; when mutated, nonfunctional Sost results in hyperactive osteoblast activity which leads to abnormal high bone mass. Previous studies have shown that Sost overexpression in transgenic mice causes reduced bone mineral density and a variety of limb phenotypes ranging from lost, fused, and split phalanges. Consistent with clinical manifestations of Sclerosteosis, Sost knockout mice exhibit increased generalized bone mineral density and syndactyly of the digits. Sostdc1 is a paralog of Sost that has also been described as an antagonist of Wnt signaling, in developing tooth buds. Unlike Sost knockouts, Sostdc1 null mice do not display any limb abnormalities. To determine if Sost and Sostdc1 have redundant functions during limb patterning, we examined Sost; Sostdc1 mice determined that they exhibit a novel preaxial polydactyly phenotype with a low penetrance. LacZ staining, skeletal preparations, and in situ hybridization experiments were used to help characterize this novel phenotype and understand how this phenotype develops. We find Sost and Sostdc1 to have complementary expression patterns during limb development, and the loss of their expression alters the transcription of several key limb regulators, such as Fgf8, Shh and Grem.

  1. Altered Sleep Homeostasis in Rev-erbα Knockout Mice

    Science.gov (United States)

    Mang, Géraldine M.; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A.; Albrecht, Urs; Franken, Paul

    2016-01-01

    Study Objectives: The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. Methods: EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Results: Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1–4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Conclusions: Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. Citation: Mang GM, La Spada F, Emmenegger Y, Chappuis S, Ripperger JA, Albrecht U, Franken P. Altered sleep homeostasis in Rev

  2. Immunosympathectomy as the first phenotypic knockout with antibodies.

    Science.gov (United States)

    Cattaneo, Antonino

    2013-03-26

    In a PNAS Classic Article published in 1960, Rita Levi-Montalcini offered formal and conclusive proof that endogenous NGF was responsible for the survival of sympathetic neurons in vivo. Thus ended an experimental tour de force lasting a decade, starting with the demonstration that a humoral factor, produced from a tumor transplanted in a chicken embryo, was responsible for stimulating outgrowth of nerve fibers from sympathetic and sensory neurons. From a more general methodological point of view, this work provided a breakthrough in the quest to achieve targeted loss of function and experimentally validate the function of biological molecules. Finally, this work provided an example of the ablation of a specific neuronal subpopulation in an otherwise intact nervous system, an immunological knife of unsurpassed effectiveness and precision. The novelty and the importance of the PNAS Classic Article is discussed here, collocating it within the context of the particular moment of the NGF discovery saga, of Rita Levi-Montalcini's scientific and academic career, and of the general scientific context of those years. This seminal work, involving the use of antibodies for phenotypic knockout in vivo, planted seeds that were to bear new fruit many years later with the advent of monoclonal antibodies and recombinant antibody technologies.

  3. Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

    Science.gov (United States)

    Shen, Jianbin; Yang, Maozhou; Jiang, Hong; Ju, Donghong; Zheng, Jian-Pu; Xu, Zhonghui; Liao, Tang-Dong; Li, Li

    2011-04-01

    Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

  4. Modeling fragile X syndrome in the Fmr1 knockout mouse

    Science.gov (United States)

    Kazdoba, Tatiana M.; Leach, Prescott T.; Silverman, Jill L.; Crawley, Jacqueline N.

    2014-01-01

    Summary Fragile X Syndrome (FXS) is a commonly inherited form of intellectual disability and one of the leading genetic causes for autism spectrum disorder. Clinical symptoms of FXS can include impaired cognition, anxiety, hyperactivity, social phobia, and repetitive behaviors. FXS is caused by a CGG repeat mutation which expands a region on the X chromosome containing the FMR1 gene. In FXS, a full mutation (> 200 repeats) leads to hypermethylation of FMR1, an epigenetic mechanism that effectively silences FMR1 gene expression and reduces levels of the FMR1 gene product, fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is important for the regulation of protein expression. In an effort to further understand how loss of FMR1 and FMRP contribute to FXS symptomology, several FXS animal models have been created. The most well characterized rodent model is the Fmr1 knockout (KO) mouse, which lacks FMRP protein due to a disruption in its Fmr1 gene. Here, we review the behavioral phenotyping of the Fmr1 KO mouse to date, and discuss the clinical relevance of this mouse model to the human FXS condition. While much remains to be learned about FXS, the Fmr1 KO mouse is a valuable tool for understanding the repercussions of functional loss of FMRP and assessing the efficacy of pharmacological compounds in ameliorating the molecular and behavioral phenotypes relevant to FXS. PMID:25606362

  5. Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.

    Science.gov (United States)

    Powers, J F; Evinger, M J; Tsokas, P; Bedri, S; Alroy, J; Shahsavari, M; Tischler, A S

    2000-12-01

    Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.

  6. Orexin knockout mice exhibit impaired spatial working memory.

    Science.gov (United States)

    Dang, Ruozhi; Chen, Qiuhan; Song, Jie; He, Chao; Zhang, Jun; Xia, Jianxia; Hu, Zhian

    2018-03-06

    Orexins play a crucial role in the maintenance of arousal and are involved in the modulation of diverse physiological process, including cognitive function. Recent data have suggested that orexins are involved in learning and memory processes. The purpose of this study was to assess the effects of orexin deficiency on working memory. A delayed non-matching-to-place T-maze task was used to evaluate spatial working memory in mice lacking orexin prepro-peptide (orexin knockout; KO) and wild-type controls. We demonstrated that the number of correct choices in the orexin KO mice became lower than that of the controls over training. In an object exploration task, the controls explored the displaced object more than the mutants did, whereas this difference was not observed for the nondisplaced objects in either group. The orexin KO mice showed locomotor activity comparable to the control mice in terms of total distance traveled across training in both the object exploration task and the open field test. These findings indicate that the orexin system plays an important role in working memory of spatial cues. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Bone growth and turnover in progesterone receptor knockout mice.

    Energy Technology Data Exchange (ETDEWEB)

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O' Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  8. Identification of a large set of rare complete human knockouts.

    Science.gov (United States)

    Sulem, Patrick; Helgason, Hannes; Oddson, Asmundur; Stefansson, Hreinn; Gudjonsson, Sigurjon A; Zink, Florian; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Jonasdottir, Adalbjorg; Jonasdottir, Aslaug; Sigurdsson, Asgeir; Magnusson, Olafur Th; Kong, Augustine; Helgason, Agnar; Holm, Hilma; Thorsteinsdottir, Unnur; Masson, Gisli; Gudbjartsson, Daniel F; Stefansson, Kari

    2015-05-01

    Loss-of-function mutations cause many mendelian diseases. Here we aimed to create a catalog of autosomal genes that are completely knocked out in humans by rare loss-of-function mutations. We sequenced the whole genomes of 2,636 Icelanders and imputed the sequence variants identified in this set into 101,584 additional chip-genotyped and phased Icelanders. We found a total of 6,795 autosomal loss-of-function SNPs and indels in 4,924 genes. Of the genotyped Icelanders, 7.7% are homozygotes or compound heterozygotes for loss-of-function mutations with a minor allele frequency (MAF) below 2% in 1,171 genes (complete knockouts). Genes that are highly expressed in the brain are less often completely knocked out than other genes. Homozygous loss-of-function offspring of two heterozygous parents occurred less frequently than expected (deficit of 136 per 10,000 transmissions for variants with MAF <2%, 95% confidence interval (CI) = 10-261).

  9. Improved cognitive flexibility in serotonin transporter knockout rats is unchanged following chronic cocaine self-administration

    NARCIS (Netherlands)

    Nonkes, L.J.; Maes, J.H.R.; Homberg, J.R.

    2013-01-01

    Cocaine dependence is associated with orbitofrontal cortex (OFC)-dependent cognitive inflexibility in both humans and laboratory animals. A critical question is whether cocaine self-administration affects pre-existing individual differences in cognitive flexibility. Serotonin transporter knockout

  10. Serotonin transporter knockout rats show improved strategy set-shifting and reduced latent inhibition

    NARCIS (Netherlands)

    Nonkes, L.J.P.; Vondervoort, I.I.G.M. van de; Leeuw, M.J.C. de; Wijlaars, L.P.; Maes, J.H.R.; Homberg, J.R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT−/−) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility,

  11. Serotonin transporter knockout rats show improved strategy set-shifting and reduced latent inhibition.

    NARCIS (Netherlands)

    Nonkes, L.J.P.; Vondervoort, I.I. van de; Leeuw, M.J. de; Wijlaars, L.P.; Maes, J.H.; Homberg, J.R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT(-/-)) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility,

  12. Altered Sleep Homeostasis in Rev-erbα Knockout Mice.

    Science.gov (United States)

    Mang, Géraldine M; La Spada, Francesco; Emmenegger, Yann; Chappuis, Sylvie; Ripperger, Jürgen A; Albrecht, Urs; Franken, Paul

    2016-03-01

    The nuclear receptor REV-ERBα is a potent, constitutive transcriptional repressor critical for the regulation of key circadian and metabolic genes. Recently, REV-ERBα's involvement in learning, neurogenesis, mood, and dopamine turnover was demonstrated suggesting a specific role in central nervous system functioning. We have previously shown that the brain expression of several core clock genes, including Rev-erbα, is modulated by sleep loss. We here test the consequences of a loss of REV-ERBα on the homeostatic regulation of sleep. EEG/EMG signals were recorded in Rev-erbα knockout (KO) mice and their wild type (WT) littermates during baseline, sleep deprivation, and recovery. Cortical gene expression measurements after sleep deprivation were contrasted to baseline. Although baseline sleep/wake duration was remarkably similar, KO mice showed an advance of the sleep/wake distribution relative to the light-dark cycle. After sleep onset in baseline and after sleep deprivation, both EEG delta power (1-4 Hz) and sleep consolidation were reduced in KO mice indicating a slower increase of homeostatic sleep need during wakefulness. This slower increase might relate to the smaller increase in theta and gamma power observed in the waking EEG prior to sleep onset under both conditions. Indeed, the increased theta activity during wakefulness predicted delta power in subsequent NREM sleep. Lack of Rev-erbα increased Bmal1, Npas2, Clock, and Fabp7 expression, confirming the direct regulation of these genes by REV-ERBα also in the brain. Our results add further proof to the notion that clock genes are involved in sleep homeostasis. Because accumulating evidence directly links REV-ERBα to dopamine signaling the altered homeostatic regulation of sleep reported here are discussed in that context. © 2016 Associated Professional Sleep Societies, LLC.

  13. Piroxicam treatment augments bone abnormalities in interleukin-10 knockout mice.

    Science.gov (United States)

    Holgersen, Kristine; Dobie, Ross; Farquharson, Colin; vanʼt Hof, Rob; Ahmed, Syed Faisal; Hansen, Axel Kornerup; Holm, Thomas L

    2015-02-01

    Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut inflammation in an experimental colitis model. Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia and focal transmural inflammation, which was consistent with Crohn's disease-like pathology. The gut inflammation was accompanied by a 14% and 12% reduction in trabecular thickness relative to piroxicam-treated wild type and untreated wild type mice, respectively (P < 0.001). The trabecular bone structure was also changed in PAC IL-10 k.o. mice, whereas no differences in cortical bone geometry were observed. The trabecular thickness was inversely correlated with serum levels of CTX (r = -0.93, P = 0.006). Moreover, numerous inflammatory mediators, including RANKL and osteoprotegerin, were significantly increased in the colon of PAC IL-10 k.o. mice. PAC IL-10 k.o. mice develop bone loss and changed trabecular structure, as a result of increased bone resorption. Thus, the PAC IL-10 k.o. model could be a useful experimental model in preclinical research of inflammatory bowel disease-associated bone loss.

  14. Analysis of microsatellite polymorphism in inbred knockout mice.

    Directory of Open Access Journals (Sweden)

    Baofen Zuo

    Full Text Available Previously, we found that the genotype of 42 out of 198 mouse microsatellite loci, which are distributed among all chromosomes except the Y chromosome, changed from monomorphism to polymorphism (CMP in a genetically modified inbred mouse strain. In this study, we further examined whether CMP also relates to the homologous recombination in gene knockout (KO mouse strains. The same 42 microsatellite loci were analyzed by polymerase chain reaction (PCR in 29 KO inbred mouse strains via short tandem sequence repeat (STR scanning and direct sequence cloning to justify microsatellite polymorphisms. The C57BL/6J and 129 mouse strains, from which these 29 KO mice were derived, were chosen as the background controls. The results indicated that 10 out of 42 (23.8% loci showed CMP in some of these mouse strains. Except for the trinucleotide repeat locus of D3Mit22, which had microsatellite CMP in strain number 9, the core sequences of the remaining 41 loci were dinucleotide repeats, and 9 out of 41 (21.95% showed CMPs among detected mouse strains. However, 11 out of 29 (37.9% KO mice strains were recognized as having CMPs. The popular dinucleotide motifs in CMP were (TG(n (50%, 2/4, followed by (GT(n (27.27%, 3/11 and (CA(n (23.08%, 3/13. The microsatellite CMP in (CT(n and (AG(n repeats were 20% (1/5. According to cloning sequencing results, 6 KO mouse strains showed insertions of nucleotides whereas 1 showed a deletion. Furthermore, 2 loci (D13Mit3 and D14Mit102 revealed CMP in 2 strains, and mouse strain number 9 showed CMPs in two loci (D3Mit22 and D13Mit3 simultaneously. Collectively, these results indicated that microsatellite polymorphisms were present in the examined inbred KO mice.

  15. Osteoprotegerin-Knockout Mice Developed Early Onset Root Resorption.

    Science.gov (United States)

    Liu, Yi; Du, Haiming; Wang, Yunfei; Liu, Mengmeng; Deng, Shijian; Fan, Linlin; Zhang, Lili; Sun, Yao; Zhang, Qi

    2016-10-01

    Recent studies indicate that the osteoprotegerin (OPG)/RANKL/RANK pathway takes part in root resorption. However, the relationship between OPG and root resorption is vague. The purpose of our study was to investigate the role of OPG in root resorption. The first molars of the mandibles of osteoprotegerin-knockout (Opg-KO) mice and wild-type (WT) mice were evaluated by micro-computed tomography, histology, and immunohistochemistry at 4, 6, 26, and 52 weeks. To detect the activity of the osteoclasts, we induced bone marrow macrophages into osteoclast-like cells from Opg-KO mice and wild-type mice in vitro and then compared their osteoclast activities. To evaluate the cementum quality, an osteoclast-cementum co-culture model was established in vitro. In Opg-KO mice, root resorption began at the age of 4 weeks. At 6 weeks the cementum damage extended to the coronal and apical regions, and at 52 weeks the damage reached the predentin. At all observed stages, more tartrate-resistant acid phosphatase (TRAP)-positive cells were found on the surface of cementum in Opg-KO mice. In vitro, the mRNA levels of cathepsin K, TRAP, matrix metalloproteinase-9, and matrix metalloproteinase-1, as well as the protein expression of nuclear factor of activated T cell 1 and TRAP, increased significantly in osteoclast-like cells from Opg-KO mice. In addition, the cementum resorption pits of Opg-KO mice were larger when co-cultured with osteoclast-like cells. Our study demonstrated that loss of OPG led to root resorption via increasing activation of osteoclasts and reducing mineralization of cementum. Copyright © 2016. Published by Elsevier Inc.

  16. Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Hanneke Korsten

    Full Text Available Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m developed at older age (>10m into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC, adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK, and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7-8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1 and tumor class 2 (TC2. TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma/intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor

  17. Maximal Oxygen Consumption is Reduced in Aquaporin-1 Knockout Mice

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    Samer Al-Samir

    2016-08-01

    Full Text Available We have measured maximal oxygen consumption (V’O2,max of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9 and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V’O2,max as determined by the Helox technique is reduced by ~ 16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V’O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2 by pulse oximetry. Neither under normoxic (inspiratory O2 21% nor under hypoxic conditions (11% O2 is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V’O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V’O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V’O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V’O2,max, which constitutes a new phenotype of these mice.

  18. Elevated body temperature during sleep in orexin knockout mice

    Science.gov (United States)

    Mochizuki, Takatoshi; Klerman, Elizabeth B.; Sakurai, Takeshi; Scammell, Thomas E.

    2008-01-01

    Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is thought to promote heat production during wakefulness, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, the Tb of orexin KO mice was 0.4°C higher than wild-type (WT) littermates during the dark period. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During sustained wakefulness, Tb was the same in both groups. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0°C, but Tb in orexin KO mice decreased only 0.4°C. Even during intense recovery sleep after 8 hr of sleep deprivation, the Tb of orexin KO mice remained 0.7°C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy. PMID:16556901

  19. Salty taste deficits in CALHM1 knockout mice.

    Science.gov (United States)

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Sildenafil restores endothelial function in the apolipoprotein E knockout mouse

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    Balarini Camille M

    2013-01-01

    Full Text Available Abstract Background Atherosclerosis is an inflammatory process of the arterial walls and is initiated by endothelial dysfunction accompanied by an imbalance in the production of reactive oxygen species (ROS and nitric oxide (NO. Sildenafil, a selective phosphodiesterase-5 (PDE5 inhibitor used for erectile dysfunction, exerts its cardiovascular effects by enhancing the effects of NO. The aim of this study was to investigate the influence of sildenafil on endothelial function and atherosclerosis progression in apolipoprotein E knockout (apoE−/− mice. Methods ApoE−/− mice treated with sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage were compared to the untreated apoE−/− and the wild-type (WT mice. Aortic rings were used to evaluate the relaxation responses to acetylcholine (ACh in all of the groups. In a separate set of experiments, the roles of NO and ROS in the relaxation response to ACh were evaluated by incubating the aortic rings with L-NAME (NO synthase inhibitor or apocynin (NADPH oxidase inhibitor. In addition, the atherosclerotic lesions were quantified and superoxide production was assessed. Results Sildenafil restored the vasodilator response to acetylcholine (ACh in the aortic rings of the apoE−/− mice. Treatment with L-NAME abolished the vasodilator responses to ACh in all three groups of mice and revealed an augmented participation of NO in the endothelium-dependent vasodilation in the sildenafil-treated animals. The normalized endothelial function in sildenafil-treated apoE−/− mice was unaffected by apocynin highlighting the low levels of ROS production in these animals. Moreover, morphological analysis showed that sildenafil treatment caused approximately a 40% decrease in plaque deposition in the aorta. Conclusion This is the first study demonstrating the beneficial effects of chronic treatment with sildenafil on endothelial dysfunction and atherosclerosis in a model of spontaneous

  1. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

    Science.gov (United States)

    Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

    2015-06-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

    Science.gov (United States)

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Sakai, Keisuke; Nakayama, Kazuhiko; Emoto, Noriaki; Yanagisawa, Masashi; Ihn, Hironobu

    2014-01-01

    Endothelin (ET)-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF)-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF)-α and connective tissue growth factor (CTGF) were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  3. Impaired social behavior in 5-HT3A receptor knockout mice

    Directory of Open Access Journals (Sweden)

    Laura A Smit-Rigter

    2010-11-01

    Full Text Available The 5-HT3 receptor is a ligand-gated ion channel expressed on interneurons throughout the brain. So far, analysis of the 5-HT3A knockout mouse revealed changes in nociceptive processing and a reduction in anxiety related behavior. Recently, it was shown that the 5-HT3 receptor is also expressed on Cajal-Retzius cells which play a key role in cortical development and that knockout mice lacking this receptor showed aberrant growth of the dendritic tree of cortical layer II/III pyramidal neurons. Other mouse models in which serotonergic signaling was disrupted during development showed similar morphological changes in the cortex, and in addition, also deficits in social behavior. Here, we subjected male and female 5-HT3A knockout mice and their non-transgenic littermates to several tests of social behavior. We found that 5-HT3A knockout mice display impaired social communication in the social transmission of food preference task. Interestingly, we showed that in the social interaction test only female 5-HT3A knockout mice spent less time in reciprocal social interaction starting after 5 minutes of testing. Moreover, we observed differences in preference for social novelty for male and female 5-HT3A knockout mice during the social approach test. However, no changes in olfaction, exploratory activity and anxiety were detected. These results indicate that the 5-HT3A knockout mouse displays impaired social behavior with specific changes in males and females, reminiscent to other mouse models in which serotonergic signaling is disturbed in the developing brain.

  4. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

    Directory of Open Access Journals (Sweden)

    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  5. Diacylglycerol lipase a knockout mice demonstrate metabolic and behavioral phenotypes similar to those of cannabinoid receptor 1 knockout mice

    Directory of Open Access Journals (Sweden)

    David R Powell

    2015-06-01

    Full Text Available After creating >4650 knockouts (KOs of independent mouse genes, we screened them by high-throughput phenotyping and found that cannabinoid receptor 1 (Cnr1 KO mice had the same lean phenotype published by others. We asked if our KOs of DAG lipase a or b (Dagla or Daglb, which catalyze biosynthesis of the endocannabinoid (EC 2-Arachidonoylglycerol (2-AG, or Napepld, which catalyzes biosynthesis of the EC anandamide, shared the lean phenotype of Cnr1 KO mice. We found that Dagla KO mice, but not Daglb or Napepld KO mice, were among the leanest of 3651 chow-fed KO lines screened. In confirmatory studies, chow- or high fat diet-fed Dagla and Cnr1 KO mice were leaner than wild type (WT littermates; when data from multiple cohorts of adult mice were combined, body fat was 47% and 45% lower in Dagla and Cnr1 KO mice, respectively, relative to WT values. In contrast, neither Daglb nor Napepld KO mice were lean. Weanling Dagla KO mice ate less than WT mice and had body weight similar to pair-fed WT mice, and adult Dagla KO mice had normal activity and VO2 levels, similar to Cnr1 KO mice. Our Dagla and Cnr1 KO mice also had low fasting insulin, triglyceride and total cholesterol levels, and after a glucose challenge had normal glucose but very low insulin levels. Dagla and Cnr1 KO mice also showed similar responses to a battery of behavioral tests. These data suggest: 1 the lean phenotype of young Dagla and Cnr1 KO mice is mainly due to hypophagia; 2 in pathways where ECs signal through Cnr1 to regulate food intake and other metabolic and behavioral phenotypes observed in Cnr1 KO mice, Dagla alone provides the 2-AG that serves as the EC signal; and 3 small molecule Dagla inhibitors with a pharmacokinetic profile similar to that of Cnr1 inverse agonists are likely to mirror the ability of these Cnr1 inverse agonists to lower body weight and improve glycemic control in obese patients with type 2 diabetes, but may also induce undesirable neuropsychiatric

  6. The Knockout of Secretin in Cerebellar Purkinje Cells Impairs Mouse Motor Coordination and Motor Learning

    Science.gov (United States)

    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-01-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells. PMID:24356714

  7. Knockouts of high-ranking males have limited impact on baboon social networks.

    Science.gov (United States)

    Franz, Mathias; Altmann, Jeanne; Alberts, Susan C

    Social network structures can crucially impact complex social processes such as collective behaviour or the transmission of information and diseases. However, currently it is poorly understood how social networks change over time. Previous studies on primates suggest that `knockouts' (due to death or dispersal) of high-ranking individuals might be important drivers for structural changes in animal social networks. Here we test this hypothesis using long-term data on a natural population of baboons, examining the effects of 29 natural knockouts of alpha or beta males on adult female social networks. We investigated whether and how knockouts affected (1) changes in grooming and association rates among adult females, and (2) changes in mean degree and global clustering coefficient in these networks. The only significant effect that we found was a decrease in mean degree in grooming networks in the first month after knockouts, but this decrease was rather small, and grooming networks rebounded to baseline levels by the second month after knockouts. Taken together our results indicate that the removal of high-ranking males has only limited or no lasting effects on social networks of adult female baboons. This finding calls into question the hypothesis that the removal of high-ranking individuals has a destabilizing effect on social network structures in social animals.

  8. Stabilization of tooth movement by administration of reveromycin A to osteoprotegerin-deficient knockout mice.

    Science.gov (United States)

    Yabumoto, Takahiro; Miyazawa, Ken; Tabuchi, Masako; Shoji, Satsuki; Tanaka, Miyuki; Kadota, Manami; Yoshizako, Mamoru; Kawatani, Makoto; Osada, Hiroyuki; Maeda, Hatsuhiko; Goto, Shigemi

    2013-09-01

    In this study, mechanical stress in the form of tooth movement was applied to osteoprotegerin-deficient knockout mice, which served as an animal model for juvenile Paget's disease. To compare and evaluate bone turnover and response of the surrounding bony tissue, we administered reveromycin A. We also investigated the ability of reveromycin A to control osteoclastic activity in juvenile Paget's disease. Eight-week-old male osteoprotegerin-deficient knockout and wild-type mice were injected with reveromycin A (15 mg/kg of body weight) intraperitoneally twice daily. An elastic module was inserted interproximally between the maxillary left first and second molars. Administration of reveromycin A to osteoprotegerin-deficient knockout mice reduced tooth movement distances, increased bone volumes at the interradicular septum, decreased osteoclast counts, and reduced serum alkaline phosphatase and tartrate resistant acid phosphatase. Reveromycin A administration also caused a temporal shift in peak Runx2 staining in osteoprotegerin-deficient knockout mice so that the overall staining time course was similar to that observed for wild-type mice. Reveromycin A administration in osteoprotegerin-deficient knockout mice inhibited bone resorption and normalized bone formation. As a result, normal bone turnover was obtained. Copyright © 2013 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

  9. Gene Knockout Identification Using an Extension of Bees Hill Flux Balance Analysis

    Directory of Open Access Journals (Sweden)

    Yee Wen Choon

    2015-01-01

    Full Text Available Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes.

  10. High-temperature expansion and knock-out properties of moulding sands with water glass

    Directory of Open Access Journals (Sweden)

    Major-Gabryś K.

    2007-01-01

    Full Text Available The article focuses on the topic of improving the knock-out properties of moulding sand with water glass and ester hardener. It is settled that the cause of worse knock-out properties of moulding sand can be brought by their thermal expansion in increased temperatures. There is a presentation of the influence of different additives, containing Al2O3, on moulding sands’ expansion in increased temperatures. Within the frames of research, there was an elaboration of the influence of authors own additive- Glassex, on the expansion phenomenon of moulding sands with water glass and ester hardener. It is concluded, that the new additive stops the expansion of moulding sands and as well it improves their knock-out properties.

  11. Impaired water maze learning performance in mu-opioid receptor knockout mice.

    Science.gov (United States)

    Jang, Choon-Gon; Lee, Seok-Yong; Yoo, Ji-Hoon; Yan, Ji-Jing; Song, Dong-Keun; Loh, Horace H; Ho, Ing K

    2003-09-10

    Previous study has demonstrated that the lack of mu-opioid receptor decreased LTP in the dentate gyrus of the hippocampus, suggesting the possibility that the lack of mu-opioid receptor may accompany a change in learning and memory. However, no behavioral study has been undertaken to correlate LTP deficits with spatial memory impairment in mu-opioid receptor knockout mice. Therefore, the present study investigated the hypothesis that mu-opioid receptors contribute to learning and memory by using the Morris water maze, and comparing responses in wild type and mu-opioid receptor gene knockout mice. Our results indicated that mu-opioid receptor knockout mice showed a significant spatial memory impairment compared to wild type in the Morris water maze. This result suggests that the expression of mu-opioid receptor plays an important role in spatial learning and memory examined by Morris water maze.

  12. Altered metabolism in the melatonin-related receptor (GPR50) knockout mouse.

    Science.gov (United States)

    Ivanova, Elena A; Bechtold, David A; Dupré, Sandrine M; Brennand, John; Barrett, Perry; Luckman, Simon M; Loudon, Andrew S I

    2008-01-01

    The X-linked orphan receptor GPR50 shares 45% homology with the melatonin receptors, yet its ligand and physiological function remain unknown. Here we report that mice lacking functional GPR50 through insertion of a lacZ gene into the coding sequence of GPR50 exhibit an altered metabolic phenotype. GPR50 knockout mice maintained on normal chow exhibit lower body weight than age-matched wild-type littermates by 10 wk of age. Furthermore, knockout mice were partially resistant to diet-induced obesity. When placed on a high-energy diet (HED) for 5 wk, knockout mice consumed significantly more food per unit body weight yet exhibited an attenuated weight gain and reduced body fat content compared with wild-type mice. Wheel-running activity records revealed that, although GPR50 knockout mice showed no alteration of circadian period, the overall levels of activity were significantly increased over wild types in both nocturnal and diurnal phases. In line with this, basal metabolic rate (O2 consumption, CO2 production, and respiratory quotient) was found to be elevated in knockout mice. Using in situ hybridization (wild-type mice) and beta-galactosidase activity (from LacZ insertion element in knockout mice), brain expression of GPR50 was found to be restricted to the ependymal layer of the third ventricle and dorsomedial nucleus of the hypothalamus. GPR50 expression was highly responsive to energy status, showing a significantly reduced expression following both fasting and 5 wk of HED. These data implicate GPR50 as an important regulator of energy metabolism.

  13. Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

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    Traver Hart

    2017-08-01

    Full Text Available The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3 library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

  14. Hyperactivity of Newborn Pten Knock-out Neurons Results from Increased Excitatory Synaptic Drive

    Science.gov (United States)

    Williams, Michael R.; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T.

    2015-01-01

    Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either “birthdate” or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. PMID:25609613

  15. Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

    Science.gov (United States)

    Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

    2017-01-01

    The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

  16. Data on the effect of knockout of cytohesin-1 in myelination-related protein kinase signaling.

    Science.gov (United States)

    Tsuneishi, Ruri; Matsumoto, Naoto; Itaoka, Misa; Urai, Yuri; Kaneko, Minami; Watanabe, Natsumi; Takashima, Shou; Seki, Yoichi; Morimoto, Takako; Sakagami, Hiroyuki; Miyamoto, Yuki; Yamauchi, Junji

    2017-12-01

    Cytohesin-1 is the guanine-nucleotide exchange factor of Arf6, a small GTPase of Arf family, and participates in cellular morphological changes. Knockout mice of cytohesin-1 exhibit decreased myelination of neuronal axons in the peripheral nervous system (PNS) "Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2012) [1]". Herein we provide the data regarding decreased phosphorylation levels of protein kinases involved in two major myelination-related kinase cascades in cytohesin-1 knockout mice.

  17. A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

    Science.gov (United States)

    Zhao, Cui; Zhang, Chen; Chen, Bin; Shi, Yanghui; Quan, Yanping; Nie, Zuoming; Zhang, Yaozhou; Yu, Wei

    2016-01-01

    A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (pviral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

  18. Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

    Science.gov (United States)

    McDaniel, Allison L.; Alger, Heather M.; Sawyer, Janet K.; Kelley, Kathryn L.; Kock, Nancy D.; Brown, J. Mark; Temel, Ryan E.; Rudel, Lawrence L.

    2014-01-01

    Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels. PMID:23380580

  19. Age-Related Changes in the Behavior of Apolipoprotein E Knockout Mice.

    Science.gov (United States)

    Fuentes, Dasha; Fernández, Nidia; García, Yenela; García, Teidy; Morales, Ana Ruth; Menéndez, Roberto

    2018-03-03

    The knockout mouse model, B6.129P2-Apoe tm1Unc is homozygotic for the Apolipoprotein E (ApoE) deletion; thus, it is capable of developing hyperlipidemia and atherosclerosis but ApoE is also a lipid-transport protein abundantly expressed in most neurons in the central nervous system, so these animals could also be models of neurodegenerative diseases. The aim of this study was to determine age-related changes in spontaneous behavior and in learning and memory of Apolipoprotein E knockout mice. Spontaneous behavioral measurements included sleeping pattern, motor coordination and balance by rotarod and open field activity, whereas learning and memory tests included forced alternation in Y-maze, novel object recognition and passive avoidance conditioning. Significant behavioral differences between aged knockout mice and age-matched wild type strain, C57Bl/6 were found in all the behavioral tests, except for the rotarod test. Genetically' modified mice exhibited less huddling contact during sleeping, decreased locomotor activity in novel environments and in learning and memory deficits. These results are consistent with the cognitive impairment and memory loss seen as the earliest clinical symptoms in neurodegenerative disorders such as Alzheimer's disease. The ApoE knockout mice might therefore be an appropriate model for studying the underlying mechanisms involved in behavioral changes caused by neurodegenerative diseases as well as for evaluating new therapies for these pathologies.

  20. Generation and basic characterization of glutamate carboxypeptidase II knock-out mice

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, Petr; Šácha, Pavel; Sedláček, Radislav; Konvalinka, Jan

    2016-01-01

    Roč. 25, č. 2 (2016), s. 267 ISSN 0962-8819. [Transgenic Technology Meeting (TT2016) /13./. 20.03.2016-23.03.2016, Praha] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : GCPII * PSMA * FolhI * knock-out mice Subject RIV: CE - Biochemistry

  1. Revealing novel functions of glutamate carboxypeptidase II using knock-out mice

    Czech Academy of Sciences Publication Activity Database

    Vorlová, Barbora; Kašpárek, P.; Šácha, Pavel; Sedláček, R.; Konvalinka, Jan

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 358 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] Institutional support: RVO:61388963 Keywords : glutamate carboxypeptidase II * knock-out mice Subject RIV: CE - Biochemistry

  2. Efficient target-selected mutagenesis in Caenorhabditis elegans : toward a knockout for every gene

    NARCIS (Netherlands)

    Cuppen, Edwin; Gort, Eelke; Hazendonk, Esther; Mudde, Josine; van de Belt, José; Nijman, Isaäc J; Guryev, Victor; Plasterk, Ronald H A

    Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we

  3. Mig-6 Gene Knockout Induces Neointimal Hyperplasia in the Vascular Smooth Muscle Cell

    Directory of Open Access Journals (Sweden)

    Ju Hee Lee

    2014-01-01

    Full Text Available Although advances in vascular interventions can reduce the mortality associated with cardiovascular disease, neointimal hyperplasia remains a clinically significant obstacle limiting the success of current interventions. Identification of signaling pathways involved in migration and proliferation of vascular smooth muscle cells (SMCs is an important approach for the development of modalities to combat this disease. Herein we investigate the role of an immediate early response gene, mitogen-inducible gene-6 (Mig-6, in the development of neointimal hyperplasia using vascular smooth muscle specific Mig-6 knockout mice. We induced endoluminal injury to one side of femoral artery by balloon dilatation in both Mig-6 knockout and control mice. Four weeks following injury, the artery of Mig-6 knockout mice demonstrated a 5.3-fold increase in the neointima/media ratio compared with control mice (P=0.04. In addition, Mig-6 knockout vascular SMCs displayed an increase in both cell migration and proliferation compared with wild-type SMCs. Taken together, our data suggest that Mig-6 plays a critical role in the development of atherosclerosis. This finding provides new insight into the development of more effective ways to treat and prevent neointimal hyperplasia, particularly in-stent restenosis after percutaneous vascular intervention.

  4. Hematopoiesis in 5-Fluorouracil-Treated Adenosine A(3) Receptor Knock-Out Mice

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Komůrková, Denisa

    2015-01-01

    Roč. 64, č. 2 (2015), s. 255-262 ISSN 0862-8408 Institutional support: RVO:68081707 Keywords : Adenosine A(3) receptor knock-out mice * Hematopoiesis * 5-fluorouracil-induced hematotoxicity Subject RIV: BO - Biophysics Impact factor: 1.643, year: 2015

  5. Pharmacological treatment of fragile X syndrome with GABAergic drugs in a knockout mouse model

    NARCIS (Netherlands)

    Heulens, Inge; D'Hulst, Charlotte; Van Dam, Debby; De Deyn, Peter P.; Kooy, R. Frank

    2012-01-01

    Molecular and electrophysiological studies have provided evidence for a general downregulation of the GABAergic system in the Fmr1 knockout mouse. GABA(A) receptors are the main inhibitory receptors in the brain and the GABA(A) receptor was proposed as a novel target for treatment of the fragile X

  6. Probing the structure of unstable nuclei through the recoiled proton tagged knockout reaction

    International Nuclear Information System (INIS)

    Ye, Y.; Cao, Z.; Jiang, D.

    2010-01-01

    Recoiled proton tagged knockout reaction experiments were carried-out for 8 He at 82,5 MeV/u in RIKEN and for 6 He at 65 MeV/u in Lanzhou. The very preliminary results for the distinguish of the reaction mechanism are presented and compared to the kinematics calculation. (authors)

  7. Hyperactivity and lack of social discrimination in the adolescent Fmr1 knockout mouse

    DEFF Research Database (Denmark)

    Sørensen, Emilie M; Bertelsen, Freja; Weikop, Pia

    2015-01-01

    The aims of this study were to investigate behaviour relevant to human autism spectrum disorder (ASD) and the fragile X syndrome in adolescent Fmr1 knockout (KO) mice and to evaluate the tissue levels of striatal monoamines. Fmr1 KO mice were evaluated in the open field, marble burying and three...

  8. Myocardial ischemia and reperfusion injury: Studies using transgenic and knockout mice

    NARCIS (Netherlands)

    Jong, W. M. C.; ten Cate, H.; Reitsma, P. H.; de Winter, R. J.

    2005-01-01

    Transgenic and knockout mice are created and used for a large variety of research objectives. This overview describes the (genetically modified) mouse models that have been used to study the development of myocardial ischemia and reperfusion injury. The role of cytokines, chemokines, leukocytes,

  9. Serotonin Transporter Knockout Rats Show Improved Strategy Set-Shifting and Reduced Latent Inhibition

    Science.gov (United States)

    Nonkes, Lourens J. P.; van de Vondervoort, Ilse I. G. M.; de Leeuw, Mark J. C.; Wijlaars, Linda P.; Maes, Joseph H. R.; Homberg, Judith R.

    2012-01-01

    Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT[superscript -/-]) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting…

  10. Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

    DEFF Research Database (Denmark)

    Zhang, Yao; Cheng, Yajun; Hansen, Gert H

    2011-01-01

    Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT...

  11. Cross sections for one-neutron knock-out from Ca-37 at intermediate energy

    Czech Academy of Sciences Publication Activity Database

    Burger, A.; Azaiez, F.; Algora, A.; Al-Khatib, A.; Bastin, B.; Benzoni, G.; Borcea, R.; Bourgeois, C.; Bringel, P.; Clement, E.; Dalouzy, J.-C.; Dlouhý, Zdeněk; Dombradi, Z.; Drouart, A.; Engelhardt, C.; Franchoo, S.; Fülöp, Zs.; Gorgen, A.; Grévy, S.; Hubel, H.; Ibrahim, F.; Korten, W.; Mrázek, Jaromír; Navin, A.; Rotaru, F.; Chomaz, P. R.; Saint-Laurent, M. G.; Sletten, G.; Sohler, D.; Sorlin, O.; Stanoiu, M.; Stefan, I.; Theisen, C.; Timis, C.; Verney, D.; Williams, S.

    2012-01-01

    Roč. 86, č. 6 (2012), 064609/1-064609/5 ISSN 0556-2813 Institutional support: RVO:61389005 Keywords : nuclear structure * knock-out reactions * shell model Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders Impact factor: 3.715, year: 2012 http://prc.aps.org/pdf/PRC/v86/i6/e064609

  12. Behavior training reverses asymmetry in hippocampal transcriptome of the cav3.2 knockout mice.

    Directory of Open Access Journals (Sweden)

    Ni-Chun Chung

    Full Text Available Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome.

  13. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

    NARCIS (Netherlands)

    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  14. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

    Science.gov (United States)

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher

    2015-05-19

    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Altered sleep and affect in the neurotensin receptor 1 knockout mouse.

    Science.gov (United States)

    Fitzpatrick, Karrie; Winrow, Christopher J; Gotter, Anthony L; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H; Renger, John J; Turek, Fred W

    2012-07-01

    Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders.

  16. Sdhd and SDHD/H19 knockout mice do not develop paraganglioma or pheochromocytoma.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Bayley

    Full Text Available BACKGROUND: Mitochondrial succinate dehydrogenase (SDH is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL and pheochromocytoma (PC. SDHD is remarkable in showing an 'imprinted' tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. CONCLUSIONS: Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.

  17. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

    2005-01-01

    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  18. Highly segmented CVD diamond detectors and high-resolution momentum measurements in knockout reactions; Hochsegmentierte CVD Diamant Detektoren und hochaufloesende Impulsmessungen in Knockout Reaktionen

    Energy Technology Data Exchange (ETDEWEB)

    Schwertel, Sabine

    2009-11-26

    In recent years knockout reactions have proven to be important tools for investigations of the structure of light exotic nuclei. In spring 2006 an experiment was performed with the fragment separator at GSI in Darmstadt to extend this method to medium-mass nuclei with energies of about 400 AMeV. An experiment with a stable and well-known {sup 48}Ca primary beam was performed as a reference. The FRS was set for the reaction {sup 56}Ti{yields}{sup 55}Ti. Because of the high acceptance of the FRS, mother and daughter nuclei of one-neutron knockout reactions in the Sc isotopes {sup 51,52,53,54,55}Sc were also transported with high efficiency. These are investigated in the first part of this thesis. Inclusive cross sections of 77(10) mbarn for one-neutron knockout from {sup 48}Ca and 78(12) mbarn, 99(15) mbarn, 101(15) mbarn, 113(17) mbarn and 72(14) mbarn for knockout from {sup 51,52,53,54,55}Sc, respectively, were measured for the first time. For the Sc isotopes the reduction factors are close to 1. For the one-neutron knockout reactions in {sup 48}Ca and the Sc isotopes, respectively, the momentum distributions could be measured with a relative resolution of 0.17-0.27 %. From the momentum distributions spectroscopic factors of the involved orbitals could be extracted. In the future, further knockout experiments should be performed using the R{sup 3}B setup at FAIR. The available beam intensity will be up to four orders of magnitude higher. As the beam has to be tracked from the dispersive plane of the Super-FRS up to the R{sup 3}B target, radiation hard detectors are needed. In the framework of this thesis extensive measurements were performed at the tandem accelerator in Munich with numerous small (10 x 10 mm{sup 2}) test detectors. Samples using new manufacturing methods were characterized. A dose of some 10{sup 11} ions/mm{sup 2} was determined as a limit for the exposure of the material with heavy ions of high ionisation density. It could be shown that even

  19. Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

    Science.gov (United States)

    Denecke, Shane; Fusetto, Roberto; Batterham, Philip

    2017-12-01

    ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available BACKGROUND Tuberculosis (TB is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH, encoded by iunH gene (Rv3393, is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a

  1. Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

    Directory of Open Access Journals (Sweden)

    Anne Drumond Villela

    Full Text Available Cytidine deaminase (MtCDA, encoded by cdd gene (Rv3315c, is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

  2. Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

    DEFF Research Database (Denmark)

    Walls, Anne Byriel; Eyjolfsson, Elvar M.; Smeland, Olav B.

    2011-01-01

    γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD......65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-(13)C]glucose and the astrocyte-specific substrate [1,2-(13)C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses...... was further investigated in GAD65 knockout and wild-type mice using [1,2-(13)C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex...

  3. Generation of knockout rabbits using transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  4. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Anna Osiak

    Full Text Available Gene knockout in murine embryonic stem cells (ESCs has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs. Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  5. Impairments in the initiation of maternal behavior in oxytocin receptor knockout mice.

    Directory of Open Access Journals (Sweden)

    Megan E Rich

    Full Text Available Oxytocin (Oxt acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt -/- mice, forebrain conditional Oxtr knockout (Oxtr FB/FB mice, and total body Oxtr knockout (Oxtr -/- mice. Since Oxtr -/- mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr -/- mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples of Oxtr +/+ mice and sham-thelectomizing Oxtr -/- mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr -/- dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr -/- dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr -/- dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr -/- dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior.

  6. Memory Impairment in Estrogen Receptor ? Knockout Mice Through Accumulation of Amyloid-? Peptides

    OpenAIRE

    Hwang, Chul Ju; Yun, Hyung-Mun; Park, Kyung-Ran; Song, Ju Kyung; Seo, Hyun Ok; Hyun, Byung Kook; Choi, Dong Young; Yoo, Hwan-Soo; Oh, Ki-Wan; Hwang, Dae Yeun; Han, Sang-Bae; Hong, Jin Tae

    2014-01-01

    Estrogen has been known to reduce the development of Alzheimer?s disease (AD). However, exact mechanisms are not clear. We investigated whether estrogen can increase amyloid-beta (A?) degradation and affects A?-induced memory impairment in an estrogen deficiency model. Estrogen receptor alpha (ER?) knockout mice and wild-type mice were intracerebroventricular (ICV) infused with A? (300?pmol) for 2?weeks. Cognitive function was then assessed by the Morris water maze test and passive avoidance ...

  7. [Propagation of prdm1 gene knockout mouse and its genotype identification].

    Science.gov (United States)

    Lu, Xiao-Yun; Chen, Chong; Pan, Xiu-Ying; Zeng, Ling-Yu; Li, Zhen-Yu; Song, Xu-Guang; Xu, Kai-Lin

    2012-08-01

    This study was aimed to propagate and identify the prdm1 gene-knockout mice, so as to lay the foundation for studying Blimp-1 protein. Two kinds of transgenic homozygous mice with B6.prdm1(flox/flox) and B6.Lck-Cre were feed and propagated; after successful propagating, the first passage mice were obtained; after the first passage mice were copulated once again, the genotypes were obtained as follows: B6. prdm1(wild/wild). Lck-Cre, B6. prdm1(wild/wild), B6.prdm1(flox/flox). Lck-Cre, B6.prdm1(flox/wild). Lck-Cre, B6.prdm1(flox/flox), B6. prdm1(flox/wild). The genomic DNA of second passage mice was extracted, the Cre and loxp gene fragments were amplified by PCR, then the size of Cre and loxp genomic DNA were detected by agarose gel electrophoresis. The mice with B6.prdm1(flow/flox). Lek-Cre were used as conditionally prdm1-knockout mice, B6.prdm1(flox/wild). Lck-Cre mice, B6.prdm1(flox/flox) and B6 mice were used as controls. The spleen T lymphocytes and B lymphocytes were sorted by using magnetic beads, the blimp-1 target protein was identified by Western blot. The results showed that the two transgenic homozygous mice had the ability to reproduce, and the separation ratio of second passage mice generated from propagation of their offspring cach other meet Mendelian laws, and the prdm1 gene-knockout mice also could successfully obtained. It is concluded that the application of Cre-loxp system may successfully obtain plentiful prdm1 gene-knockout mice.

  8. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

    Science.gov (United States)

    Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

  9. Fetal growth retardation and lack of hypotaurine in ezrin knockout mice.

    Directory of Open Access Journals (Sweden)

    Tomohiro Nishimura

    Full Text Available Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/- were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine and taurine were not affected. Lack of hypotaurine in Ez(-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

  10. Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum.

    Science.gov (United States)

    Zhang, Yali; Yu, Mingrui; Yang, Shang-Tian

    2012-01-01

    Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3-53.8% and ≈ 11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28-35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h(-1) vs. 0.15 h(-1) on glucose and 0.14 h(-1) vs. 0.10 h(-1) on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26-48% increase for butyrate, 81-100% increase for acetate, and 38-46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  11. Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice.

    Science.gov (United States)

    Wood, Tyler T; Winden, Duane R; Marlor, Derek R; Wright, Alex J; Jones, Cameron M; Chavarria, Michael; Rogers, Geraldine D; Reynolds, Paul R

    2014-11-15

    The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in proinflammatory signaling, and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short-term secondhand smoke (SHS) exposure in RAGE knockout and control mice compared with identical animals exposed to room air only. Quantitative PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after 4 wk of SHS exposure and an anticipated absence of RAGE expression in RAGE knockout mice regardless of smoke exposure. Ras activation, NF-κB activity, and cytokine elaboration were assessed to characterize the molecular basis of SHS-induced inflammation in the mouse lung. Furthermore, bronchoalveolar lavage fluid was procured from RAGE knockout and control animals for the assessment of inflammatory cells and molecules. As a general theme, inflammation coincident with leukocyte recruitment was induced by SHS exposure and significantly influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to SHS. However, ongoing research is still warranted to fully explain roles for RAGE and other receptors in cells coping with involuntary smoke exposure for prolonged periods of time. Copyright © 2014 the American Physiological Society.

  12. Impact of temporal variation on design and analysis of mouse knockout phenotyping studies.

    Directory of Open Access Journals (Sweden)

    Natasha A Karp

    Full Text Available A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies.

  13. Proximal gut mucosal epithelial homeostasis in aged IL-1 type I receptor knockout mice after starvation.

    Science.gov (United States)

    Song, Juquan; Wolf, Steven E; Wu, Xiao-Wu; Finnerty, Celeste C; Herndon, David N; Jeschke, Marc G

    2011-08-01

    Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. Aged IL-1R knockout mice were larger than aged-matched wild-type mice (P starvation (P starvation (P Starvation decreased cell proliferation in IL-1R knockout mice (P starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. FANCA knockout in human embryonic stem cells causes a severe growth disadvantage.

    Science.gov (United States)

    Vanuytsel, Kim; Cai, Qing; Nair, Nisha; Khurana, Satish; Shetty, Swati; Vermeesch, Joris R; Ordovas, Laura; Verfaillie, Catherine M

    2014-09-01

    Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. Copyright © 2014. Published by Elsevier B.V.

  15. Myo5b knockout mice as a model of microvillus inclusion disease

    Science.gov (United States)

    Cartón-García, Fernando; Overeem, Arend W.; Nieto, Rocio; Bazzocco, Sarah; Dopeso, Higinio; Macaya, Irati; Bilic, Josipa; Landolfi, Stefania; Hernandez-Losa, Javier; Schwartz, Simo; Ramon y Cajal, Santiago; van Ijzendoorn, Sven C. D.; Arango, Diego

    2015-01-01

    Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches. PMID:26201991

  16. Knockout of the Nogo-B Gene Attenuates Tumor Growth and Metastasis in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Bo Zhu

    2017-07-01

    Full Text Available Human hepatocellular carcinoma (HCC is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4 family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells. Nogo-B knockout, via the CRISPR-Cas9 technique, resulted in significant suppression of HCC cell proliferation and tumor growth. Next-generation sequencing analysis showed that Nogo-B knockout have effects on interleukin-6 (IL-6 signaling pathway. Furthermore, we observed that IL-6 induced phosphorylation of STAT3 (pSTAT3 in wild-type HCC cells, but Nogo-B knockout could reduce IL-6–induced increase of pSTAT3, supporting that Nogo-B affects HCC tumor progression possibly via regulating the IL-6/STAT3 signaling pathway. In conclusion, Nogo-B is expressed aberrantly in HCCs and plays an oncogenic role. These findings support that Nogo-B may be a novel anti-HCC therapeutic target.

  17. Characterization of Phototransduction Gene Knockouts Revealed Important Signaling Networks in the Light-Induced Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Jayalakshmi Krishnan

    2008-01-01

    Full Text Available Understanding the molecular pathways mediating neuronal function in retinas can be greatly facilitated by the identification of genes regulated in the retinas of different mutants under various light conditions. We attempted to conduct a gene chip analysis study on the genes regulated during rhodopsin kinase (Rhok-/- and arrestin (Sag-/- knockout and double knockouts in mice retina. Hence, mice were exposed to constant illumination of 450 lux or 6,000 lux on dilated pupils for indicated periods. The retinas were removed after the exposure and processed for microarray analysis. Double knockout was associated with immense changes in gene expression regulating a number of apoptosis inducing transcription factors. Subsequently, network analysis revealed that during early exposure the transcription factors, p53, c-MYC, c-FOS, JUN, and, in late phase, NF-B, appeared to be essential for the initiation of light-induced retinal rod loss, and some other classical pro- and antipoptotic genes appeared to be significantly important as well.

  18. Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.

    Science.gov (United States)

    Baym, Michael; Shaket, Lev; Anzai, Isao A; Adesina, Oluwakemi; Barstow, Buz

    2016-11-10

    Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

  19. Generation of ERα-floxed and knockout mice using the Cre/LoxP system

    International Nuclear Information System (INIS)

    Antonson, P.; Omoto, Y.; Humire, P.; Gustafsson, J.-Å.

    2012-01-01

    Highlights: ► ERα floxed and knockout mice were generated. ► Disruption of the ERα gene results in sterility in both male and female mice. ► ERα −/− mice have ovaries with hemorrhagic follicles and hypoplastic uterus. ► Female ERα −/− mice develop obesity. -- Abstract: Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

  20. Probing short-range correlations in asymmetric nuclei with quasi-free pair knockout reactions

    Science.gov (United States)

    Stevens, Sam; Ryckebusch, Jan; Cosyn, Wim; Waets, Andreas

    2018-02-01

    Short-range correlations (SRC) in asymmetric nuclei with an unusual neutron-to-proton ratio can be studied with quasi-free two-nucleon knockout processes following the collision between accelerated ions and a proton target. We derive an approximate factorized cross section for those SRC-driven p (A ,p‧N1N2) reactions. Our reaction model hinges on the factorization properties of SRC-driven A (e ,e‧N1N2) reactions for which strong indications are found in theory-experiment comparisons. In order to put our model to the test we compare its predictions with results of 12C (p ,p‧ pn) measurements conducted at Brookhaven National Laboratory (BNL) and find a fair agreement. The model can also reproduce characteristic features of SRC-driven two-nucleon knockout reactions, like back-to-back emission of the correlated nucleons. We study the asymmetry dependence of nuclear SRC by providing predictions for the ratio of proton-proton to proton-neutron knockout cross sections for the carbon isotopes 9-15C thereby covering neutron excess values (N - Z) / Z between -0.5 and +0.5.

  1. Txnip ablation reduces vascular smooth muscle cell inflammation and ameliorates atherosclerosis in apolipoprotein E knockout mice.

    Science.gov (United States)

    Byon, Chang Hyun; Han, Tieyan; Wu, Judy; Hui, Simon T

    2015-08-01

    Inflammation of vascular smooth muscle cells (VSMC) is intimately linked to atherosclerosis and other vascular inflammatory disease. Thioredoxin interacting protein (Txnip) is a key regulator of cellular sulfhydryl redox and a mediator of inflammasome activation. The goals of the present study were to examine the impact of Txnip ablation on inflammatory response to oxidative stress in VSMC and to determine the effect of Txnip ablation on atherosclerosis in vivo. Using cultured VSMC, we showed that ablation of Txnip reduced cellular oxidative stress and increased protection from oxidative stress when challenged with oxidized phospholipids and hydrogen peroxide. Correspondingly, expression of inflammatory markers and adhesion molecules were diminished in both VSMC and macrophages from Txnip knockout mice. The blunted inflammatory response was associated with a decrease in NF-ĸB nuclear translocation. Loss of Txnip in VSMC also led to a dramatic reduction in macrophage adhesion to VSMC. In vivo data from Txnip-ApoE double knockout mice showed that Txnip ablation led to 49% reduction in atherosclerotic lesion in the aortic root and 71% reduction in the abdominal aorta, compared to control ApoE knockout mice. Our data show that Txnip plays an important role in oxidative inflammatory response and atherosclerotic lesion development in mice. The atheroprotective effect of Txnip ablation implicates that modulation of Txnip expression may serve as a potential target for intervention of atherosclerosis and inflammatory vascular disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting.

    Science.gov (United States)

    Kang, J-T; Ryu, J; Cho, B; Lee, E-J; Yun, Y-J; Ahn, S; Lee, J; Ji, D-Y; Lee, K; Park, K-W

    2016-12-01

    Pigs are an attractive animal model to study the progression of cancer because of their anatomical and physiological similarities to human. However, the use of pig models for cancer research has been limited by availability of genetically engineered pigs which can recapitulate human cancer progression. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, specific CRISPR/Cas9 systems were used to target RUNX3, a known tumour suppressor gene, to generate a pig model that can induce gastric cancer in human. First, RUNX3 knockout cell lines carrying genetic modification (monoallelic or biallelic) of RUNX3 were generated by introducing engineered CRISPR/Cas9 system specific to RUNX3 into foetal fibroblast cells. Then, the genetically modified foetal fibroblast cells were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer. We successfully obtained four live RUNX3 knockout piglets from two surrogates. The piglets showed the lack of RUNX3 protein in their internal organ system. Our results demonstrate that the CRISPR/Cas9 system is effective in inducing mutations on a specific locus of genome and the RUNX3 knockout pigs can be useful resources for human cancer research and to develop novel cancer therapies. © 2016 Blackwell Verlag GmbH.

  3. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease.

    Science.gov (United States)

    Hisamatsu, Shin; Sakaue, Motoharu; Takizawa, Akiko; Kato, Tsubasa; Kamoshita, Maki; Ito, Junya; Kashiwazaki, Naomi

    2015-02-01

    Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs. © 2014 Japanese Society of Animal Science.

  4. Less is More: unveiling the functional core of hematopoietic stem cells through knockout mice

    Science.gov (United States)

    Rossi, Lara; Lin, Kuanyin K.; Boles, Nathan C.; Yang, Liubin; King, Katherine Y.; Jeong, Mira; Mayle, Allison; Goodell, Margaret A.

    2012-01-01

    Summary Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cells. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, TGF-β signaling, Pten/AKT signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology. In the field of design, the minimalist movement stripped down buildings and objects to their most basic features, a sentiment that architect Ludwig Mies van der Rohe summarized in his motto “less is more”. By depleting HSCs of specific genes, knockout studies transpose the minimalist approach into research biology, providing insights into the essential core of genetic features that is indispensable for a well-functioning hematopoietic system. PMID:22958929

  5. Hippocampal long-term potentiation is enhanced in urethane-anesthetized RGS2 knockout mice.

    Science.gov (United States)

    Hutchison, R Matthew; Chidiac, Peter; Leung, L Stan

    2009-08-01

    RGS2 is a member of the regulator of G-protein signaling (RGS) family and has been implicated in cellular mechanisms associated with neuronal plasticity. Long-term potentiation (LTP) of RGS2 knockout and wild-type mice was examined at the Schaffer collaterals to CA1 pathway in urethane-anesthetized mice in vivo to examine RGS2's possible role in the regulation of potentiation. As compared to wild-type mice, RGS2 knockouts demonstrated much stronger LTP of the extracellular population spikes at the somatic and dendritic layers in CA1 region and more pronounced LTP of the population excitatory postsynaptic current sink. Under baseline conditions, RGS2 knockouts showed lower paired-pulse facilitation of the excitatory postsynaptic potentials and associated current sinks in vivo as compared with wild-type mice. The data show for the first time that RGS2 deficient mice in vivo differ from wild-type mice in both short-term and long-term synaptic plasticity suggesting that RGS2 serves as a negative regulator of long-term synaptic plasticity. Copyright (c) 2009 Wiley-Liss, Inc.

  6. Adaptations in pre- and postsynaptic 5-HT(1A) receptor function and cocaine supersensitivity in serotonin transporter knockout rats

    NARCIS (Netherlands)

    Homberg, Judith R; De Boer, Sietse F; Raasø, Halfdan S; Olivier, Jocelien D A; Verheul, Mark; Ronken, Eric; Cools, Alexander R; Ellenbroek, Bart A; Schoffelmeer, Anton N M; Vanderschuren, Louk J M J; De Vries, Taco J; Cuppen, Edwin

    2008-01-01

    RATIONALE: While individual differences in vulnerability to psychostimulants have been largely attributed to dopaminergic neurotransmission, the role of serotonin is not fully understood. OBJECTIVES: To study the rewarding and motivational properties of cocaine in the serotonin transporter knockout

  7. The serotonin transporter plays an important role in male sexual behavior: a study in serotonin transporter knockout rats

    NARCIS (Netherlands)

    Chan, J.Y.; Snoeren, E.; Cuppen, E.; Waldinger, M.; Olivier, B.; Oosting, R.

    2011-01-01

    INTRODUCTION: Serotonin (5-HT) is an important neurotransmitter for sexual behaviors. Heterozygous (+/-) serotonin transporter (SERT) rats and SERT knockout rats (-/-) have serotonergic disturbances with significant elevations of basal extracellular 5-HT levels. AIM: To investigate the putative role

  8. TNF receptor 1, IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection.

    Science.gov (United States)

    Kalns, John; Scruggs, Julie; Millenbaugh, Nancy; Vivekananda, Jeeva; Shealy, David; Eggers, Jeffrey; Kiel, Johnathan

    2002-03-22

    Anthrax produces at least two toxins that cause an intense systemic inflammatory response, edema, shock, and eventually death. The relative contributions of various elements of the immune response to mortality and course of disease progression are poorly understood. We hypothesized that knockout mice missing components of the immune system will have an altered response to infection. Parent strain mice and knockouts were challenged with LD95 of anthrax spores (5 x 10(6)) administered subcutaneously. Our results show that all genetic knockouts succumbed to anthrax infection at the same frequency as the parent. TNF antibody delayed death but TNF receptor 1 knockout had no effect. IL-1 receptor or iNOS knockouts died sooner. Anthrax was more abundant in the injection site of TNF-alpha and iNOS knockouts compared to parent suggesting that attenuated cellular response increases rate of disease progression. With the exception of edema and necrosis at the injection site pathological changes in internal organs were not observed. (C)2002 Elsevier Science (USA).

  9. An In Silico Knockout Model for Gastrointestinal Absorption Using a Systems Pharmacology Approach - Development and Application for Ketones.

    Directory of Open Access Journals (Sweden)

    Vittal Shivva

    Full Text Available Gastrointestinal absorption and disposition of ketones is complex. Recent work describing the pharmacokinetics (PK of d-β-hydroxybutyrate (BHB following oral ingestion of a ketone monoester ((R-3-hydroxybutyl (R-3-hydroxybutyrate found multiple input sites, nonlinear disposition and feedback on endogenous production. In the current work, a human systems pharmacology model for gastrointestinal absorption and subsequent disposition of small molecules (monocarboxylic acids with molecular weight < 200 Da was developed with an application to a ketone monoester. The systems model was developed by collating the information from the literature and knowledge gained from empirical population modelling of the clinical data. In silico knockout variants of this systems model were used to explore the mechanism of gastrointestinal absorption of ketones. The knockouts included active absorption across different regions in the gut and also a passive diffusion knockout, giving 10 gut knockouts in total. Exploration of knockout variants has suggested that there are at least three distinct regions in the gut that contribute to absorption of ketones. Passive diffusion predominates in the proximal gut and active processes contribute to the absorption of ketones in the distal gut. Low doses are predominantly absorbed from the proximal gut by passive diffusion whereas high doses are absorbed across all sites in the gut. This work has provided mechanistic insight into the absorption process of ketones, in the form of unique in silico knockouts that have potential for application with other therapeutics. Future studies on absorption process of ketones are suggested to substantiate findings in this study.

  10. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Shoji Suzuki

    2017-01-01

    Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

  11. Generation of Hoxc13 knockout pigs recapitulates human ectodermal dysplasia-9.

    Science.gov (United States)

    Han, Kai; Liang, Liuping; Li, Li; Ouyang, Zhen; Zhao, Bentian; Wang, Qi; Liu, Zhaoming; Zhao, Yu; Ren, Xiaoshuai; Jiang, Fei; Lai, Chengdan; Wang, Kepin; Yan, Sen; Huang, Liang; Guo, Lin; Zeng, Kang; Lai, Liangxue; Fan, Nana

    2017-01-01

    Atrichia and sparse hair phenotype cause distress to many patients. Ectodermal dysplasia-9 (ED-9) is a congenital condition characterized by hypotrichosis and nail dystrophy without other disorders, and Hoxc13 is a pathogenic gene for ED-9. However, mice carrying Hoxc13 mutation present several other serious disorders, such as skeletal defects, progressive weight loss and low viability. Mouse models cannot faithfully mimic human ED-9. In this study, we generated an ED-9 pig model via Hoxc13 gene knockout through single-stranded oligonucleotides (c.396C > A) combined with CRISPR/Cas9 and somatic cell nuclear transfer. Eight cloned piglets with three types of biallelic mutations (five piglets with Hoxc13c.396C > A/c.396C > A, two piglets with Hoxc13c.396C > A/c.396C > A + 1 and one piglet with Hoxc13Δ40/Δ40) were obtained. Hoxc13 was not expressed in pigs with all three mutation types, and the expression levels of Hoxc13-regulated genes, namely, Foxn1, Krt85 and Krt35, were decreased. The hair follicles displayed various abnormal phenotypes, such as reduced number of follicles and disarrayed hair follicle cable without normal hair all over the body. By contrast, the skin structure, skeleton phenotype, body weight gain and growth of Hoxc13 knockout pigs were apparently normal. The phenotypes of Hoxc13 mutation in pigs were similar to those in ED-9 patients. Therefore, Hoxc13 knockout pigs could be utilized as a model for ED-9 pathogenesis and as a hairless model for hair regeneration research. Moreover, the hairless pigs without other major abnormal phenotypes generated in this study could be effective models for other dermatological research because of the similarity between pig and human skins. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Unintentional miRNA ablation is a risk factor in gene knockout studies: a short report.

    Directory of Open Access Journals (Sweden)

    Ivan Osokine

    2008-02-01

    Full Text Available One of the most powerful techniques for studying the function of a gene is to disrupt the expression of that gene using genetic engineering strategies such as targeted recombination or viral integration of gene trap cassettes. The tremendous utility of these tools was recognized this year with the awarding of the Nobel Prize in Physiology or Medicine to Capecchi, Evans, and Smithies for their pioneering work in targeted recombination mutagenesis in mammals. Another noteworthy discovery made nearly a decade ago was the identification of a novel class of non-coding genes called microRNAs. MicroRNAs are among the largest known classes of regulatory elements with more than 1000 predicted to exist in the mouse genome. Over 50% of known microRNAs are located within introns of coding genes. Given that currently about half of the genes in mouse have been knocked out, we investigated the possibility that intronic microRNAs may have been coincidentally deleted or disrupted in some of these mouse models. We searched published murine knockout studies and gene trap embryonic stem cell line databases for cases where a microRNA was located within or near the manipulated genomic loci, finding almost 200 cases where microRNA expression may have been disrupted along with another gene. Our results draw attention to the need for careful planning in future knockout studies to minimize the unintentional disruption of microRNAs. These data also raise the possibility that many knockout studies may need to be reexamined to determine if loss of a microRNA contributes to the phenotypic consequences attributed to loss of a protein-encoding gene.

  13. Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

    Energy Technology Data Exchange (ETDEWEB)

    Antonson, P., E-mail: per.antonson@ki.se [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Omoto, Y.; Humire, P. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Gustafsson, J.-A. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established a new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

  14. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9

    Science.gov (United States)

    Zhou, Wenjun; Wan, Yongjie; Guo, Rihong; Deng, Mingtian; Deng, Kaiping; Wang, Zhen; Zhang, Yanli; Wang, Feng

    2017-01-01

    Goat’s milk, considered a substitute for cow’s milk, has a high nutritional value. However, goat’s milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%–9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research. PMID:29016691

  15. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

    Science.gov (United States)

    Zhou, Wenjun; Wan, Yongjie; Guo, Rihong; Deng, Mingtian; Deng, Kaiping; Wang, Zhen; Zhang, Yanli; Wang, Feng

    2017-01-01

    Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1) and 0% (10 ng/μL sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group) and 28.6% (50 ng/μL sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

  16. Generation of beta-lactoglobulin knock-out goats using CRISPR/Cas9.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhou

    Full Text Available Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly β-lactoglobulin (BLG. In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/μL sg1 and 0% (10 ng/μL sg2 and efficiencies of dual sgRNAs were 25.0% (25 ng/μL sg2+sg3 group and 28.6% (50 ng/μL sg2+sg3 group. Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01 decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05. As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4, and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.

  17. IKKε knockout prevents high fat diet induced arterial atherosclerosis and NF-κB signaling in mice.

    Directory of Open Access Journals (Sweden)

    Changchun Cao

    Full Text Available AIMS: Atherosclerosis is a public health concern affecting many worldwide, but its pathogenesis remains unclear. In this study we investigated the role of IKKε during the formation of atherosclerosis and its molecular mechanism in the mouse aortic vessel wall. METHODS AND RESULTS: C57BL/6 wild-type or IKKε knockout mice bred into the ApoE knockout genetic background were divided into 4 groups: (1 wild-type (WT, (2 ApoE knockout (AK, (3 IKKε knockout (IK, (4 or both ApoE and IKKε knockout (DK. Each group of mice were fed with a high fat diet (HFD for 12 weeks from 8 weeks of age. Immunohistochemistry and Western blotting analysis demonstrated obvious increases in the expression of IKKε in the AK group compared with the WT group, especially in the intima. Serum lipid levels were significantly higher in the AK and DK groups than in the other two groups. Staining with hematoxylin-eosin and Oil Red, as well as scanning electron microscopy revealed less severe atherosclerotic lesions in the DK group than in the AK group. Immunofluorescence and Western blot analysis demonstrated obvious increases in the expression of NF-κB pathway components and downstream factors in the AK group, especially in the intima, while these increases were blocked in the DK group. CONCLUSION: The knockout of IKKε prevented significant atherosclerosis lesions in the mouse aorta from in both wild-type and ApoE knockout mice fed a HFD, suggesting that IKKε may play a vital role in HFD-induced atherosclerosis and would be an important target for the treatment of atherosclerosis.

  18. Increased Salt-Sensitivity in Endothelial Nitric Oxide Synthase-Knockout Mice

    OpenAIRE

    Leonard, Allison M.; Chafe, Linda L.; Montani, Jean-Pierre; Van Vliet, Bruce N.

    2017-01-01

    Background: Although impaired nitric oxide production contributes importantly to salt-sensitivity, the role of the endothelial isoform of nitric oxide synthase (eNOS) has received little attention. In the present study we compared the effects of a high-salt diet on the blood pressure response of eNOS knockout (eNOS−/−) and control (eNOS+/+) mice. Methods: Mean arterial pressure (MAP), heart rate, pulse pressure, and activity levels were recorded by telemetry in mice fed a regular-salt diet (0...

  19. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2015-09-01

    Full Text Available To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524. The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

  20. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs.

    Science.gov (United States)

    Liu, Ming; Wang, Xiu-Ping

    2015-09-01

    To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015].

  1. Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice

    Directory of Open Access Journals (Sweden)

    Karin eTein

    2015-08-01

    Full Text Available BackgroundMutations in WFS1 gene cause Wolfram syndrome, which is a rare autosomal recessive disorder, characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy and deafness (DIDMOAD. The WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene exhibit disturbances in the processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of the wolframin protein have been observed in the hippocampus, amygdala and limbic structures. The aim of this study was to investigate the effect of Wfs1 knockout on peptide processing in mouse hippocampus. A peptidomic approach was used to characterize individual peptides in the hippocampus of wild-type and Wfs1 knockout mice. ResultsWe identified 126 peptides in hippocampal extracts and the levels of 10 peptides differed between Wfs1 KO and wild-type mice at P<0.05. The peptide with the largest alteration was little-LEN, which level was 25 times higher in the hippocampus of Wfs1 KO mice compared to wild-type mice. Processing (cleavage of little-LEN from the Pcsk1n gene product proSAAS involves prohormone convertase 2 (PC2. Thus, PC2 activity was measured in extracts prepared from the hippocampus of Wfs1 knockout mice. The activity of PC2 in Wfs1 mutant mice was significantly higher (149.9±2.3%, p<0.0001, n=8 than in wild-type mice (100.0±7.0%, n=8. However, Western blot analysis showed that protein levels of 7B2, proPC2 and PC2 were same in both groups, and so were gene expression levels.ConclusionsProcessing of proSAAS is altered in the hippocampus of Wfs1-KO mice, which is caused by increased activity of PC2. Increased activity of PC2 in Wfs1 knockout mice is not caused by alteration in the levels of PC2 protein. Our results suggest a functional link between Wfs1 and PC2. Thus, the detailed molecular mechanism of the role of Wfs1 in the regulation of PC2 activity needs further investigation.

  2. Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yiting [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Liu, Lan [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Sheng, Ming [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Xiong, Kai [Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Grønnegårdsvej 7, 1870 Frederiksberg C (Denmark); Huang, Lei; Gao, Qian; Wei, Jingliang; Wu, Tianwen; Yang, Shulin [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Liu, Honglin, E-mail: liuhonglinnjau@163.com [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Mu, Yulian, E-mail: muyulian76@iascaas.net.cn [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China); Li, Kui [State Key Laboratory of Animal Nutrition and Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193 (China)

    2015-06-10

    Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1{sup −/−} MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1{sup −/−} MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1{sup −/−} MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1{sup −/−} MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1

  3. Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice

    Czech Academy of Sciences Publication Activity Database

    Vrbacký, Marek; Kovalčíková, Jana; Chawengsaksophak, Kallayanee; Beck, Inken; Mráček, Tomáš; Nůsková, Hana; Sedmera, David; Papoušek, František; Kolář, František; Sobol, Margaryta; Hozák, Pavel; Sedláček, Radislav; Houštěk, Josef

    2016-01-01

    Roč. 25, č. 21 (2016), s. 4674-4685 ISSN 0964-6906 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LL1204; GA MŠk(CZ) ED1.1.00/02.0109; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) LM2015062; GA MZd(CZ) NV16-33018A; GA MŠk(CZ) LM2015040 Institutional support: RVO:67985823 ; RVO:68378050 Keywords : mouse knockout * mitochondria * ATP synthase * TMEM70 * biogenesis * mitochondrial disease s Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.340, year: 2016

  4. Attenuated vasodilator effectiveness of protease-activated receptor 2 agonist in heterozygous par2 knockout mice.

    Directory of Open Access Journals (Sweden)

    John C Hennessey

    Full Text Available Studies of homozygous PAR2 gene knockout mice have described a mix of phenotypic effects in vitro and in vivo. However, there have been few studies of PAR2 heterozygous (wild-type/knockout; PAR2-HET mice. The phenotypes of many hemi and heterozygous transgenic mice have been described as intermediates between those of wild-type and knockout animals. In our study we aimed to determine the effects of intermediary par2 gene zygosity on vascular tissue responses to PAR2 activation. Specifically, we compared the vasodilator effectiveness of the PAR2 activating peptide 2-furoyl-LIGRLO-amide in aortas of wild-type PAR2 homozygous (PAR2-WT and PAR2-HET mice. In myographs under isometric tension conditions, isolated aortic rings were contracted by alpha 1-adrenoeceptor agonist (phenylephrine, and thromboxane receptor agonist (U46619 and then relaxation responses by the additions of 2-furoyl-LIGRLO-amide, acetylcholine, and nitroprusside were recorded. A Schild regression analysis of the inhibition by a PAR2 antagonist (GB-83 of PAR2 agonist-induced aortic ring relaxations was used to compare receptor expression in PAR2-WT to PAR2-HET. PAR2 mRNA in aortas was measured by quantitative real-time PCR. In aortas contracted by either phenylephrine or U46619, the maximum relaxations induced by 2-furoyl-LIGRLO-amide were less in PAR2-HET than in the gender-matched PAR2-WT. GB-83 was 3- to 4-fold more potent for inhibition of 2fly in PAR2-HET than in PAR2-WT. PAR2 mRNA content of aortas from PAR2-HET was not significantly different than in PAR2-WT. Acetylcholine- and nitroprusside-induced relaxations of aortas from PAR2-HET were not significantly different than in PAR2-WT and PAR2 knockout. An interesting secondary finding was that relaxations induced by agonists of PAR2 and muscarinic receptors were larger in females than in males. We conclude that the lower PAR2-mediated responses in PAR2-HET aortas are consistent with evidence of a lower quantity of functional

  5. Wip1 knockout inhibits the proliferation and enhances the migration of bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Tang, Yiting; Liu, Lan; Sheng, Ming; Xiong, Kai; Huang, Lei; Gao, Qian; Wei, Jingliang; Wu, Tianwen; Yang, Shulin; Liu, Honglin; Mu, Yulian; Li, Kui

    2015-01-01

    Mesenchymal stem cells (MSCs), a unique population of multipotent adult progenitor cells originally found in bone marrow (BM), are extremely useful for multifunctional therapeutic approaches. However, the growth arrest and premature senescence of MSCs in vitro prevent the in-depth characterization of these cells. In addition, the regulatory factors involved in MSCs migration remain largely unknown. Given that protein phosphorylation is associated with the processes of MSCs proliferation and migration, we focused on wild-type p53-inducible phosphatase-1 (Wip1), a well-studied modulator of phosphorylation, in this study. Our results showed that Wip1 knockout significantly inhibited MSCs proliferation and induced G2-phase cell-cycle arrest by reducing cyclinB1 expression. Compared with WT-MSCs, Wip1 −/− MSCs displayed premature growth arrest after six passages in culture. Transwell and scratch assays revealed that Wip1 −/− MSCs migrate more effectively than WT-MSCs. Moreover, the enhanced migratory response of Wip1 −/− MSCs may be attributed to increases in the induction of Rac1-GTP activity, the pAKT/AKT ratio, the rearrangement of filamentous-actin (f-actin), and filopodia formation. Based on these results, we then examined the effect of treatment with a PI3K/AKT and Rac1 inhibitor, both of which impaired the migratory activity of MSCs. Therefore, we propose that the PI3K/AKT/Rac1 signaling axis mediates the Wip1 knockout-induced migration of MSCs. Our findings indicate that the principal function of Wip1 in MSCs transformation is the maintenance of proliferative capacity. Nevertheless, knocking out Wip1 increases the migratory capacity of MSCs. This dual effect of Wip1 provides the potential for purposeful routing of MSCs. - Highlights: • Wip1 knockout inhibited MSCs proliferation through reducing cyclinB1 expression. • Wip1 −/− MSCs displayed premature growth arrest in vitro after six passages. • Knocking out Wip1 increases the migratory

  6. Increased susceptibility to diet-induced obesity in GPRC6A receptor knockout mice

    DEFF Research Database (Denmark)

    Clemmensen, Christoffer; Smajilovic, Sanela; Madsen, Andreas N

    2013-01-01

    locomotor activity. Moreover, diet-induced obese Gprc6a KO mice had increased circulating insulin and leptin levels relative to WT animals, thereby demonstrating that endocrine abnormalities associate with the reported disturbances in energy balance. The phenotype was further accompanied by disruptions...... complications is still elusive. In the present study, we investigated the impact of GPRC6A deficiency in a murine model of diet-induced obesity (DIO). Male Gprc6a knockout (KO) mice and WT littermates were subjected to a high-fat diet (HFD) for 25 weeks and exposed to comprehensive metabolic phenotyping...

  7. Hepatic caveolin-1 is enhanced in Cyp27a1/ApoE double knockout mice

    OpenAIRE

    Escher, Geneviève; Zurkinden, Line; Mistry, H; Mansour, Y; Rohrbach, B; Vogt, Bruno

    2016-01-01

    Sterol 27?hydroxylase (CYP27A1) is involved in bile acid synthesis and cholesterol homoeostasis. Cyp27a1 (?/?)/Apolipoprotein E (?/?) double knockout mice (DKO) fed a western diet failed to develop atherosclerosis. Caveolin?1 (CAV?1), the main component of caveolae, is associated with lipid homoeostasis and has regulatory roles in vascular diseases. We hypothesized that liver CAV?1 would contribute to the athero?protective mechanism in DKO mice. Cyp27a1 (+/+)/ApoE (?/?) (ApoE KO), Cyp27a1 (+/...

  8. Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout

    Directory of Open Access Journals (Sweden)

    О. P. Kanyuka

    2014-04-01

    Full Text Available A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein – β-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of ery­throcyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed under pttg gene knockout.

  9. Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy.

    Science.gov (United States)

    Ejdesjö, Andreas; Brings, Sebastian; Fleming, Thomas; Fred, Rikard G; Nawroth, Peter P; Eriksson, Ulf J

    2016-07-01

    The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. One-neutron knockout from light neutron-rich nuclei at relativistic energies

    International Nuclear Information System (INIS)

    Rodriguez-Tajes, C.; Alvarez-Pol, H.; Benjamim, E.; Benlliure, J.; Caamano, M.; Casarejos, E.; Cortina-Gil, D.; Gascon, M.; Kurtukian, T.; Perez-Loureiro, D.; Aumann, T.; Chatillon, A.; Geissel, H.; Nociforo, C.; Prochazka, A.; Simon, H.; Suemmerer, K.; Weick, H.; Winkler, M.; Borge, M. J. G.

    2010-01-01

    One-neutron knockout reactions from neutron-rich nuclei, with Z=6-13 and N=8-22, were studied at the Fragment Separator (GSI) at high beam energies, around 700 MeV/nucleon. Structural phenomena such as the formation of one-neutron halos in odd-mass carbon isotopes ( 15,17,19 C) will be discussed. In addition, one-neutron knockout measurements from 22 N were carried out for the first time and demonstrate clearly the change from a 0d 5/2 to a 1s 1/2 orbital for the valence neutron, an effect that is expected above N=14 and that was also observed in 23 O and 24 F. The possibility of an anomalous structure of 26 F, due to a significant 1s 1/2 neutron admixture, will also be discussed in the light of the experimental data obtained in this work. Finally, the ground-state configuration of neutron-rich neon isotopes ( 24-28 Ne) was studied, providing new information in a region that is relatively close to the island of inversion.

  11. Endothelin-1 Overexpression Improves Renal Function in eNOS Knockout Mice

    Directory of Open Access Journals (Sweden)

    Oleg Tsuprykov

    2015-10-01

    Full Text Available Background/Aims: To investigate the renal phenotype under conditions of an activated renal ET-1 system in the status of nitric oxide deficiency, we compared kidney function and morphology in wild-type, ET-1 transgenic (ET+/+, endothelial nitric oxide synthase knockout (eNOS-/- and ET+/+eNOS-/- mice. Methods: We assessed blood pressure, parameters of renal morphology, plasma cystatin C, urinary protein excretion, expression of genes associated with glomerular filtration barrier and tissue remodeling, and plasma metabolites using metabolomics. Results: eNOS-/- and ET+/+eNOS-/- mice developed hypertension. Osteopontin, albumin and protein excretion were increased in eNOS-/- and restored in ET+/+eNOS-/- animals. All genetically modified mice developed renal interstitial fibrosis and glomerulosclerosis. Genes involved in tissue remodeling (serpine1, TIMP1, Col1a1, CCL2 were up-regulated in eNOS-/-, but not in ET+/+eNOS-/- mice. Plasma levels of free carnitine and acylcarnitines, amino acids, diacyl phosphatidylcholines, lysophosphatidylcholines and hexoses were descreased in eNOS-/- and were in the normal range in ET+/+eNOS-/- mice. Conclusion: eNOS-/- mice developed renal dysfunction, which was partially rescued by ET-1 overexpression in eNOS-/- mice. The metabolomics results suggest that ET-1 overexpression on top of eNOS knockout is associated with a functional recovery of mitochondria (rescue effect in β-oxidation of fatty acids and an increase in antioxidative properties (normalization of monounsaturated fatty acids levels.

  12. Fmr1 and Nlgn3 knockout rats: novel tools for investigating autism spectrum disorders.

    Science.gov (United States)

    Hamilton, Shannon M; Green, Jennie R; Veeraragavan, Surabi; Yuva, Lisa; McCoy, Aaron; Wu, Yumei; Warren, Joe; Little, Lara; Ji, Diana; Cui, Xiaoxia; Weinstein, Edward; Paylor, Richard

    2014-04-01

    Animal models are critical for gaining insights into autism spectrum disorder (ASD). Despite their apparent advantages to mice for neural studies, rats have not been widely used for disorders of the human CNS, such as ASD, for the lack of convenient genome manipulation tools. Here we describe two of the first transgenic rat models for ASD, developed using zinc-finger nuclease (ZFN) methodologies, and their initial behavioral assessment using a rapid juvenile test battery. A syndromic and nonsyndromic rat model for ASD were created as two separate knockout rat lines with heritable disruptions in the genes encoding Fragile X mental retardation protein (FMRP) and Neuroligin3 (NLGN3). FMRP, a protein with numerous proposed functions including regulation of mRNA and synaptic protein synthesis, and NLGN3, a member of the neuroligin synaptic cell-adhesion protein family, have been implicated in human ASD. Juvenile subjects from both knockout rat lines exhibited abnormalities in ASD-relevant phenotypes including juvenile play, perseverative behaviors, and sensorimotor gating. These data provide important first evidence regarding the utility of rats as genetic models for investigating ASD-relevant genes.

  13. A Knockout Experiment: Disciplinary Divides and Experimental Skill in Animal Behaviour Genetics.

    Science.gov (United States)

    Nelson, Nicole C

    2015-07-01

    In the early 1990s, a set of new techniques for manipulating mouse DNA allowed researchers to 'knock out' specific genes and observe the effects of removing them on a live mouse. In animal behaviour genetics, questions about how to deploy these techniques to study the molecular basis of behaviour became quite controversial, with a number of key methodological issues dissecting the interdisciplinary research field along disciplinary lines. This paper examines debates that took place during the 1990s between a predominately North American group of molecular biologists and animal behaviourists around how to design, conduct, and interpret behavioural knockout experiments. Drawing from and extending Harry Collins's work on how research communities negotiate what counts as a 'well-done experiment,' I argue that the positions practitioners took on questions of experimental skill reflected not only the experimental traditions they were trained in but also their differing ontological and epistemological commitments. Different assumptions about the nature of gene action, eg., were tied to different positions in the knockout mouse debates on how to implement experimental controls. I conclude by showing that examining representations of skill in the context of a community's knowledge commitments sheds light on some of the contradictory ways in which contemporary animal behaviour geneticists talk about their own laboratory work as a highly skilled endeavour that also could be mechanised, as easy to perform and yet difficult to perform well.

  14. Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice

    Directory of Open Access Journals (Sweden)

    José Belizário

    2015-01-01

    Full Text Available Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents or intracellular (DNA damage and Ca2+ overload stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER, cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1, RIPK3, and the mixed kinase domain-like (MLKL for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice.

  15. Characterization of nasal potential difference in cftr knockout and F508del-CFTR mice.

    Directory of Open Access Journals (Sweden)

    Emilie Lyne Saussereau

    Full Text Available BACKGROUND: Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF. Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V(TE to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V(TE in CF mice must be well characterized for correct interpretation. METHODS: We performed V(TE measurements in large-scale studies of two mouse models of CF--B6;129 cftr knockout and FVB F508del-CFTR--and their respective wild-type (WT littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice. RESULTS: We determined the typical V(TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl(- solution was considered to indicate a normal response. CONCLUSIONS: These data will make it possible to interpret changes in nasal V(TE in mouse models of CF, in future preclinical studies.

  16. Characterization of TG2 and TG1-TG2 double knock-out mouse epidermis.

    Science.gov (United States)

    Pitolli, Consuelo; Pietroni, Valentina; Marekov, Lyuben; Terrinoni, Alessandro; Yamanishi, Kiyofumi; Mazzanti, Cinzia; Melino, Gerry; Candi, Eleonora

    2017-03-01

    Transglutaminases (TGs) are a family of enzymes that catalyse the formation of isopeptide bonds between the γ-carboxamide groups of glutamine residues and the ε-amino groups of lysine residues leading to cross-linking reactions among proteins. Four members, TG1, TG2, TG3, and TG5, of the nine mammalian enzymes are expressed in the skin. TG1, TG3 and TG5 crosslinking properties are fundamental for cornified envelope assembly. In contrast, the role of TG2 in keratinization has never been studied at biochemical level in vivo. In this study, taking advantage of the TG2 knock-out (KO) and TG1 heterozygous mice, we generated and characterized the epidermis of TG1-TG2 double knock-out (DKO) mice. We performed morphological analysis of the epidermis and evaluation of the expression of differentiation markers. In addition, we performed analysis of the amino acid composition from isolated corneocytes. We found a significant change in amino acid composition in TG1KO cornified cell envelopes (CEs) while TG2KO amino acid composition was similar to wild-type CEs. Our results confirm a key role of TG1 in skin differentiation and CE assembly and demonstrate that TG2 is not essential for CE assembly and skin formation.

  17. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-γ receptor knockout mice

    International Nuclear Information System (INIS)

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Bjoerkman, Maria; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Berg, Anna-Lena; Bohlooly-Y, Mohammad

    2006-01-01

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-γ. HNF4-γ is expressed in the kidneys, gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-γ +/+ ), the HNF4-γ knockout (HNF4-γ -/- ) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-γ -/- mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test

  18. Molecular mechanisms of liver ischemia reperfusion injury: Insights from transgenic knockout models

    Science.gov (United States)

    Datta, Gourab; Fuller, Barry J; Davidson, Brian R

    2013-01-01

    Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery. Understanding the mechanisms of liver ischemia reperfusion injury (IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation, as well as expanding the potential pool of usable donor grafts. The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes, increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis. Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury. IRI involves a complex interplay between neutrophils, natural killer T-cells cells, CD4+ T cell subtypes, cytokines, nitric oxide synthases, haem oxygenase-1, survival kinases such as the signal transducer and activator of transcription, Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways. Transgenic animals, particularly genetic knockout models, have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies. Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein. This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI. PMID:23555157

  19. Application of Monoclonal Antibodies against Bioactive Natural Products: Eastern Blotting and Preparation of Knockout Extract

    Directory of Open Access Journals (Sweden)

    Hiroyuki Tanaka

    2012-01-01

    Full Text Available Matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was used for the confirmation of hapten number in synthesized antigen. As application of MAb, the MAbs against ginsenosides and glycyrrhizin have been prepared resulting in the development of two new techniques that we named the eastern blotting method and the knockout extract preparation. In eastern blotting technique, glycosides like ginsenosides and glycyrrhizin separated by silica gel TLC were blotted to PVDF membrane that was treated with a NaIO4 solution followed by BSA resulted in glycoside-BSA conjugate on a PVDF membrane. The blotted spots were stained by MAb. Double staining of eastern blotting for ginsenosides using antiginsenoside Rb1 and Rg1 MAbs promoted complete identification of ginsenosides in Panax species. The immunoaffinity concentration of glycyrrhizin was determined by immunoaffinity column conjugated with antiglycyrrhizin MAb resulting in the glycyrrhizin-knockout extract, which was determined by the synergic effect with glycyrrhizin on NO production using the cell line.

  20. Idebenone and resveratrol extend lifespan and improve motor function of HtrA2 knockout mice.

    Directory of Open Access Journals (Sweden)

    Ellen Gerhardt

    Full Text Available Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntington's disease (HD and Parkinson's disease (PD. Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD.

  1. Quasi-free one nucleon knockout reactions on neutron-rich oxygen isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Atar, Leyla; Aumann, Thomas [TU Darmstadt, Darmstadt (Germany); GSI, Darmstadt (Germany); Bertulani, Carlos [Texas A and M University-Commerce, Commerse (United States); Paschalis, Stefanos [TU Darmstadt, Darmstadt (Germany); Nociforo, Chiara [GSI, Darmstadt (Germany); Collaboration: R3B-Collaboration

    2015-07-01

    Recent experiments have shown a reduction of spectroscopic strengths to about 60-70% for stable nuclei. When going to drip lines this tendency is changing, loosely bound nucleons have spectroscopic strengths close unity while deeply bound nucleons have a large reduction of the strength. We aim to make a systematic study of spectroscopic factors (SF) of the Oxygen isotopes using quasi-free (p,2p) and (p,pn) knockout reactions in inverse kinematics. Quasi-free knockout reactions are a direct tool to study the occupancy and the location of valance and deeply bound single particle states. The Oxygen isotopes offer a large variation of separation energies which will allow us to obtain a qualitative and quantitative understanding of SF in a large variation of isospin asymmetry. For this we performed an experiment at the R3B-LAND setup at the GSI with secondary beams containing {sup 14-24}O. The {sup 16-18}O and {sup 21-23}O isotopes have been analyzed and the preliminary results will be presented. The results include the partial cross sections, gamma ray spectra of the residual fragments in coincidence, and the SF obtained via comparison with theory.

  2. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-gamma receptor knockout mice.

    Science.gov (United States)

    Gerdin, Anna Karin; Surve, Vikas V; Jönsson, Marie; Bjursell, Mikael; Björkman, Maria; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurström, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Törnell, Jan; Berg, Anna-Lena; Bohlooly-Y, Mohammad

    2006-10-20

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-gamma. HNF4-gamma is expressed in the kidneys, gut, pancreas, and testis. The first level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-gamma(+/+)), the HNF4-gamma knockout (HNF4-gamma(-/-)) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-gamma(-/-) mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.

  3. Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

    Science.gov (United States)

    Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

    2016-11-10

    Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Obese Neuronal PPARγ Knockout Mice Are Leptin Sensitive but Show Impaired Glucose Tolerance and Fertility.

    Science.gov (United States)

    Fernandez, Marina O; Sharma, Shweta; Kim, Sun; Rickert, Emily; Hsueh, Katherine; Hwang, Vicky; Olefsky, Jerrold M; Webster, Nicholas J G

    2017-01-01

    The peroxisome-proliferator activated receptor γ (PPARγ) is expressed in the hypothalamus in areas involved in energy homeostasis and glucose metabolism. In this study, we created a deletion of PPARγ brain-knockout (BKO) in mature neurons in female mice to investigate its involvement in metabolism and reproduction. We observed that there was no difference in age at puberty onset between female BKOs and littermate controls, but the BKOs gave smaller litters when mated and fewer oocytes when ovulated. The female BKO mice had regular cycles but showed an increase in the number of cycles with prolonged estrus. The mice also had increased luteinizing hormone (LH) levels during the LH surge and histological examination showed hemorrhagic corpora lutea. The mice were challenged with a 60% high-fat diet (HFD). Metabolically, the female BKO mice showed normal body weight, glucose and insulin tolerance, and leptin levels but were protected from obesity-induced leptin resistance. The neuronal knockout also prevented the reduction in estrous cycles due to the HFD. Examination of ovarian histology showed a decrease in the number of primary and secondary follicles in both genotypes due to the HFD, but the BKO ovaries showed an increase in the number of hemorrhagic follicles. In summary, our results show that neuronal PPARγ is required for optimal female fertility but is also involved in the adverse effects of diet-induced obesity by creating leptin resistance potentially through induction of the repressor Socs3. Copyright © 2017 by the Endocrine Society.

  5. Obesity occurring in apolipoprotein E-knockout mice has mild effects on fertility.

    Science.gov (United States)

    Zhang, Ting; Dai, Pengyuan; Cheng, Dong; Zhang, Liang; Chen, Zijiang; Meng, Xiaoqian; Zhang, Fumiao; Han, Xiaoying; Liu, Jianwei; Pan, Jie; Yang, Guiwen; Zhang, Cong

    2014-02-01

    The Apolipoprotein (Apo) family is implicated in lipid metabolism. There are five types of Apo: Apoa, Apob, Apoc, Apod, and Apoe. Apoe has been demonstrated to play a central role in lipoprotein metabolism and to be essential for efficient receptor-mediated plasma clearance of chylomicron remnants and VLDL remnant particles by the liver. Apoe-deficient (Apoe(-/-)) mice develop atherosclerotic plaques spontaneously, followed by obesity. In this study, we investigated whether lipid deposition caused by Apoe knockout affects reproduction in female mice. The results demonstrated that Apoe(-/-) mice were severely hypercholesterolemic, with their cholesterol metabolism disordered, and lipid accumulating in the ovaries causing the ovaries to be heavier compared with the WT counterparts. In addition, estrogen and progesterone decreased significantly at D 100. Quantitative PCR analysis demonstrated that at D 100 the expression of cytochromeP450 aromatase (Cyp19a1), 3β-hydroxysteroid dehydrogenase (Hsd3b), mechanistic target of rapamycin (Mtor), and nuclear factor-κB (Nfkb) decreased significantly, while that of BCL2-associated agonist of cell death (Bad) and tuberous sclerosis complex 2 (Tsc2) increased significantly in the Apoe(-/-) mice. However, there was no difference in the fertility rates of the Apoe(-/-) and WT mice; that is, obesity induced by Apoe knockout has no significant effect on reproduction. However, the deletion of Apoe increased the number of ovarian follicles and the ratio of ovarian follicle atresia and apoptosis. We believe that this work will augment our understanding of the role of Apoe in reproduction.

  6. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

    Science.gov (United States)

    Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

  7. Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

    Science.gov (United States)

    Osaki, Yoshinori; Nakagawa, Yoshimi; Miyahara, Shoko; Iwasaki, Hitoshi; Ishii, Akiko; Matsuzaka, Takashi; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Sone, Hirohito; Ohashi, Ken; Ishibashi, Shun; Yamada, Nobuhiro; Shimano, Hitoshi

    2015-10-23

    HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. ARGINASE ENZYMES IN ISOLATED AIRWAYS FROM NORMAL AND NITRIC OXIDE SYNTHASE 2-KNOCKOUT MICE EXPOSED TO OVALBUMIN

    Science.gov (United States)

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J.; Last, Jerold A.

    2009-01-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses---inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration--were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2

  9. Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

    DEFF Research Database (Denmark)

    Honoré, Bent; Buus, Søren; Claësson, Mogens H

    2008-01-01

    ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensiona......ABSTRACT: BACKGROUND: Knockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two...

  10. Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

    OpenAIRE

    Teng, Bunyen; Ansari, Habib R.; Oldenburg, Peter J.; Schnermann, J.; Mustafa, S. Jamal

    2005-01-01

    Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A1 receptor (A1AR)-knockout (A1KO) mice and their wild-type (A1WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiot...

  11. Effect of Cyp27A1 gene dosage on atherosclerosis development in ApoE-knockout mice.

    OpenAIRE

    Zurkinden L Solcà C Vögeli IA Vogt B Ackermann D Erickson SK Frey FJ Sviridov D Escher G.

    2014-01-01

    In humans, sterol 27-hydroxylase (CYP27A1) deficiency leads to cholesterol deposition in tendons and vasculature. Thus, in addition to its role in bile acid synthesis, where it converts cholesterol to 27-hydroxycholesterol (27-OHC), CYP27A1 may also be atheroprotective. Cyp27A1-deficient (Cyp27A1−/−) mice were crossed with apolipoprotein E (apoE)-deficient mice. Cyp27A1+/+/apoE−/− [ApoE-knockout (KO)], Cyp27A1+/−/apoE−/− heterozygous (het), and Cyp27A1−/−/apoE−/− [double-knockout (DKO)] mice ...

  12. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey, E-mail: carey.pope@okstate.edu

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  13. RNA-seq reveals transcriptome changes in goats following myostatin gene knockout

    Science.gov (United States)

    Cai, Bei; Zhou, Shiwei; Zhu, Haijing; Qu, Lei; Wang, Xiaolong

    2017-01-01

    Myostatin (MSTN) is a powerful negative regulator of skeletal muscle mass in mammalian species that is primarily expressed in skeletal muscles, and mutations of its encoding gene can result in the double-muscling trait. In this study, the CRISPR/Cas9 technique was used to edit MSTN in Shaanbei Cashmere goats and generate knockout animals. RNA sequencing was used to determine and compare the transcriptome profiles of the muscles from three wild-type (WT) goats, three fibroblast growth factor 5 (FGF5) knockout goats (FGF5+/- group) and three goats with disrupted expression of both the FGF5 and MSTN genes (FM+/- group). The sequence reads were obtained using the Illumina HiSeq 2000 system and mapped to the Capra hircus reference genome using TopHat (v2.0.9). In total, 68.93, 62.04 and 66.26 million clean sequencing reads were obtained from the WT, FM+/- and FGF5+/- groups, respectively. There were 201 differentially expressed genes (DEGs) between the WT and FGF5+/- groups, with 86 down- and 115 up-regulated genes in the FGF5+/- group. Between the WT and FM+/- groups, 121 DEGs were identified, including 81 down- and 40 up-regulated genes in the FM+/- group. A total of 198 DEGs were detected between the FGF5+/- group and FM+/- group, with 128 down- and 70 up-regulated genes in the FM+/- group. At the transcriptome level, we found substantial changes in genes involved in fatty acid metabolism and the biosynthesis of unsaturated fatty acids, such as stearoyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydratase 2, ELOVL fatty acid elongase 6 and fatty acid synthase, suggesting that the expression levels of these genes may be directly regulated by MSTN and that these genes are likely downstream targets of MSTN with potential roles in lipid metabolism in goats. Moreover, five randomly selected DEGs were further validated with qRT-PCR, and the results were consistent with the transcriptome analysis. The present study provides insight into the unique transcriptome profile of the

  14. RNA-seq reveals transcriptome changes in goats following myostatin gene knockout.

    Directory of Open Access Journals (Sweden)

    Lamei Wang

    Full Text Available Myostatin (MSTN is a powerful negative regulator of skeletal muscle mass in mammalian species that is primarily expressed in skeletal muscles, and mutations of its encoding gene can result in the double-muscling trait. In this study, the CRISPR/Cas9 technique was used to edit MSTN in Shaanbei Cashmere goats and generate knockout animals. RNA sequencing was used to determine and compare the transcriptome profiles of the muscles from three wild-type (WT goats, three fibroblast growth factor 5 (FGF5 knockout goats (FGF5+/- group and three goats with disrupted expression of both the FGF5 and MSTN genes (FM+/- group. The sequence reads were obtained using the Illumina HiSeq 2000 system and mapped to the Capra hircus reference genome using TopHat (v2.0.9. In total, 68.93, 62.04 and 66.26 million clean sequencing reads were obtained from the WT, FM+/- and FGF5+/- groups, respectively. There were 201 differentially expressed genes (DEGs between the WT and FGF5+/- groups, with 86 down- and 115 up-regulated genes in the FGF5+/- group. Between the WT and FM+/- groups, 121 DEGs were identified, including 81 down- and 40 up-regulated genes in the FM+/- group. A total of 198 DEGs were detected between the FGF5+/- group and FM+/- group, with 128 down- and 70 up-regulated genes in the FM+/- group. At the transcriptome level, we found substantial changes in genes involved in fatty acid metabolism and the biosynthesis of unsaturated fatty acids, such as stearoyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydratase 2, ELOVL fatty acid elongase 6 and fatty acid synthase, suggesting that the expression levels of these genes may be directly regulated by MSTN and that these genes are likely downstream targets of MSTN with potential roles in lipid metabolism in goats. Moreover, five randomly selected DEGs were further validated with qRT-PCR, and the results were consistent with the transcriptome analysis. The present study provides insight into the unique transcriptome

  15. Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice

    DEFF Research Database (Denmark)

    Schmidt, Lene Sørensen; Thomsen, Morgane; Weikop, Pia

    2011-01-01

    Rationale The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M4 receptor subtype plays an important role in regulation of dopaminergic neurotransmission. Objectives Here we investigated...... for the first time the involvement of M4 receptors in the reinforcing effects of cocaine using chronic intravenous cocaine self-administration in extensively backcrossed M4 receptor knockout (M4 -/-) mice. Methods We evaluated acquisition of cocaine self-administration in experimentally naïve mice. Both cocaine...... self-administration and food-maintained operant behavior were evaluated under fixed ratio 1 (FR 1) and progressive ratio (PR) schedules of reinforcement. In addition, cocaine-induced dopamine release and cocaine-induced hyperactivity were evaluated. Results M4 -/- mice earned significantly more cocaine...

  16. Management Of Hanford KW Basin Knockout Pot Sludge As Spent Nuclear Fuel

    Energy Technology Data Exchange (ETDEWEB)

    Raymond, R. E. [CH2M HIll Plateau Remediation Company, Richland, WA (United States); Evans, K. M. [AREVA, Avignon (France)

    2012-10-22

    CH2M HILL Plateau Remediation Company (CHPRC) and AREVA Federal Services, LLC (AFS) have been working collaboratively to develop and deploy technologies to remove, transport, and interim store remote-handled sludge from the 10S-K West Reactor Fuel Storage Basin on the U.S. Department of Energy (DOE) Hanford Site near Richland, WA, USA. Two disposal paths exist for the different types of sludge found in the K West (KW) Basin. One path is to be managed as Spent Nuclear Fuel (SNF) with eventual disposal at an SNF at a yet to be licensed repository. The second path will be disposed as remote-handled transuranic (RH-TRU) waste at the Waste Isolation Pilot Plant (WIPP) in Carlsbad, NM. This paper describes the systems developed and executed by the Knockout Pot (KOP) Disposition Subproject for processing and interim storage of the sludge managed as SNF, (i.e., KOP material).

  17. The effect of insulin deficiency on tau and neurofilament in the insulin knockout mouse

    International Nuclear Information System (INIS)

    Schechter, Ruben; Beju, Delia; Miller, Kenneth E.

    2005-01-01

    Complications of diabetes mellitus within the nervous system are peripheral and central neuropathy. In peripheral neuropathy, defects in neurofilament and microtubules have been demonstrated. In this study, we examined the effects of insulin deficiency within the brain in insulin knockout mice (I(-/-)). The I(-/-) exhibited hyperphosphorylation of tau, at threonine 231, and neurofilament. In addition, we showed hyperphosphorylation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 β (GSK-3 β) at serine 9. Extracellular signal-regulated kinase 1 (ERK 1) showed decrease in phosphorylation, whereas ERK 2 showed no changes. Ultrastructural examination demonstrated swollen mitochondria, endoplasmic reticulum, and Golgi apparatus, and dispersion of the nuclear chromatin. Microtubules showed decrease in the number of intermicrotubule bridges and neurofilament presented as bunches. Thus, lack of insulin brain stimulation induces JNK hyperphosphorylation followed by hyperphosphorylation of tau and neurofilament, and ultrastructural cellular damage, that over time may induce decrease in cognition and learning disabilities

  18. Protein knockouts in living eukaryotes using deGradFP and green fluorescent protein fusion targets.

    Science.gov (United States)

    Caussinus, Emmanuel; Kanca, Oguz; Affolter, Markus

    2013-09-24

    This unit describes deGradFP (degrade Green Fluorescent Protein), an easy-to-implement protein knockout method applicable in any eukaryotic genetic system. Depleting a protein in order to study its function in a living organism is usually achieved at the gene level (genetic mutations) or at the RNA level (RNA interference and morpholinos). However, any system that acts upstream of the proteic level depends on the turnover rate of the existing target protein, which can be extremely slow. In contrast, deGradFP is a fast method that directly depletes GFP fusion proteins. In particular, deGradFP is able to counteract maternal effects in embryos and causes early and fast onset loss-of-function phenotypes of maternally contributed proteins. Copyright © 2013 John Wiley & Sons, Inc.

  19. Management Of Hanford KW Basin Knockout Pot Sludge As Spent Nuclear Fuel

    International Nuclear Information System (INIS)

    Raymond, R. E.; Evans, K. M.

    2012-01-01

    CH2M Hill Plateau Remediation Company (CHPRC) and AREVA Federal Services, LLC (AFS) have been working collaboratively to develop and deploy technologies to remove, transport, and interim store remote-handled sludge from the 10S-K West Reactor Fuel Storage Basin on the U.S. Department of Energy (DOE) Hanford Site near Richland, WA, USA. Two disposal paths exist for the different types of sludge found in the K West (KW) Basin. One path is to be managed as Spent Nuclear Fuel (SNF) with eventual disposal at an SNF at a yet to be licensed repository. The second path will be disposed as remote-handled transuranic (RH-TRU) waste at the Waste Isolation Pilot Plant (WIPP) in Carlsbad, NM. This paper describes the systems developed and executed by the Knockout Pot (KOP) Disposition Subproject for processing and interim storage of the sludge managed as SNF, (i.e., KOP material)

  20. Zika virus infection of adult and fetal STAT2 knock-out hamsters.

    Science.gov (United States)

    Siddharthan, Venkatraman; Van Wettere, Arnaud J; Li, Rong; Miao, Jinxin; Wang, Zhongde; Morrey, John D; Julander, Justin G

    2017-07-01

    Zika virus (ZIKV) infection was investigated in adult and fetal STAT2 knock-out (KO) hamsters. Subcutaneous injection of ZIKV of adults resulted in morbidity, mortality, and infection of the uterus, placenta, brain, spinal cord, and testicles, thus providing an opportunity to evaluate congenital ZIKV infection in a second rodent species besides mice. ZIKV-infected cells with morphologies of Sertoli cells and spermatogonia were observed in the testes, which may have implications for sexual transmission and male sterility. Neonates exposed as fetuses to ZIKV at 8 days post-coitus were not smaller than controls. Nevertheless, infectious virus and ZIKV RNA was detected in some, but not all, placentas and fetal brains of KO hamsters. STAT2 KO hamsters may be useful for addressing sexual transmission, pathogenesis, routes of fetal infection, and neurological disease outcomes, and may also be used in antiviral or vaccine studies to identify intervention strategies. Copyright © 2017. Published by Elsevier Inc.

  1. Increased radiosensitivity and radiation-induced apoptosis in SRC-3 knockout mice

    International Nuclear Information System (INIS)

    Jin Jie; Wang Yu; Xu Yang; Chen Shilei; Wang Junping; Ran Xinze; Su Yongping; Wang Jin

    2014-01-01

    Steroid receptor coactivator-3 (SRC-3), a multifunctional transcriptional coactivator, plays an important role in regulation of cell apoptosis in chemoresistant cancer cells. However, its role in radiation-induced apoptosis in hematopoietic cells is still unclear. In this study, we used SRC-3 knockout (SRC-3 -/- ) mice to assess the role of SRC-3 in radiation-induced hematopoietic injury in vivo. After a range of doses of irradiation, SRC-3 -/- mice exhibited lower counts of peripheral blood cells and bone marrow (BM) mononuclear cells and excessive BM depression, which resulted in a significantly higher mortality compared with wildtype mice. Moreover, BM mononuclear cells obtained from SRC-3 -/- mice showed a remarkable increase in radiation-induced apoptosis. Collectively, our data demonstrate that SRC-3 plays a role in radiation-induced apoptosis of BM hematopoietic cells. Regulation of SRC-3 might influence the radiosensitivity of hematopoietic cells, which highlights a potential therapeutic target for radiation-induced hematopoietic injury. (author)

  2. PTEN gene knock-out effect of radiosensitivity and its mechanism

    International Nuclear Information System (INIS)

    Fu Chunling; Huo Yanying; Hu Yingchun; Li Gang; Wu Dechang; Gou Qiao; Yang Liu; Mi Can

    2008-01-01

    Objective: To analyze the effect of PTEN gene on radiosensitivity and its mechanism. Methods: The reactive oxygen species levels of MEF1 and MEF1/PTEN -/- cell were determined with flow cytometry. The AKT activity pretreated with diphenyleneiodonium chloride or hydrogen peroxide (H 2 O 2 ) was detected by Western blot. Cell cloning efficiency test was used to detect the radiosensitivity. Results: Deletion of PTEN increased the level of basal reactive oxygen species and decreased the radiosensitivity. Pretreatment with diphenyleneiodonium chloride or hydrogen peroxide influenced the AKT activity of control MEF1 cells but not MEF1/Pten -/- cells. Conclusions: Knock-out of PTEN gene could make AKT constitutively active and block H 2 O 2 mediated PI3K/AKT signal transduction pathway, which should be the most reason of radioresistance. (authors)

  3. Estrogens and Spermiogenesis: New Insights from Type 1 Cannabinoid Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Giovanna Cacciola

    2013-01-01

    Full Text Available Spermatogenesis is a complex mechanism which allows the production of male gametes; it consists of mitotic, meiotic, and differentiation phases. Spermiogenesis is the terminal differentiation process during which haploid round spermatids undergo several biochemical and morphological changes, including extensive remodelling of chromatin and nuclear shape. Spermiogenesis is under control of endocrine, paracrine, and autocrine factors, like gonadotropins and testosterone. More recently, emerging pieces of evidence are suggesting that, among these factors, estrogens may have a role. To date, this is a matter of debate and concern because of the agonistic and antagonistic estrogenic effects that environmental chemicals may have on animal and human with damaging outcome on fertility. In this review, we summarize data which fuel this debate, with a particular attention to our recent results, obtained using type 1 cannabinoid receptor knockout male mice as animal model.

  4. Connecting tubule-selective knockout of AQP2 causes a mild urinary concentrating defect

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen; Pedersen, Nis Borbye; Fenton, Robert A.

    2011-01-01

    Aquaporin-2 (AQP2) is the main vasopressin-regulated water channel in the kidney connecting tubule and collecting ducts and is responsible for the regulation of final urine output. Previous studies on transgenic mice have demonstrated a crucial role of AQP2 in water handling in the collecting duct....... After 2 days of acclimatization, body weight, food and water intake and 24 hr urine were measured for 2 days. Animals where then challenged by a 24 hr water restriction, providing around 55% of baseline water intake in gelled food. Confocal laser scanning immunofluorescence microsopy demonstrated......Osm/l), suggesting a mild urinary concentrating defect. There was no difference in bodyweight, food intake or osmolar excretion. The mean drinking volume was higher in the knockout group. However, this difference was not statistically significant. A 24-hr water restriction decreased urine volume in both the wildtype...

  5. Sortilin 1 knockout alters basal adipose glucose metabolism but not diet-induced obesity in mice.

    Science.gov (United States)

    Li, Jibiao; Matye, David J; Wang, Yifeng; Li, Tiangang

    2017-04-01

    Sortilin 1 (Sort1) is a trafficking receptor that has been implicated in the regulation of plasma cholesterol in humans and mice. Here, we use metabolomics and hyperinsulinemic-euglycemic clamp approaches to obtain further understanding of the in vivo effects of Sort1 deletion on diet-induced obesity as well as on adipose lipid and glucose metabolism. Results show that Sort1 knockout (KO) does not affect Western diet-induced obesity nor adipose fatty acid and ceramide concentrations. Under the basal fasting state, chow-fed Sort1 KO mice have decreased adipose glycolytic metabolites, but Sort1 deletion does not affect insulin-stimulated tissue glucose uptake during the insulin clamp. These results suggest that Sort1 loss-of-function in vivo does not affect obesity development, but differentially modulates adipose glucose metabolism under fasting and insulin-stimulated states. © 2017 Federation of European Biochemical Societies.

  6. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins.

    Science.gov (United States)

    Baek, Kwangryul; Kim, Duk Hyoung; Jeong, Jooyeon; Sim, Sang Jun; Melis, Anastasios; Kim, Jin-Soo; Jin, EonSeon; Bae, Sangsu

    2016-07-28

    Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.

  7. AVE 0991-angiotensin-(1-7) receptor agonist, inhibits atherogenesis in apoE-knockout mice.

    Science.gov (United States)

    Toton-Zuranska, J; Gajda, M; Pyka-Fosciak, G; Kus, K; Pawlowska, M; Niepsuj, A; Wolkow, P; Olszanecki, R; Jawien, J; Korbut, R

    2010-04-01

    Recent evidence shows that the renin-angiotensin system is a crucial player in atherosclerotic processes. It was also proved that Ang II promotes atherogenesis. Angiotensin-(1-7) [Ang-(1-7)] opposites Ang II action. Therefore, we would like to find out whether Ang-(1-7) receptor agonist: AVE 0991, could ameliorate atherosclerosis progression in an experimental model of atherosclerosis: apolipoprotein E (apoE) - knockout mice. AVE 0991 inhibited atherogenesis, measured both by "en face" method (7.63+/-1.6% vs. 14.6+/-2.1%) and "cross-section" method (47 235+/-7 546 microm(2) vs. 91 416+/-8 357 microm(2)). This is the first report showing the effect of AVE 0991 on atherogenesis in gene-targeted mice.

  8. Analyzing AbrB-Knockout Effects through Genome and Transcriptome Sequencing of Bacillus licheniformis DW2

    Science.gov (United States)

    Shu, Cheng-Cheng; Wang, Dong; Guo, Jing; Song, Jia-Ming; Chen, Shou-Wen; Chen, Ling-Ling; Gao, Jun-Xiang

    2018-01-01

    As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (ΔAbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102∼4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ΔAbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ΔAbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ΔAbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression. PMID:29599755

  9. 12/15-Lipoxygenase Inhibition or Knockout Reduces Warfarin-Associated Hemorrhagic Transformation After Experimental Stroke.

    Science.gov (United States)

    Liu, Yu; Zheng, Yi; Karatas, Hulya; Wang, Xiaoying; Foerch, Christian; Lo, Eng H; van Leyen, Klaus

    2017-02-01

    For stroke prevention, patients with atrial fibrillation typically receive oral anticoagulation. The commonly used anticoagulant warfarin increases the risk of hemorrhagic transformation (HT) when a stroke occurs; tissue-type plasminogen activator treatment is therefore restricted in these patients. This study was designed to test the hypothesis that 12/15-lipoxygenase (12/15-LOX) inhibition would reduce HT in warfarin-treated mice subjected to experimental stroke. Warfarin was dosed orally in drinking water, and international normalized ratio values were determined using a Coaguchek device. C57BL6J mice or 12/15-LOX knockout mice were subjected to transient middle cerebral artery occlusion with 3 hours severe ischemia (model A) or 2 hours ischemia and tissue-type plasminogen activator infusion (model B), with or without the 12/15-LOX inhibitor ML351. Hemoglobin was determined in brain homogenates, and hemorrhage areas on the brain surface and in brain sections were measured. 12/15-LOX expression was detected by immunohistochemistry. Warfarin treatment resulted in reproducible increased international normalized ratio values and significant HT in both models. 12/15-LOX knockout mice suffered less HT after severe ischemia, and ML351 reduced HT in wild-type mice. When normalized to infarct size, ML351 still independently reduced hemorrhage. HT after tissue-type plasminogen activator was similarly reduced by ML351. In addition to its benefits in infarct size reduction, 12/15-LOX inhibition also may independently reduce HT in warfarin-treated mice. ML351 should be further evaluated as stroke treatment in anticoagulated patients suffering a stroke, either alone or in conjunction with tissue-type plasminogen activator. © 2017 American Heart Association, Inc.

  10. Feasibility of a Conditional Knockout System for Chlamydia Based on CRISPR Interference

    Directory of Open Access Journals (Sweden)

    Scot P. Ouellette

    2018-02-01

    Full Text Available Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi based on the catalytically inactive Cas9 variant (dCas9 of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C. trachomatis. CRISPRi has been developed and used successfully in a variety of bacterial organisms including E. coli and Mycobacterium tuberculosis. I first describe the creation of a single plasmid system for CRISPRi in Chlamydia, targeted to a non-essential gene, incA, that expresses a dispensable inclusion membrane protein. Control transformations of C. trachomatis serovar L2 with plasmids encoding only the dCas9, under the control of an inducible promoter, or only the guide RNA (gRNA targeted to the 5' UTR of incA, expressed constitutively, failed to prevent expression of IncA. Importantly, expression of dCas9 alone did not have a deleterious effect on chlamydiae. Transformation of C. trachomatis with a plasmid combining the dCas9 and a gRNA targeting incA and induction of expression of the dCas9 resulted in the reversible inhibition of IncA expression. Consequently, conditional knockout mediated by CRISPRi is feasible in Chlamydia. Potential improvements and experimental concerns in using the system are also discussed.

  11. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

    2010-01-01

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  12. Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice.

    Science.gov (United States)

    Ito, Junitsu; Otsuki, Noriyuki; Zhang, Xuhong; Konno, Tasuku; Kurahashi, Toshihiro; Takahashi, Motoko; Yamato, Mayumi; Matsuoka, Yuta; Yamada, Ken-ichi; Miyata, Satoshi; Fujii, Junichi

    2014-01-24

    Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a(-/-)) mice and human Akrla-transgenic (Akr1a(tg/+)) mice experience different anesthetic actions from pentobarbital-prolonged in Akr1a-knockout (Akr1a(-/-)) mice and shortened in human Akrla-transgenic (Akr1a(tg/+)) mice. We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice. Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a(-/-) mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a(-/-) mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics. Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients. Copyright © 2013. Published by Elsevier Inc.

  13. Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-20

    Directory of Open Access Journals (Sweden)

    Deeti K. Shetty

    2016-03-01

    Full Text Available Ovarian carcinoma immuno-reactive antigen domain containing 1(OCIAD1 single copy was knocked out generating an OCIAD1 heterozygous knockout human embryonic stem line named BJNhem20-OCIAD1-CRISPR-20. The line was generated using CRISPR-Cas9D10A double nickase knockout strategy (Mali et al., 2013.

  14. A study in male and female 5-HT transporter knockout rats : An animal model for anxiety and depression disorders

    NARCIS (Netherlands)

    Olivier, J D A; Van Der Hart, M G C; Van Swelm, R P L; Dederen, P J; Homberg, J R; Cremers, T; Deen, P M T; Cuppen, E; Cools, A R; Ellenbroek, B A

    2008-01-01

    Human studies have shown that a reduction of 5-HT transporter (SERT) increases the vulnerability for anxiety and depression. Moreover, women are more vulnerable to develop depression and anxiety disorders than men. For that reason we hypothesized that homozygous 5-HT transporter knockout rat

  15. Characterization of the serotonin transporter knockout rat : A selective change in the functioning of the serotonergic system

    NARCIS (Netherlands)

    Homberg, J. R.; Olivier, J.D.A.; Smits, B. M. G.; Mul, J. D.; Mudde, J.; Verheul, M.; Nieuwenhuizen, O. F. M.; Cools, A. R.; Ronken, E; Cremers, Thomas; Schoffelmeere, A. N. M.; Ellenbroeik, B. A.; Cuppen, E.

    2007-01-01

    Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by

  16. Altered expression and modulation of activity-regulated cytoskeletal associated protein (Arc) in serotonin transporter knockout rats.

    NARCIS (Netherlands)

    Molteni, R.; Calabrese, F.; Maj, P.F.; Olivier, J.D.A.; Racagni, G.; Ellenbroek, A.A.; Riva, M.A.

    2009-01-01

    A gene variant in the human serotonin transporter (SERT) can increase the vulnerability to mood disorders. SERT knockout animals show similarities to the human condition and represent an important tool to investigate the mechanisms underlying the pathologic condition in humans. Along this line of

  17. Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

    NARCIS (Netherlands)

    Homberg, J.R.; Olivier, J.D.A.; Smits, B.M.; Mul, J.D.; Mudde, J.; Verheul, M.; Nieuwenhuizen, O.F.; Cools, A.R.; Ronken, E.; Cremers, T.; Schoffelmeer, A.N.; Ellenbroek, B.A.; Cuppen, E.

    2007-01-01

    Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by

  18. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

    NARCIS (Netherlands)

    McCarthy, J.; O'Mahony, L.; O'Callaghan, L.; Sheil, B.; Vaughan, E.E.; Fitzsimons, N.A.; Fitzgibbon, J.; O'Sullivan, G.C.; Kiely, B.; Collins, J.K.; Shanahan, F.

    2003-01-01

    Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout ( KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. Aims: ( 1) To compare the effect of feeding

  19. Alterations in apical dendrite bundling in the somatosensory cortex of 5-HT3A receptor knockout mice.

    NARCIS (Netherlands)

    Smit-Rigter, L.A.; Wadman, W.J.; van Hooft, J.A.

    2011-01-01

    In various species and areas of the cerebral cortex, apical dendrites of pyramidal neurons form clusters which extend through several layers of the cortex also known as dendritic bundles. Previously, it has been shown that 5-HT3A receptor knockout mice show hypercomplex apical dendrites of cortical

  20. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only

    Science.gov (United States)

    Pagani, Jerome H.; Williams Avram, Sarah K.; Cui, Zhenzhong; Song, June; Mezey, Éva; Senerth, Julia M.; Baumann, Michael H.; Young, W. Scott

    2015-01-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically-mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to eight of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. PMID:25677455

  1. A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders.

    NARCIS (Netherlands)

    Olivier, J.; Van Der Hart, M.G.C.; Van Swelm, R.P.L.; Dederen, P.J.; Homberg, J.R.; Cremers, T.; Deen, P.M.T.; Cuppen, E.; Cools, A.R.; Ellenbroek, B.A.

    2008-01-01

    Human studies have shown that a reduction of 5-HT transporter (SERT) increases the vulnerability for anxiety and depression. Moreover, women are more vulnerable to develop depression and anxiety disorders than men. For that reason we hypothesized that homozygous 5-HT transporter knockout rat

  2. No further loss of dorsal root ganglion cells after axotomy in p75 neurotrophin receptor knockout mice

    DEFF Research Database (Denmark)

    Sørensen, Bodil; Tandrup, Trine; Koltzenburg, Martin

    2003-01-01

    disector techniques. At birth, the total number of DRG neurons was 10,000 ±2,600 in control mice compared with 5,100 ±1,300 in p75 knockout mice. During postnatal development, 1,400 neuronal B-cell bodies were lost in p75 knockouts (2P ± 0.±05) and 1,100 in controls (NS), whereas the A-cell population......The role of the p75 neurotrophin receptor for neuronal survival after nerve crush was studied in L5 dorsal root ganglia (DRG) of knockout mice and controls with assumption-free stereological methods. Numbers of neuronal A- and B-cells were obtained using the optical fractionator and optical...... remained stable. After a sciatic nerve crush, the total neuron loss in controls was 15.4% ±3.5% (2P ±0.05) and 22.7% 5.1% (2P neurons after crush in p75 knockout mice. Neuronal A-cell number was unchanged after...

  3. Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model.

    Directory of Open Access Journals (Sweden)

    Tilman Jobst-Schwan

    Full Text Available Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.

  4. Studies of an Androgen-Binding Protein Knockout Corroborate a Role for Salivary ABP in Mouse Communication

    Czech Academy of Sciences Publication Activity Database

    Chung, A. G.; Belone, P. M.; Vošlajerová Bímová, Barbora; Karn, R. C.; Laukaitis, C. M.

    2017-01-01

    Roč. 205, č. 4 (2017), s. 1517-1527 ISSN 0016-6731 R&D Projects: GA ČR GA15-13265S Institutional support: RVO:67985904 Keywords : androgen-binding protein * knockout mouse * preference testing Subject RIV: EA - Cell Biology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 4.556, year: 2016

  5. CD8 Knockout Mice Are Protected from Challenge by Vaccination with WR201, a Live Attenuated Mutant of Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Samuel L. Yingst

    2013-01-01

    Full Text Available CD8+ T cells have been reported to play an important role in defense against B. abortus infection in mouse models. In the present report, we use CD8 knockout mice to further elucidate the role of these cells in protection from B. melitensis infection. Mice were immunized orally by administration of B. melitensis WR201, a purine auxotrophic attenuated vaccine strain, then challenged intranasally with B. melitensis 16M. In some experiments, persistence of WR201 in the spleens of CD8 knockout mice was slightly longer than that in the spleens of normal mice. However, development of anti-LPS serum antibody, antigen-induced production of γ-interferon (IFN-γ by immune splenic lymphocytes, protection against intranasal challenge, and recovery of nonimmunized animals from intranasal challenge were similar between normal and knockout animals. Further, primary Brucella infection was not exacerbated in perforin knockout and Fas-deficient mice and these animals’ anti-Brucella immune responses were indistinguishable from those of normal mice. These results indicate that CD8+ T cells do not play an essential role as either cytotoxic cells or IFN-γ producers, yet they do participate in a specific immune response to immunization and challenge in this murine model of B. melitensis infection.

  6. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures

    NARCIS (Netherlands)

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; Söder, Olle; Stukenborg, Jan-Bernd

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have

  7. Enhanced proliferation of astrocytes from beta(2)-adrenergic receptor knockout mice is influenced by the IGF system

    NARCIS (Netherlands)

    Chesik, Daniel; Glazenburg, Lisa; De Keyser, Jacques; Wilczak, Nadine

    In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (beta(2)AR). beta(2)AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as

  8. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  9. Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats

    Science.gov (United States)

    Sun, Jianjun; Kouranova, Evguenia; Cui, Xiaoxia; Mach, Robert H.; Xu, Jinbin

    2014-01-01

    Pathogenic autosomal recessive mutations in the DJ-1 (Park7) or the PTEN-induced putative kinase 1 (Pink1 or PARK6) genes are associated with familial Parkinson’s disease (PD). It is not well known regarding the pathological mechanisms involving the DJ-1 and Pink1 mutations. Here we characterized DJ-1 and Pink1 knockout rats both through expression profiling and using quantitative autoradiography to measure the densities of the dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) in the striatum of transgenic rats and wild type controls. Expression profiling with a commercially available array of 84 genes known to be involved in PD indicated that only the target gene was significantly downregulated in each transgenic rat model. D1 receptor, VMAT2, and DAT were measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. No significant changes were observed in the density of DAT in either model. Although the densities of VMAT2 and D1 receptor were unchanged in Pink1 knockout, but both were increased in DJ-1 knockout rats. The densities of D2 and D3 receptors, determined by mathematical analysis of binding of radioligands [3H]WC-10 and [3H]raclopride, were significantly increased in both knockout models. These distinctive changes in the expression of dopamine presynaptic markers and receptors in the striatum may reflect different compensatory regulation of dopamine system in DJ-1 versus Pink1 knockout rat models of familial PD. PMID:24157858

  10. Remyelination in experimentally demyelinated connexin 32 KnockOut mice Remielinização em camundongos KnockOut para conexina 32 desmielinizados experimentalmente

    Directory of Open Access Journals (Sweden)

    Adriano Tony Ramos

    2009-06-01

    Full Text Available The aim of this study was to evaluate the role of connexin 32 (Cx 32 during remyelination of the peripheral nervous system, through a local injection of either 0,1% ethidium bromide solution or saline in the sciatic nerve of Cx 32 knockout mice. Euthanasia was performed ranging from 1, 2, 3, 7, 15, 21 to 30 days after injection. Histochemical, immunohistochemical, immunofluorescence and transmission electron microscopical techniques were used to analyze the development of the lesions. Within the sciatic nerves, Schwann cells initially showed signs of intoxication and rejected their sheaths; after seven days, some thin newly formed myelin sheaths with uneven compactness and redundant loops (tomacula were conspicuous. We concluded that the regeneration of lost myelin sheaths within the PNS followed the pattern already reported for this model in other laboratory species. Therefore, these results suggest that absence of Cx 32 did not interfere with the normal pattern of remyelination in this model in young mice.Este estudo visou avaliar o papel da conexina 32 (Cx 32 durante a remielinização no sistema nervoso periférico. Uma injeção local de 0,1% de solução de brometo de etídio foi realizada no nervo ciático de camundongos deletados para a Cx 32, com eutanásia dos animais aos 1, 2, 3, 7, 15, 21 e 30 dias pós-injeção. Avaliações histoquímicas, imunoistoquímicas, por imunofluorescência e por microscopia eletrônica de transmissão foram utilizadas na análise do desenvolvimento das lesões. Nos nervos ciáticos, células de Schwann mostraram inicialmente sinais de intoxicação e rejeitaram suas bainhas. Após sete dias, observaram-se finas bainhas neoformadas, com compactação desigual e alças redundantes (tomácula. Conclui-se que a regeneração de bainhas de mielina perdidas no SNP seguiu o padrão já relatado deste modelo em outras espécies de laboratório. Portanto, estes resultados sugerem que a ausência da Cx 32 n

  11. Unique nuclear vacuoles in the motor neurons of conditional ADAR2-knockout mice.

    Science.gov (United States)

    Sasaki, Shoichi; Takenari Yamashita; Hideyama, Takuto; Kwak, Shin

    2014-03-06

    A reduction in adenosine deaminase acting on RNA 2 (ADAR2) activity causes the death of spinal motor neurons specifically via the GluA2 Q/R site-RNA editing failure in sporadic amyotrophic lateral sclerosis (ALS). We studied, over time, the spinal cords of ADAR2-knockout mice, which are the mechanistic model mice for sporadic ALS, using homozygous ADAR2(flox/flox)/VAChT-Cre.Fast (AR2), homozygous ADAR2(flox/flox)/VAChT-Cre.Slow (AR2Slow), and heterozygous ADAR2(flox/+)/VAChT-Cre.Fast (AR2H) mice. The conditional ADAR2-knockout mice were divided into 3 groups by stage: presymptomatic (AR2H mice), early symptomatic (AR2 mice, AR2H mice) and late symptomatic (AR2Slow mice). Light-microscopically, some motor neurons in AR2 and AR2H mice (presymptomatic) showed simple neuronal atrophy and astrogliosis, and AR2H (early symptomatic) and AR2Slow mice often showed vacuoles predominantly in motor neurons. The number of vacuole-bearing anterior horn neurons decreased with the loss of anterior horn neurons in AR2H mice after 40 weeks of age. Electron-microscopically, in AR2 mice, while the cytoplasm of normal-looking motor neurons was almost always normal-appearing, the interior of dendrites was frequently loose and disorganized. In AR2H and AR2Slow mice, large vacuoles without a limiting membrane were observed in the anterior horns, preferentially in the nuclei of motor neurons, astrocytes and oligodendrocytes. Nuclear vacuoles were not observed in AR2res (ADAR2(flox/flox)/VAChT-Cre.Fast/GluR-B(R/R)) mice, in which motor neurons express edited GluA2 in the absence of ADAR2. These findings suggest that ADAR2-reduction is associated with progressive deterioration of nuclear architecture, resulting in vacuolated nuclei due to a Ca(2+)-permeable AMPA receptor-mediated mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. The Pain Genes Database: An interactive web browser of pain-related transgenic knockout studies.

    Science.gov (United States)

    Lacroix-Fralish, Michael L; Ledoux, Jean B; Mogil, Jeffrey S

    2007-09-01

    The transgenic knockout mouse is one of the most important tools of modern biology, and commonly employed by pain researchers to examine the function of genes of interest. Over 400 papers, at a current rate of >60 papers per year, have been published to date describing a statistically significant behavioral pain "phenotype" resulting from the null mutation of a single gene. The standard literature review format is incapable of providing a sufficiently broad and up-to-date overview of the field. We have therefore constructed the Pain Genes Database, an interactive, web-based data browser designed to allow easy access to and analysis of the published pain-related phenotypes of mutant mice (over 200 different mutants at the date of submission). Manuscripts describing results of pain-relevant knockout studies were identified via Medline search. Manuscripts were included in the database if they described the testing of a spontaneous or genetically engineered mutant mouse with null expression of a single gene on a behavioral assay of acute or tonic nociception, injury- or stimulus-induced hypersensitivity (i.e., allodynia or hyperalgesia), or drug- or stress-induced inhibition of nociception (i.e., analgesia), and reported at least one statistically significant difference between the mutant mice and their simultaneously tested wildtype controls. The database features two levels of exploration, one allowing the identification of genes by name, acronym, genomic position or "summary" phenotype, and the other allowing in-depth browsing, paper-by-paper, of specific phenotypes and test parameters. Links to genetic databases and Medline abstracts are provided for each gene and paper. It is our intention to update the database continually based on weekly Medline searches. This database should provide pain researchers with a useful and easy-to-use tool for the generation of novel hypotheses regarding the roles of genes and their protein products in pain processing and modulation

  13. Global analysis of gene expression in the developing brain of Gtf2ird1 knockout mice.

    Directory of Open Access Journals (Sweden)

    Jennifer O'Leary

    Full Text Available Williams-Beuren Syndrome (WBS is a neurodevelopmental disorder caused by a hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23 encompassing 26 genes. One of these genes, GTF2IRD1, codes for a putative transcription factor that is expressed throughout the brain during development. Genotype-phenotype studies in patients with atypical deletions of 7q11.23 implicate this gene in the neurological features of WBS, and Gtf2ird1 knockout mice show reduced innate fear and increased sociability, consistent with features of WBS. Multiple studies have identified in vitro target genes of GTF2IRD1, but we sought to identify in vivo targets in the mouse brain.We performed the first in vivo microarray screen for transcriptional targets of Gtf2ird1 in brain tissue from Gtf2ird1 knockout and wildtype mice at embryonic day 15.5 and at birth. Changes in gene expression in the mutant mice were moderate (0.5 to 2.5 fold and of candidate genes with altered expression verified using real-time PCR, most were located on chromosome 5, within 10 Mb of Gtf2ird1. siRNA knock-down of Gtf2ird1 in two mouse neuronal cell lines failed to identify changes in expression of any of the genes identified from the microarray and subsequent analysis showed that differences in expression of genes on chromosome 5 were the result of retention of that chromosome region from the targeted embryonic stem cell line, and so were dependent upon strain rather than Gtf2ird1 genotype. In addition, specific analysis of genes previously identified as direct in vitro targets of GTF2IRD1 failed to show altered expression.We have been unable to identify any in vivo neuronal targets of GTF2IRD1 through genome-wide expression analysis, despite widespread and robust expression of this protein in the developing rodent brain.

  14. Reconstructing gene-regulatory networks from time series, knock-out data, and prior knowledge

    Directory of Open Access Journals (Sweden)

    Timmer Jens

    2007-02-01

    Full Text Available Abstract Background Cellular processes are controlled by gene-regulatory networks. Several computational methods are currently used to learn the structure of gene-regulatory networks from data. This study focusses on time series gene expression and gene knock-out data in order to identify the underlying network structure. We compare the performance of different network reconstruction methods using synthetic data generated from an ensemble of reference networks. Data requirements as well as optimal experiments for the reconstruction of gene-regulatory networks are investigated. Additionally, the impact of prior knowledge on network reconstruction as well as the effect of unobserved cellular processes is studied. Results We identify linear Gaussian dynamic Bayesian networks and variable selection based on F-statistics as suitable methods for the reconstruction of gene-regulatory networks from time series data. Commonly used discrete dynamic Bayesian networks perform inferior and this result can be attributed to the inevitable information loss by discretization of expression data. It is shown that short time series generated under transcription factor knock-out are optimal experiments in order to reveal the structure of gene regulatory networks. Relative to the level of observational noise, we give estimates for the required amount of gene expression data in order to accurately reconstruct gene-regulatory networks. The benefit of using of prior knowledge within a Bayesian learning framework is found to be limited to conditions of small gene expression data size. Unobserved processes, like protein-protein interactions, induce dependencies between gene expression levels similar to direct transcriptional regulation. We show that these dependencies cannot be distinguished from transcription factor mediated gene regulation on the basis of gene expression data alone. Conclusion Currently available data size and data quality make the reconstruction of

  15. Exposure to low-dose rotenone precipitates synaptic plasticity alterations in PINK1 heterozygous knockout mice.

    Science.gov (United States)

    Martella, G; Madeo, G; Maltese, M; Vanni, V; Puglisi, F; Ferraro, E; Schirinzi, T; Valente, E M; Bonanni, L; Shen, J; Mandolesi, G; Mercuri, N B; Bonsi, P; Pisani, A

    2016-07-01

    Heterozygous mutations in the PINK1 gene are considered a susceptibility factor to develop early-onset Parkinson's disease (PD), as supported by dopamine hypometabolism in asymptomatic mutation carriers and subtle alterations of dopamine-dependent striatal synaptic plasticity in heterozygous PINK1 knockout (PINK1(+/-)) mice. The aim of the present study was to investigate whether exposure to low-dose rotenone of heterozygous PINK1(+/-) mice, compared to their wild-type PINK1(+/+) littermates, could impact on dopamine-dependent striatal synaptic plasticity, in the absence of apparent structural alterations. Mice were exposed to a range of concentrations of rotenone (0.01-1mg/kg). Chronic treatment with concentrations of rotenone up to 0.8mg/kg did not cause manifest neuronal loss or changes in ATP levels both in the striatum or substantia nigra of PINK1(+/-) and PINK1(+/+) mice. Moreover, rotenone (up to 0.8mg/kg) treatment did not induce mislocalization of the mitochondrial membrane protein Tom20 and release of cytochrome c in PINK1(+/-) striata. Accordingly, basic electrophysiological properties of nigral dopaminergic and striatal medium spiny neurons (MSNs) were normal. Despite the lack of gross alterations in neuronal viability in chronically-treated PINK1(+/-), a complete loss of both long-term depression (LTD) and long-term potentiation (LTP) was recorded in MSNs from PINK1(+/-) mice treated with a low rotenone (0.1mg/kg) concentration. Even lower concentrations (0.01mg/kg) blocked LTP induction in heterozygous PINK1(+/-) MSNs compared to PINK1(+/+) mice. Of interest, chronic pretreatment with the antioxidants alpha-tocopherol and Trolox, a water-soluble analog of vitamin E and powerful antioxidant, rescued synaptic plasticity impairment, confirming that, at the doses we utilized, rotenone did not induce irreversible alterations. In this model, chronic exposure to low-doses of rotenone was not sufficient to alter mitochondrial integrity and ATP production, but

  16. New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

    Directory of Open Access Journals (Sweden)

    Hileman Stanley M

    2007-10-01

    Full Text Available Abstract Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed. Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

  17. Testing the role of predicted gene knockouts in human anthropometric trait variation

    Science.gov (United States)

    Lessard, Samuel; Manning, Alisa K.; Low-Kam, Cécile; Auer, Paul L.; Giri, Ayush; Graff, Mariaelisa; Schurmann, Claudia; Yaghootkar, Hanieh; Luan, Jian'an; Esko, Tonu; Karaderi, Tugce; Bottinger, Erwin P.; Lu, Yingchang; Carlson, Chris; Caulfield, Mark; Dubé, Marie-Pierre; Jackson, Rebecca D.; Kooperberg, Charles; McKnight, Barbara; Mongrain, Ian; Peters, Ulrike; Reiner, Alex P.; Rhainds, David; Sotoodehnia, Nona; Hirschhorn, Joel N.; Scott, Robert A.; Munroe, Patricia B.; Frayling, Timothy M.; Loos, Ruth J.F.; North, Kari E.; Edwards, Todd L.; Tardif, Jean-Claude; Lindgren, Cecilia M.; Lettre, Guillaume

    2016-01-01

    Although the role of complete gene inactivation by two loss-of-function mutations inherited in trans is well-established in recessive Mendelian diseases, we have not yet explored how such gene knockouts (KOs) could influence complex human phenotypes. Here, we developed a statistical framework to test the association between gene KOs and quantitative human traits. Our method is flexible, publicly available, and compatible with common genotype format files (e.g. PLINK and vcf). We characterized gene KOs in 4498 participants from the NHLBI Exome Sequence Project (ESP) sequenced at high coverage (>100×), 1976 French Canadians from the Montreal Heart Institute Biobank sequenced at low coverage (5.7×), and >100 000 participants from the Genetic Investigation of ANthropometric Traits (GIANT) Consortium genotyped on an exome array. We tested associations between gene KOs and three anthropometric traits: body mass index (BMI), height and BMI-adjusted waist-to-hip ratio (WHR). Despite our large sample size and multiple datasets available, we could not detect robust associations between specific gene KOs and quantitative anthropometric traits. Our results highlight several limitations and challenges for future gene KO studies in humans, in particular when there is no prior knowledge on the phenotypes that might be affected by the tested gene KOs. They also suggest that gene KOs identified with current DNA sequencing methodologies probably do not strongly influence normal variation in BMI, height, and WHR in the general human population. PMID:26908616

  18. Pressor responses to ephedrine are not impaired in dopamine β-hydroxylase knockout mice

    Science.gov (United States)

    Liles, J T; Baber, S R; Deng, W; Porter, J R; Corll, C; Murthy, S N; Thomas, S A; Kadowitz, P J

    2006-01-01

    Background and Purpose: Ephedrine and amphetamine can cause substantial increases in systemic arterial pressure. However, the role of endogenous noradrenaline release in mediating the pressor response to ephedrine is controversial. Studies using pharmacologic agents to decrease the synthesis, storage, and release of catecholamines have supported both a direct and an indirect mechanism of action for ephedrine. The purpose of the present study was to determine if endogenous noradrenaline release is required for cardiovascular responses to ephedrine and amphetamine using a genetic mouse model. Experimental Approach: Increases in systemic arterial pressure and heart rate in response to ephedrine and amphetamine were investigated and compared in dopamine β-hydroxylase knockout (Dbh -/-) mice that cannot synthesize noradrenaline. Dbh +/- littermates have normal noradrenaline and adrenaline tissue levels, and served as controls in all experiments. Key Results: In Dbh -/- mice the increases in systemic arterial pressure and heart rate in response to i.v. injections of ephedrine were not impaired whereas responses to amphetamine were markedly reduced, when compared with responses in Dbh +/- mice. The pressor response to tyramine was abolished whereas pressor responses to noradrenaline, phenylephrine, dopamine, and angiotensin II were similar in Dbh -/- and Dbh +/- mice. Conclusions and Implications: The present results in Dbh -/- mice provide support for the hypothesis that pressor responses to ephedrine are directly mediated whereas responses to amphetamine are dependent on the release of noradrenaline and suggest that Dbh +/- and Dbh -/- mice are useful for the study of direct and indirect mechanisms. PMID:17099719

  19. Pressor responses to ephedrine are not impaired in dopamine beta-hydroxylase knockout mice.

    Science.gov (United States)

    Liles, J T; Baber, S R; Deng, W; Porter, J R; Corll, C; Murthy, S N; Thomas, S A; Kadowitz, P J

    2007-01-01

    Ephedrine and amphetamine can cause substantial increases in systemic arterial pressure. However, the role of endogenous noradrenaline release in mediating the pressor response to ephedrine is controversial. Studies using pharmacologic agents to decrease the synthesis, storage, and release of catecholamines have supported both a direct and an indirect mechanism of action for ephedrine. The purpose of the present study was to determine if endogenous noradrenaline release is required for cardiovascular responses to ephedrine and amphetamine using a genetic mouse model. Increases in systemic arterial pressure and heart rate in response to ephedrine and amphetamine were investigated and compared in dopamine beta-hydroxylase knockout (Dbh -/-) mice that cannot synthesize noradrenaline. Dbh +/- littermates have normal noradrenaline and adrenaline tissue levels, and served as controls in all experiments. In Dbh -/- mice the increases in systemic arterial pressure and heart rate in response to i.v. injections of ephedrine were not impaired whereas responses to amphetamine were markedly reduced, when compared with responses in Dbh +/- mice. The pressor response to tyramine was abolished whereas pressor responses to noradrenaline, phenylephrine, dopamine, and angiotensin II were similar in Dbh -/- and Dbh +/- mice. The present results in Dbh -/- mice provide support for the hypothesis that pressor responses to ephedrine are directly mediated whereas responses to amphetamine are dependent on the release of noradrenaline and suggest that Dbh +/- and Dbh -/- mice are useful for the study of direct and indirect mechanisms.

  20. Soy milk versus simvastatin for preventing atherosclerosis and left ventricle remodeling in LDL receptor knockout mice.

    Science.gov (United States)

    Santos, L; Davel, A P; Almeida, T I R; Almeida, M R; Soares, E A; Fernandes, G J M; Magalhães, S F; Barauna, V G; Garcia, J A D

    2017-02-20

    Functional food intake has been highlighted as a strategy for the prevention of cardiovascular diseases by reducing risk factors. In this study, we compared the effects of oral treatment with soy milk and simvastatin on dyslipidemia, left ventricle remodeling and atherosclerotic lesion of LDL receptor knockout mice (LDLr-/-) fed a hyperlipidic diet. Forty 3-month old male LDLr-/- mice were distributed into four groups: control group (C), in which animals received standard diet; HL group, in which animals were fed a hyperlipidic diet; HL+SM or HL+S groups, in which animals were submitted to a hyperlipidic diet plus soy milk or simvastatin, respectively. After 60 days, both soy milk and simvastatin treatment prevented dyslipidemia, atherosclerotic lesion progression and left ventricle hypertrophy in LDLr-/- mice. These beneficial effects of soy milk and simvastatin were associated with reduced oxidative stress and inflammatory state in the heart and aorta caused by the hyperlipidic diet. Treatment with soy milk was more effective in preventing HDLc reduction and triacylglycerol and VLDLc increase. On the other hand, simvastatin was more effective in preventing an increase in total cholesterol, LDLc and superoxide production in aorta, as well as CD40L both in aorta and left ventricle of LDLr-/-. In conclusion, our results suggest a cardioprotective effect of soy milk in LDLr-/- mice comparable to the well-known effects of simvastatin.

  1. Diacylglycerol kinase β knockout mice exhibit lithium-sensitive behavioral abnormalities.

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    Kenichi Kakefuda

    Full Text Available BACKGROUND: Diacylglycerol kinase (DGK is an enzyme that phosphorylates diacylglycerol (DG to produce phosphatidic acid (PA. DGKβ is widely distributed in the central nervous system, such as the olfactory bulb, cerebral cortex, striatum, and hippocampus. Recent studies reported that the splice variant at the COOH-terminal of DGKβ was related to bipolar disorder, but its detailed mechanism is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we performed behavioral tests using DGKβ knockout (KO mice to investigate the effects of DGKβ deficits on psychomotor behavior. DGKβ KO mice exhibited some behavioral abnormalities, such as hyperactivity, reduced anxiety, and reduced depression. Additionally, hyperactivity and reduced anxiety were attenuated by the administration of the mood stabilizer, lithium, but not haloperidol, diazepam, or imipramine. Moreover, DGKβ KO mice showed impairment in Akt-glycogen synthesis kinase (GSK 3β signaling and cortical spine formation. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKβ KO mice exhibit lithium-sensitive behavioral abnormalities that are, at least in part, due to the impairment of Akt-GSK3β signaling and cortical spine formation.

  2. Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

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    Xingsheng Hou

    Full Text Available FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7 and a flcA deletion mutant (Sp7-flcAΔ revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot. The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase, nitrogen metabolism (Glutamine synthetase and nitric oxide synthase, stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit and morphological transformation (transducer coupling protein. The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

  3. Chemical Transport Knockout for Oxidized Vitamin C, Dehydroascorbic Acid, Reveals Its Functions in vivo

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    Hongbin Tu

    2017-09-01

    Full Text Available Despite its transport by glucose transporters (GLUTs in vitro, it is unknown whether dehydroascorbic acid (oxidized vitamin C, DHA has any in vivo function. To investigate, we created a chemical transport knockout model using the vitamin C analog 6-bromo-ascorbate. This analog is transported on sodium-dependent vitamin C transporters but its oxidized form, 6-bromo-dehydroascorbic acid, is not transported by GLUTs. Mice (gulo−/− unable to synthesize ascorbate (vitamin C were raised on 6-bromo-ascorbate. Despite normal survival, centrifugation of blood produced hemolysis secondary to near absence of red blood cell (RBC ascorbate/6-bromo-ascorbate. Key findings with clinical implications were that RBCs in vitro transported dehydroascorbic acid but not bromo-dehydroascorbic acid; RBC ascorbate in vivo was obtained only via DHA transport; ascorbate via DHA transport in vivo was necessary for RBC structural integrity; and internal RBC ascorbate was essential to maintain ascorbate plasma concentrations in vitro/in vivo.

  4. Cellular responses during morphological transformation in Azospirillum brasilense and Its flcA knockout mutant.

    Science.gov (United States)

    Hou, Xingsheng; McMillan, Mary; Coumans, Joëlle V F; Poljak, Anne; Raftery, Mark J; Pereg, Lily

    2014-01-01

    FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA- strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.

  5. Utilising the resources of the International Knockout Mouse Consortium: the Australian experience.

    Science.gov (United States)

    Cotton, Leanne M; Meilak, Michelle L; Templeton, Tanya; Gonzales, Jose G; Nenci, Arianna; Cooney, Melissa; Truman, Dirk; Rodda, Fleur; Lynas, Alyce; Viney, Elizabeth; Rosenthal, Nadia; Bianco, Deborah M; O'Bryan, Moira K; Smyth, Ian M

    2015-04-01

    Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service.

  6. Impairment in extinction of cued fear memory in syntenin-1 knockout mice.

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    Talukdar, Gourango; Inoue, Ran; Yoshida, Tomoyuki; Mori, Hisashi

    2018-03-01

    Syntenin-1 is a PDZ domain-containing intracellular scaffold protein involved in exosome production, synapse formation, and synaptic plasticity. We tested whether syntenin-1 can regulate learning and memory through its effects on synaptic plasticity. Specifically, we investigated the role of syntenin-1 in contextual and cued fear conditioning and extinction of conditioned fear using syntenin-1 knockout (KO) mice. Genetic disruption of syntenin-1 had little effect on contextual and cued fear memory. However, syntenin-1 KO mice exhibited selective impairment in cued fear extinction retention. This extinction retention deficit in syntenin-1 KO mice was associated with reduced c-Fos-positive neurons in the basolateral amygdala (BLA) and infralimbic cortex (IL) after extinction training and increased c-Fos-positive neurons in the BLA after an extinction retention test. Our results suggest that syntenin-1 plays an important role in extinction of cued fear memory by modulating neuronal activity in the BLA and IL. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Sexual dimorphism in the glucose homeostasis phenotype of the Aromatase Knockout (ArKO) mice.

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    Van Sinderen, Michelle; Steinberg, Gregory; Jorgensen, Sebastian B; Honeyman, Jane; Chow, Jenny D Y; Simpson, Evan R; Jones, Margaret E E; Boon, Wah Chin

    2017-06-01

    We investigated the effects of estrogens on glucose homeostasis using the Aromatase Knockout (ArKO) mouse, which is unable to convert androgens into estrogens. The ArKO mouse is a model of total estrogen ablation which develops symptoms of metabolic syndrome. To determine the development and progression of whole body state of insulin resistance of ArKO mice, comprehensive whole body tolerance tests were performed on WT, ArKO and estrogen administrated mice at 3 and 12 months of age. The absence of estrogens in the male ArKO mice leads to hepatic insulin resistance, glucose and pyruvate intolerance from 3 to 12 months with consistent improvement upon estrogen treatment. Estrogen absence in the female ArKO mice leads to glucose intolerance without pyruvate intolerance or insulin resistance. The replacement of estrogens in the female WT and ArKO mice exhibited both insulin sensitizing and resistance effects depending on age and dosage. In conclusion, this study presents information on the sexually dimorphic roles of estrogens on glucose homeostasis regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. [ADAR1 Knockout Inhibits Notch1-induced T-ALL in Mice].

    Science.gov (United States)

    Gao, Hui-Er; Peng, Lu-Yun; Yang, Xin; Zhang, Ying-Chi; Hu, Tian-Yuan; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao; Gao, Ying-Dai

    2016-06-01

    To investigate the effect of ADAR1 on the occurrence and development of mouse T cell acute lymphoblastic leukemia (T-ALL). Lck-Cre; ADAR1lox/lox mice were generated through interbreeding. The lineage-cells of Lck-Cre; ADAR1lox/lox mice and the control were enriched respectively by the means of MACS, and the lin- cells were transfected with retrovirus carrying MSCV-ICN1-IRES-GFP fusion gene. Then the transfection efficiency was detected by the means of FACS, and the same number of GFP+ cells were transplanted into lethally irradiated recipient mice to observe the survival of mice in 2 recipient group after transplantation. T cell-specific knockout ADAR1 mice were generated, and Notch1-induced T-ALL mouse model was established successfully. The leukemia with T-ALL characteristics occured in the mice of control group, but did not in the ADAR1 kmockout mice after transplantation. ADAR1 plays a key role in the incidence and development of Notch1-induced T-ALL.

  9. Serum CTX levels and histomorphometric analysis in Src versus RANKL knockout mice.

    Science.gov (United States)

    Takeshita, Sunao; Fumoto, Toshio; Ito, Masako; Ikeda, Kyoji

    2017-06-06

    Src knockout (KO) and RANKL KO mice both exhibit near complete osteopetrosis in terms of 3D-bone volume (BV) fraction by micro-CT, whereas the serum CTX concentration of Src KO is apparently normal and that of RANKL KO is 30% of wild-type (WT) despite the fact that they lack osteoclasts. By histomorphometry we found that, whereas eroded surface (ES) and osteoid surface (OS) are zero values in RANKL KO, they are indistinguishable from WT in Src KO; because of marked increase in bone surface (BS), ES/BS and OS/BS of Src KO are 30-40% of WT. While RANKL KO lack both osteoclasts and osteoblasts, Src KO reveal increased numbers of osteoclasts and indistinguishable numbers of osteoblasts compared with WT; again, on the basis of BS, N.Oc/BS is comparable to WT and N.Ob/BS is markedly decreased in Src KO. The apparently increased number of total osteoclasts may be due to increased expression of RANKL found in Src KO bone in vivo. Src has a gene dosage-dependent effect on osteoclast function in vitro, with Src -/- osteoclasts completely lacking bone-resorbing function as determined by CTX release on dentin. Thus, Src KO osteoclasts retain some bone-resorbing function in vivo. The number of osteocytes is proportionally increased in RANKL KO, while Src KO mice have relative osteocyte deficiency, raising the possibility that RANKL and Src has an unrecognized role in osteocyte survival.

  10. MicroRNA-155 knockout mice are susceptible to Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Iwai, Hiroki; Funatogawa, Keiji; Matsumura, Kazunori; Kato-Miyazawa, Masako; Kirikae, Fumiko; Kiga, Kotaro; Sasakawa, Chihiro; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2015-05-01

    MicroRNAs (miRNAs) are short, conserved, non-coding RNA molecules that repress translation, followed by the decay of miRNA-targeted mRNAs that encode molecules involved in cell differentiation, development, immunity and apoptosis. At least six miRNAs, including microRNA-155 (miR-155), were up-regulated when born marrow-derived macrophages from C57BL/6 mice were infected with Mycobacterium tuberculosis Erdman. C57BL/6 mice intravenously infected with Erdman showed up-regulation of miR-155 in livers and lungs. Following infection, miR-155-deficient C57BL/6 mice died significantly earlier and had significantly higher numbers of CFU in lungs than wild-type mice. Moreover, fewer CD4(+) T cells, but higher numbers of monocytes and neutrophils, were present in the lungs of Erdman-infected miR-155 knockout (miR-155(-/-)) than of wild-type mice. These findings indicated that miR-155 plays a critical role in immune responses to M. tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice

    Science.gov (United States)

    Roman, Brian B.; Meyer, Ronald A.; Wiseman, Robert W.

    2002-01-01

    Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.

  12. Examination of MARCO activity on dendritic cell phenotype and function using a gene knockout mouse.

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    Hiroshi Komine

    Full Text Available We have reported the upregulation of MARCO, a member of the class A scavenger receptor family, on the surface of murine and human dendritic cells (DCs pulsed with tumor lysates. Exposure of murine tumor lysate-pulsed DCs to an anti-MARCO antibody led to loss of dendritic-like processes and enhanced migratory capacity. In this study, we have further examined the biological and therapeutic implications of MARCO expression by DCs. DCs generated from the bone marrow (bm of MARCO knockout (MARCO⁻/⁻ mice were phenotypically similar to DCs generated from the bm of wild-type mice and produced normal levels of IL-12 and TNF-α when exposed to LPS. MARCO⁻/⁻ DCs demonstrated enhanced migratory capacity in response to CCL-21 in vitro. After subcutaneous injection into mice, MARCO⁻/⁻ TP-DCs migrated more efficiently to the draining lymph node leading to enhanced generation of tumor-specific IFN-γ producing T cells and improved tumor regression and survival in B16 melanoma-bearing mice. These results support targeting MARCO on the surface of DCs to improve trafficking and induction of anti-tumor immunity.

  13. Effects of Three Lipidated Oxytocin Analogs on Behavioral Deficits in CD38 Knockout Mice

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    Stanislav M. Cherepanov

    2017-10-01

    Full Text Available Oxytocin (OT is a nonapeptide that plays an important role in social behavior. Nasal administration of OT has been shown to improve trust in healthy humans and social interaction in autistic subjects. As is consistent with the nature of a peptide, OT has some unfavorable characteristics: it has a short half-life in plasma and shows poor permeability across the blood-brain barrier. Analogs with long-lasting effects may overcome these drawbacks. To this end, we have synthesized three analogs: lipo-oxytocin-1 (LOT-1, in which two palmitoyl groups are conjugated to the cysteine and tyrosine residues, lipo-oxytocin-2 (LOT-2 and lipo-oxytocin-3 (LOT-3, which include one palmitoyl group conjugated at the cysteine or tyrosine residue, respectively. The following behavioral deficits were observed in CD38 knockout (CD38−/− mice: a lack of paternal nurturing in CD38−/− sires, decreased ability for social recognition, and decreased sucrose consumption. OT demonstrated the ability to recover these disturbances to the level of wild-type mice for 30 min after injection. LOT-2 and LOT-3 partially recovered the behaviors for a short period. Conversely, LOT-1 restored the behavioral parameters, not for 30 min, but for 24 h. These data suggest that the lipidation of OT has some therapeutic benefits, and LOT-1 would be most useful because of its long-last activity.

  14. Interactions of the opioid and cannabinoid systems in reward: Insights from knockout studies

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    Katia eBefort

    2015-02-01

    Full Text Available The opioid system consists of three receptors, mu, delta, and kappa, which are activated by endogenous opioid peptides (enkephalins, endorphins and dynorphins. The endogenous cannabinoid system comprises lipid neuromodulators (endocannabinoids, enzymes for their synthesis and their degradation and two well-characterized receptors, cannabinoid receptors CB1 and CB2. These systems play a major role in the control of pain as well as in mood regulation, reward processing and the development of addiction. Both opioid and cannabinoid receptors are coupled to G proteins and are expressed throughout the brain reinforcement circuitry. Extending classical pharmacology, research using genetically modified mice has provided important progress in the identification of the specific contribution of each component of these endogenous systems in vivo on reward process. This review will summarize available genetic tools and our present knowledge on the consequences of gene knockout on reinforced behaviors in both systems, with a focus on their potential interactions. A better understanding of opioid-cannabinoid interactions may provide novel strategies for therapies in addicted individuals.

  15. ChREBP-Knockout Mice Show Sucrose Intolerance and Fructose Malabsorption.

    Science.gov (United States)

    Kato, Takehiro; Iizuka, Katsumi; Takao, Ken; Horikawa, Yukio; Kitamura, Tadahiro; Takeda, Jun

    2018-03-12

    We have previously reported that 60% sucrose diet-fed ChREBP knockout mice (KO) showed body weight loss resulting in lethality. We aimed to elucidate whether sucrose and fructose metabolism are impaired in KO. Wild-type mice (WT) and KO were fed a diet containing 30% sucrose with/without 0.08% miglitol, an α-glucosidase inhibitor, and these effects on phenotypes were tested. Furthermore, we compared metabolic changes of oral and peritoneal fructose injection. A thirty percent sucrose diet feeding did not affect phenotypes in KO. However, miglitol induced lethality in 30% sucrose-fed KO. Thirty percent sucrose plus miglitol diet-fed KO showed increased cecal contents, increased fecal lactate contents, increased growth of lactobacillales and Bifidobacterium and decreased growth of clostridium cluster XIVa. ChREBP gene deletion suppressed the mRNA levels of sucrose and fructose related genes. Next, oral fructose injection did not affect plasma glucose levels and liver fructose contents; however, intestinal sucrose and fructose related mRNA levels were increased only in WT. In contrast, peritoneal fructose injection increased plasma glucose levels in both mice; however, the hepatic fructose content in KO was much higher owing to decreased hepatic Khk mRNA expression. Taken together, KO showed sucrose intolerance and fructose malabsorption owing to decreased gene expression.

  16. ChREBP-Knockout Mice Show Sucrose Intolerance and Fructose Malabsorption

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    Takehiro Kato

    2018-03-01

    Full Text Available We have previously reported that 60% sucrose diet-fed ChREBP knockout mice (KO showed body weight loss resulting in lethality. We aimed to elucidate whether sucrose and fructose metabolism are impaired in KO. Wild-type mice (WT and KO were fed a diet containing 30% sucrose with/without 0.08% miglitol, an α-glucosidase inhibitor, and these effects on phenotypes were tested. Furthermore, we compared metabolic changes of oral and peritoneal fructose injection. A thirty percent sucrose diet feeding did not affect phenotypes in KO. However, miglitol induced lethality in 30% sucrose-fed KO. Thirty percent sucrose plus miglitol diet-fed KO showed increased cecal contents, increased fecal lactate contents, increased growth of lactobacillales and Bifidobacterium and decreased growth of clostridium cluster XIVa. ChREBP gene deletion suppressed the mRNA levels of sucrose and fructose related genes. Next, oral fructose injection did not affect plasma glucose levels and liver fructose contents; however, intestinal sucrose and fructose related mRNA levels were increased only in WT. In contrast, peritoneal fructose injection increased plasma glucose levels in both mice; however, the hepatic fructose content in KO was much higher owing to decreased hepatic Khk mRNA expression. Taken together, KO showed sucrose intolerance and fructose malabsorption owing to decreased gene expression.

  17. Effects of Eaf2 gene knockout on cataract induced by ultraviolet irradiation in mice

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    Yan-Hua Jiang

    2016-02-01

    Full Text Available AIM:To evaluate the effects of Eaf2 gene knockout on cataract in mice induced by ultraviolet irradiation.METHODS:Fifteen wild type mice were used as the control group, and 10 Eaf2 KO mice were used as the experimental group. The 14-week mice were taken as the research objects in the two groups. So the subgroups were: WT -nonUV, WT -UV, Eaf2 KO-nonUV and Eaf2 KO-UV, a total of 4 groups. Observe the lens of mice in vivo with slit lamp microscope, grade the lens opacity with Lens Opacities Classification System II(LOCSII. Then the mice were sacrificed by breaking the neck, the lens were removed and were observed by dark field microscopy. According to the captured images, the proportion of cataract region was analyzed using Image J software. The data of the two groups were statistically analyzed.RESULTS: The results detected by the two methods were similar. In WT-UV group and Eaf2 KO-UV group, the degree of lens opacity was significantly higher than those of WT-nonUV group and Eaf2 KO-nonUV group. The lens opacity of WT-UV group was significantly higher than that in Eaf2 KO-UV group, and the difference was statistically significant(PCONCLUSION: Ultraviolet radiation can lead to the formation of cataract in mice. Eaf2 protein can promote the formation of cataract in mice caused by ultraviolet.

  18. Impaired functional organization in the visual cortex of muscarinic receptor knock-out mice.

    Science.gov (United States)

    Groleau, Marianne; Nguyen, Hoang Nam; Vanni, Matthieu P; Huppé-Gourgues, Frédéric; Casanova, Christian; Vaucher, Elvire

    2014-09-01

    Acetylcholine modulates maturation and neuronal activity through muscarinic and nicotinic receptors in the primary visual cortex. However, the specific contribution of different muscarinic receptor subtypes in these neuromodulatory mechanisms is not fully understood. The present study evaluates in vivo the functional organization and the properties of the visual cortex of different groups of muscarinic receptor knock-out (KO) mice. Optical imaging of intrinsic signals coupled to continuous and episodic visual stimulation paradigms was used. Retinotopic maps along elevation and azimuth were preserved among the different groups of mice. However, compared to their wild-type counterparts, the apparent visual field along elevation was larger in M2/M4-KO mice but smaller in M1-KO. There was a reduction in the estimated relative receptive field size of V1 neurons in M1/M3-KO and M1-KO mice. Spatial frequency and contrast selectivity of V1 neuronal populations were affected only in M1/M3-KO and M1-KO mice. Finally, the neuronal connectivity was altered by the absence of M2/M4 muscarinic receptors. All these effects suggest the distinct roles of different subtypes of muscarinic receptors in the intrinsic organization of V1 and a strong involvement of the muscarinic transmission in the detectability of visual stimuli. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Tensor-based morphometry and stereology reveal brain pathology in the complexin1 knockout mouse.

    Science.gov (United States)

    Kielar, Catherine; Sawiak, Stephen J; Navarro Negredo, Paloma; Tse, Desmond H Y; Morton, A Jennifer

    2012-01-01

    Complexins (Cplxs) are small, soluble, regulatory proteins that bind reversibly to the SNARE complex and modulate synaptic vesicle release. Cplx1 knockout mice (Cplx1(-/-)) have the earliest known onset of ataxia seen in a mouse model, although hitherto no histopathology has been described in these mice. Nevertheless, the profound neurological phenotype displayed by Cplx1(-/-) mutants suggests that significant functional abnormalities must be present in these animals. In this study, MRI was used to automatically detect regions where structural differences were not obvious when using a traditional histological approach. Tensor-based morphometry of Cplx1(-/-) mouse brains showed selective volume loss from the thalamus and cerebellum. Stereological analysis of Cplx1(-/-) and Cplx1(+/+) mice brain slices confirmed the volume loss in the thalamus as well as loss in some lobules of the cerebellum. Finally, stereology was used to show that there was loss of cerebellar granule cells in Cplx1(-/-) mice when compared to Cplx1(+/+) animals. Our study is the first to describe pathological changes in Cplx1(-/-) mouse brain. We suggest that the ataxia in Cplx1(-/-) mice is likely to be due to pathological changes in both cerebellum and thalamus. Reduced levels of Cplx proteins have been reported in brains of patients with neurodegenerative diseases. Therefore, understanding the effects of Cplx depletion in brains from Cplx1(-/-) mice may also shed light on the mechanisms underlying pathophysiology in disorders in which loss of Cplx1 occurs.

  20. Tensor-based morphometry and stereology reveal brain pathology in the complexin1 knockout mouse.

    Directory of Open Access Journals (Sweden)

    Catherine Kielar

    Full Text Available Complexins (Cplxs are small, soluble, regulatory proteins that bind reversibly to the SNARE complex and modulate synaptic vesicle release. Cplx1 knockout mice (Cplx1(-/- have the earliest known onset of ataxia seen in a mouse model, although hitherto no histopathology has been described in these mice. Nevertheless, the profound neurological phenotype displayed by Cplx1(-/- mutants suggests that significant functional abnormalities must be present in these animals. In this study, MRI was used to automatically detect regions where structural differences were not obvious when using a traditional histological approach. Tensor-based morphometry of Cplx1(-/- mouse brains showed selective volume loss from the thalamus and cerebellum. Stereological analysis of Cplx1(-/- and Cplx1(+/+ mice brain slices confirmed the volume loss in the thalamus as well as loss in some lobules of the cerebellum. Finally, stereology was used to show that there was loss of cerebellar granule cells in Cplx1(-/- mice when compared to Cplx1(+/+ animals. Our study is the first to describe pathological changes in Cplx1(-/- mouse brain. We suggest that the ataxia in Cplx1(-/- mice is likely to be due to pathological changes in both cerebellum and thalamus. Reduced levels of Cplx proteins have been reported in brains of patients with neurodegenerative diseases. Therefore, understanding the effects of Cplx depletion in brains from Cplx1(-/- mice may also shed light on the mechanisms underlying pathophysiology in disorders in which loss of Cplx1 occurs.

  1. Single-neutron knockout from 20C and the structure of 19C

    Science.gov (United States)

    Hwang, J. W.; Kim, S.; Satou, Y.; Orr, N. A.; Kondo, Y.; Nakamura, T.; Gibelin, J.; Achouri, N. L.; Aumann, T.; Baba, H.; Delaunay, F.; Doornenbal, P.; Fukuda, N.; Inabe, N.; Isobe, T.; Kameda, D.; Kanno, D.; Kobayashi, N.; Kobayashi, T.; Kubo, T.; Leblond, S.; Lee, J.; Marqués, F. M.; Minakata, R.; Motobayashi, T.; Murai, D.; Murakami, T.; Muto, K.; Nakashima, T.; Nakatsuka, N.; Navin, A.; Nishi, S.; Ogoshi, S.; Otsu, H.; Sato, H.; Shimizu, Y.; Suzuki, H.; Takahashi, K.; Takeda, H.; Takeuchi, S.; Tanaka, R.; Togano, Y.; Tuff, A. G.; Vandebrouck, M.; Yoneda, K.

    2017-06-01

    The low-lying unbound level structure of the halo nucleus 19C has been investigated using single-neutron knockout from 20C on a carbon target at 280 MeV/nucleon. The invariant mass spectrum, derived from the momenta of the forward going beam velocity 18C fragment and neutrons, was found to be dominated by a very narrow near threshold (Erel = 0.036 (1) MeV) peak. Two less strongly populated resonance-like features were also observed at Erel = 0.84 (4) and 2.31 (3) MeV, both of which exhibit characteristics consistent with neutron p-shell hole states. Comparisons of the energies, measured cross sections and parallel momentum distributions to the results of shell-model and eikonal reaction calculations lead to spin-parity assignments of 5 /21+ and 1 /21- for the levels at Ex = 0.62 (9) and 2.89 (10) MeV with Sn = 0.58 (9) MeV. Spectroscopic factors were also deduced and found to be in reasonable accord with shell-model calculations. The valence neutron configuration of the 20C ground state is thus seen to include, in addition to the known 1s1/22 component, a significant 0d5/22 contribution. The level scheme of 19C, including significantly the 1 /21- cross-shell state, is well accounted for by the YSOX shell-model interaction developed from the monopole-based universal interaction.

  2. Age-Related Hearing Loss in Mn-SOD Heterozygous Knockout Mice

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    Makoto Kinoshita

    2013-01-01

    Full Text Available Age-related hearing loss (AHL reduces the quality of life for many elderly individuals. Manganese superoxide dismutase (Mn-SOD, one of the antioxidant enzymes acting within the mitochondria, plays a crucial role in scavenging reactive oxygen species (ROS. To determine whether reduction in Mn-SOD accelerates AHL, we evaluated auditory function in Mn-SOD heterozygous knockout (HET mice and their littermate wild-type (WT C57BL/6 mice by means of auditory brainstem response (ABR. Mean ABR thresholds were significantly increased at 16 months when compared to those at 4 months in both WT and HET mice, but they did not significantly differ between them at either age. The extent of hair cell loss, spiral ganglion cell density, and thickness of the stria vascularis also did not differ between WT and HET mice at either age. At 16 months, immunoreactivity of 8-hydroxydeoxyguanosine was significantly greater in the SGC and SV in HET mice compared to WT mice, but that of 4-hydroxynonenal did not differ between them. These findings suggest that, although decrease of Mn-SOD by half may increase oxidative stress in the cochlea to some extent, it may not be sufficient to accelerate age-related cochlear damage under physiological aging process.

  3. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    Science.gov (United States)

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system.

  4. Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Tadafumi Yokoyama

    Full Text Available The Wiskott-Aldrich syndrome (WAS is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg obtained from Was gene knockout (WKO mice and found that their numbers were significantly lower in these mice compared to wild type (WT controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

  5. Metabolism and tissue distribution of sulforaphane in Nrf2 knockout and wild-type mice.

    Science.gov (United States)

    Clarke, John D; Hsu, Anna; Williams, David E; Dashwood, Roderick H; Stevens, Jan F; Yamamoto, Masayuki; Ho, Emily

    2011-12-01

    To determine the metabolism and tissue distribution of the dietary chemoprotective agent sulforaphane following oral administration to wild-type and Nrf2 knockout (Nrf2(-/-)) mice. Male and female wild-type and Nrf2(-/-) mice were given sulforaphane (5 or 20 μmoles) by oral gavage; plasma, liver, kidney, small intestine, colon, lung, brain and prostate were collected at 2, 6 and 24 h (h). The five major metabolites of sulforaphane were measured in tissues by high performance liquid chromatography coupled with tandem mass spectrometry. Sulforaphane metabolites were detected in all tissues at 2 and 6 h post gavage, with the highest concentrations in the small intestine, prostate, kidney and lung. A dose-dependent increase in sulforaphane concentrations was observed in all tissues except prostate. At 5 μmole, Nrf2(-/-) genotype had no effect on sulforaphane metabolism. Only Nrf2(-/-) females given 20 μmoles sulforaphane for 6 h exhibited a marked increase in tissue sulforaphane metabolite concentrations. The relative abundance of each metabolite was not strikingly different between genders and genotypes. Sulforaphane is metabolized and reaches target tissues in wild-type and Nrf2(-/-) mice. These data provide further evidence that sulforaphane is bioavailable and may be an effective dietary chemoprevention agent for several tissue sites.

  6. Comprehensive behavioral analysis of calcium/calmodulin-dependent protein kinase IV knockout mice.

    Directory of Open Access Journals (Sweden)

    Keizo Takao

    Full Text Available Calcium-calmodulin dependent protein kinase IV (CaMKIV is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency.

  7. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    Science.gov (United States)

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  8. Conditional Knockout of Telomerase Reverse Transcriptase in Mesenchymal Cells Impairs Mouse Pulmonary Fibrosis.

    Directory of Open Access Journals (Sweden)

    Tianju Liu

    Full Text Available Telomerase is typically expressed in cellular populations capable of extended replication, such as germ cells, tumor cells, and stem cells, but is also induced in tissue injury, repair and fibrosis. Its catalytic component, telomerase reverse transcriptase (TERT is induced in lung fibroblasts from patients with fibrotic interstitial lung disease and in rodents with bleomycin-induced pulmonary fibrosis. To evaluate the fibroblast specific role of TERT in pulmonary fibrosis, transgenic mice bearing a floxed TERT allele were generated, and then crossed with an inducible collagen α2(I-Cre mouse line to generate fibroblast specific TERT conditional knockout mice. TERT-specific deficiency in mesenchymal cells caused attenuation of pulmonary fibrosis as manifested by reduced lung hydroxyproline content, type I collagen and α-smooth muscle actin mRNA levels. The TERT-deficient mouse lung fibroblasts displayed decreased cell proliferative capacity and higher susceptibility to induced apoptosis compared with control cells. Additionally TERT deficiency was associated with heightened α-smooth muscle actin expression indicative of myofibroblast differentiation. However the impairment of cell proliferation and increased susceptibility to apoptosis would cause a reduction in the myofibroblast progenitor population necessary to mount a successful myofibroblast-dependent fibrotic response. These findings identified a key role for TERT in fibroblast proliferation and survival essential for pulmonary fibrosis.

  9. Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency

    Directory of Open Access Journals (Sweden)

    Jun Song

    2018-03-01

    Full Text Available Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0 and three male IL2RG null (−/y F1 animals demonstrated severe combined immunodeficiency (SCID, characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

  10. Persistent lung inflammation and fibrosis in serum amyloid P component (APCs-/- knockout mice.

    Directory of Open Access Journals (Sweden)

    Darrell Pilling

    Full Text Available Fibrosing diseases, such as pulmonary fibrosis, cardiac fibrosis, myelofibrosis, liver fibrosis, and renal fibrosis are chronic and debilitating conditions and are an increasing burden for the healthcare system. Fibrosis involves the accumulation and differentiation of many immune cells, including macrophages and fibroblast-like cells called fibrocytes. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2 inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP also promotes the formation of immuno-regulatory Mreg macrophages. To elucidate the endogenous function of SAP, we used bleomycin aspiration to induce pulmonary inflammation and fibrosis in mice lacking SAP. Compared to wildtype C57BL/6 mice, we find that in Apcs-/- "SAP knock-out" mice, bleomycin induces a more persistent inflammatory response and increased fibrosis. In both C57BL/6 and Apcs-/- mice, injections of exogenous SAP reduce the accumulation of inflammatory macrophages and prevent fibrosis. The types of inflammatory cells present in the lungs following bleomycin-aspiration appear similar between C57BL/6 and Apcs-/- mice, suggesting that the initial immune response is normal in the Apcs-/- mice, and that a key endogenous function of SAP is to promote the resolution of inflammation and fibrosis.

  11. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-Ryool

    2016-06-13

    Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

  12. Abolition of lemniscal barrellette patterning in Prrxl1 knockout mice: Effects upon ingestive behavior.

    Science.gov (United States)

    Bakalar, Dana; Tamaiev, Jonathan; Zeigler, H Philip; Feinstein, Paul

    2015-01-01

    Ingestive behaviors in mice are dependent on orosensory cues transmitted via the trigeminal nerve, as confirmed by transection studies. However, these studies cannot differentiate between deficits caused by the loss of the lemniscal pathway vs. the parallel paralemniscal pathway. The paired-like homeodomain protein Prrxl1 is expressed widely in the brain and spinal cord, including the trigeminal system. A knockout of Prrxl1 abolishes somatotopic barrellette patterning in the lemniscal brainstem nucleus, but not in the parallel paralemniscal nucleus. Null animals are significantly smaller than littermates by postnatal day 5, but reach developmental landmarks at appropriate times, and survive to adulthood on liquid diet. A careful analysis of infant and adult ingestive behavior reveals subtle impairments in suckling, increases in time spent feeding and the duration of feeding bouts, feeding during inappropriate times of the day, and difficulties in the mechanics of feeding. During liquid diet feeding, null mice display abnormal behaviors including extensive use of the paws to move food into the mouth, submerging the snout in the diet, changes in licking, and also have difficulty consuming solid chow pellets. We suggest that our Prrxl1(-/-) animal is a valuable model system for examining the genetic assembly and functional role of trigeminal lemniscal circuits in the normal control of eating in mammals and for understanding feeding abnormalities in humans resulting from the abnormal development of these circuits.

  13. Hepatic caveolin-1 is enhanced in Cyp27a1/ApoE double knockout mice.

    Science.gov (United States)

    Zurkinden, Line; Mansour, Yosef T; Rohrbach, Beatrice; Vogt, Bruno; Mistry, Hiten D; Escher, Geneviève

    2016-10-01

    Sterol 27-hydroxylase (CYP27A1) is involved in bile acid synthesis and cholesterol homoeostasis. Cyp27a1 (-/-) / Apolipoprotein E (-/-) double knockout mice (DKO) fed a western diet failed to develop atherosclerosis. Caveolin-1 (CAV-1), the main component of caveolae, is associated with lipid homoeostasis and has regulatory roles in vascular diseases. We hypothesized that liver CAV-1 would contribute to the athero-protective mechanism in DKO mice. Cyp27a1 (+/+) / ApoE (-/-) (ApoE KO), Cyp27a1 (+/-) / ApoE (-/-) (het), and DKO mice were fed a western diet for 2 months. Atherosclerotic plaque and CAV-1 protein were quantified in aortas. Hepatic Cav-1 mRNA was assessed using qPCR, CAV-1 protein by immunohistochemistry and western blotting. Total hepatic and plasma cholesterol was measured using chemiluminescence. Cholesterol efflux was performed in RAW264.7 cells, using mice plasma as acceptor. CAV-1 protein expression in aortas was increased in endothelial cells of DKO mice and negatively correlated with plaque surface ( P CYP27A1, CAV-1 overexpression might have an additional athero-protective role by partly overcoming the defect in CYP27A1-mediated cholesterol efflux.

  14. Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

    Science.gov (United States)

    Vicente-Rodríguez, Marta; Gramage, Esther; Herradón, Gonzalo; Pérez-García, Carmen

    2013-12-06

    The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain.

    Science.gov (United States)

    Wang, Huimin; Shimizu, Eiji; Tang, Ya-Ping; Cho, Min; Kyin, Maureen; Zuo, Wenqi; Robinson, Daphne A; Alaimo, Peter J; Zhang, Chao; Morimoto, Hiromi; Zhuo, Min; Feng, Ruiben; Shokat, Kevan M; Tsien, Joe Z

    2003-04-01

    By integrating convergent protein engineering and rational inhibitor design, we have developed an in vivo conditional protein knockout andor manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as alpha-Ca(2+)calmodulin-dependent protein kinase II (alphaCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.

  16. Gamma-ray measurements in the one-neutron knockout of 17C, 19N, 21O and 25F

    International Nuclear Information System (INIS)

    Rodriguez-Tajes, C.; Cortina-Gil, D.; Alvarez-Pol, H.; Benjamim, E.; Benlliure, J.; Caamano, M.; Casarejos, E.; Gascon, M.; Kurtukian, T.; Perez-Loureiro, D.; Aumann, T.; Chatillon, A.; Geissel, H.; Nociforo, C.; Prochazka, A.; Simon, H.; Suemmerer, K.; Weick, H.; Winkler, M.; Borge, M.J.G.; Pascual-Izarra, C.; Perea, A.; Tengblad, O.; Chulkov, L.V.; Eppinger, K.; Faestermann, T.; Gernhaeuser, R.; Kruecken, R.; Maierbeck, P.; Schwertel, S.; Jonson, B.; Kanungo, R.; Nilsson, T.; Zhukov, M.V.

    2012-01-01

    One-neutron knockout reactions in a 9 Be target have been investigated at relativistic energies, near 700 MeV/u, for a set of sd-shell, neutron-rich nuclei. The experiment was performed in the FRS spectrometer, at GSI. γ-ray measurements were carried out by means of the MINIBALL γ-ray spectrometer and allowed the determination of partial cross-sections and branching ratios corresponding to the final states of the emerging knockout fragments. Experimental results are presented for 17 C, 19 N, 21 O and 25 F projectiles. The role of excited states of the N - 1 fragments in the composition of the ground state of these neutron-rich projectiles is outlined in this work. (orig.)

  17. A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice.

    Science.gov (United States)

    Crawley, J N; Paylor, R

    1997-06-01

    Behavioral phenotyping of transgenic and knockout mice requires rigorous, formal analyses. Well-characterized paradigms can be chosen from the established behavioral neuroscience literature. This review describes (1) a series of neurological and neuropsychological tests which are effectively used as a first screen for behavioral abnormalities in mutant mice, and (2) a series of specific behavioral paradigms, clustered by category. Included are multiple paradigms for each category, including learning and memory, feeding, analgesia, aggression, anxiety, depression, schizophrenia, and drug abuse models. Examples are given from the experiences of the authors, in applying these experimental designs to transgenic and knockout mice. Extensive references for each behavioral paradigm are provided, to allow new investigators to access the relevant literature on behavioral methodology.

  18. Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs.

    Science.gov (United States)

    Watanabe, Takahito; Noji, Sumihare; Mito, Taro

    2014-08-15

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically basal. These insects include many deleterious species. The cricket, Gryllus bimaculatus, is an emerging model for hemimetabolous insects, based on the success of RNA interference (RNAi)-based gene-functional analyses and transgenic technology. Taking advantage of genome-editing technologies in this species would greatly promote functional genomics studies. Genome editing using transcription activator-like effector nucleases (TALENs) has proven to be an effective method for site-specific genome manipulation in various species. TALENs are artificial nucleases that are capable of inducing DNA double-strand breaks into specified target sequences. Here, we describe a protocol for TALEN-based gene knockout in G. bimaculatus, including a mutant selection scheme via mutation detection assays, for generating homozygous knockout organisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Lack of susceptibility of heterozygous p53-knockout CBA and CIEA mice to phenolphthalein in a 6-month carcinogenicity study.

    Science.gov (United States)

    Okamura, Miwa; Kashida, Yoko; Watanabe, Takao; Yasuhara, Kazuo; Onodera, Hiroshi; Hirose, Masao; Usui, Toshimi; Tamaoki, Norikazu; Mitsumori, Kunitoshi

    2003-03-14

    Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in female heterozygous p53-knockout TSG mice (C57BL/6 background) at the same dose in a 6-month study. To examine whether carcinogenic potential of phenolphthalein can be detected in other p53-knockout mouse [p53 (+/-)] strains such as p53 (+/-) CBA mice or p53 (+/-) CIEA mice (C57BL/6 background) and their littermates, they were given a diet containing 0, 6000 or 12000 ppm for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that p53 (+/-) CBA mice and p53 (+/-) CIEA mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.

  20. Behavioral responses of dopamine β-hydroxylase knockout mice to modafinil suggest a dual noradrenergic-dopaminergic mechanism of action

    OpenAIRE

    Mitchell, Heather A.; Bogenpohl, James W.; Liles, L. Cameron; Epstein, Michael P.; Bozyczko-Coyne, Donna; Williams, Michael; Weinshenker, David

    2008-01-01

    Modafinil is approved for use in the treatment of excessive daytime sleepiness. The precise mechanism of modafinil action has not been elucidated, although both dopamine (DA) and norepinephrine (NE) systems have been implicated. To explore the roles of DA and NE in the mechanism of modafinil-induced arousal, dopamine β-hydroxylase knockout (Dbh −/−) mice were examined in behavioral paradigms of arousal (photobeam breaks and behavioral scoring of sleep latency). Dbh −/− mice completely lack NE...

  1. Protective activity ethanol extract of the fruits of Illicium verum against atherogenesis in apolipoprotein E knockout mice

    OpenAIRE

    Park, Sun Haeng; Sung, Yoon-Young; Nho, Kyoung Jin; Kim, Ho Kyoung

    2015-01-01

    Background Illicium verum Hook. fil. Illiciaceae (Illicium v.) has been traditionally used in herbal medicine for treating many inflammatory diseases, including skin inflammation and rheumatism. We investigated its use as a preventive agent against inflammatory and vascular diseases in a murine model of atherosclerosis using apolipoprotein E-knockout (ApoE ?/?) mice fed on a high-fat diet (HFD). Methods We investigated the effect of Illicium v. on cytotoxicity, NF-?B activity, and adhesion mo...

  2. Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

    Science.gov (United States)

    Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

    2018-04-04

    Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

  3. Estimate of the theoretical uncertainty of the cross sections for nucleon knockout in neutral-current neutrino-oxygen interactions

    OpenAIRE

    Ankowski, Artur M.; Barbaro, M. B.; Benhar, Omar; Caballero Carretero, Juan Antonio; Giusti, Carlotta; González Jiménez, Raúl; Megías Vázquez, Guillermo Daniel; Meucci, Andrea

    2015-01-01

    Free nucleons propagating in water are known to produce γ rays, which form a background to the searches for diffuse supernova neutrinos and sterile neutrinos carried out with Cherenkov detectors. As a consequence, the process of nucleon knockout induced by neutral-current quasielastic interactions of atmospheric (anti)neutrinos with oxygen needs to be under control at the quantitative level in the background simulations of ongoing and future experiments. In this paper, we provide a quantitati...

  4. Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9

    Directory of Open Access Journals (Sweden)

    Anne M. Bara

    2016-09-01

    Full Text Available Here, we generated a monoallelic mutation in the TLE3 (Transducin Like Enhancer of Split 3 gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC line WA01. The heterozygous knockout cell line, TLE3-447-D08-A01, displays partial loss of TLE3 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.

  5. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures

    OpenAIRE

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; S?der, Olle; Stukenborg, Jan-Bernd

    2017-01-01

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Te...

  6. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only.

    Science.gov (United States)

    Pagani, J H; Williams Avram, S K; Cui, Z; Song, J; Mezey, É; Senerth, J M; Baumann, M H; Young, W S

    2015-02-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to 8 of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  7. Altered Neuronal Dynamics in the Striatum on the Behavior of Huntingtin Interacting Protein 14 (HIP14 Knockout Mice

    Directory of Open Access Journals (Sweden)

    Ana María Estrada-Sánchez

    2013-11-01

    Full Text Available Huntington’s disease (HD, a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene, impairs information processing in the striatum, which, as part of the basal ganglia, modulates motor output. Growing evidence suggests that huntingtin interacting protein 14 (HIP14 contributes to HD neuropathology. Here, we recorded local field potentials (LFPs in the striatum as HIP14 knockout mice and wild-type controls freely navigated a plus-shaped maze. Upon entering the choice point of the maze, HIP14 knockouts tend to continue in a straight line, turning left or right significantly less often than wild-types, a sign of motor inflexibility that also occurs in HD mice. Striatal LFP activity anticipates this difference. In wild-types, the power spectral density pattern associated with entry into the choice point differs significantly from the pattern immediately before entry, especially at low frequencies (≤13 Hz, whereas HIP14 knockouts show no change in LFP activity as they enter the choice point. The lack of change in striatal activity may explain the turning deficit in the plus maze. Our results suggest that HIP14 plays a critical role in the aberrant behavioral modulation of striatal neuronal activity underlying motor inflexibility, including the motor signs of HD.

  8. Knock-out transmembrane prostate androgen-induced protein gene suppressed triple-negative breast cancer cell proliferation

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    Bantari W.K. Wardhani

    2017-11-01

    Full Text Available Background: Triple negative breast cancer (TNBC tends to grow more rapidly and has poorer prognosis compared to others. High expression of transmembrane prostate androgen-induced protein (TMEPAI correlates with poor prognosis in TNBC patients. However, the mechanistic role of TMEPAI in tumorigenic remains unknown. This study aimed to knock-out TMEPAI in TNBC cell line to determine its function further in cells proliferation.Methods: CRISPR-Cas9 has been used previously to knock-out TMEPAI in Hs857T TNBC cell line. Hs587T TNBC parental cell line (wild-type/WT and TMEPAI knock out Hs 586T cell lines were cultured in Dulbecco’s modified eagle medium (DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and amphotericin B. Both cell lines were seeded in 24-well plates and counted every two days, then proliferation rates were plotted. Afterwards, total RNA were isolated from the cells and Ki-67, and TGF-β mRNA expression levels as proliferation markers were determined.Results: Cell proliferation rates as displayed in growth curve plots showed that WT-TMEPAI cell line grew more rapidly than KO-TMEPAI. In accordance, mRNA expression levels of  Ki-67 and TGF-β  were significantly decreased KO-TMEPAI as compare to TMEPAI-WT.Conclusion: Knock-out of TMEPAI attenuates cell proliferation in TNBC.

  9. CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

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    Naser Jafari

    Full Text Available Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

  10. Rescue of the Friedreich ataxia knockout mutation in transgenic mice containing an FXN-EGFP genomic reporter.

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    Joseph P Sarsero

    Full Text Available Friedreich ataxia (FRDA is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. We previously generated BAC-based transgenic mice containing an FXN-EGFP genomic reporter construct in which the EGFP gene is fused in-frame immediately following the final codon of exon 5a of the human FXN gene. These transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Fxn gene, to generate mice homozygous for the Fxn knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Fxn knockout mutation was observed. Rescue mice displayed normal behavioral and histological parameters with normal viability, fertility and life span and without any signs of aberrant phenotype. Immunoblotting demonstrated the production of full-length frataxin-EGFP fusion protein that appears to act as a bifunctional hybrid protein. This study shows frataxin replacement may be a viable therapeutic option. Further, these mice should provide a useful resource for the study of human FXN gene expression, frataxin function, the evaluation of pharmacologic inducers of FXN expression in a whole-animal model and provide a useful source of cells for stem cell transplantation studies.

  11. ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level

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    Kexiu Song

    2014-01-01

    Full Text Available HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

  12. FOCuS: a metaheuristic algorithm for computing knockouts from genome-scale models for strain optimization.

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    Mutturi, Sarma

    2017-06-27

    Although handful tools are available for constraint-based flux analysis to generate knockout strains, most of these are either based on bilevel-MIP or its modifications. However, metaheuristic approaches that are known for their flexibility and scalability have been less studied. Moreover, in the existing tools, sectioning of search space to find optimal knocks has not been considered. Herein, a novel computational procedure, termed as FOCuS (Flower-pOllination coupled Clonal Selection algorithm), was developed to find the optimal reaction knockouts from a metabolic network to maximize the production of specific metabolites. FOCuS derives its benefits from nature-inspired flower pollination algorithm and artificial immune system-inspired clonal selection algorithm to converge to an optimal solution. To evaluate the performance of FOCuS, reported results obtained from both MIP and other metaheuristic-based tools were compared in selected case studies. The results demonstrated the robustness of FOCuS irrespective of the size of metabolic network and number of knockouts. Moreover, sectioning of search space coupled with pooling of priority reactions based on their contribution to objective function for generating smaller search space significantly reduced the computational time.

  13. Imaging colon cancer development in mice: IL-6 deficiency prevents adenoma in azoxymethane-treated Smad3 knockouts

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    Harpel, Kaitlin; Leung, Sarah; Faith Rice, Photini; Jones, Mykella; Barton, Jennifer K.; Bommireddy, Ramireddy

    2016-02-01

    The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

  14. LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

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    Hinkle Kelly M

    2012-05-01

    Full Text Available Abstract Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

  15. Atherosclerotic plaque disruption induced by stress and lipopolysaccharide in apolipoprotein E knockout mice.

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    Ni, Mei; Wang, Yan; Zhang, Mei; Zhang, Peng Fei; Ding, Shi Fang; Liu, Chun Xi; Liu, Xiao Ling; Zhao, Yu Xia; Zhang, Yun

    2009-05-01

    To establish an animal model with disruptions of atherosclerotic plaques, 96 male apolipoprotein E knockout (apoE(-/-)) mice were randomly divided into stress, lipopolysaccharide (LPS), stress+LPS, and control groups (n = 24 each). All mice were fed a high-fat diet throughout the experiment, and carotid atherosclerotic lesions were induced by placement of a constrictive perivascular collar. Four weeks after surgery, mice in the LPS and stress+LPS groups were intraperitoneally injected with LPS (1 mg/kg twice per week for 8 wk). Eight weeks after surgery, mice in the stress and stress+LPS groups were treated with intermittent physical stress (electric foot shock and noise stimulation) for 4 wk. Morphological analysis revealed a plaque disruption rate of 16.7% in control, 34.8% in LPS, 54.2% in stress, and 60.9% in stress+LPS groups. The disruption rates in stress and stress+LPS groups were both significantly higher than those of controls (P = 0.007 and P = 0.002, respectively). Luminal thrombosis secondary to plaque disruption was observed only in the stress+LPS group. Both stress and LPS stimulation significantly decreased fibrous cap thickness and increased macrophage and lipid contents in plaques. Moreover, the combination of stress and LPS stimulation further lowered cap thickness and enhanced accumulation of macrophages and expression of inflammatory cytokines and matrix metalloproteinases. Stress activated the sympathetic nervous system, as manifested by increased blood pressure and flow velocity. Plasma fibrinogen levels were remarkably elevated in the stress and stress+LPS groups. In conclusion, stress- and LPS-costimulated apoE(-/-) mice provide a useful model for studies of plaque vulnerability and interventions.

  16. Calcium current homeostasis and synaptic deficits in hippocampal neurons from Kelch-like 1 knockout mice

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    Paula Patricia Perissinotti

    2015-01-01

    Full Text Available Kelch-like 1 (KLHL1 is a neuronal actin-binding protein that modulates voltage-gated CaV2.1 (P/Q-type and CaV3.2 (α1H T-type calcium channels; KLHL1 knockdown experiments (KD cause down-regulation of both channel types and altered synaptic properties in cultured rat hippocampal neurons (Perissinotti et al., 2014. Here, we studied the effect of ablation of KLHL1 on calcium channel function and synaptic properties in cultured hippocampal neurons from KLHL1 knockout (KO mice. Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT; and PQ-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current. In contrast, T-type currents did not change in culture. However, biophysical properties and western blot analysis revealed a differential contribution of T-type channel isoforms in the KO, with CaV3.2 α1H subunit being down-regulated and CaV3.1 α1G up-regulated. Synapsin I levels were reduced in the KO hippocampus; cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC frequency. In summary, genetic ablation of the calcium channel modulator resulted in compensatory mechanisms to maintain calcium current homeostasis in hippocampal KO neurons; however, synaptic alterations resulted in a reduction of excitatory synapse number, causing an imbalance of the excitatory-inhibitory synaptic input ratio favoring inhibition.

  17. Running exercise alleviates trabecular bone loss and osteopenia in hemizygous β-globin knockout thalassemic mice.

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    Thongchote, Kanogwun; Svasti, Saovaros; Teerapornpuntakit, Jarinthorn; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2014-06-15

    A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice and to determine whether endurance running (60 min/day, 5 days/wk for 12 wk in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1-2 mo old) showed growth retardation as indicated by smaller body weight and femoral length than their wild-type littermates. A decrease in BMD was more severe in female than in male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines IL-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice compared with those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, whereas running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia. Copyright © 2014 the American Physiological Society.

  18. Prenatal nicotine exposure increases hyperventilation in α4-knock-out mice during mild asphyxia.

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    Avraam, Joanne; Cohen, Gary; Drago, John; Frappell, Peter B

    2015-03-01

    Prenatal nicotine exposure alters breathing and ventilatory responses to stress through stimulation of nicotine acetylcholine receptors (nAChRs). We tested the hypothesis that α4-containing nAChRs are involved in mediating the effects of prenatal nicotine exposure on ventilatory and metabolic responses to intermittent mild asphyxia (MA). Using open-flow plethysmography, we measured ventilation (V̇(E)) and rate of O2 consumption ( V̇(O2)) of wild-type (WT) and α4-knock-out (KO) mice, at postnatal (P) days 1-2 and 7-8, with and without prenatal nicotine exposure (6 mg kg(-1) day(-1) beginning on embryonic day 14). Mice were exposed to seven 2 min cycles of mild asphyxia (10% O2 and 5% CO2), each interspersed with 2 min of air. Compared to WT, α4 KO mice had increased air V̇(E) and V̇(O2) at P7-8, but not P1-2. Irrespective of age, genotype had no effect on the hyperventilatory response (increase in V̇(E)/V̇(O2)) to MA. At P1-2, nicotine suppressed air V̇(E) and V̇(O2) in both genotypes but did not affect the hyperventilatory response to MA. At P7-8 nicotine suppressed air V̇(E) and V̇(O2) of only α4 KO's but also significantly enhanced V̇(E) during MA (nearly double that of WT; pprenatal nicotine exposure on ventilatory and metabolic interactions and responses to stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Sleeve gastrectomy leads to weight loss in the Magel2 knockout mouse.

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    Arble, Deanna M; Pressler, Joshua W; Sorrell, Joyce; Wevrick, Rachel; Sandoval, Darleen A

    2016-12-01

    Prader-Willi syndrome (PWS) is a genetic disorder characterized by hyperphagia, obesity, cardiopulmonary diseases, and increased mortality. Although successful weight loss improves health in PWS, few treatments cause sustained weight loss in obese patients let alone obese individuals with PWS. The present study uses the Magel2 knockout (KO) mouse, an animal model of PWS, to conduct a preclinical study on the efficacy of sleeve gastrectomy (SG) in PWS. Academic research laboratory, United States. We performed sham or SG surgeries in 24- to 28-week-old male Magel2 KO and wild-type littermate control mice (WT) who had been maintained on a high-fat diet for 10 weeks. We monitored weight, food intake, and fat and lean mass pre- and postoperatively. Fasting glucose, glucose tolerance, and counter-regulation were measured postoperatively. Magel2 KO animals had similar recovery and mortality rates compared with WT. SG resulted in similar weight loss, specifically loss of fat but not lean mass, in both Magel2 KO and WT mice. SG also resulted in significantly lower fasting glucose levels and a reduction in fat intake in both Magel2 KO and WT mice. We also found that Magel2 KO mice failed to increase their food intake in response to the glucoprivic agent 2-deoxy-D-glucose, suggesting impaired glucose counter-regulation, but this occurred regardless of surgical status. All results were considered significant when P< .05. We find in this mouse model of PWS, SG is a well-tolerated, effective strategy for weight and fat loss. Copyright © 2016 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

  20. Resources for methylome analysis suitable for gene knockout studies of potential epigenome modifiers

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    Wilson Gareth A

    2012-07-01

    Full Text Available Abstract Background Methylated DNA immunoprecipitation (MeDIP is a popular enrichment based method and can be combined with sequencing (termed MeDIP-seq to interrogate the methylation status of cytosines across entire genomes. However, quality control and analysis of MeDIP-seq data have remained to be a challenge. Results We report genome-wide DNA methylation profiles of wild type (wt and mutant mouse cells, comprising 3 biological replicates of Thymine DNA glycosylase (Tdg knockout (KO embryonic stem cells (ESCs, in vitro differentiated neural precursor cells (NPCs and embryonic fibroblasts (MEFs. The resulting 18 methylomes were analysed with MeDUSA (Methylated DNA Utility for Sequence Analysis, a novel MeDIP-seq computational analysis pipeline for the identification of differentially methylated regions (DMRs. The observed increase of hypermethylation in MEF promoter-associated CpG islands supports a previously proposed role for Tdg in the protection of regulatory regions from epigenetic silencing. Further analysis of genes and regions associated with the DMRs by gene ontology, pathway, and ChIP analyses revealed further insights into Tdg function, including an association of TDG with low-methylated distal regulatory regions. Conclusions We demonstrate that MeDUSA is able to detect both large-scale changes between cells from different stages of differentiation and also small but significant changes between the methylomes of cells that only differ in the KO of a single gene. These changes were validated utilising publicly available datasets and confirm TDG's function in the protection of regulatory regions from epigenetic silencing.

  1. Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

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    Dubey, J P; Verma, S K; Dunams, D; Calero-Bernal, R; Rosenthal, B M

    2015-11-01

    The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri.

  2. CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

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    Kang, Yu; Zheng, Bo; Shen, Bin; Chen, Yongchang; Wang, Lei; Wang, Jianying; Niu, Yuyu; Cui, Yiqiang; Zhou, Jiankui; Wang, Hong; Guo, Xuejiang; Hu, Bian; Zhou, Qi; Sha, Jiahao; Ji, Weizhi; Huang, Xingxu

    2015-12-20

    Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/β-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Gut microbiota is a key modulator of insulin resistance in TLR 2 knockout mice.

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    Andréa M Caricilli

    2011-12-01

    Full Text Available Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics, the metabolic characteristics, and insulin signaling in TLR2 knockout (KO mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes

  4. Safrole-2',3'-oxide induces atherosclerotic plaque vulnerability in apolipoprotein E-knockout mice.

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    Su, Le; Zhang, Haiyan; Zhao, Jing; Zhang, Shangli; Zhang, Yun; Zhao, Baoxiang; Miao, Junying

    2013-02-27

    Safrole-2',3'-oxide (SFO) is the major electrophilic metabolite of safrole (4-allyl-1, 2-methylenedioxybenzene), a natural plant constituent found in essential oils of numerous edible herbs and spices and in food containing these herbs, such as pesto sauce, cola beverages and bologna sausages. The effects of SFO in mammalian systems, especially the cardiovascular system, are little known. Disruption of vulnerable atherosclerotic plaques in atherosclerosis, a chronic inflammatory disease, is the main cause of cardiovascular events. In this study, we investigated SFO-induced atherosclerotic plaque vulnerability (possibility of rupture) in apolipoprotein E-knockout (apoE(-/-)) mice. Lipid area in vessel wall reached 59.8% in high dose SFO (SFO-HD) treated group, which is only 31.2% in control group. SFO treatment changed the lesion composition to an unstable phenotype, increased the number of apoptotic cells in plaque and the endothelium in plaques was damaged after SFO treatment. Furthermore, compared with control groups, the plaque endothelium level of p75(NTR) was 3-fold increased and the liver level of p75(NTR) was 17.4-fold increased by SFO-HD. Meanwhile, the serum level of KC (a functional homolog of IL-8 and the main proinflammatory alpha chemokine in mice) in apoE(-/-) mice was up to 357pg/ml in SFO-HD treated group. Thus, SFO contributes to the instability of atherosclerotic plaque in apoE(-/-) mice through activating p75(NTR) and IL-8 and cell apoptosis in plaque. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  5. Pregnenolone rescues schizophrenia-like behavior in dopamine transporter knockout mice.

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    Peiyan Wong

    Full Text Available Pregnenolone belongs to a class of endogenous neurosteroids in the central nervous system (CNS, which has been suggested to enhance cognitive functions through GABA(A receptor signaling by its metabolites. It has been shown that the level of pregnenolone is altered in certain brain areas of schizophrenic patients, and clozapine enhances pregnenolone in the CNS in rats, suggesting that pregnenolone could be used to treat certain symptoms of schizophrenia. In addition, early phase proof-of-concept clinical trials have indicated that pregnenolone is effective in reducing the negative symptoms and cognitive deficits of schizophrenia patients. Here, we evaluate the actions of pregnenolone on a mouse model for schizophrenia, the dopamine transporter knockout mouse (DAT KO. DAT KO mice mirror certain symptoms evident in patients with schizophrenia, such as the psychomotor agitation, stereotypy, deficits of prepulse inhibition and cognitive impairments. Following acute treatment, pregnenolone was found to reduce the hyperlocomotion, stereotypic bouts and pre-pulse inhibition (PPI deficits in DAT KO mice in a dose-dependent manner. At 60 mg/kg of pregnenolone, there were no significant differences in locomotor activities and stereotypy between wild-type and DAT KO mice. Similarly, acute treatment of 60 mg/kg of pregnenolone fully rescued PPI deficits of DAT KO mice. Following chronic treatment with pregnenolone at 60 mg/kg, the cognitive deficits of DAT KO mice were rescued in the paradigms of novel object recognition test and social transmission of food preference test. Pregnenolone thus holds promise as a therapeutic candidate in schizophrenia.

  6. Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice.

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    Tein, Karin; Kasvandik, Sergo; Kõks, Sulev; Vasar, Eero; Terasmaa, Anton

    2015-01-01

    Mutations in WFS1 gene cause Wolfram syndrome, which is a rare autosomal recessive disorder, characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy, and deafness. The WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene exhibit disturbances in the processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of the wolframin protein have been observed in the hippocampus, amygdala, and limbic structures. The aim of this study was to investigate the effect of Wfs1 knockout (KO) on peptide processing in mouse hippocampus. A peptidomic approach was used to characterize individual peptides in the hippocampus of wild-type and Wfs1 KO mice. We identified 126 peptides in hippocampal extracts and the levels of 10 peptides differed between Wfs1 KO and wild-type mice at P Wfs1 KO mice compared to wild-type mice. Processing (cleavage) of little-LEN from the Pcsk1n gene product proSAAS involves prohormone convertase 2 (PC2). Thus, PC2 activity was measured in extracts prepared from the hippocampus of Wfs1 KO mice. The activity of PC2 in Wfs1 mutant mice was significantly higher (149.9 ± 2.3%, p Wfs1-KO mice, which is caused by increased activity of PC2. Increased activity of PC2 in Wfs1 KO mice is not caused by alteration in the levels of PC2 protein. Our results suggest a functional link between Wfs1 and PC2. Thus, the detailed molecular mechanism of the role of Wfs1 in the regulation of PC2 activity needs further investigation.

  7. Tissue inhibitor of metalloproteinase-3 knockout mice exhibit enhanced energy expenditure through thermogenesis.

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    Yohsuke Hanaoka

    Full Text Available Tissue inhibitors of metalloproteinases (TIMPs regulate matrix metalloproteinase activity and maintain extracellular matrix homeostasis. Although TIMP-3 has multiple functions (e.g., apoptosis, inhibition of VEGF binding to VEGF receptor, and inhibition of TNFα converting enzyme, its roles in thermogenesis and metabolism, which influence energy expenditure and can lead to the development of metabolic disorders when dysregulated, are poorly understood. This study aimed to determine whether TIMP-3 is implicated in metabolism by analyzing TIMP-3 knockout (KO mice. TIMP-3 KO mice had higher body temperature, oxygen consumption, and carbon dioxide production than wild-type (WT mice, although there were no differences in food intake and locomotor activity. These results suggest that metabolism is enhanced in TIMP-3 KO mice. Real-time PCR analysis showed that the expression of PPAR-δ, UCP-2, NRF-1 and NRF-2 in soleus muscle, and PGC-1α and UCP-2 in gastrocnemius muscle, was higher in TIMP-3 KO mice than in WT mice, suggesting that TIMP-3 deficiency may increase mitochondrial activity. When exposed to cold for 8 hours to induce thermogenesis, TIMP-3 KO mice had a higher body temperature than WT mice. In the treadmill test, oxygen consumption and carbon dioxide production were higher in TIMP-3 KO mice both before and after starting exercise, and the difference was more pronounced after starting exercise. Our findings suggest that TIMP-3 KO mice exhibit enhanced metabolism, as reflected by a higher body temperature than WT mice, possibly due to increased mitochondrial activity. Given that TIMP-3 deficiency increases energy expenditure, TIMP-3 may present a novel therapeutic target for preventing metabolic disorders.

  8. Muscarinic acetylcholine receptor knockout mice show distinct synaptic plasticity impairments in the visual cortex

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    Origlia, Nicola; Kuczewski, Nicola; Aztiria, Eugenio; Gautam, Dinesh; Wess, Jürgen; Domenici, Luciano

    2006-01-01

    In the present report, we focused our attention on the role played by the muscarinic acetylcholine receptors (mAChRs) in different forms of long-term synaptic plasticity. Specifically, we investigated long-term potentiation (LTP) and long-term depression (LTD) expression elicited by theta-burst stimulation (TBS) and low-frequency stimulation (LFS), respectively, in visual cortical slices obtained from different mAChR knockout (KO) mice. A normal LTP was evoked in M1/M3 double KO mice, while LTP was impaired in the M2/M4 double KO animals. On the other hand, LFS induced LTD in M2/M4 double KO mice, but failed to do so in M1/M3 KO mice. Interestingly, LFS produced LTP instead of LTD in M1/M3 KO mice. Analysis of mAChR single KO mice revealed that LTP was affected only by the simultaneous absence of both M2 and M4 receptors. A LFS-dependent shift from LTD to LTP was also observed in slices from M1 KO mice, while LTD was simply abolished in slices from M3 KO mice. Using pharmacological tools, we showed that LTP in control mice was blocked by pertussis toxin, an inhibitor of Gi/o proteins, but not by raising intracellular cAMP levels. In addition, the inhibition of phospholipase C by U73122 induced the same shift from LTD to LTP after LFS observed in M1 single KO and M1/M3 double KO mice. Our results indicate that different mAChR subtypes regulate different forms of long-term synaptic plasticity in the mouse visual cortex, activating specific G proteins and downstream intracellular mechanisms. PMID:17023506

  9. Moderate Continuous Aerobic Exercise Training Improves Cardiomyocyte Contractility in Β1 Adrenergic Receptor Knockout Mice

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    Aurora Corrêa Rodrigues

    2018-02-01

    Full Text Available Abstract Background: The lack of cardiac β1-adrenergic receptors (β1-AR negatively affects the regulation of both cardiac inotropy and lusitropy, leading, in the long term, to heart failure (HF. Moderate-intensity aerobic exercise (MCAE is recommended as an adjunctive therapy for patients with HF. Objective: We tested the effects of MCAE on the contractile properties of left ventricular (LV myocytes from β1 adrenergic receptor knockout (β1ARKO mice. Methods: Four- to five-month-old male wild type (WT and β1ARKO mice were divided into groups: WT control (WTc and trained (WTt; and β1ARKO control (β1ARKOc and trained (β1ARKOt. Animals from trained groups were submitted to a MCAE regimen (60 min/day; 60% of maximal speed, 5 days/week on a treadmill, for 8 weeks. P ≤ 0.05 was considered significant in all comparisons. Results: The β1ARKO and exercised mice exhibited a higher (p < 0.05 running capacity than WT and sedentary ones, respectively. The β1ARKO mice showed higher body (BW, heart (HW and left ventricle (LVW weights, as well as the HW/BW and LVW/BW than WT mice. However, the MCAE did not affect these parameters. Left ventricular myocytes from β1ARKO mice showed increased (p < 0.05 amplitude and velocities of contraction and relaxation than those from WT. In addition, MCAE increased (p < 0.05 amplitude and velocities of contraction and relaxation in β1ARKO mice. Conclusion: MCAE improves myocyte contractility in the left ventricle of β1ARKO mice. This is evidence to support the therapeutic value of this type of exercise training in the treatment of heart diseases involving β1-AR desensitization or reduction.

  10. Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice

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    Bleich, David; Chen, Songyuan; Zipser, Brian; Sun, Duxin; Funk, Colin D.; Nadler, Jerry L.

    1999-01-01

    Leukocyte 12-lipoxygenase (12-LO) gene expression in pancreatic β cells is upregulated by cytotoxic cytokines like IL-1β. Recent studies have demonstrated that 12-LO inhibitors can prevent glutamate-induced neuronal cell death when intracellular glutathione stores are depleted. Therefore, 12-LO pathway inhibition may prevent β-cell cytotoxicity. To evaluate the role of 12-LO gene expression in immune-mediated islet destruction, we used 12-LO knockout (12-LO KO) mice. Male homozygous 12-LO KO mice and control C57BL/6 mice received 5 consecutive daily injections of low-dose streptozotocin to induce immune-mediated diabetes. Fasting serum glucose and insulin levels were measured at 7-day intervals, and the mice were followed up for 28 days. 12-LO KO mice were highly resistant to diabetes development compared with control mice and had higher serum insulin levels on day 28. Isolated pancreatic islets were treated with IL-1β, TNF-α, and IFN-γ for 18 hours. Glucose-stimulated insulin secretion in cytokine-treated islets from C57/BL6 mice decreased 54% from that of untreated islets. In marked contrast, the same cytokine mix led to only a 26% decrease in islets from 12-LO KO mice. Furthermore, cytokine-induced 12-hydroxyeicosatetraenoic acid (12-HETE) production was absent in 12-LO KO islets but present in C57/BL6 islets. Isolated peritoneal macrophages were stimulated for 48 hours with IFN-γ + LPS and compared for nitrate/nitrite generation. 12-LO KO macrophages generated 50% less nitrate/nitrite when compared with C57BL/6 macrophages. In summary, elimination of leukocyte 12-LO in mice ameliorates low dose streptozotocin–induced diabetes by increasing islet resistance to cytokines and decreasing macrophage production of nitric oxide. PMID:10330425

  11. Experimental evidence for the involvement of PDLIM5 in mood disorders in hetero knockout mice.

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    Yasue Horiuchi

    Full Text Available BACKGROUND: Reports indicate that PDLIM5 is involved in mood disorders. The PDLIM5 (PDZ and LIM domain 5 gene has been genetically associated with mood disorders; it's expression is upregulated in the postmortem brains of patients with bipolar disorder and downregulated in the peripheral lymphocytes of patients with major depression. Acute and chronic methamphetamine (METH administration may model mania and the evolution of mania into psychotic mania or schizophrenia-like behavioral changes, respectively. METHODS: To address whether the downregulation of PDLIM5 protects against manic symptoms and cause susceptibility to depressive symptoms, we evaluated the effects of reduced Pdlim5 levels on acute and chronic METH-induced locomotor hyperactivity, prepulse inhibition, and forced swimming by using Pdlim5 hetero knockout (KO mice. RESULTS: The homozygous KO of Pdlim5 is embryonic lethal. The effects of METH administration on locomotor hyperactivity and the impairment of prepulse inhibition were lower in Pdlim5 hetero KO mice than in wild-type mice. The transient inhibition of PDLIM5 (achieved by blocking the translocation of protein kinase C epsilon before the METH challenge had a similar effect on behavior. Pdlim5 hetero KO mice showed increased immobility time in the forced swimming test, which was diminished after the chronic administration of imipramine. Chronic METH treatment increased, whereas chronic haloperidol treatment decreased, Pdlim5 mRNA levels in the prefrontal cortex. Imipramine increased Pdlim5 mRNA levels in the hippocampus. CONCLUSION: These findings are partially compatible with reported observations in humans, indicating that PDLIM5 is involved in psychiatric disorders, including mood disorders.

  12. Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.

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    Donowitz, M; Singh, S; Singh, P; Salahuddin, F F; Chen, Y; Chakraborty, M; Murtazina, R; Gucek, M; Cole, R N; Zachos, N C; Kovbasnjuk, O; Broere, N; Smalley-Freed, W G; Reynolds, A B; Hubbard, A L; Seidler, U; Weinman, E; de Jonge, H R; Hogema, B M; Li, X

    2010-11-15

    Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in

  13. Features of emotional and social behavioral phenotypes of calsyntenin2 knockout mice.

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    Ranneva, S V; Pavlov, K S; Gromova, A V; Amstislavskaya, T G; Lipina, T V

    2017-08-14

    Calsyntenin-2 (Clstn2) is the synaptic protein that belongs to the super family of cadherins, playing an important role in learning and memory. We recently reported that Clstn2 knockout mice (Clstn2-KO) have a deficit of GABAergic interneurons coupled with hyperactivity and deficient spatial memory. Given, that impaired functioning of GABA receptors is linked to several psychopathologies, including anxiety and autism, we sought to further characterize Clstn2-KO mice with respect to emotional and social behavior. Clstn2-KO males and females were tested in the elevated plus-maze (EPM), open field (OF), forced swim test, social affiliation and recognition test, social transmission of food preference (STFP), dyadic social interactions and marble burying test. Clstn2-KO mice demonstrated high exploration and hyperactivity in the dimly lit EPM that affect anxiety parameters. In contrast, in a more adverse situation in the OF have increased emotionality in Clstn2-KO males, not females. Assessment of hyperactivity for prolong period in the OF showed that Clstn2-KO animals were able to decline their hyperactivity, but their ambulation still remained higher than in WT littermates. Additionally, Clstn2-KO mice expressed stereotyped behavior. Strikingly, analysis of social behavior identified deficient social motivation and social recognition only in Clstn2-KO males, but not in females. Further analysis of social communication in the STFP and direct observation of agonistic interactions confirmed the reduced social behavior in Clstn2-KO males. Altogether, current results showed Clstn2 gene and sex interactions on socio-emotional performance in mice, suggesting a possible role of calsyntenin2 in psychopathological mechanisms of autism. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Cdh13 and AdipoQ gene knockout alter instrumental and Pavlovian drug conditioning.

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    King, C P; Militello, L; Hart, A; St Pierre, C L; Leung, E; Versaggi, C L; Roberson, N; Catlin, J; Palmer, A A; Richards, J B; Meyer, P J

    2017-09-01

    Genome-wide association studies in humans have suggested that variants of the cadherin-13 (CDH13) gene are associated with substance use disorder, subjective response to amphetamine, and attention deficit hyperactivity disorder. To examine the role of the Cdh13 and its peptide ligand adiponectin (AdipoQ) in addiction-related behaviors, we assessed Cdh13 knockout (KO) rats and AdipoQ KO mice using intravenous cocaine self-administration and conditioned place preference (CPP) paradigms. During intravenous cocaine self-administration, male Cdh13 heterozygous (+/-) and KO (-/-) rats showed increased cue-induced reinstatement compared with wild-type (WT) rats when presented with a cocaine-paired stimulus, whereas female Cdh13 rats showed no differences across genotype. Cdh13 -/- rats showed higher responding for a saccharin reinforcer and learned the choice reaction time (RT) task more slowly than WTs. However, we found no differences between Cdh13 -/- and +/+ rats in responding for sensory reinforcement, number of premature responses in the RT task, tendency to approach a Pavlovian food cue, CPP and locomotor activation to cocaine (10 or 20 mg/kg). In AdipoQ -/- mice, there was a significant increase in CPP to methamphetamine (1 mg/kg) but not to a range of d-amphetamine doses (0.5, 1, 2 and 4 mg/kg). Taken together, these data suggest that Cdh13 and AdipoQ regulate sensitivity to psychomotor stimulants and palatable rewards without producing major changes in other behaviors. In humans, these two genes may regulate sensitivity to natural and drug rewards, thus influencing susceptibility to the conditioned drug effects and relapse. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  15. Aspirin but not meloxicam attenuates early atherosclerosis in apolipoprotein E knockout mice.

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    Kraus, Sarah; Naumov, Inna; Shapira, Shiran; Kazanov, Dina; Aroch, Ilan; Afek, Arnon; Eisenberg, Oded; George, Jacob; Arber, Nadir; Finkelstein, Ariel

    2014-04-01

    Atherosclerosis is a complex vascular inflammatory disease. In the last decade it was suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) and in particular inhibition of cyclooxygenase (COX)-2 are associated with an increase in cardiovascular morbidity and mortality. Aspirin is known to reduce the incidence and mortality from ischemic heart disease and is a mainstay in the prevention of vascular complications of atherosclerosis. To examine the effect of meloxicam, a selective COX-2 inhibitor, or low dose aspirin on the development of experimental atherosclerosis in apoE knockout (KO) compared to wild-type (WT) mice. We aimed to test the hypothesis that meloxicam, a potential vasculitis inducer, would exacerbate atherosclerotic lesions while aspirin, which is known to reduce the incidence of thrombosis occlusive events, would increase protection in this model. We randomly divided 36 male apoE KO and 36 WT mice, 8 weeks old. Mice were treated for 10 weeks with 0.1 mg/ml aspirin, or 0.05 mg/ml meloxicam, dissolved in their drinking water. Control groups received regular drinking water. At sacrifice, the hearts were removed for histochemical staining and plaque size and composition were examined. Aspirin-treated animals displayed a decreased atherosclerotic lesion area compared to the untreated control mice, while meloxicam had a null effect on the extent of atherosclerosis in Apo E KO mice. These results suggest that low dose aspirin reduces early atherosclerosis, while inhibition of COX-2 by meloxicam is not associated with an increase in atherosclerotic plaque size in this mouse model.

  16. Mechanism of hyperphagia contributing to obesity in brain-derived neurotrophic factor knockout mice.

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    Fox, E A; Biddinger, J E; Jones, K R; McAdams, J; Worman, A

    2013-01-15

    Global-heterozygous and brain-specific homozygous knockouts (KOs) of brain-derived neurotrophic factor (BDNF) cause late- and early-onset obesity, respectively, both involving hyperphagia. Little is known about the mechanism underlying this hyperphagia or whether BDNF loss from peripheral tissues could contribute to overeating. Since global-homozygous BDNF-KO is perinatal lethal, a BDNF-KO that spared sufficient brainstem BDNF to support normal health was utilized to begin to address these issues. Meal pattern and microstructure analyses suggested overeating of BDNF-KO mice was mediated by deficits in both satiation and satiety that resulted in increased meal size and frequency and implicated a reduction of vagal signaling from the gut to the brain. Meal-induced c-Fos activation in the nucleus of the solitary tract, a more direct measure of vagal afferent signaling, however, was not decreased in BDNF-KO mice, and thus was not consistent with a vagal afferent role. Interestingly though, meal-induced c-Fos activation was increased in the dorsal motor nucleus of the vagus nerve (DMV) of BDNF-KO mice. This could imply that augmentation of vago-vagal digestive reflexes occurred (e.g., accommodation), which would support increased meal size and possibly increased meal number by reducing the increase in intragastric pressure produced by a given amount of ingesta. Additionally, vagal sensory neuron number in BDNF-KO mice was altered in a manner consistent with the increased meal-induced activation of the DMV. These results suggest reduced BDNF causes satiety and satiation deficits that support hyperphagia, possibly involving augmentation of vago-vagal reflexes mediated by central pathways or vagal afferents regulated by BDNF levels. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Altered gene expression in early postnatal monoamine oxidase A knockout mice.

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    Chen, Kevin; Kardys, Abbey; Chen, Yibu; Flink, Stephen; Tabakoff, Boris; Shih, Jean C

    2017-08-15

    We reported previously that monoamine oxidase (MAO) A knockout (KO) mice show increased serotonin (5-hydroxytryptamine, 5-HT) levels and autistic-like behaviors characterized by repetitive behaviors, and anti-social behaviors. We showed that administration of the serotonin synthesis inhibitor para-chlorophenylalanine (pCPA) from post-natal day 1 (P1) through 7 (P7) in MAO A KO mice reduced the serotonin level to normal and reverses the repetitive behavior. These results suggested that the altered gene expression at P1 and P7 may be important for the autistic-like behaviors seen in MAO A KO mice and was studied here. In this study, Affymetrix mRNA array data for P1 and P7 MAO A KO mice were analyzed using Partek Genomics Suite and Ingenuity Pathways Analysis to identify genes differentially expressed versus wild-type and assess their functions and relationships. The number of significant differentially expressed genes (DEGs) varied with age: P1 (664) and P7 (3307) [false discovery rate (FDR) 1.5 for autism-linked genes and >2.0 for functionally categorized genes]. Eight autism-linked genes were differentially expressed in P1 (upregulated: NLGN3, SLC6A2; down-regulated: HTR2C, MET, ADSL, MECP2, ALDH5A1, GRIN3B) while four autism-linked genes were differentially expressed at P7 (upregulated: HTR2B; downregulated: GRIN2D, GRIN2B, CHRNA4). Many other genes involved in neurodevelopment, apoptosis, neurotransmission, and cognitive function were differentially expressed at P7 in MAO A KO mice. This result suggests that modulation of these genes by the increased serotonin may lead to neurodevelopmental alteration in MAO A KO mice and results in autistic-like behaviors. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Somatostatin receptor 1 and 5 double knockout mice mimic neurochemical changes of Huntington's disease transgenic mice.

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    Padmesh S Rajput

    Full Text Available Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD. However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5.To delineate subtype selective biological responses we have here investigated changes in SSTR1 and 5 double knockout mice brain and compared with HD transgenic mouse model (R6/2. Our study revealed significant loss of dopamine and cAMP regulated phosphoprotein of 32 kDa (DARPP-32 and comparable changes in SST, N-methyl-D-aspartic acid receptors subtypes, calbindin and brain nitric oxide synthase expression as well as in key signaling proteins including calpain, phospho-extracellular-signal-regulated kinases1/2, synapsin-IIa, protein kinase C-α and calcineurin in SSTR1/5(-/- and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease.

  19. In vivo epicardial force and strain characterisation in normal and MLP-knockout murine hearts.

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    Michaelides, M; Georgiadou, S; Constantinides, C

    2015-07-01

    The study's objective is to quantify in vivo epicardial force and strain in the normal and transgenic myocardium using microsensors.Male mice (n = 39), including C57BL/6 (n = 26), 129/Sv (n = 5), wild-type (WT) C57  ×  129Sv (n = 5), and muscle LIM protein (MLP) knock-out (n = 3), were studied under 1.5% isoflurane anaesthesia. Microsurgery allowed the placement of two piezoelectric crystals at longitudinal epicardial loci at the basal, middle, and apical LV regions, and the independent (and/or concurrent) placement of a cantilever force sensor. The findings demonstrate longitudinal contractile and relaxation strains that ranged between 4.8-9.3% in the basal, middle, and apical regions of C57BL/6 mice, and in the mid-ventricular regions of 129/Sv, WT, and MLP mice. Measured forces ranged between 3.1-8.9 mN. The technique's feasibility is also demonstrated in normal mice following afterload, occlusion-reperfusion challenges.Furthermore, the total mid-ventricular forces developed in MLP mice were significantly reduced compared to the WT controls (5.9  ±  0.4 versus 8.9  ±  0.2 mN, p MLP mice.The possibility of quantifying in vivo force and strain from the normal murine heart is demonstrated with a potential usefulness in the characterisation of transgenic and diseased mice, where regional myocardial function may be significantly altered.

  20. Impact of chocolate liquor on vascular lesions in apoE-knockout mice.

    Science.gov (United States)

    Yazdekhasti, Narges; Brandsch, Corinna; Hirche, Frank; Kühn, Julia; Schloesser, Anke; Esatbeyoglu, Tuba; Huebbe, Patricia; Wolffram, Siegfried; Rimbach, Gerald; Stangl, Gabriele I

    2017-10-15

    Cocoa polyphenols are thought to reduce the risk of cardiovascular diseases. Thus, cocoa-containing foods may have significant health benefits. Here, we studied the impact of chocolate liquor on vascular lesion development and plaque composition in a mouse model of atherosclerosis. Apolipoprotein E (apoE)-knockout mice were assigned to two groups and fed a Western diet that contained 250 g/kg of either chocolate liquor or a polyphenol-free isoenergetic control paste for 16 weeks. In addition to fat, protein, and fibers, the chocolate liquor contained 2 g/kg of polyphenols. Compared with the control group, mice fed the chocolate liquor had larger plaque areas in the descending aorta and aortic root, which were attributed to a higher mass of vascular smooth muscle cells (VSMCs) and collagen. Vascular lipid deposits and calcification areas did not differ between the two groups. The aortic tissue level of interleukin-6 (IL-6) mRNA was 5-fold higher in the mice fed chocolate liquor than in the control mice. Chocolate-fed mice exhibited an increased hepatic saturated to polyunsaturated fatty acid ratio than the controls. Although the chocolate liquor contained 14 µg/kg of vitamin D 2 , the chocolate liquor-fed mice did not have measurable 25-hydroxyvitamin D 2 in the serum. These mice even showed a 25% reduction in the level of 25-hydroxyvitamin D 3 compared with the control mice. Overall, present data may contribute to our understanding how chocolate constituents can impact vascular lesion development. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  1. Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

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    Jige Xin

    Full Text Available Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs and successfully generated α-1,3-galactosyltransferase (GGTA1 gene biallelic knockout (KO pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26 among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209 among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209 of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT. Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

  2. Behavioral Phenotyping of Dopamine Transporter Knockout Rats: Compulsive Traits, Motor Stereotypies, and Anhedonia

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    Stefano Cinque

    2018-02-01

    Full Text Available Alterations in dopamine neurotransmission are generally associated with diseases such as attention-deficit/hyperactivity disorder (ADHD and obsessive-compulsive disorder (OCD. Such diseases typically feature poor decision making and lack of control on executive functions and have been studied through the years using many animal models. Dopamine transporter (DAT knockout (KO and heterozygous (HET mice, in particular, have been widely used to study ADHD. Recently, a strain of DAT KO rats has been developed (1. Here, we provide a phenotypic characterization of reward sensitivity and compulsive choice by adult rats born from DAT–HET dams bred with DAT–HET males, in order to further validate DAT KO rats as an animal model for preclinical research. We first tested DAT KO rats’ sensitivity to rewarding stimuli, provided by highly appetitive food or sweet water; then, we tested their choice behavior with an Intolerance-to-Delay Task (IDT. During these tests, DAT KO rats appeared less sensitive to rewarding stimuli than wild-type (WT and HET rats: they also showed a prominent hyperactive behavior with a rigid choice pattern and a wide number of compulsive stereotypies. Moreover, during the IDT, we tested the effects of amphetamine (AMPH and RO-5203648, a trace amine-associated receptor 1 (TAAR1 partial agonist. AMPH accentuated impulsive behaviors in WT and HET rats, while it had no effect in DAT KO rats. Finally, we measured the levels of tyrosine hydroxylase, dopamine receptor 2 (D2, serotonin transporter, and TAAR1 mRNA transcripts in samples of ventral striatum, finding no significant differences between WT and KO genotypes. Throughout this study, DAT KO rats showed alterations in decision-making processes and in motivational states, as well as prominent motor and oral stereotypies: more studies are warranted to fully characterize and efficiently use them in preclinical research.

  3. Elevated Anxiety and Impaired Attention in Super-Smeller, Kv1.3 Knockout Mice

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    Zhenbo Huang

    2018-03-01

    Full Text Available It has long been recognized that olfaction and emotion are linked. While chemosensory research using both human and rodent models have indicated a change in emotion can contribute to olfactory dysfunction, there are few studies addressing the contribution of olfaction to a modulation in emotion. In mice, olfactory deficits have been linked with heightened anxiety levels, suggesting that there could be an inverse relationship between olfaction and anxiety. Furthermore, increased anxiety is often co-morbid with psychiatric conditions such as attention disorders. Our study aimed to investigate the roles of olfaction in modulating anxiety. Voltage-gated potassium ion channel Kv1.3 knockout mice (Kv1.3−/−, which have heightened olfaction, and wild-type (WT mice were examined for anxiety-like behaviors using marble burying (MB, light-dark box (LDB and elevated-plus maze (EPM tests. Because Kv1.3−/− mice have increased locomotor activity, inattentive and hyperactive behaviors were quantified for both genotypes. Kv1.3−/− mice showed increased anxiety levels compared to their WT counterparts and administration of methylphenidate (MPH via oral gavage alleviated their increased anxiety. Object-based attention testing indicated young and older Kv1.3−/− mice had attention deficits and treatment with MPH also ameliorated this condition. Locomotor testing through use of a metabolic chamber indicated that Kv1.3−/− mice were not significantly hyperactive and MPH treatment failed to modify this activity. Our data suggest that heightened olfaction does not necessarily lead to decreased anxiety levels, and that Kv1.3−/− mice may have behaviors associated with inattentiveness.

  4. Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference.

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    Bahi, Amine; Tolle, Virginie; Fehrentz, Jean-Alain; Brunel, Luc; Martinez, Jean; Tomasetto, Catherine-Laure; Karam, Sherif M

    2013-05-01

    Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist "JMV2959". We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

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    Satoko eHattori

    2012-10-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

  6. A conditioned aversion study of sucrose and SC45647 taste in TRPM5 knockout mice.

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    Eddy, Meghan C; Eschle, Benjamin K; Peterson, Darlene; Lauras, Nathan; Margolskee, Robert F; Delay, Eugene R

    2012-06-01

    Previously, published studies have reported mixed results regarding the role of the TRPM5 cation channel in signaling sweet taste by taste sensory cells. Some studies have reported a complete loss of sweet taste preference in TRPM5 knockout (KO) mice, whereas others have reported only a partial loss of sweet taste preference. This study reports the results of conditioned aversion studies designed to motivate wild-type (WT) and KO mice to respond to sweet substances. In conditioned taste aversion experiments, WT mice showed nearly complete LiCl-induced response suppression to sucrose and SC45647. In contrast, TRPM5 KO mice showed a much smaller conditioned aversion to either sweet substance, suggesting a compromised, but not absent, ability to detect sweet taste. A subsequent conditioned flavor aversion experiment was conducted to determine if TRPM5 KO mice were impaired in their ability to learn a conditioned aversion. In this experiment, KO and WT mice were conditioned to a mixture of SC45647 and amyl acetate (an odor cue). Although WT mice avoided both components of the stimulus mixture, they avoided SC45647 more than the odor cue. The KO mice also avoided both stimuli, but they avoided the odor component more than SC45647, suggesting that while the KO mice are capable of learning an aversion, to them the odor cue was more salient than the taste cue. Collectively, these findings suggest the TRPM5 KO mice have some residual ability to detect SC45647 and sucrose, and, like bitter, there may be a TRPM5-independent transduction pathway for detecting these substances.

  7. Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity.

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    Ohkuri, Tadahiro; Yasumatsu, Keiko; Horio, Nao; Jyotaki, Masafumi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-04-01

    Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Galpha subunit (Galphagust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Galphagust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Galphagust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Galphagust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue.

  8. Large-conductance Ca2+-activated K+ channel β1-subunit knockout mice are not hypertensive

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    Garver, Hannah; Galligan, James J.; Fink, Gregory D.

    2011-01-01

    Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ∼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (∼5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca2+ channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension. PMID:21131476

  9. Repetitive grooming and sensorimotor abnormalities in an ephrin-A knockout model for Autism Spectrum Disorders.

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    Wurzman, Rachel; Forcelli, Patrick A; Griffey, Christopher J; Kromer, Lawrence F

    2015-02-01

    EphA receptors and ephrin-A ligands play important roles in neural development and synaptic plasticity in brain regions where expression persists into adulthood. Recently, EPHA3 and EPHA7 gene mutations were linked with Autism Spectrum Disorders (ASDs) and developmental neurological delays, respectively. Furthermore, deletions of ephrin-A2 or ephrin-A3, which exhibit high binding affinity for EphA3 and EphA7 receptors, are associated with subtle deficits in learning and memory behavior and abnormalities in dendritic spine morphology in the cortex and hippocampus in mice. To better characterize a potential role for these ligands in ASDs, we performed a comprehensive behavioral characterization of anxiety-like, sensorimotor, learning, and social behaviors in ephrin-A2/-A3 double knockout (DKO) mice. The predominant phenotype in DKO mice was repetitive and self-injurious grooming behaviors such as have been associated with corticostriatal circuit abnormalities in other rodent models of neuropsychiatric disorders. Consistent with ASDs specifically, DKO mice exhibited decreased preference for social interaction in the social approach assay, decreased locomotor activity in the open field, increased prepulse inhibition of acoustic startle, and a shift towards self-directed activity (e.g., grooming) in novel environments, such as marble burying. Although there were no gross deficits in cognitive assays, subtle differences in performance on fear conditioning and in the Morris water maze resembled traits observed in other rodent models of ASD. We therefore conclude that ephrin-A2/-A3 DKO mice have utility as a novel ASD model with an emphasis on sensory abnormalities and restricted, repetitive behavioral symptoms. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease.

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    Niemann, Axel; Huber, Nina; Wagner, Konstanze M; Somandin, Christian; Horn, Michael; Lebrun-Julien, Frédéric; Angst, Brigitte; Pereira, Jorge A; Halfter, Hartmut; Welzl, Hans; Feltri, M Laura; Wrabetz, Lawrence; Young, Peter; Wessig, Carsten; Toyka, Klaus V; Suter, Ueli

    2014-03-01

    The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. We found that Gdap1 knockout mice (Gdap1(-/-)), mimicking genetic alterations of patients suffering from severe forms of Charcot-Marie-Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1(-/-) mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1(-/-) mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1(-/-) mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1(-/-) mice compared with controls. Our findings demonstrate that Charcot-Marie-Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

  11. The Gdap1 knockout mouse mechanistically links redox control to Charcot–Marie–Tooth disease

    Science.gov (United States)

    Huber, Nina; Wagner, Konstanze M.; Somandin, Christian; Horn, Michael; Lebrun-Julien, Frédéric; Pereira, Jorge A.; Halfter, Hartmut; Welzl, Hans; Feltri, M. Laura; Wrabetz, Lawrence; Young, Peter; Wessig, Carsten; Toyka, Klaus V.; Suter, Ueli

    2014-01-01

    The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot–Marie–Tooth disease. We found that Gdap1 knockout mice (Gdap1−/−), mimicking genetic alterations of patients suffering from severe forms of Charcot–Marie–Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1−/− mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1−/− mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1−/− mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1−/− mice compared with controls. Our findings demonstrate that Charcot–Marie–Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione. PMID:24480485

  12. Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Youfeng Shen

    2017-11-01

    Full Text Available Abstract Background Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Methods Transcription activator-like effector nucleases (TALENs were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. Results The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19 were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean

  13. The influence of angiotensin-(1-7) peptidomimetic (AVE 0991) and nebivolol on angiotensin I metabolism in aorta of apoE-knockout mice.

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    Olszanecki, R; Suski, M; Gebska, A; Toton-Zuranska, J; Kus, K; Madej, J; Bujak-Gizycka, B; Jawien, J; Korbut, R

    2013-06-01

    The detrimental role of over activation of renin-angiotensin system (RAS) in atherogenesis is widely recognized. Recently, we have demonstrated that Ang-(1-7) peptidomimetic - AVE0991, as well as known beta-adrenolytic agent nebivolol, exert anti-atherogenic actions in mouse model of atherosclerosis - apoE-knockout mice. Here, using LC-ESI-MS ex vivo system, we tested whether prolonged treatment of apoE-knockout mice by these drugs can influence RAS in aorta of apoE-knockout mice in regard to generation of most active metabolites of Ang I-Ang II and Ang-(1-7). As compared to wild type animals there was increased generation of Ang II in aorta of apoE-knockout mice, while the formation of Ang-(1-7) did not differ between both groups. Either treatment with AVE0991 or nebivolol resulted in significant attenuation of Ang II production in aorta of apoE-knockout mice. In conclusion, for the first time we directly demonstrated that there is increase in ability of aortic tissue to generate Ang II in mouse model of atherosclerosis of apoE knockout mice, and that such effect could be efficiently attenuated either by treatment of nebivolol or Ang-(1-7) peptidomimetic - AVE0991. The exact mechanism(s) responsible for interference of both drugs with RAS require further investigation.

  14. CD4 T cell knockout does not protect against kidney injury and worsens cancer.

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    Ravichandran, Kameswaran; Wang, Qian; Ozkok, Abdullah; Jani, Alkesh; Li, Howard; He, Zhibin; Ljubanovic, Danica; Weiser-Evans, Mary C; Nemenoff, Raphael A; Edelstein, Charles L

    2016-04-01

    Most previous studies of cisplatin-induced acute kidney injury (AKI) have been in models of acute, high-dose cisplatin administration that leads to mortality in non-tumor-bearing mice. The aim of the study was to determine whether CD4 T cell knockout protects against AKI and cancer in a clinically relevant model of low-dose cisplatin-induced AKI in mice with cancer. Kidney function, serum neutrophil gelatinase-associated lipocalin (NGAL), acute tubular necrosis (ATN), and tubular apoptosis score were the same in wild-type and CD4 -/- mice with AKI. The lack of protection against AKI in CD4 -/- mice was associated with an increase in extracellular signal-regulated kinase (ERK), p38, CXCL1, and TNF-α, mediators of AKI and fibrosis, in both cisplatin-treated CD4 -/- mice and wild-type mice. The lack of protection was independent of the presence of cancer or not. Tumor size was double, and cisplatin had an impaired therapeutic effect on the tumors in CD4 -/- vs. wild-type mice. Mice depleted of CD4 T cells using the GK1.5 antibody were not protected against AKI and had larger tumors and lesser response to cisplatin. In summary, in a clinically relevant model of cisplatin-induced AKI in mice with cancer, (1) CD4 -/- mice were not protected against AKI; (2) ERK, p38, CXCL1, and TNF-α, known mediators of AKI, and interstitial fibrosis were increased in CD4 -/- kidneys; and (3) CD4 -/- mice had faster tumor growth and an impaired therapeutic effect of cisplatin on the tumors. The data warns against the use of CD4 T cell inhibition to attenuate cisplatin-induced AKI in patients with cancer. A clinically relevant low-dose cisplatin model of AKI in mice with cancer was used. CD4 -/- mice were not functionally or histologically protected against AKI. CD4 -/- mice had faster tumor growth. CD4 -/- mice had an impaired therapeutic effect of cisplatin on the tumors. Mice depleted of CD4 T cells were not protected against AKI and had larger tumors.

  15. Microarray analysis of gene expression in the cyclooxygenase knockout mice - a connection to autism spectrum disorder.

    Science.gov (United States)

    Rai-Bhogal, Ravneet; Ahmad, Eizaaz; Li, Hongyan; Crawford, Dorota A

    2017-11-21

    The cellular and molecular events that take place during brain development play an important role in governing function of the mature brain. Lipid-signalling molecules such as prostaglandin E 2 (PGE 2 ) play an important role in healthy brain development. Abnormalities along the COX-PGE 2 signalling pathway due to genetic or environmental causes have been linked to autism spectrum disorder (ASD). This study aims to evaluate the effect of altered COX-PGE 2 signalling on development and function of the prenatal brain using male mice lacking cyclooxygenase-1 and cyclooxygenase-2 (COX-1 -/- and COX-2 -/- ) as potential model systems of ASD. Microarray analysis was used to determine global changes in gene expression during embryonic days 16 (E16) and 19 (E19). Gene Ontology: Biological Process (GO:BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were implemented to identify affected developmental genes and cellular processes. We found that in both knockouts the brain at E16 had nearly twice as many differentially expressed genes, and affected biological pathways containing various ASD-associated genes important in neuronal function. Interestingly, using GeneMANIA and Cytoscape we also show that the ASD-risk genes identified in both COX-1 -/- and COX-2 -/- models belong to protein-interaction networks important for brain development despite of different cellular localization of these enzymes. Lastly, we identified eight genes that belong to the Wnt signalling pathways exclusively in the COX-2 -/- mice at E16. The level of PKA-phosphorylated β-catenin (S552), a major activator of the Wnt pathway, was increased in this model, suggesting crosstalk between the COX-2-PGE 2 and Wnt pathways during early brain development. Overall, these results provide further molecular insight into the contribution of the COX-PGE 2 pathways to ASD and demonstrate that COX-1 -/- and COX-2 -/- animals might be suitable new model systems for studying the disorders. © 2017 Federation of

  16. Flavor preference conditioning by different sugars in sweet ageusic Trpm5 knockout mice.

    Science.gov (United States)

    Sclafani, Anthony; Ackroff, Karen

    2015-03-01

    Knockout (KO) mice missing the taste signaling protein Trpm5 have greatly attenuated sweetener preferences but develop strong preferences for glucose in 24-h tests, which is attributed to post-oral sugar conditioning. Trpm5 KO mice express mild preferences for galactose but no preferences for fructose in 24-h tests, which suggests that these sugars differ in their post-oral reinforcing effects. Here we investigated sugar-conditioned flavor preferences in Trpm5 KO and C57BL/6J wildtype (B6) mice. The mice were trained to consume a flavored (CS+, e.g. grape) 8% sugar solution and flavored (CS-, e.g., cherry) water on alternating days followed by two-bottle choice tests with CS+ vs. CS- flavors in water and with unflavored sugar vs. water. The KO mice displayed strong preferences (>80%) for the CS+ glucose and CS+ galactose but not for the CS+ fructose flavor. They also preferred glucose and galactose, but not fructose to water. In contrast, the B6 mice preferred all three CS+ flavors to the CS- flavor, and all three sugars to water. In tests with the non-metabolizable sugar α-methyl-d-glucopyranoside (MDG), the KO and B6 mice preferred 8% MDG to water but did not prefer the CS+ 8% MDG to CS-. However, they preferred a CS+ flavor mixed with 4% MDG over the CS- flavor. Trpm5 KO mice also preferred galactose and MDG to fructose in direct choice tests. The Trpm5 KO data indicate that glucose and, to a lesser extent, galactose and MDG have post-oral reinforcing actions that stimulate intake and preference while fructose has a much weaker effect. The CS+ flavor and sugar preferences of B6 mice may be mediated by the sweet taste and/or post-oral actions of the various sugars. Glucose, galactose, and MDG, but not fructose, are ligands for the sodium-glucose transporter 1 (SGLT1) which is implicated in post-oral sugar conditioning in B6 mice. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Gal-knockout bioprostheses exhibit less immune stimulation compared to standard biological heart valves.

    Science.gov (United States)

    McGregor, Christopher G A; Kogelberg, Heide; Vlasin, Michal; Byrne, Guerard W

    2013-05-01

    Current biological heart valves (BHVs) contain the major xenogeneic antigen Gal. Recipient anti-Gal antibody binding to such an implanted BHV may contribute to valve degeneration. The study aim was to compare, by implantation in non-human primates, the immune consequences of BHVs from Gal-positive wild-type (WT) pigs and those from alpha-galactosyltransferase knockout (GTKO) pigs. Recipients were immunized prior to implant with keyhole limpet hemocyanin (KLH) conjugated to alphaGal to match the anti-Gal levels and isotypes found in humans. Stented glutaraldehyde-fixed BHVs from WT (n = 4) and GTKO (n = 3) pigs were commercially manufactured and implanted in the mitral position in non-human primates. Recipients were treated with enoxaparin (1 mg/kg b.i.d.) for five weeks which was tapered, and then discontinued. Serum antibody levels to Gal and KLH were measured using ELISA. Overall anti-Gal and anti-KLH antibody levels were decreased in both WT and GTKO BHV recipients after implantation. Serum anti-Gal IgG levels in GTKO BHV recipients fell rapidly within one month, matching the loss of anti-KLH reactivity. There was no significant difference in retention of anti-KLH antibody between the groups. WT BHV recipients retained significantly elevated levels of anti-Gal IgG during the first year post implant. Area under the curve analysis showed that anti-Gal IgG was significantly increased in the WT BHV group compared to GTKO BHV recipients (p < 0.01). Persistent and significantly (p < 0.01) elevated levels of anti-Gal IgG were observed in WT but not GTKO BHV non-human primate recipients, and indicated a continuing BHV-specific immune stimulation to the alphaGal antigen. These data support the hypothesis that the clinical use of Gal-positive xenogeneic bioprosthetic materials can induce an anti-Gal antibody response. Bioprosthetic devices prepared from GTKO pig tissue would eliminate immune stimulation to this major xenoreactive antigen, which may reduce the potential of

  18. Normal radial migration and lamination are maintained in dyslexia-susceptibility candidate gene homolog Kiaa0319 knockout mice.

    Science.gov (United States)

    Martinez-Garay, Isabel; Guidi, Luiz G; Holloway, Zoe G; Bailey, Melissa A G; Lyngholm, Daniel; Schneider, Tomasz; Donnison, Timothy; Butt, Simon J B; Monaco, Anthony P; Molnár, Zoltán; Velayos-Baeza, Antonio

    2017-04-01

    Developmental dyslexia is a common disorder with a strong genetic component, but the underlying molecular mechanisms are still unknown. Several candidate dyslexia-susceptibility genes, including KIAA0319, DYX1C1, and DCDC2, have been identified in humans. RNA interference experiments targeting these genes in rat embryos have shown impairments in neuronal migration, suggesting that defects in radial cortical migration could be involved in the disease mechanism of dyslexia. Here we present the first characterisation of a Kiaa0319 knockout mouse line. Animals lacking KIAA0319 protein do not show anatomical abnormalities in any of the layered structures of the brain. Neurogenesis and radial migration of cortical projection neurons are not altered, and the intrinsic electrophysiological properties of Kiaa0319-deficient neurons do not differ from those of wild-type neurons. Kiaa0319 overexpression in cortex delays radial migration, but does not affect final neuronal position. However, knockout animals show subtle differences suggesting possible alterations in anxiety-related behaviour and in sensorimotor gating. Our results do not reveal a migration disorder in the mouse model, adding to the body of evidence available for Dcdc2 and Dyx1c1 that, unlike in the rat in utero knockdown models, the dyslexia-susceptibility candidate mouse homolog genes do not play an evident role in neuronal migration. However, KIAA0319 protein expression seems to be restricted to the brain, not only in early developmental stages but also in adult mice, indicative of a role of this protein in brain function. The constitutive and conditional knockout lines reported here will be useful tools for further functional analyses of Kiaa0319.

  19. Random phenotypic variation of yeast (Saccharomyces cerevisiae single-gene knockouts fits a double pareto-lognormal distribution.

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    John H Graham

    Full Text Available Distributed robustness is thought to influence the buffering of random phenotypic variation through the scale-free topology of gene regulatory, metabolic, and protein-protein interaction networks. If this hypothesis is true, then the phenotypic response to the perturbation of particular nodes in such a network should be proportional to the number of links those nodes make with neighboring nodes. This suggests a probability distribution approximating an inverse power-law of random phenotypic variation. Zero phenotypic variation, however, is impossible, because random molecular and cellular processes are essential to normal development. Consequently, a more realistic distribution should have a y-intercept close to zero in the lower tail, a mode greater than zero, and a long (fat upper tail. The double Pareto-lognormal (DPLN distribution is an ideal candidate distribution. It consists of a mixture of a lognormal body and upper and lower power-law tails.If our assumptions are true, the DPLN distribution should provide a better fit to random phenotypic variation in a large series of single-gene knockout lines than other skewed or symmetrical distributions. We fit a large published data set of single-gene knockout lines in Saccharomyces cerevisiae to seven different probability distributions: DPLN, right Pareto-lognormal (RPLN, left Pareto-lognormal (LPLN, normal, lognormal, exponential, and Pareto. The best model was judged by the Akaike Information Criterion (AIC.Phenotypic variation among gene knockouts in S. cerevisiae fits a double Pareto-lognormal (DPLN distribution better than any of the alternative distributions, including the right Pareto-lognormal and lognormal distributions.A DPLN distribution is consistent with the hypothesis that developmental stability is mediated, in part, by distributed robustness, the resilience of gene regulatory, metabolic, and protein-protein interaction networks. Alternatively, multiplicative cell growth, and the

  20. Communication Impairment in Ultrasonic Vocal Repertoire during the Suckling Period of Cd157 Knockout Mice: Transient Improvement by Oxytocin

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    Olga L. Lopatina

    2017-05-01

    Full Text Available Communication consists of social interaction, recognition, and information transmission. Communication ability is the most affected component in children with autism spectrum disorder (ASD. Recently, we reported that the CD157/BST1 gene is associated with ASD, and that CD157 knockout (Cd157−/− mice display severe impairments in social behavior that are improved by oxytocin (OXT treatment. Here, we sought to determine whether Cd157−/− mice can be used as a suitable model for communication deficits by measuring ultrasonic vocalizations (USVs, especially in the early developmental stage. Call number produced in pups due to isolation from dams was higher at postnatal day (PND 3 in knockout pups than wild-type mice, but was lower at PNDs 7 and 10. Pups of both genotypes had similarly limited voice repertoires at PND 3. Later on, at PNDs 7 and 10, while wild-type pups emitted USVs consisting of six different syllable types, knockout pups vocalized with only two types. This developmental impairment in USV emission was rescued within 30 min by intraperitoneal OXT treatment, but quickly returned to control levels after 120 min, showing a transient effect of OXT. USV impairment was partially observed in Cd157+/− heterozygous mice, but not in Cd157−/− adult male mice examined while under courtship. These results demonstrate that CD157 gene deletion results in social communication insufficiencies, and suggests that CD157 is likely involved in acoustic communication. This unique OXT-sensitive developmental delay in Cd157−/− pups may be a useful model of communicative interaction impairment in ASD.

  1. Random phenotypic variation of yeast (Saccharomyces cerevisiae) single-gene knockouts fits a double pareto-lognormal distribution.

    Science.gov (United States)

    Graham, John H; Robb, Daniel T; Poe, Amy R

    2012-01-01

    Distributed robustness is thought to influence the buffering of random phenotypic variation through the scale-free topology of gene regulatory, metabolic, and protein-protein interaction networks. If this hypothesis is true, then the phenotypic response to the perturbation of particular nodes in such a network should be proportional to the number of links those nodes make with neighboring nodes. This suggests a probability distribution approximating an inverse power-law of random phenotypic variation. Zero phenotypic variation, however, is impossible, because random molecular and cellular processes are essential to normal development. Consequently, a more realistic distribution should have a y-intercept close to zero in the lower tail, a mode greater than zero, and a long (fat) upper tail. The double Pareto-lognormal (DPLN) distribution is an ideal candidate distribution. It consists of a mixture of a lognormal body and upper and lower power-law tails. If our assumptions are true, the DPLN distribution should provide a better fit to random phenotypic variation in a large series of single-gene knockout lines than other skewed or symmetrical distributions. We fit a large published data set of single-gene knockout lines in Saccharomyces cerevisiae to seven different probability distributions: DPLN, right Pareto-lognormal (RPLN), left Pareto-lognormal (LPLN), normal, lognormal, exponential, and Pareto. The best model was judged by the Akaike Information Criterion (AIC). Phenotypic variation among gene knockouts in S. cerevisiae fits a double Pareto-lognormal (DPLN) distribution better than any of the alternative distributions, including the right Pareto-lognormal and lognormal distributions. A DPLN distribution is consistent with the hypothesis that developmental stability is mediated, in part, by distributed robustness, the resilience of gene regulatory, metabolic, and protein-protein interaction networks. Alternatively, multiplicative cell growth, and the mixing of

  2. CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants ofPlasmodium falciparuminvasion.

    Science.gov (United States)

    Kanjee, Usheer; Grüring, Christof; Chaand, Mudit; Lin, Kai-Min; Egan, Elizabeth; Manzo, Jale; Jones, Patrick L; Yu, Tiffany; Barker, Robert; Weekes, Michael P; Duraisingh, Manoj T

    2017-10-31

    During malaria blood-stage infections, Plasmodium parasites interact with the RBC surface to enable invasion followed by intracellular proliferation. Critical factors involved in invasion have been identified using biochemical and genetic approaches including specific knockdowns of genes of interest from primary CD34 + hematopoietic stem cells (cRBCs). Here we report the development of a robust in vitro culture system to produce RBCs that allow the generation of gene knockouts via CRISPR/Cas9 using the immortal JK-1 erythroleukemia line. JK-1 cells spontaneously differentiate, generating cells at different stages of erythropoiesis, including terminally differentiated nucleated RBCs that we term "jkRBCs." A screen of small-molecule epigenetic regulators identified several bromodomain-specific inhibitors that promote differentiation and enable production of synchronous populations of jkRBCs. Global surface proteomic profiling revealed that jkRBCs express all known P falciparum host receptors in a similar fashion to cRBCs and that multiple P falciparum strains invade jkRBCs at comparable levels to cRBCs and RBCs. Using CRISPR/Cas9, we deleted two host factors, basigin (BSG) and CD44, for which no natural nulls exist. BSG interacts with the parasite ligand Rh5, a prominent vaccine candidate. A BSG knockout was completely refractory to parasite invasion in a strain-transcendent manner, confirming the essential role for BSG during invasion. CD44 was recently identified in an RNAi screen of blood group genes as a host factor for invasion, and we show that CD44 knockout results in strain-transcendent reduction in invasion. Furthermore, we demonstrate a functional interaction between these two determinants in mediating P falciparum erythrocyte invasion. Published under the PNAS license.

  3. Differential gene expression in the EphA4 knockout spinal cord and analysis of the inflammatory response following spinal cord injury.

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    Kathryn M Munro

    Full Text Available Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips™. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury.

  4. Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

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    Hanna Witwicka

    Full Text Available The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized

  5. Generation of two H1 hESC sublines carrying a heterozygous and homozygous knock-out of RB1

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    Laura Steenpass

    2017-12-01

    Full Text Available Retinoblastoma is a childhood cancer of the retina caused by biallelic inactivation of the tumor suppressor gene RB1. In heritable retinoblastoma, one allele is inherited in mutant form via one of the parental germ cells. To study molecular mechanisms in retinoblastoma, two sublines of H1 hESCs were generated, carrying a knock-out allele of RB1 in the heterozygous or homozygous state. Exon 3 of RB1 was targeted and modified by nucleotide deletions using the CRISPR/Cas9 nuclease system. Based on a nearby single nucleotide polymorphism, the modification could be assigned to one allele.

  6. Predictive validity and immune cell involvement in the pathogenesis of piroxicam-accelerated colitis in interleukin-10 knockout mice

    DEFF Research Database (Denmark)

    Holgersen, Kristine; Kvist, Peter Helding; Hansen, Axel Jacob Kornerup

    2014-01-01

    Piroxicam administration is a method for induction of enterocolitis in interleukin-10 knockout (IL-10 k.o.) mice. The piroxicam-accelerated colitis (PAC) IL-10 k.o. model combines a dysregulated immune response against the gut microbiota with a decreased mucosal integrity. The predictive validity...... and pathogenic mechanisms of the model have not been thoroughly investigated. In this study, IL-10 k.o. mice received piroxicam in the chow, and model qualification was performed by examining the efficacy of prophylactic anti-IL-12/23p40 monoclonal antibody (mAb), anti-TNFαmAb, cyclosporine A (CsA) and oral...

  7. A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

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    Thomas J Lampert

    Full Text Available Although G-protein coupled receptors (GPCRs are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²⁺ and K⁺, suggesting a decrease in basal excitability (decrease in Ca²⁺ channel activity. The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA and proteose peptone (PP, two known chemoattractants in Tetrahymena. Using microsomal [³⁵S]GTPγS binding assays, we found that wild-type (CU427 have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor, addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.

  8. Data on Arc and Zif268 expression in the brain of the α-2A adrenergic receptor knockout mouse

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    Jeff Sanders

    2016-06-01

    Full Text Available The α2-adrenergic receptor (α2-AR is widely distributed in the brain with distinct roles for α2-AR subtypes (A, B and C. In this article, data are provided on Activity Regulated Cytoskeleton Associated Protein (Arc and Zif268 expression in the brain of the α2A-AR knockout (α2A-AR KO mouse. These data are supplemental to an original research article examining Arc and Zif268 expression in rats injected with the α2-AR antagonist, RX821002 (http://dx.doi.org/10.1016/j.neulet.2015.12.002. [1].

  9. ZO-1 Knockout by TALEN-Mediated Gene Targeting in MDCK Cells: Involvement of ZO-1 in the Regulation of Cytoskeleton and Cell Shape

    Science.gov (United States)

    Tokuda, Shinsaku; Higashi, Tomohito; Furuse, Mikio

    2014-01-01

    ZO-1, ZO-2 and ZO-3 are tight junction-associated scaffold proteins that bind to transmembrane proteins of tight junctions and the underlying cytoskeleton. ZO-1 is involved in the regulation of cytoskeletal organization, but its detailed molecular mechanism is less well understood. Gene knockout is an ideal method to investigate the functions of proteins that might have redundant functions such as ZO proteins, when compared with methods such as RNA interference-mediated suppression of gene expression. In this study we applied transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout, and established ZO-1 knockout clones in Madin-Darby canine kidney (MDCK) cells. ZO-1 knockout induced striking changes in myosin organization at cell–cell contacts and disrupted the localization of tight junction proteins; these findings were previously unseen in studies of ZO-1 knockdown by RNA interference. Rescue experiments revealed that trace ZO-1 expression reversed these changes while excessive ZO-1 expression induced an intensive zigzag shape of cell–cell junctions. These results suggest a role for ZO-1 in the regulation of cytoskeleton and shape of cell–cell junctions in MDCK cells and indicate the advantage of knockout analysis in cultured cells. PMID:25157572

  10. Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect

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    Jeong Hyo Lee

    2017-05-01

    Full Text Available Objective Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9 to modulate the specific target gene in chicken DF1 cells. Methods Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase. After co-transfection of the nickase expression vector with green fluorescent gene (GFP gene and targeted multiplex guide RNAs (gRNAs, the GFP-positive cells were sorted out by fluorescence-activated cell sorting procedure. Results Through the genotyping analysis of the knockout cells, the mutant induction efficiency was 100% in the targeted site. Number of the deleted nucleotides ranged from 2 to 39 nucleotide deletion. There was no phenotypic difference between regular cells and knockout cells. However, myostatin protein was not apparently detected in the knockout cells by Western blotting. Additionally, six off-target sites were predicted and analyzed but any non-specific mutation in the off-target sites was not observed. Conclusion The knockout technical platform with the nickase and multiplex gRNAs can be efficiently and stablely applied to functional genomics study in poultry and finally adapted to generate the knockout poultry for agribio industry.

  11. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    Science.gov (United States)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  12. Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

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    Nor ‘Aini, A. R.

    2006-01-01

    Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

  13. Enhanced brain disposition and effects of Δ9-tetrahydrocannabinol in P-glycoprotein and breast cancer resistance protein knockout mice.

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    Adena S Spiro

    Full Text Available The ABC transporters P-glycoprotein (P-gp, Abcb1 and breast cancer resistance protein (Bcrp, Abcg2 regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis Δ(9-tetrahydrocannabinol (THC has affinity for P-gp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that mice devoid of Abcb1 and Abcg2 retain higher brain THC levels and are more sensitive to cannabinoid-induced hypothermia than wild-type (WT mice. Abcb1a/b (-/-, Abcg2 (-/- and wild-type (WT mice were injected with THC before brain and blood were collected and THC concentrations determined. Another cohort of mice was examined for THC-induced hypothermia by measuring rectal body temperature. Brain THC concentrations were higher in both Abcb1a/b (-/- and Abcg2 (-/- mice than WT mice. ABC transporter knockout mice exhibited delayed elimination of THC from the brain with the effect being more prominent in Abcg2 (-/- mice. ABC transporter knockout mice were more sensitive to THC-induced hypothermia compared to WT mice. These results show P-gp and Bcrp prolong the brain disposition and hypothermic effects of THC and offer a novel mechanism for both genetic vulnerability to the psychoactive effects of cannabis and drug interactions between CNS therapies and cannabis.

  14. Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis.

    Science.gov (United States)

    Poulsen, Christian Bjørn; Penkowa, Milena; Borup, Rehannah; Nielsen, Finn Cilius; Cáceres, Mario; Quintana, Albert; Molinero, Amalia; Carrasco, Javier; Giralt, Mercedes; Hidalgo, Juan

    2005-01-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays in wild-type and IL-6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post-lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with approximately 12,400 probe sets corresponding to approximately 10,000 different murine genes (MG_U74Av2). A robust, conventional statistical method (two-way anova) was employed to select the genes significantly affected. An orderly pattern of gene responses was clearly detected, with genes being up- or down-regulated at specific timings consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight the importance of IL-6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study.

  15. Behavioral phenotyping of calcium channel (CACN) subunit α2δ3 knockout mice: Consequences of sensory cross-modal activation.

    Science.gov (United States)

    Landmann, Julia; Richter, Franziska; Classen, Joseph; Richter, Angelika; Penninger, Josef M; Bechmann, Ingo

    2018-01-02

    Sensory cross-activation is still ill-defined and research concerning the consequences of sensory mergence on normal brain function is very limited. Human studies describe behavioral benefits of people with synesthesia- a peculiar form of perception possibly due to cross-modal activation- regarding sensory and memory abilities. Here, we studied behavioral alterations in calcium channel (CACN) subunit α2δ3 knockout (KO) mice exhibiting pain-induced cortical cross-modal activation. Knockout mice exhibited an increased response upon touch of a pinna and impaired audition, while elementary olfaction, vision, somatosensation and motor function were not altered. In contrast to synesthetic humans for whom enhanced memory function had been described, α2δ3 KO mice might have developed defects for object-based memory. However, in a task requiring use of multiple modalities, mutant mice revealed an enhanced performance compared to wild-type controls. Furthermore, several tests revealed evidence for increased anxiety-like behavior of α2δ3 KO animals. In summary, deficits in single sensory abilities and a potential gain in processing simultaneous sensory information in α2δ3 KO mice might represent behavioral correlates of sensory cross-activation. Further, our data suggest a role of CACNα2δ3 within the functionality of the sensory system, but not the motor system and general health. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice.

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    Jeanne Farrell

    Full Text Available BACKGROUND: Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy(tm1Lex/Sacy(tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference. PRINCIPAL FINDINGS: We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which 'escapes' the design of the Sacy(tm1Lex knockout allele. CONCLUSIONS/SIGNIFICANCE: These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.

  17. Ammonium acrylate biomanufacturing by an engineered Rhodococcus ruber with nitrilase overexpression and double-knockout of nitrile hydratase and amidase.

    Science.gov (United States)

    Sun, Jizhe; Yu, Huimin; Chen, Jie; Luo, Hui; Shen, Zhongyao

    2016-12-01

    Rhodococcus ruber TH was selected as a parent strain to engineer for biomanufacturing of ammonium acrylate; the characteristics of this strain included accelerated growth rate, high cell tolerance and natively overexpressed nitrile hydratase (NHase). Transcriptome analysis revealed that the transcription levels of the native NHase, amidase and nitrilase were extremely high, moderate and extremely low, respectively. Through NHase-amidase double-knockout and amidase single-knockout, the engineered strains R. ruber THdAdN and R. ruber THdA were obtained for overexpression of a heterologous nitrilase from R. rhodochrous tg1-A6 using a urea-induced Pa2 promoter. The nitrilase activity toward substrate acrylonitrile in the engineered THdAdN(Nit) reached 187.0 U/mL at 42 h, threefold of that R. rhodochrous tg1-A6 and 2.3-fold of that of THdA(Nit). The optimal catalysis temperature and pH of the nitrilases in different cells exhibited no significant difference. Using the cells as catalysts, biomanufacturing of ammonium acrylate was performed under room temperature. When catalyzed by the engineered THdAdN(Nit), the titer and productivity of ammonium acrylate dramatically increased to 741.0 g/L and 344.9 g/L/h, which are the highest results reported to date.

  18. Skeletal Muscle Fibre-Specific Knockout of p53 Does Not Reduce Mitochondrial Content or Enzyme Activity

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    Ben Stocks

    2017-12-01

    Full Text Available Tumour protein 53 (p53 has been implicated in the regulation of mitochondrial biogenesis in skeletal muscle, with whole-body p53 knockout mice displaying impairments in basal mitochondrial content, respiratory capacity, and enzyme activity. This study aimed to determine the effect of skeletal muscle-specific loss of p53 on mitochondrial content and enzyme activity. Mitochondrial protein content, enzyme activity and mRNA profiles were assessed in skeletal muscle of 8-week-old male muscle fibre-specific p53 knockout mice (p53 mKO and floxed littermate controls (WT under basal conditions. p53 mKO and WT mice displayed similar content of electron transport chain proteins I-V and citrate synthase enzyme activity in skeletal muscle. In addition, the content of proteins regulating mitochondrial morphology (MFN2, mitofillin, OPA1, DRP1, FIS1, fatty acid metabolism (β-HAD, ACADM, ACADL, ACADVL, carbohydrate metabolism (HKII, PDH, energy sensing (AMPKα2, AMPKβ2, and gene transcription (NRF1, PGC-1α, and TFAM were comparable in p53 mKO and WT mice (p > 0.05. Furthermore, p53 mKO mice exhibited normal mRNA profiles of targeted mitochondrial, metabolic and transcriptional proteins (p > 0.05. Thus, it appears that p53 expression in skeletal muscle fibres is not required to develop or maintain mitochondrial protein content or enzyme function in skeletal muscle under basal conditions.

  19. Shorter duration of non-rapid eye movement sleep slow waves in EphA4 knockout mice.

    Science.gov (United States)

    Freyburger, Marlène; Poirier, Gaétan; Carrier, Julie; Mongrain, Valérie

    2017-10-01

    Slow waves occurring during non-rapid eye movement sleep have been associated with neurobehavioural performance and memory. In addition, the duration of previous wakefulness and sleep impacts characteristics of these slow waves. However, molecular mechanisms regulating the dynamics of slow-wave characteristics remain poorly understood. The EphA4 receptor regulates glutamatergic transmission and synaptic plasticity, which have both been linked to sleep slow waves. To investigate if EphA4 regulates slow-wave characteristics during non-rapid eye movement sleep, we compared individual parameters of slow waves between EphA4 knockout mice and wild-type littermates under baseline conditions and after a 6-h sleep deprivation. We observed that, compared with wild-type mice, knockout mice display a shorter duration of positive and negative phases of slow waves under baseline conditions and after sleep deprivation. However, the mutation did not change slow-wave density, amplitude and slope, and did not affect the sleep deprivation-dependent changes in slow-wave characteristics, suggesting that EphA4 is not involved in the response to elevated sleep pressure. Our present findings suggest a role for EphA4 in shaping cortical oscillations during sleep that is independent from sleep need. © 2017 European Sleep Research Society.

  20. Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance.

    Science.gov (United States)

    Tanahashi, Hiroshi; Tian, Qing-Bao; Hara, Yoshinobu; Sakagami, Hiroyuki; Endo, Shogo; Suzuki, Tatsuo

    2016-02-05

    Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.

  1. A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development.

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    Christine P Diggle

    Full Text Available Tubulin alpha 8 (Tuba8 is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

  2. Behavioral analysis of NR2C knockout mouse reveals deficit in acquisition of conditioned fear and working memory.

    Science.gov (United States)

    Hillman, Brandon G; Gupta, Subhash C; Stairs, Dustin J; Buonanno, Andres; Dravid, Shashank M

    2011-05-01

    N-methyl-D-aspartate (NMDA) receptors play an important role in excitatory neurotransmission and mediate synaptic plasticity associated with learning and memory. NMDA receptors are composed of two NR1 and two NR2 subunits and the identity of the NR2 subunit confers unique electrophysiologic and pharmacologic properties to the receptor. The precise role of NR2C-containing receptors in vivo is poorly understood. We have performed a battery of behavioral tests on NR2C knockout/nβ-galactosidase knock-in mice and found no difference in spontaneous activity, basal anxiety, forced-swim immobility, novel object recognition, pain sensitivity and reference memory in comparison to wildtype counterparts. However, NR2C knockout mice were found to exhibit deficits in fear acquisition and working memory compared to wildtype mice. Deficit in fear acquisition correlated with lack of fear conditioning-induced plasticity at the thalamo-amygdala synapse. These findings suggest a unique role of NR2C-containing receptors in associative and executive learning representing a novel therapeutic target for deficits in cognition. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus

    DEFF Research Database (Denmark)

    Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte

    2015-01-01

    Background: Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. Methods: In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail...... to the forced swim test, as measured by Western-blot analysis. Results: In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB...... mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn2+-sensing receptor in the pathophysiology of depression with component of anxiety....

  4. Proteomic analysis of tissue from α1,3-galactosyltransferase knockout mice reveals that a wide variety of proteins and protein fragments change expression level.

    Directory of Open Access Journals (Sweden)

    Louise Thorlacius-Ussing

    Full Text Available A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient's blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes.

  5. One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

    Science.gov (United States)

    Zuo, Erwei; Cai, Yi-Jun; Li, Kui; Wei, Yu; Wang, Bang-An; Sun, Yidi; Liu, Zhen; Liu, Jiwei; Hu, Xinde; Wei, Wei; Huo, Xiaona; Shi, Linyu; Tang, Cheng; Liang, Dan; Wang, Yan; Nie, Yan-Hong; Zhang, Chen-Chen; Yao, Xuan; Wang, Xing; Zhou, Changyang; Ying, Wenqin; Wang, Qifang; Chen, Ren-Chao; Shen, Qi; Xu, Guo-Liang; Li, Jinsong; Sun, Qiang; Xiong, Zhi-Qi; Yang, Hui

    2017-01-01

    The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models. PMID:28585534

  6. Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse.

    Science.gov (United States)

    Mohr, Franziska; Krejci, Eric; Zimmermann, Martina; Klein, Jochen

    2015-01-01

    The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.

  7. Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements

    Directory of Open Access Journals (Sweden)

    Sanders Anne M

    2010-03-01

    Full Text Available Abstract Background Spontaneous Regression/Complete Resistant (SR/CR mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-, superoxide (Cybb-/, or inducible nitric oxide (Nos2-/. Methods SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180. Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. Results When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Conclusion Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required

  8. Cannabinoid 1 receptor knockout mice display cold allodynia, but enhanced recovery from spared-nerve injury-induced mechanical hypersensitivity.

    Science.gov (United States)

    Sideris, Alexandra; Piskoun, Boris; Russo, Lori; Norcini, Monica; Blanck, Thomas; Recio-Pinto, Esperanza

    2016-01-01

    The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. Cold allodynia and

  9. Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins

    NARCIS (Netherlands)

    Kaba, S.A.; Salcedo, A.M.; Wafula, P.O.; Vlak, J.M.; Oers, van M.M.

    2004-01-01

    The application of the baculovirus-in sect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67.

  10. Double knockout of Bax and Bak from kidney proximal tubules reduces unilateral urethral obstruction associated apoptosis and renal interstitial fibrosis.

    Science.gov (United States)

    Mei, Shuqin; Li, Lin; Wei, Qingqing; Hao, Jielu; Su, Yunchao; Mei, Changlin; Dong, Zheng

    2017-03-20

    Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases.

  11. Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in Escherichia coli

    DEFF Research Database (Denmark)

    Long, Christopher P.; Gonzalez, Jacqueline E.; Feist, Adam M.

    2018-01-01

    robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and highresolution C-13-metabolic flux analysis were performed on 10 adaptively evolved pgi knockouts of Escherichia coli. Pgi catalyzes the first reaction in glycolysis, and its loss results in major......Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic......, which corresponded to elevated flux from pyruvate to phosphoenolpyruvate. The overall energy metabolism was found to be strikingly robust, and what have been previously described as latently activated Entner-Doudoroff and glyoxylate shunt pathways are shown here to represent no real increases...

  12. Perturbed Hippocampal Synaptic Inhibition and γ-Oscillations in a Neuroligin-4 Knockout Mouse Model of Autism

    Directory of Open Access Journals (Sweden)

    Matthieu Hammer

    2015-10-01

    Full Text Available Loss-of-function mutations in the synaptic adhesion protein Neuroligin-4 are among the most common genetic abnormalities associated with autism spectrum disorders, but little is known about the function of Neuroligin-4 and the consequences of its loss. We assessed synaptic and network characteristics in Neuroligin-4 knockout mice, focusing on the hippocampus as a model brain region with a critical role in cognition and memory, and found that Neuroligin-4 deletion causes subtle defects of the protein composition and function of GABAergic synapses in the hippocampal CA3 region. Interestingly, these subtle synaptic changes are accompanied by pronounced perturbations of γ-oscillatory network activity, which has been implicated in cognitive function and is altered in multiple psychiatric and neurodevelopmental disorders. Our data provide important insights into the mechanisms by which Neuroligin-4-dependent GABAergic synapses may contribute to autism phenotypes and indicate new strategies for therapeutic approaches.

  13. Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

    DEFF Research Database (Denmark)

    Poulsen, Christian Bjørn; Penkowa, Milena; Borup, Rehannah

    2005-01-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays...... in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight...... the importance of IL-6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study....

  14. Lessons from Hepatocyte-Specific Cyp51 Knockout Mice: Impaired Cholesterol Synthesis Leads to Oval Cell-Driven Liver Injury

    Science.gov (United States)

    Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Gutierrez-Mariscal, Francisco M.; Lewinska, Monika; Gebhardt, Rolf; Keber, Rok; Horvat, Simon; Björkhem, Ingemar; Rozman, Damjana

    2015-03-01

    We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies.

  15. Increased amphetamine-induced locomotor activity, sensitization, and accumbal dopamine release in M5 muscarinic receptor knockout mice

    DEFF Research Database (Denmark)

    Schmidt, Lene S; Miller, Anthony D; Lester, Deranda B

    2010-01-01

    showed that M(5) receptor knockout (M (5) (-/-) ) mice are less sensitive to the reinforcing properties of addictive drugs. MATERIALS AND METHODS: Here, we investigate the role of M(5) receptors in the effects of amphetamine and cocaine on locomotor activity, locomotor sensitization, and dopamine release...... and locomotor sensitization were enhanced in M (5) (-/-) mice, while the effects of cocaine were similar in M (5) (-/-) and wild-type mice. RESULTS: Consistent with the behavioral results, amphetamine-, but not cocaine, -elicited dopamine release in nucleus accumbens was enhanced in M (5) (-/-) mice. DISCUSSION......: The different effects of amphetamine and cocaine in M (5) (-/-) mice may be due to the divergent pharmacological profile of the two drugs, where amphetamine, but not cocaine, is able to release intracellular stores of dopamine. In conclusion, we show here for the first time that amphetamine...

  16. Galactonojirimycin derivatives restore mutant human beta-galactosidase activities expressed in fibroblasts from enzyme-deficient knockout mouse.

    Science.gov (United States)

    Tominaga, L; Ogawa, Y; Taniguchi, M; Ohno, K; Matsuda, J; Oshima, A; Suzuki, Y; Nanba, E

    2001-08-01

    Ten low molecular compounds analogous to galactose were screened for inhibition of human beta-galactosidase activity. Among them, 1-deoxy-galactonojirimycin and N-(n-butyl)-deoxy-galactonojirimycin showed an inhibitory effect at high concentrations. However, they restored mutant enzyme activities expressed in enzyme-deficient knockout mouse fibroblasts and human beta-galactosidosis fibroblasts at lower intracellular concentrations. This effect was more remarkable on G(M1)-gangliosidosis mutations (R201C, I51T, R201H, R457Q) than Morquio B disease mutations (W273L, Y83H). These low molecular compounds pass though the blood-brain barrier in mice. We hope that this new therapeutic approach will become clinically applicable in the near future.

  17. Minimal Effects of Age and Exposure to a Noisy Environment on Hearing in Alpha9 Nicotinic Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Amanda M. Lauer

    2017-06-01

    Full Text Available Studies have suggested a role of weakened medial olivocochlear (OC efferent feedback in accelerated hearing loss and increased susceptibility to noise. The present study investigated the progression of hearing loss with age and exposure to a noisy environment in medial OC-deficient mice. Alpha9 nicotinic acetylcholine receptor knockout (α9KO and wild types were screened for hearing loss using auditory brainstem responses. α9KO mice housed in a quiet environment did not show increased hearing loss compared to wild types in young adulthood and middle age. Challenging the medial OC system by housing in a noisy environment did not increase hearing loss in α9KO mice compared to wild types. ABR wave 1 amplitudes also did not show differences between α9KO mice and wild types. These data suggest that deficient medial OC feedback does not result in early onset of hearing loss.

  18. Toward a molecular understanding of psychostimulant actions using genetically engineered dopamine receptor knockout mice as model systems.

    Science.gov (United States)

    Zhang, J; Xu, M

    2001-01-01

    A major focus in studying the progression and prevention of addictive diseases has been to understand the molecular and cellular mechanisms underlying drug addiction. The brain dopaminergic system plays a central role in reward and motivation and is thought to be the main neural substrate for the actions of abusive drugs. We have used the gene targeting technology to generate dopamine D1 and D3 receptor knockout mice and used these mice as model systems to gain a molecular understanding of acute effects of psychostimulants cocaine and amphetamine. The use of a combined approach involving behavioral, electrophysiological as well as molecular studies has allowed us to define initially the roles of dopamine D1 and D3 receptors in the acute effects of psychostimulants and will enable us to understand mechanisms underlying their chronic actions in the future.

  19. A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting

    Directory of Open Access Journals (Sweden)

    So-Jung Kim

    2017-03-01

    Full Text Available Kelch-like ECH-associated protein 1 (keap1 is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a. Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC. The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential.

  20. Smooth muscle cells healing atherosclerotic plaque disruptions are of local, not blood, origin in apolipoprotein E knockout mice

    DEFF Research Database (Denmark)

    Bentzon, Jacob F.; Sondergaard, Claus S.; Kassem, Moustapha

    2007-01-01

    GFP+ SMCs were detected. To examine the origin of healing SMCs in a model that recapitulates more features of human plaque rupture and healing, we developed a mechanical technique that produced consistent plaque disruption, superimposed thrombosis, and SMC-mediated plaque healing in apoE-/- mice. Mechanical......BACKGROUND: Signs of preceding episodes of plaque rupture and smooth muscle cell (SMC)-mediated healing are common in atherosclerotic plaques, but the source of the healing SMCs is unknown. Recent studies suggest that activated platelets adhering to sites of injury recruit neointimal SMCs from...... circulating bone marrow-derived progenitor cells. Here, we analyzed the contribution of this mechanism to plaque healing after spontaneous and mechanical plaque disruption in apolipoprotein E knockout (apoE-/-) mice. METHODS AND RESULTS: To determine the origin of SMCs after spontaneous plaque disruption...

  1. Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice.

    Science.gov (United States)

    Irimia, Jose M; Tagliabracci, Vincent S; Meyer, Catalina M; Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

    2015-09-11

    Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Muscle Glycogen Remodeling and Glycogen Phosphate Metabolism following Exhaustive Exercise of Wild Type and Laforin Knockout Mice*

    Science.gov (United States)

    Irimia, Jose M.; Tagliabracci, Vincent S.; Meyer, Catalina M.; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.

    2015-01-01

    Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease. PMID:26216881

  3. Orp8 deficiency in bone marrow-derived cells reduces atherosclerotic lesion progression in LDL receptor knockout mice.

    Directory of Open Access Journals (Sweden)

    Erik van Kampen

    Full Text Available INTRODUCTION: Oxysterol binding protein Related Proteins (ORPs mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown. METHODS AND RESULTS: LDL receptor knockout (KO mice were transplanted with bone marrow (BM from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity. CONCLUSIONS: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.

  4. Adipocyte SIRT1 knockout promotes PPARγ activity, adipogenesis and insulin sensitivity in chronic-HFD and obesity.

    Science.gov (United States)

    Mayoral, Rafael; Osborn, Olivia; McNelis, Joanne; Johnson, Andrew M; Oh, Da Young; Izquierdo, Cristina Llorente; Chung, Heekyung; Li, Pingping; Traves, Paqui G; Bandyopadhyay, Gautam; Pessentheiner, Ariane R; Ofrecio, Jachelle M; Cook, Joshua R; Qiang, Li; Accili, Domenico; Olefsky, Jerrold M

    2015-05-01

    Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ). To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1 (flx/flx) aP2Cre (+) (ATKO) and Sirt1 (flx/flx) aP2Cre (-) (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue. On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity. Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.

  5. Comparative functional genomics analysis of NNK tobacco-carcinogen induced lung adenocarcinoma development in Gprc5a-knockout mice.

    Directory of Open Access Journals (Sweden)

    Junya Fujimoto

    2010-07-01

    Full Text Available Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. Previously, we found that mice with a knockout (KO of G-protein coupled receptor 5A (Gprc5a develop lung tumors after a long latent period (12 to 24 months.To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n=5 of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A gene expression signature, NNK-ADC, of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively.Our findings demonstrate that lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK and that gene expression changes induced by tobacco carcinogen(s may be conserved between mouse and human lung epithelial cells. Further experimentation to prove the reliability of the Gprc5a knockout mouse model for the study of tobacco-induced lung carcinogenesis is warranted.

  6. Novel therapeutic targets in osteoarthritis: Narrative review on knock-out genes involved in disease development in mouse animal models.

    Science.gov (United States)

    Veronesi, Francesca; Della Bella, Elena; Cepollaro, Simona; Brogini, Silvia; Martini, Lucia; Fini, Milena

    2016-05-01

    Osteoarthritis (OA) can affect every joint, especially the knee. Given the complexity of this pathology, OA is difficult to treat with current therapies, which only relieve pain and inflammation and are not capable of restoring tissues once OA has started. Currently, researchers focus on finding a therapeutic strategy that may help to arrest disease progression. The present narrative review gives an overview of the genes involved in the development and progression of OA, assessing in vivo studies performed in knock-out mice affected by OA, to suggest new therapeutic strategies. The article search was performed on the PubMed database and www.webofknowledge.com website with the following keywords: "knee osteoarthritis" AND "knockout mice". The included studies were in English and published from 2005 to 2015. Additional papers were found within the references of the selected articles. In the 55 analyzed in vivo studies, genes mainly affected chondrocyte homeostasis, inflammatory processes, extracellular matrix and the relationship between obesity and OA. Genes are defined as inducing, preventing and not influencing OA. This review shows that joint homeostasis depends on a variety of genetic factors, and preventing or restoring the loss of a gene encoding for protective proteins, or inhibiting the expression of proteins that induce OA, might be a potential therapeutic approach. However, conclusions cannot be drawn because of the wide variability concerning the technique used for OA induction, the role of the genes, the method for tissue evaluations and the lack of assessments of all joint tissues. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias.

    Science.gov (United States)

    Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

    2013-05-01

    PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ER(T) under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors.

  8. Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.

    Science.gov (United States)

    Teng, Bunyen; Ansari, Habib R; Oldenburg, Peter J; Schnermann, J; Mustafa, S Jamal

    2006-04-01

    Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.

  9. STARS knockout attenuates hypoxia-induced pulmonary arterial hypertension by suppressing pulmonary arterial smooth muscle cell proliferation.

    Science.gov (United States)

    Shi, Zhaoling; Wu, Huajie; Luo, Jianfeng; Sun, Xin

    2017-03-01

    STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein, which expressed early in cardiac development and involved in pathological remodeling. Abundant evidence indicated that STARS could regulate cell proliferation, but it's exact function remains unclear. In this study, we aimed to investigate the role of STARS in the proliferation of pulmonary arterial smooth muscle cells (PASMC) and the potential effect on the progression of pulmonary arterial hypertension (PAH). In this study, we established a PAH mouse model through chronic hypoxia exposure as reflected by the increased RVSP and RVHI. Western blot and RT-qPCR detected the increased STARS protein and mRNA levels in PAH mice. Next, we cultured the primary PASMC from PAH mice. After STARS overexpression in PASMC, STARS, SRF and Egr-1 were up-regulated significantly. The MTT assay revealed an increase in cell proliferation. Flow cytometry showed a marked inhibition of cell apoptosis. However, STARS silence in PASMC exerted opposite effects with STARS overexpression. SRF siRNA transfection blocked the effects of STARS overexpression in PASMC. In order to further confirm the role of STARS in PAH mice in vivo, we exposed STARS knockout mice to hypoxia and found lower RVSP and RVHI in knockout mice as compared with controls. Our results not only suggest that STARS plays a crucial role in the development of PAH by increasing the proliferation of PASMC through activation of the SRF/Egr-1 pathway, but also provides a new mechanism for hypoxia-induced PAH. In addition, STARS may represent a potential treatment target. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  10. Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice

    Directory of Open Access Journals (Sweden)

    Tao Yuan-Xiang

    2007-10-01

    Full Text Available Abstract Neuronal nitric oxide synthase (nNOS is a key enzyme for nitric oxide production in neuronal tissues and contributes to the spinal central sensitization in inflammatory pain. However, the role of nNOS in neuropathic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to examine the effects of genetic knockout and pharmacologic inhibition of nNOS on neuropathic pain induced by unilateral fifth lumbar spinal nerve injury in mice. In contrast to wildtype mice, nNOS knockout mice failed to display nerve injury-induced mechanical hypersensitivity. Furthermore, either intraperitoneal (100 mg/kg or intrathecal (30 μg/5 μl administration of L-NG-nitro-arginine methyl ester, a nonspecific NOS inhibitor, significantly reversed nerve injury-induced mechanical hypersensitivity on day 7 post-nerve injury in wildtype mice. Intrathecal injection of 7-nitroindazole (8.15 μg/5 μl, a selective nNOS inhibitor, also dramatically attenuated nerve injury-induced mechanical hypersensitivity. Western blot analysis showed that the expression of nNOS protein was significantly increased in ipsilateral L5 dorsal root ganglion but not in ipsilateral L5 lumbar spinal cord on day 7 post-nerve injury. The expression of inducible NOS and endothelial NOS proteins was not markedly altered after nerve injury in either the dorsal root ganglion or spinal cord. Our findings suggest that nNOS, especially in the dorsal root ganglion, may participate in the development and/or maintenance of mechanical hypersensitivity after nerve injury.

  11. Effect of Cyp27A1 gene dosage on atherosclerosis development in ApoE-knockout mice.

    Science.gov (United States)

    Zurkinden, Line; Solcà, Curzio; Vögeli, Isabelle A; Vogt, Bruno; Ackermann, Daniel; Erickson, Sandra K; Frey, Felix J; Sviridov, Dmitri; Escher, Geneviève

    2014-03-01

    In humans, sterol 27-hydroxylase (CYP27A1) deficiency leads to cholesterol deposition in tendons and vasculature. Thus, in addition to its role in bile acid synthesis, where it converts cholesterol to 27-hydroxycholesterol (27-OHC), CYP27A1 may also be atheroprotective. Cyp27A1-deficient (Cyp27A1(-/-)) mice were crossed with apolipoprotein E (apoE)-deficient mice. Cyp27A1(+/+)/apoE(-/-) [ApoE-knockout (KO)], Cyp27A1(+/-)/apoE(-/-) heterozygous (het), and Cyp27A1(-/-)/apoE(-/-) [double-knockout (DKO)] mice were challenged with a Western diet (WD) for 3 and 6 mo. ApoE-KO mice fed a chow diet or a WD were used as the control. The severity of atherosclerosis in DKO mice was reduced 10-fold. Compared with the control, the DKO mice had no 27-OHC, total plasma cholesterol and low-density lipoprotein and very low density lipoprotein (LDL/VLDL) concentrations were reduced 2-fold, and HDL was elevated 2-fold. Expression of hepatic CYP7A1, CYP3A, and CYP8B1 were 5- to 10-fold higher. 3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) activity increased 4-fold. Fecal cholesterol was increased. In contrast, het mice fed a WD developed accelerated atherosclerosis and severe skin lesions, possibly because of reduced reverse cholesterol transport due to diminished 27-OHC production. CYP27A1 activity is involved in the control of cholesterol homeostasis and development of atherosclerosis with a distinct gene dose-dependent effect.

  12. Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after amino acid feeding.

    NARCIS (Netherlands)

    Rojek, A.; Fuchtbauer, E.M.; Fuchtbauer, A.; Jelen, S.K.; Malmendal, A.; Fenton, R.A.; Nielsen, S.

    2013-01-01

    Aquaporin 11 (AQP11) is a protein channel expressed intracellularly in multiple organs, yet its physiological function is unclear. Aqp11 knockout (KO) mice die early due to malfunction of the kidney, a result of hydropic degeneration of proximal tubule cells. Here we report the generation of

  13. Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice

    NARCIS (Netherlands)

    Jong, M.C.; Rensen, P.C.N.; Dahlmans, V.E.H.; Boom, H. van der; Berkel, T.J.C. van; Havekes, L.M.

    2001-01-01

    Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3-/-) mice

  14. Protease activity of legumain is inhibited by an increase of cystatin E/M in the DJ-1-knockout mouse spleen, cerebrum and heart

    Directory of Open Access Journals (Sweden)

    Takuya Yamane

    2017-03-01

    Full Text Available Legumain (EC 3.4.22.34 is an asparaginyl endopeptidase. Legumain activity has been detected in various mouse tissues including the kidney, spleen and epididymis. Legumain is overexpressed in the majority of human solid tumors and transcription of the legumain gene is regulated by the p53 tumor suppressor in HCT116 cells. The legumain activity is also increased under acid conditions in Alzheimer's disease brains. DJ-1/PARK7, a cancer- and Parkinson's disease-associated protein, works as a coactivator to various transcription factors, including the androgen receptor, p53, PSF, Nrf2, SREBP and RREB1. Recently, we found that legumain expression, activation and cleavage of annexin A2 are regulated by DJ-1 through p53. In this study, we found that the expression levels of legumain mRNA were increased in the cerebrum, kidney, spleen, heart, lung, epididymis, stomach, small intestine and pancreas from DJ-1-knockout mice, although legumain activity levels were decreased in the cerebrum, spleen and heart from DJ-1-knockout mice. Furthermore, we found that cystatin E/M expression was increased in the spleen, cerebrum and heart from DJ-1-knockout mice. These results suggest that reduction of legumain activity is caused by an increase of cystatin E/M expression in the spleen, cerebrum and heart from DJ-1-knockout mice.

  15. In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

    DEFF Research Database (Denmark)

    Holmgaard, Andreas; Askou, Anne Louise; Benckendorff, Josephine Natalia Esther

    2017-01-01

    Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp) Cas9, delivered by lentiviral vectors (LVs), can be used...

  16. The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yanyan [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zheng, Hongzhi [The First Affiliated Hospital, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States); Fu, Jingqi; Hou, Yongyong; Wang, Huihui [School of Public Health, China Medical University, Shenyang, Liaoning (China); Zhang, Qiang [Rollins School of Public Health, Emory University, Atlanta, GA (United States); Yamamoto, Masayuki [Graduate School of Medicine, Tohoku University, Sendai (Japan); Pi, Jingbo, E-mail: jbpi@cmu.edu.cn [School of Public Health, China Medical University, Shenyang, Liaoning (China); The Hamner Institutes for Health Sciences, Research Triangle Park, NC (United States)

    2016-09-09

    Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

  17. The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

    International Nuclear Information System (INIS)

    Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

    2016-01-01

    Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.

  18. Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development

    NARCIS (Netherlands)

    T. Baardman (Taco); M.V. Zwier (Mathijs V.); L.J. Wisse (Lambertus); A.C. Gittenberger-De Groot (Adriana); W.S. Kerstjens-Frederikse (Wilhelmina); R.M.W. Hofstra (Robert); A. Jurdzinski (Angelika); B.P. Hierck (Beerend); M.R.M. Jongbloed (Monique); R.M.F. Berger (Rolf); T. Plösch (Torsten); M.C. DeRuiter (Marco)

    2016-01-01

    textabstractLipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the

  19. Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development

    NARCIS (Netherlands)

    Baardman, Maria E.; Zwier, Mathijs V.; Wisse, Lambertus J.; Gittenberger-de Groot, Adriana C.; Kerstjens-Frederikse, Wilhelmina S.; Hofstra, Robert M. W.; Jurdzinski, Angelika; Hierck, Beerend P.; Jongbloed, Monique R. M.; Berger, Rolf M. F.; Plosch, Torsten; DeRuiter, Marco C.

    2016-01-01

    Lipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the

  20. Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice

    NARCIS (Netherlands)

    Kortekaas, Jeroen; Vloet, Rianka P M; McAuley, Alexander J; Shen, Xiaoli; Bosch, Berend Jan; de Vries, Laura; Moormann, Rob J M; Bente, Dennis A

    2015-01-01

    Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout

  1. RETINOIC ACID INDUCTION OF CLEFT PALATE IN EGF AND TGF-ALPHA KNOCKOUT MICE: STAGE SPECIFIC INFLUENCES OF GROWTH FACTOR EXPRESSION

    Science.gov (United States)

    ABBOTT, B. D., LEFFLER, K.E. AND BUCKALEW, A.R, Reproductive Toxicology Division, NHEERL, ORD, US EPA, Research Triangle Park, North Carolina. Retinoic acid induction of cleft palate (CP) in EGF and TGF knockout mice: Stage specific influences of growth factor expression.<...

  2. Role of interferon-gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-gamma knock-out mice

    DEFF Research Database (Denmark)

    Nansen, A; Christensen, Jan Pravsgaard; Röpke, C

    1998-01-01

    , a viral peptide could also elicit a T cell mediated inflammatory response in virus-primed IFN-gamma knock-out mice, indicating that redundancy of this cytokine as a proinflammatory mediator is not restricted to inflammatory reactions triggered by an active infection. Thus, T cell mediated inflammation may...

  3. Sarcocystis pantherophis, n. sp. from eastern rat snakes (Pantherophis alleghaniensis) definitive hosts and interferongamma gene knockout mice as experimental intermediate hosts

    Science.gov (United States)

    Here we report a new species, Sarcocystis pantherophisi with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n=15) from intestinal contents of the snake were 17.3 x 10....

  4. Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor in experimental autoimmune encephalomyelitis models of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Sofia Sisay

    Full Text Available Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1 receptor and the orphan G protein receptor fifty-five (GPR55. Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational

  5. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons.

    Science.gov (United States)

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British

  6. Gene knockouts, in vivo site-directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae.

    Science.gov (United States)

    Stuckey, Samantha; Storici, Francesca

    2013-01-01

    Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene mutations, or the generation of random mutations in a gene are all essential processes for gene manipulation. The genome of the yeast Saccharomyces cerevisiae is relatively easy to modify, owing to its efficient homologous recombination (HR) system. Gene knockout can be a very simple, one-step approach to eliminate a gene by substituting its DNA sequence with that of a genetic marker. Differently, desired mutations can be introduced into a gene by replacing the sequence of the normal gene with that of the mutated gene. Recombinant DNA can be created in vitro and then introduced into cells, most often exploiting the endogenous recombination system of the cells. However, unless the desired mutation gives a particular phenotype, a bottleneck of 'recombineering' is the requirement of a selection system to identify the recombinant clones among those unmodified. Even in an organism like yeast where the level of HR is highly above the incidence of random integration, the frequency of homologous targeting is in the range of 10(-4)-10(-6) depending on the length of the homology used (Wach et al., 1994). Thus, a selection system is always required to identify the targeted clones. Counterselectable markers, such as URA3, LYS2, LYS5, MET15, and TRP1 (Bach and LaCroute, 1972; Chattoo et al., 1979; Singh and Sherman, 1974; Toyn et al., 2000), are widely utilized in yeast and can be recycled for additional usage in the same yeast strain. If the marker is not eliminated or it is popped out via site-specific recombination between direct repeats, such as in the Flp/FRT or Cre/Lox systems, a heterologous sequence is left as a scar at the site of the modified DNA

  7. Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yuehai [Cardiovascular Department, Liaocheng People’s Hospital of Shandong University, Liaocheng, Shandong 252000 (China); Cardiovascular Department, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China); Lu, Huixia [The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong 250012 (China); Huang, Ziyang, E-mail: huangziyang666@126.com [Cardiovascular Department, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China); Lin, Huili [Cardiovascular Department, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China); Lei, Zhenmin [Department of OB/GYN, University of Louisville School of Medicine, Louisville, KY 40292 (United States); Chen, Xiaoqing [Department of Rheumatism and Immunology, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China); Tang, Mengxiong; Gao, Fei; Dong, Mei [The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong 250012 (China); Li, Rongda [Department of Rheumatism and Immunology, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China); Lin, Ling, E-mail: qzlinl@163.com [Department of Rheumatism and Immunology, The Second Clinical Medical College of Fujian Medical University, Quanzhou, Fujian 362000 (China)

    2014-07-18

    Highlights: • Titers of ANA and anti-dsDNA antibodies were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • The spleen weights and glomerular areas were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • Expressions of IgG and C3 in glomeruli were similar in ApoE{sup −/−} and Fas{sup −/−} mice. • IgG, C3 and macrophage infiltration in aortic plaques were found in ApoE{sup −/−} mice. - Abstract: Background: Apolipoprotein E-knockout (ApoE{sup −/−}) mice is a classic model of atherosclerosis. We have found that ApoE{sup −/−} mice showed splenomegaly, higher titers of serum anti-nuclear antibody (ANA) and anti-dsDNA antibody compared with C57B6/L (B6) mice. However, whether ApoE{sup −/−} mice show autoimmune injury remains unclear. Methods and results: Six females and six males in each group, ApoE{sup −/−}, Fas{sup −/−} and B6 mice, were used in this study. The titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein were measured by ELISA after 4 months of high-fat diet. The spleen weight and the glomerular area were determined. The expressions of IgG, C3 and macrophage in kidney and atherosclerotic plaque were detected by immunostaining followed by morphometric analysis. Similar to the characteristics of Fas{sup −/−} mice, a model of systemic lupus erythematosus (SLE), ApoE{sup −/−} mice, especially female, displayed significant increases of spleen weight and glomerular area when compared to B6 mice. Also, elevated titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein. Moreover, the expressions of IgG, C3 and macrophage in glomeruli and aortic plaques were found in ApoE{sup −/−} mice. In addition, the IgG and C3 expressions in glomeruli and plaques significantly increased (or a trend of increase) in female ApoE{sup −/−} mice compared with males. Conclusions: Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta.

  8. Tactile Defensiveness and Impaired Adaptation of Neuronal Activity in the Fmr1 Knock-Out Mouse Model of Autism.

    Science.gov (United States)

    He, Cynthia X; Cantu, Daniel A; Mantri, Shilpa S; Zeiger, William A; Goel, Anubhuti; Portera-Cailliau, Carlos

    2017-07-05

    Sensory hypersensitivity is a common symptom in autism spectrum disorders (ASDs), including fragile X syndrome (FXS), and frequently leads to tactile defensiveness. In mouse models of ASDs, there is mounting evidence of neuronal and circuit hyperexcitability in several brain regions, which could contribute to sensory hypersensitivity. However, it is not yet known whether or how sensory stimulation might trigger abnormal sensory processing at the circuit level or abnormal behavioral responses in ASD mouse models, especially during an early developmental time when experience-dependent plasticity shapes such circuits. Using a novel assay, we discovered exaggerated motor responses to whisker stimulation in young Fmr1 knock-out (KO) mice (postnatal days 14-16), a model of FXS. Adult Fmr1 KO mice actively avoided a stimulus that was innocuous to wild-type controls, a sign of tactile defensiveness. Using in vivo two-photon calcium imaging of layer 2/3 barrel cortex neurons expressing GCaMP6s, we found no differences between wild-type and Fmr1 KO mice in overall whisker-evoked activity, though 45% fewer neurons in young Fmr1 KO mice responded in a time-locked manner. Notably, we identified a pronounced deficit in neuronal adaptation to repetitive whisker stimulation in both young and adult Fmr1 KO mice. Thus, impaired adaptation in cortical sensory circuits is a potential cause of tactile defensiveness in autism. SIGNIFICANCE STATEMENT We use a novel paradigm of repetitive whisker stimulation and in vivo calcium imaging to assess tactile defensiveness and barrel cortex activity in young and adult Fmr1 knock-out mice, the mouse model of fragile X syndrome (FXS). We describe evidence of tactile defensiveness, as well as a lack of L2/3 neuronal adaptation in barrel cortex, during whisker stimulation. We propose that a defect in sensory adaptation within local neuronal networks, beginning at a young age and continuing into adulthood, likely contributes to sensory

  9. Light-induced fos expression in intrinsically photosensitive retinal ganglion cells in melanopsin knockout (opn4 mice.

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    Gary E Pickard

    Full Text Available Retinal ganglion cells that express the photopigment melanopsin are intrinsically photosensitive (ipRGCs and exhibit robust synaptically driven ON-responses to light, yet they will continue to depolarize in response to light when all synaptic input from rod and cone photoreceptors is removed. The light-evoked increase in firing of classical ganglion cells is determined by synaptic input from ON-bipolar cells in the proximal sublamina of the inner plexiform layer. OFF-bipolar cells synapse with ganglion cell dendrites in the distal sublamina of the inner plexiform layer. Of the several types of ipRGC that have been described, M1 ipRGCs send dendrites exclusively into the OFF region of the inner plexiform layer where they stratify near the border of the inner nuclear layer. We tested whether M1 ipRGCs with dendrites restricted to the OFF sublamina of the inner plexiform layer receive synaptic ON-bipolar input by examining light-induced gene expression in vivo using melanopsin knockout mice. Mice in which both copies of the melanopsin gene (opn4 have been replaced with the tau-lacZ gene (homozygous tau-lacZ(+/+ knockin mice are melanopsin knockouts (opn4(-/- but M1 ipRGCs are specifically identified by their expression of beta-galactosidase. Approximately 60% of M1 ipRGCs in Opn4(-/- mice exposed to 3 hrs of light expressed c-Fos; no beta-galactosidase-positive RGCs expressed c-Fos in the dark. Intraocular application of L-AP4, a compound which blocks transmission of visual signals between photoreceptors and ON-bipolar cells significantly reduced light-evoked c-Fos expression in M1 ipRGCs compared to saline injected eyes (66% saline vs 27% L-AP4. The results are the first description of a light-evoked response in an ipRGC lacking melanopsin and provide in vivo confirmation of previous in vitro observations illustrating an unusual circuit in the retina in which ganglion cells sending dendrites to the OFF sublamina of the inner plexiform layer

  10. Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity.

    Science.gov (United States)

    Yamaleyeva, Liliya M; Pulgar, Victor M; Lindsey, Sarah H; Yamane, Larissa; Varagic, Jasmina; McGee, Carolynne; daSilva, Mauro; Lopes Bonfa, Paula; Gurley, Susan B; Brosnihan, K Bridget

    2015-07-01

    Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype. Copyright © 2015 the American Physiological Society.

  11. Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin

    Science.gov (United States)

    Migliarini, Sara; Pacini, Giulia; Pasqualetti, Massimo

    2015-01-01

    Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84–178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice. Finally, we set up

  12. The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Natalia Madrid-López

    2017-10-01

    Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

  13. Selective Attention to Visual Stimuli Using Auditory Distractors Is Altered in Alpha-9 Nicotinic Receptor Subunit Knock-Out Mice.

    Science.gov (United States)

    Terreros, Gonzalo; Jorratt, Pascal; Aedo, Cristian; Elgoyhen, Ana Belén; Delano, Paul H

    2016-07-06

    During selective attention, subjects voluntarily focus their cognitive resources on a specific stimulus while ignoring others. Top-down filtering of peripheral sensory responses by higher structures of the brain has been proposed as one of the mechanisms responsible for selective attention. A prerequisite to accomplish top-down modulation of the activity of peripheral structures is the presence of corticofugal pathways. The mammalian auditory efferent system is a unique neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear bundle, and it has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear neurons in selective attention paradigms. Here, we trained wild-type and α-9 nicotinic receptor subunit knock-out (KO) mice, which lack cholinergic transmission between medial olivocochlear neurons and outer hair cells, in a two-choice visual discrimination task and studied the behavioral consequences of adding different types of auditory distractors. In addition, we evaluated the effects of contralateral noise on auditory nerve responses as a measure of the individual strength of the olivocochlear reflex. We demonstrate that KO mice have a reduced olivocochlear reflex strength and perform poorly in a visual selective attention paradigm. These results confirm that an intact medial olivocochlear transmission aids in ignoring auditory distraction during selective attention to visual stimuli. The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear system. It has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear

  14. Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin.

    Directory of Open Access Journals (Sweden)

    Barbara Pelosi

    Full Text Available Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84-178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice

  15. Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

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    Malur, Anagha; Huizar, Isham [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Wells, Greg [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Barna, Barbara P. [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Malur, Achut G. [Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States); Thomassen, Mary Jane, E-mail: thomassenm@ecu.edu [Program in Lung Cell Biology and Translational Research, Division of Pulmonary, Critical Care and Sleep Medicine, East Carolina University, Greenville, NC (United States); Department of Microbiology and Immunology, East Carolina University, Greenville, NC (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Lentivirus-ABCG1 reduces lipid accumulation in lungs of GM-CSF knock-out mice. Black-Right-Pointing-Pointer Up-regulation of ABCG1 improves lung function. Black-Right-Pointing-Pointer Upregulation of ABCG1 improves surfactant metabolism. -- Abstract: We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR{gamma}) and the PPAR{gamma}-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte-macrophage colony stimulating factor (GM-CSF), an upregulator of PPAR{gamma}. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPAR{gamma} plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10 days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p = 0.0005) increase in ABCG1 expression with no change of PPAR{gamma} or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by

  16. Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

    International Nuclear Information System (INIS)

    Wang, Yuehai; Lu, Huixia; Huang, Ziyang; Lin, Huili; Lei, Zhenmin; Chen, Xiaoqing; Tang, Mengxiong; Gao, Fei; Dong, Mei; Li, Rongda; Lin, Ling

    2014-01-01

    Highlights: • Titers of ANA and anti-dsDNA antibodies were similar in ApoE −/− and Fas −/− mice. • The spleen weights and glomerular areas were similar in ApoE −/− and Fas −/− mice. • Expressions of IgG and C3 in glomeruli were similar in ApoE −/− and Fas −/− mice. • IgG, C3 and macrophage infiltration in aortic plaques were found in ApoE −/− mice. - Abstract: Background: Apolipoprotein E-knockout (ApoE −/− ) mice is a classic model of atherosclerosis. We have found that ApoE −/− mice showed splenomegaly, higher titers of serum anti-nuclear antibody (ANA) and anti-dsDNA antibody compared with C57B6/L (B6) mice. However, whether ApoE −/− mice show autoimmune injury remains unclear. Methods and results: Six females and six males in each group, ApoE −/− , Fas −/− and B6 mice, were used in this study. The titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein were measured by ELISA after 4 months of high-fat diet. The spleen weight and the glomerular area were determined. The expressions of IgG, C3 and macrophage in kidney and atherosclerotic plaque were detected by immunostaining followed by morphometric analysis. Similar to the characteristics of Fas −/− mice, a model of systemic lupus erythematosus (SLE), ApoE −/− mice, especially female, displayed significant increases of spleen weight and glomerular area when compared to B6 mice. Also, elevated titers of serum ANA, anti-dsDNA antibody and creatinine and urine protein. Moreover, the expressions of IgG, C3 and macrophage in glomeruli and aortic plaques were found in ApoE −/− mice. In addition, the IgG and C3 expressions in glomeruli and plaques significantly increased (or a trend of increase) in female ApoE −/− mice compared with males. Conclusions: Apolipoprotein E-knockout mice on high-fat diet show autoimmune injury on kidney and aorta

  17. Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

    Science.gov (United States)

    Jagoda, E M; Kiesewetter, D O; Shimoji, K; Ravasi, L; Yamada, M; Gomeza, J; Wess, J; Eckelman, W C

    2003-04-01

    A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; Pcortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.

  18. Knockout of Amyloid β Protein Precursor (APP) Expression Alters Synaptogenesis, Neurite Branching and Axonal Morphology of Hippocampal Neurons.

    Science.gov (United States)

    Southam, Katherine A; Stennard, Fiona; Pavez, Cassandra; Small, David H

    2018-03-23

    The function of the β-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

  19. Participation of calbindin-D28K in nociception: results from calbindin-D28K knockout mice.

    Science.gov (United States)

    Egea, Javier; Malmierca, Eduardo; Rosa, Angelo O; del Barrio, Laura; Negredo, Pilar; Nuñez, Angel; López, Manuela G

    2012-03-01

    Since calbindin-D(28K) (CB-D(28K))-positive neurons have been related to nociceptive sensory processing, we have hypothesized that altered CB-D(28K) expression could alter nociceptive transmission. We have used +/+ and -/- knockout (KO) mice for CB-D(28k) in different behavioral models of pain and sensory responses at the caudalis subdivision of the trigeminal spinal nucleus in order to understand how this protein may participate in nociception. Behavioral responses to formalin injection in the hind paw or at the whisker pad or in the hind paw glutamate or i.p. acetic acid tests showed an increase of the pain threshold in CB-D(28k) -/- mice. KO mice showed a diminution of the inhibitory activity at Sp5C nucleus and a marked reduction of GABA content. Sp5C neurons from CB-D(28k) -/- mice did not change their spontaneous activity or tactile response after formalin injection in the whisker pad. In contrast, Sp5C neurons increased their spontaneous firing rate and tactile response after formalin injection in their receptive field in CB-D(28k) +/+ mice. The results of this study demonstrate the active role played by CB-D(28k) in nociceptive sensory transmission. The lack of this calcium binding protein, associated to deficient GABAergic neurotransmission, translates into dysfunction of sensory processing of nociceptive stimuli.

  20. Motor and memory testing of long-lived pregnancy-associated plasma protein--a knock-out mice.

    Science.gov (United States)

    Mason, Emily J; Grell, Jacquelyn A; West, Sally A; Conover, Cheryl A

    2014-12-01

    Mice deficient in pregnancy-associated plasma protein-A (PAPP-A), an IGF binding protein protease, have been shown to be resistant to experimentally induced atherosclerosis and diabetic nephropathy, and, in the laboratory environment, live 30-40% longer than wild-type littermates in association with delayed incidence and occurrence of age-related neoplasms and degenerative diseases. PAPP-A is highly expressed in the cerebellum and hippocampus of the mouse brain. Therefore, the studies presented here were aimed at determining motor behavior, learning and retention in PAPP-A knock-out (KO) mice compared to wild-type (WT) littermates with age. Balance and coordination were assessed using an accelerating rotarod; learning and memory were assessed in a Stone T-maze. Time on the rotarod decreased with age but there was no significant difference between PAPP-A KO and WT mice at any of the testing ages. Latency to reach the goal box and number of errors committed in the Stone T-maze did not change with age and there were no significant differences between PAPP-A KO and WT mice. Lack of PAPP-A in mice did not impact central regulation of coordination, learning or memory. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Weekly Treatment of 2-Hydroxypropyl-β-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice

    Directory of Open Access Journals (Sweden)

    Sofie M. A. Walenbergh

    2015-09-01

    Full Text Available Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD. Two-hydroxypropyl-β-cyclodextrin (HP-B-CD is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs. Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr−/− mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

  2. Double knockout of pendrin and Na-Cl cotransporter (NCC) causes severe salt wasting, volume depletion, and renal failure

    Science.gov (United States)

    Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Shull, Gary E.; Siddiqui, Faraz; Zahedi, Kamyar; Amlal, Hassane

    2012-01-01

    The Na-Cl cotransporter (NCC), which is the target of inhibition by thiazides, is located in close proximity to the chloride-absorbing transporter pendrin in the kidney distal nephron. Single deletion of pendrin or NCC does not cause salt wasting or excessive diuresis under basal conditions, raising the possibility that these transporters are predominantly active during salt depletion or in response to excess aldosterone. We hypothesized that pendrin and NCC compensate for loss of function of the other under basal conditions, thereby masking the role that each plays in salt absorption. To test our hypothesis, we generated pendrin/NCC double knockout (KO) mice by crossing pendrin KO mice with NCC KO mice. Pendrin/NCC double KO mice displayed severe salt wasting and sharp increase in urine output under basal conditions. As a result, animals developed profound volume depletion, renal failure, and metabolic alkalosis without hypokalemia, which were all corrected with salt replacement. We propose that the combined inhibition of pendrin and NCC can provide a strong diuretic regimen without causing hypokalemia for patients with fluid overload, including patients with congestive heart failure, nephrotic syndrome, diuretic resistance, or generalized edema. PMID:22847418

  3. Effects of insulin sensitizers on plaque vulnerability associated with elevated lipid content in atheroma in ApoE-knockout mice.

    Science.gov (United States)

    Cefalu, W T; Wang, Z Q; Schneider, D J; Absher, P M; Baldor, L C; Taatjes, D J; Sobel, B E

    2004-03-01

    Acute coronary syndromes are generally precipitated by rupture of lipid-laden, relatively acellular, vulnerable atherosclerotic plaques with thin fibrous caps. We investigated whether a high-fat diet alters insulin sensitivity and whether insulin sensitizers (troglitazone and rosiglitazone) alter the composition of otherwise lipidladen atherosclerotic plaques in mice deficient in apolipoprotein E (ApoE). ApoE-knockout mice were fed a high-fat (n=30) or standard chow (n=10) diet for two weeks. Thereafter, those fed the high-fat diet were treated with troglitazone (n=10), rosiglitazone (n=10) or no drug (n=10) for 16 weeks beginning at 8 weeks of age. Carbohydrate metabolism was assessed with intraperitoneal glucose tolerance tests and insulin tolerance tests. Plaque composition was characterised with confocal laser scanning microscopy. The high-fat diet induced insulin resistance in the absence of weight gain. Compared with control animals on the high-fat diet, animals given troglitazone (400 mg/kg/day) or rosiglitazone (4 mg/kg/day) had significantly less area under the curve (AUC) for insulin ( p<0.05) and glucose disposal ( p<0.05). Despite significant increases in insulin sensitivity with drug treatment, no change in HDL-cholesterol and triglyceride levels, nor reduction in atheroma size or lipid content was noted. Thus, improvement in insulin resistance induced by a high-fat diet in this animal model of vasculopathy did not alter plaque composition.

  4. Layer- and column-specific knockout of NMDA receptors in pyramidal neurons of the mouse barrel cortex.

    Directory of Open Access Journals (Sweden)

    Rachel Aronoff

    2007-11-01

    Full Text Available Viral vectors injected into the mouse brain offer the possibility for localized genetic modifications in a highly controlled manner. Lentivector injection into mouse neocortex transduces cells within a diameter of approximately 200µm, which closely matches the lateral scale of a column in barrel cortex. The depth and volume of the injection determines which cortical layer is transduced. Furthermore, transduced gene expression from the lentivector can be limited to predominantly pyramidal neurons by using a 1.3kb fragment of the αCaMKII promoter. This technique therefore allows genetic manipulation of a specific cell type in defined columns and layers of the neocortex. By expressing Cre recombinase from such a lentivector in gene-targeted mice carrying a floxed gene, highly specific genetic lesions can be induced. Here, we demonstrate the utility of this approach by specifically knocking out NMDA receptors (NMDARs in pyramidal neurons in the somatosensory barrel cortex of gene-targeted mice carrying floxed NMDAR 1 genes. Neurons transduced with lentivector encoding GFP and Cre recombinase exhibit not only reductions in NMDAR 1 mRNA levels, but reduced NMDAR-dependent currents and pairing-induced synaptic potentiation. This technique for knockout of NMDARs in a cell type, column- and layer-specific manner in the mouse somatosensory cortex may help further our understanding of the functional roles of NMDARs in vivo during sensory perception and learning.

  5. Lactobacillus acidophilus ATCC 4356 Prevents Atherosclerosis via Inhibition of Intestinal Cholesterol Absorption in Apolipoprotein E-Knockout Mice

    Science.gov (United States)

    Wang, Jinfeng; Quan, Guihua; Wang, Xiaojun; Yang, Longfei; Zhong, Lili

    2014-01-01

    The objective of this study was to investigate the effect of Lactobacillus acidophilus ATCC 4356 on the development of atherosclerosis in apolipoprotein E-knockout (ApoE−/−) mice. Eight-week-old ApoE−/− mice were fed a Western diet with or without L. acidophilus ATCC 4356 daily for 16 weeks. L. acidophilus ATCC 4356 protected ApoE−/− mice from atherosclerosis by reducing their plasma cholesterol levels from 923 ± 44 to 581 ± 18 mg/dl, likely via a marked decrease in cholesterol absorption caused by modulation of Niemann-Pick C1-like 1 (NPC1L1). In addition, suppression of cholesterol absorption induced reverse cholesterol transport (RCT) in macrophages through the peroxisome proliferator-activated receptor/liver X receptor (PPAR/LXR) pathway. Fecal lactobacillus and bifidobacterium counts were significantly (P intestine, colon, and feces during the feeding trial. The bacterial levels remained high even after the administration of lactic acid bacteria had been stopped for 2 weeks. These results suggest that administration of L. acidophilus ATCC 4356 can protect against atherosclerosis through the inhibition of intestinal cholesterol absorption. Therefore, L. acidophilus ATCC 4356 may be a potential therapeutic material for preventing the progression of atherosclerosis. PMID:25261526

  6. Whole body analysis of the knockout gene mouse model for cystic fibrosis using thermal and fast neutron activation analysis

    International Nuclear Information System (INIS)

    Mason, M.M.; Morris, J.S.; Derenzy, B.A.; Spate, V.L.; Horsman, T.L.; Baskett, C.K.; Nichols, T.A.; Colbert, J.W.; Clarke, L.L.; Gawenis, L.R.; Hillman, L.S.

    1998-01-01

    A genetically engineered 'knockout gene' mouse model for human cystic fibrosis (CF) has been utilized to study bone mineralization. In CF, the so-called cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride ion channel, is either absent or defective. To produce the animal model the murine CFTR gene has been inactivated producing CF symptoms in the homozygotic progeny. CF results in abnormal intestinal absorption of minerals and nutrients which presumably results in substandard bone mineralization. The objective of this study was to determine the feasibility of using whole-body thermal and fast neutron activation analysis to determine mineral and trace-element differences between homozygote controls (+/+) and CF (-/-), murine siblings. Gender-matched juvenile +/+ and -/- litter mates were lyophilized and placed in a BN capsule to reduce thermal-neutron activation and irradiated for 10 seconds at φ fast ∼ 1 x 10 13 n x cm -2 x s -1 using the MURR pneumatic-tube facility. Phosphorus was measured via the 31 P 15 (n,α) 28 Al 13 reaction. After several days decay, the whole-body specimens were re-irradiated in the same facility, but without thermal-neutron shielding, for 5 seconds and the gamma-ray spectrum was recorded at two different decay periods allowing measurement of 77m Se, 24 Na, 27m g, 38 Cl, 42k , 49 Ca, 56 Mn, 66 Cu and 80 Br from the corresponding radiative-capture reactions. (author)

  7. Reduced Inhibition within Layer IV of Sert Knockout Rat Barrel Cortex is Associated with Faster Sensory Integration.

    Science.gov (United States)

    Miceli, Stéphanie; Nadif Kasri, Nael; Joosten, Joep; Huang, Chao; Kepser, Lara; Proville, Rémi; Selten, Martijn M; van Eijs, Fenneke; Azarfar, Alireza; Homberg, Judith R; Celikel, Tansu; Schubert, Dirk

    2017-02-01

    Neural activity is essential for the maturation of sensory systems. In the rodent primary somatosensory cortex (S1), high extracellular serotonin (5-HT) levels during development impair neural transmission between the thalamus and cortical input layer IV (LIV). Rodent models of impaired 5-HT transporter (SERT) function show disruption in their topological organization of S1 and in the expression of activity-regulated genes essential for inhibitory cortical network formation. It remains unclear how such alterations affect the sensory information processing within cortical LIV. Using serotonin transporter knockout (Sert-/-) rats, we demonstrate that high extracellular serotonin levels are associated with impaired feedforward inhibition (FFI), fewer perisomatic inhibitory synapses, a depolarized GABA reversal potential and reduced expression of KCC2 transporters in juvenile animals. At the neural population level, reduced FFI increases the excitatory drive originating from LIV, facilitating evoked representations in the supragranular layers II/III. The behavioral consequence of these changes in network excitability is faster integration of the sensory information during whisker-based tactile navigation, as Sert-/- rats require fewer whisker contacts with tactile targets and perform object localization with faster reaction times. These results highlight the association of serotonergic homeostasis with formation and excitability of sensory cortical networks, and consequently with sensory perception. © The Author 2017. Published by Oxford University Press.

  8. Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Dan, E-mail: y.dan@lacdr.leidenuniv.nl [Division of Biopharmaceutics, LACDR, Leiden University (Netherlands); Meurs, Illiana [Division of Biopharmaceutics, LACDR, Leiden University (Netherlands); Ohigashi, Megumi [Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University (Japan); Calpe-Berdiel, Laura; Habets, Kim L.L.; Zhao, Ying [Division of Biopharmaceutics, LACDR, Leiden University (Netherlands); Kubo, Yoshiyuki [Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University (Japan); Yamaguchi, Akihito [Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University (Japan); Van Berkel, Theo J.C. [Division of Biopharmaceutics, LACDR, Leiden University (Netherlands); Nishi, Tsuyoshi [Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University (Japan); Van Eck, Miranda [Division of Biopharmaceutics, LACDR, Leiden University (Netherlands)

    2010-05-07

    Objectives: To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development. Methods and results: Chimeras with dysfunctional macrophage ABCA5 (ABCA5{sup -M/-M}) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5{sup -/-}) mice into irradiated LDLr{sup -/-} mice. In vitro, bone marrow-derived macrophages from ABCA5{sup -M/-M} chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr{sup -/-} mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5{sup -M/-M} chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5{sup -M/-M} chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding. Conclusions: ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr{sup -/-} mice.

  9. Anticancer Effects of the Marine SpongeLipastrotethyasp. Extract on Wild-Type and p53 Knockout HCT116 Cells.

    Science.gov (United States)

    Choi, Kiheon; Lim, Hyun Kyung; Oh, Sung Ryong; Chung, Woo-Hyun; Jung, Joohee

    2017-01-01

    Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE) was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT) or p53 knockout (KO) HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

  10. Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild-Type and p53 Knockout HCT116 Cells

    Science.gov (United States)

    Choi, Kiheon; Lim, Hyun Kyung; Oh, Sung Ryong; Chung, Woo-Hyun

    2017-01-01

    Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE) was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT) or p53 knockout (KO) HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status. PMID:28127380

  11. Anticancer Effects of the Marine Sponge Lipastrotethya sp. Extract on Wild-Type and p53 Knockout HCT116 Cells

    Directory of Open Access Journals (Sweden)

    Kiheon Choi

    2017-01-01

    Full Text Available Interest in marine bioresources is increasing in the drug development sector. In particular, marine sponges produce a wide range of unique metabolites that enable them to survive in challenging environments, which makes them attractive sources of candidate pharmaceuticals. In previous study, we investigated over 40 marine specimens collected in Micronesia and provided by the Korean Institute of Ocean Science and Technology, for their antiproliferative effects on various cancer cell lines, and Lipastrotethya sp. extract (LSSE was found to have a marked antiproliferative effect. In the present study, we investigated the mechanism responsible for its anticancer effect on wild-type p53 (WT or p53 knockout (KO HCT116 cells. LSSE inhibited cell viability and induced apoptotic cell death more so in HCT116 p53 KO cells than the WT. HCT116 WT cells treated with LSSE underwent apoptosis associated with the induction of p53 and its target genes. On the other hand, in HCT116 p53 KO cells, LSSE reduced mTOR and Bcl-2 and increased Beclin-1 and LC3-II protein levels, suggesting autophagy induction. These results indicate that the mechanisms responsible for the anticancer effect of LSSE depend on p53 status.

  12. Studies on functional roles of the histaminergic neuron system by using pharmacological agents, knockout mice and positron emission tomography

    International Nuclear Information System (INIS)

    Watanabe, Takehiko; Yanai, Kazuhiko

    2001-01-01

    Since one of us, Takehiko Watanabe (TW), elucidated the location and distribution of the histaminergic neuron system in the brain with antibody raised against L-histidine decarboxylase (a histamine-forming enzyme, HDC) as a marker in 1984 and came to Tohoku University School of Medicine in Sendai, we have been collaborating on the functions of this neuron system by using pharmacological agents, knockout mice of the histamine-related genes, and, in some cases, positron emission tomography (PET). Many of our graduate students and colleagues have been actively involved in histamine research since 1985. Our extensive studies have clarified some of the functions of histamine neurons using methods from molecular techniques to non-invasive human PET imaging. Histamine neurons are involved in many brain functions, such as spontaneous locomotion, arousal in wake-sleep cycle, appetite control, seizures, learning and memory, aggressive behavior and emotion. Particularly, the histaminergic neuron system is one of the most important neuron systems to maintain and stimulate wakefulness. Histamine also functions as a biprotection system against various noxious and unfavorable stimuli (for examples, convulsion, nociception, drug sensitization, ischemic lesions, and stress). Although activators of histamine neurons have not been clinically available until now, we would like to point out that the activation of the histaminergic neuron system is important to maintain mental health. Here, we summarize the newly-discovered functions of histamine neurons mainly on the basis of results from our research groups. (author)

  13. Mamu-A*01/Kb transgenic and MHC Class I knockout mice as a tool for HIV vaccine development

    International Nuclear Information System (INIS)

    Li Jinliang; Srivastava, Tumul; Rawal, Ravindra; Manuel, Edwin; Isbell, Donna; Tsark, Walter; La Rosa, Corinna; Wang Zhongde; Li Zhongqi; Barry, Peter A.; Hagen, Katharine D.; Longmate, Jeffrey; Diamond, Don J.

    2009-01-01

    We have developed a murine model expressing the rhesus macaque (RM) Mamu-A*01 MHC allele to characterize immune responses and vaccines based on antigens of importance to human disease processes. Towards that goal, transgenic (Tg) mice expressing chimeric RM (α1 and α2 Mamu-A*01 domains) and murine (α3, transmembrane, and cytoplasmic H-2K b domains) MHC Class I molecules were derived by transgenesis of the H-2K b D b double MHC Class I knockout strain. After immunization of Mamu-A*01/K b Tg mice with rVV-SIVGag-Pol, the mice generated CD8 + T-cell IFN-γ responses to several known Mamu-A*01 restricted epitopes from the SIV Gag and Pol antigen sequence. Fusion peptides of highly recognized CTL epitopes from SIV Pol and Gag and a strong T-help epitope were shown to be immunogenic and capable of limiting an rVV-SIVGag-Pol challenge. Mamu-A*01/K b Tg mice provide a model system to study the Mamu-A*01 restricted T-cell response for various infectious diseases which are applicable to a study in RM.

  14. Hawthorn (Crataegus pinnatifida Bunge) leave flavonoids attenuate atherosclerosis development in apoE knock-out mice.

    Science.gov (United States)

    Dong, Pengzhi; Pan, Lanlan; Zhang, Xiting; Zhang, Wenwen; Wang, Xue; Jiang, Meixiu; Chen, Yuanli; Duan, Yajun; Wu, Honghua; Xu, Yantong; Zhang, Peng; Zhu, Yan

    2017-02-23

    Hawthorn (Crataegus pinnatifida Bunge) leave have been used to treat cardiovascular diseases in China and Europe. Hawthorn leave flavonoids (HLF) are the main part of extraction. Whether hawthorn leave flavonoids could attenuate the development of atherosclerosis and the possible mechanism remain unknown. High-fat diet (HFD) mixed with HLF at concentrations of 5mg/kg and 20mg/kg were administered to apolipoprotein E (apoE) knock out mice. 16 weeks later, mouse serum was collected to determine the lipid profile while the mouse aorta dissected was prepared to measure the lesion area. Hepatic mRNA of genes involved in lipid metabolism were determined. Peritoneal macrophages were collected to study the impact of HLF on cholesterol efflux, formation of foam cell and the expression of ATP binding cassette transporter A1 (ABCA1). Besides, in vivo reverse cholesterol transport (RCT) was conducted. HLF attenuated the development of atherosclerosis that the mean atherosclerotic lesion area in en face aortas was reduced by 23.1% (Pflavonoids can slow down the development of atherosclerosis in apoE knockout mice via induced expression of genes involved in antioxidant activities, inhibition of the foam cell formation and promotion of RCT in vivo, which implies the potential use in the prevention of atherosclerosis. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  15. Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2015-01-01

    Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

  16. Knockout of the TauT gene predisposes C57BL/6 mice to streptozotocin-induced diabetic nephropathy.

    Directory of Open Access Journals (Sweden)

    Xiaobin Han

    Full Text Available Diabetic nephropathy is the leading cause of end stage renal disease in the world. Although tremendous efforts have been made, scientists have yet to identify an ideal animal model that can reproduce the characteristics of human diabetic nephropathy. In this study, we hypothesize that taurine insufficiency is a critical risk factor for development of diabetic nephropathy associated with diabetes mellitus. This hypothesis was tested in vivo in TauT heterozygous (TauT+/- and homozygous (TauT-/- knockout in C57BL/6 background mice. We have shown that alteration of the TauT gene (also known as SLC6A6 has a substantial effect on the susceptibility to development of extensive diabetic kidney disease in both TauT+/- and TauT-/-mouse models of diabetes. These animals developed histological changes characteristic of human diabetic nephropathy that included glomerulosclerosis, nodular lesions, arteriosclerosis, arteriolar dilation, and tubulointerstitial fibrosis. Immunohistochemical staining of molecular markers of smooth muscle actin, CD34, Ki67 and collagen IV further confirmed these observations. Our results demonstrated that both homozygous and heterozygous TauT gene deletion predispose C57BL/6 mice to develop end-stage diabetic kidney disease, which closely replicates the pathological features of diabetic nephropathy in human diabetic patients.

  17. Curcumin Protects against Atherosclerosis in Apolipoprotein E-Knockout Mice by Inhibiting Toll-like Receptor 4 Expression.

    Science.gov (United States)

    Zhang, Shanshan; Zou, Jun; Li, Peiyang; Zheng, Xiumei; Feng, Dan

    2018-01-17

    Toll-like receptor 4 (TLR4) has been reported to play a critical role in the pathogenesis of atherosclerosis, the current study aimed to investigate whether curcumin suppresses atherosclerosis development in ApoE-knockout (ApoE -/- ) mice by inhibiting TLR4 expression. ApoE -/- mice were fed a high-fat diet supplemented with or without curcumin (0.1% w/w) for 16 weeks. Curcumin supplementation significantly reduced TLR4 expression and macrophage infiltration in atherosclerotic plaques. Curcumin also reduced aortic interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression, nuclear factor-κB (NF-κB) activity, and plasma IL-1β, TNF-α, soluble VCAM-1 and ICAM-1 levels. In addition, aortic sinus sections revealed that curcumin treatment reduced the extent of atherosclerotic lesions and inhibited atherosclerosis development. In vitro, curcumin inhibited NF-κB activation in macrophages and reduced TLR4 expression induced by lipopolysaccharide. Our results indicate that curcumin protects against atherosclerosis at least partially by inhibiting TLR4 expression and its related inflammatory reaction.

  18. Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability.

    Science.gov (United States)

    Liu, Dong; Zhang, Lu; Xue, Wen; Wang, Yaping; Ju, Jiansong; Zhao, Baohua

    2015-07-01

    This study focused on the alanine racemase gene (alr-2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr-2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr-2 does not impose an auxotrophic requirement for d-alanine. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

    Directory of Open Access Journals (Sweden)

    Nathalie Geurts

    Full Text Available The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO and knock-in mice, has become an established tool to study gene function in many animal models for human diseases. However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

  20. Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice.

    Science.gov (United States)

    Guidotti, J E; Mignon, A; Haase, G; Caillaud, C; McDonell, N; Kahn, A; Poenaru, L

    1999-05-01

    The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.

  1. Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

    Directory of Open Access Journals (Sweden)

    Luis Segu

    Full Text Available Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4, but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4 knock-out (KO and wild-type (WT mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4 control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4. Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4 KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT, the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4 KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4 KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4 to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4 aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4 mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

  2. [Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs].

    Science.gov (United States)

    Li, Zhi-fang; Feng, Chong; Ji, Hui-li; Shi, Ning-ning; Song, Xiao-feng; Zhao, Qin-li; Long, Chuan; Pan, Deng-ke; Yang, Xiao-gan

    2015-12-01

    The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

  3. Dietary Flaxseed Oil Prevents Western-Type Diet-Induced Nonalcoholic Fatty Liver Disease in Apolipoprotein-E Knockout Mice

    Directory of Open Access Journals (Sweden)

    Hao Han

    2017-01-01

    Full Text Available The prevalence of nonalcoholic fatty liver disease (NAFLD has dramatically increased globally during recent decades. Intake of n-3 polyunsaturated fatty acids (PUFAs, mainly eicosapentaenoic acid (EPA, C20:5n-3 and docosahexaenoic acid (DHA, C22:6n-3, is believed to be beneficial to the development of NAFLD. However, little information is available with regard to the effect of flaxseed oil rich in α-linolenic acid (ALA, C18:3n-3, a plant-derived n-3 PUFA, in improving NAFLD. This study was to gain the effect of flaxseed oil on NAFLD and further investigate the underlying mechanisms. Apolipoprotein-E knockout (apoE-KO mice were given a normal chow diet, a western-type high-fat and high-cholesterol diet (WTD, or a WTD diet containing 10% flaxseed oil (WTD + FO for 12 weeks. Our data showed that consumption of flaxseed oil significantly improved WTD-induced NAFLD, as well as ameliorated impaired lipid homeostasis, attenuated oxidative stress, and inhibited inflammation. These data were associated with the modification effects on expression levels of genes involved in de novo fat synthesis (SREBP-1c, ACC, triacylglycerol catabolism (PPARα, CPT1A, and ACOX1, inflammation (NF-κB, IL-6, TNF-α, and MCP-1, and oxidative stress (ROS, MDA, GSH, and SOD.

  4. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases.

    Science.gov (United States)

    Mashimo, Tomoji; Takizawa, Akiko; Voigt, Birger; Yoshimi, Kazuto; Hiai, Hiroshi; Kuramoto, Takashi; Serikawa, Tadao

    2010-01-25

    Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  5. Properties of extensor digitorum longus muscle and skinned fibers from adult and aged male and female Actn3 knockout mice.

    Science.gov (United States)

    Chan, Stephen; Seto, Jane T; Houweling, Peter J; Yang, Nan; North, Kathryn N; Head, Stewart I

    2011-01-01

    Absence of α-actinin-3, encoded by the ACTN3 "speed gene," is associated with poorer sprinting performance in athletes and a slowing of relaxation in fast-twitch muscles of Actn3 knockout (KO) mice. Our first aim was to investigate, at the individual-fiber level, possible mechanisms for this slowed relaxation. Our second aim was to characterize the contractile properties of whole extensor digitorum longus (EDL) muscles from KO mice by age and gender. We examined caffeine-induced Ca(2+) release in mechanically skinned EDL fibers from KO mice, and measured isolated whole EDL contractile properties. The sarcoplasmic reticulum of KO muscle fibers loaded Ca(2+) more slowly than that of wild-types (WTs). Whole KO EDL muscles had longer twitch and tetanus relaxation times than WTs, and reduced mass and cross-sectional area. These effects occurred in both male and female mice, but they diminished with age. These changes in KO muscles and fibers help to explain the effects of α-actinin-3 deficiency observed in athletes. Copyright © 2010 Wiley Periodicals, Inc.

  6. Central nervous system-specific knockout of steroidogenic factor 1 results in increased anxiety-like behavior.

    Science.gov (United States)

    Zhao, Liping; Kim, Ki Woo; Ikeda, Yayoi; Anderson, Kimberly K; Beck, Laurel; Chase, Stephanie; Tobet, Stuart A; Parker, Keith L

    2008-06-01

    Steroidogenic factor 1 (SF-1) plays key roles in adrenal and gonadal development, expression of pituitary gonadotropins, and development of the ventromedial hypothalamic nucleus (VMH). If kept alive by adrenal transplants, global knockout (KO) mice lacking SF-1 exhibit delayed-onset obesity and decreased locomotor activity. To define specific roles of SF-1 in the VMH, we used the Cre-loxP system to inactivate SF-1 in a central nervous system (CNS)-specific manner. These mice largely recapitulated the VMH structural defect seen in mice lacking SF-1 in all tissues. In multiple behavioral tests, mice with CNS-specific KO of SF-1 had significantly more anxiety-like behavior than wild-type littermates. The CNS-specific SF-1 KO mice had diminished expression or altered distribution in the mediobasal hypothalamus of several genes whose expression has been linked to stress and anxiety-like behavior, including brain-derived neurotrophic factor, the type 2 receptor for CRH (Crhr2), and Ucn 3. Moreover, transfection and EMSAs support a direct role of SF-1 in Crhr2 regulation. These findings reveal important roles of SF-1 in the hypothalamic expression of key regulators of anxiety-like behavior, providing a plausible molecular basis for the behavioral effect of CNS-specific KO of this nuclear receptor.

  7. A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism.

    Science.gov (United States)

    Chen, Wei; Zhang, Xiaoting; Birsoy, Kivanc; Roeder, Robert G

    2010-06-01

    As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

  8. IL-23 p19 knockout mice exhibit minimal defects in responses to primary and secondary infection with Francisella tularensis LVS.

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    Sherry L Kurtz

    Full Text Available Our laboratory's investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.

  9. Deficiency of Serotonin in Raphe Neurons and Altered Behavioral Responses in Tryptophan Hydroxylase 2-Knockout Medaka (Oryzias latipes).

    Science.gov (United States)

    Ansai, Satoshi; Hosokawa, Hiroshi; Maegawa, Shingo; Naruse, Kiyoshi; Washio, Youhei; Sato, Kenji; Kinoshita, Masato

    2017-12-01

    Serotonin (5-hydroxytryptamine [5-HT]) is a bioactive monoamine that acts as a neurotransmitter in the central and peripheral nervous system of animals. Teleost fish species have serotonergic neurons in the raphe nuclei of the brainstem; however, the role of 5-HT in the raphe neurons in teleost fish remains largely unknown. Here, we established a medaka (Oryzias latipes) strain with targeted disruption of tryptophan hydroxylase 2 (tph2) gene that is involved in the 5-HT synthesis in the raphe nuclei. Immunohistochemistry and mass spectrometry analysis revealed that the homozygous mutants (tph2 Δ13/Δ13 ) lacked the ability to synthesize 5-HT in the raphe neurons. To investigate the effects of 5-HT deficiency in adult behaviors, the mutant fish were subjected to five behavioral paradigms (diving, open-field, light-dark transition, mirror-biting, and two-fish social interaction). The homozygous mutation caused a longer duration of freezing response in all examined paradigms and reduced the number of entries to the top area in the diving test. In addition, the mutants exhibited a decreased number of mirror-biting in the males and an increased contact time in direct social interaction between the females. These results indicate that this tph2-knockout medaka serves as a good model to analyze the effects of 5-HT deficiency in the raphe neurons.

  10. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

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    Dejia Li

    2010-12-01

    Full Text Available Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

  11. Upregulated Expression of Cytotoxicity-Related Genes in IFN-γ Knockout Mice with Schistosoma japonicum Infection

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    Xiaotang Du

    2011-01-01

    Full Text Available It is well accepted that IFN-γ is important to the development of acquired resistance against murine schistosomiasis. However, the in vivo role of this immunoregulatory cytokine in helminth infection needs to be further investigated. In this study, parasite burden and host immune response were observed in IFN-γ knockout mice (IFNg KO infected with Schistosoma japonicum for 6 weeks. The results suggested that deficiency in IFN-γ led to decreased egg burden in mice, with low schistosome-specific IgG antibody response and enhanced activation of T cells during acute infection. Microarray and qRT-PCR data analyses showed significant upregulation of some cytotoxicity-related genes, including those from the granzyme family, tumor necrosis factor, Fas Ligand, and chemokines, in the spleen cells of IFNg KO mice. Furthermore, CD8+ cells instead of NK cells of IFNg KO mice exhibited increased transcription of cytotoxic genes compared with WT mice. Additionally, Schistosoma japonicum-specific egg antigen immunization also could activate CD8+ T cells to upregulate the expression of cytotoxic genes in IFNg KO mice. Our data suggest that IFN-γ is not always a positive regulator of immune responses. In certain situations, the disruption of IFN-γ signaling may up-regulate the cytotoxic T-cell-mediated immune responses to the parasite.

  12. Predicting the names of the best teams after the knock-out phase of a cricket series.

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    Lemmer, Hermanus Hofmeyr

    2014-01-01

    Cricket players' performances can best be judged after a large number of matches had been played. For test or one-day international (ODI) players, career data are normally used to calculate performance measures. These are normally good indicators of future performances, although various factors influence the performance of a player in a specific match. It is often necessary to judge players' performances based on a small number of scores, e.g. to identify the best players after a short series of matches. The challenge then is to use the best available criteria in order to assess performances as accurately and fairly as possible. In the present study the results of the knock-out phase of an International Cricket Council (ICC) World Cup ODI Series are used to predict the names of the best teams by means of a suitably formulated logistic regression model. Despite using very sparse data, the methods used are reasonably successful. It is also shown that if the same technique is applied to career ratings, very good results are obtained.

  13. High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.

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    Luo, Yonglun; Li, Juan; Liu, Ying; Lin, Lin; Du, Yutao; Li, Shengting; Yang, Huanming; Vajta, Gábor; Callesen, Henrik; Bolund, Lars; Sørensen, Charlotte Brandt

    2011-10-01

    Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

  14. Contribution of PPARγ in modulation of acrolein-induced inflammatory signaling in gp91phox knock-out mice.

    Science.gov (United States)

    Yousefipour, Zivar; Chug, Neha; Marek, Katarzyna; Nesbary, Alicia; Mathew, Joseph; Ranganna, Kasturi; Newaz, Mohammad A

    2017-08-01

    Oxidative stress and inflammation are major contributors to acrolein toxicity. Peroxisome proliferator activated receptor gamma (PPARγ) has antioxidant and anti-inflammatory effects. We investigated the contribution of PPARγ ligand GW1929 to the attenuation of oxidative stress in acrolein-induced insult. Male gp91 phox knock-out (KO) mice were treated with acrolein (0.5 mg·(kg body mass) -1 by intraperitoneal injection for 7 days) with or without GW1929 (GW; 0.5 mg·(kg body mass) -1 ·day -1 , orally, for 10 days). The livers were processed for further analyses. Acrolein significantly increased 8-isoprostane and reduced PPARγ activity (P acrolein-treated WT mice, and was reduced by GW1929 (by 65%). KO mice exhibited higher xanthine oxidase (XO). Acrolein increased XO and COX in WT mice and XO in KO mice. GW1929 significantly reduced COX in WT and KO mice and reduced XO in KO mice. Acrolein significantly reduced the total antioxidant status in WT and KO mice (P acrolein-treated WT mice. GW1929 reduced NF-κB levels (by 51%) in KO mice. Acrolein increased CD36 in KO mice (by 43%), which was blunted with GW1929. Data confirms that the generation of free radicals by acrolein is mainly through NAD(P)H, but other oxygenates play a role too. GW1929 may alleviate the toxicity of acrolein by attenuating NF-κB, COX, and CD36.

  15. Global gene expression profiles of MT knockout and wild-type mice in the condition of doxorubicin-induced cardiomyopathy.

    Science.gov (United States)

    Shuai, Yi; Guo, Jun; Dong, Yansheng; Zhong, Weijian; Xiao, Ping; Zhou, Tong; Zhang, Lishi; Peng, Shuangqing

    2011-01-15

    Increasing evidence from in vivo and in vitro studies has indicated that MT exerts protective effects against DOX-induced cardiotoxicity; however the underlying precise mechanisms still remain an enigma. Therefore, the present study was designed using MT knockout mice in concert with genomic approaches to explore the possible molecular and cellular mechanisms in terms of the genetic network changes. MT-I/II null (MT⁻/⁻) mice and corresponding wild-type mice (MT+/+) were administrated with a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. Global gene expression profiles of cardiac mRNA from two genotype mice revealed that 381 characteristically MT-responsive genes were identified between MT+/+ mice and MT⁻/⁻ mice in response to DOX, including fos, ucp3, car3, atf3, map3k6, etc. Functional analysis implied MAPK signaling pathway, p53 signaling pathway, Jak-STAT signaling pathway, PPAR signaling pathway, Wnt signaling pathway, etc. might be involved to mediate the protection of DOX cardiomyopathy by MT. Results from the present study not only validated the previously reported possible mechanisms of MT protection against DOX toxicity, but also provided new clues into the molecular mechanisms involved in this process. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  16. Inhalation exposure of gas-metal arc stainless steel welding fume increased atherosclerotic lesions in apolipoprotein E knockout mice.

    Science.gov (United States)

    Erdely, Aaron; Hulderman, Tracy; Salmen-Muniz, Rebecca; Liston, Angie; Zeidler-Erdely, Patti C; Chen, Bean T; Stone, Samuel; Frazer, David G; Antonini, James M; Simeonova, Petia P

    2011-07-04

    Epidemiological studies suggest that welding, a process which generates an aerosol of inhalable gases and metal rich particulates, increases the risk for cardiovascular disease. In this study we analyzed systemic inflammation and atherosclerotic lesions following gas metal arc-stainless steel (GMA-SS) welding fume exposure. Apolipoprotein E knockout (apoE(-/-)) mice, fed a Western diet, were exposed to GMA-SS at 40mg/m(3) for 3h/day for ten days (∼8.26μg daily alveolar deposition). Mice were sacrificed two weeks after exposure and serum chemistry, serum protein profiling and aortic lesion area were determined. There were no significant changes in serum total cholesterol, triglycerides or alanine aminotransferase. Serum levels of uric acid, a potent antioxidant, were decreased perhaps suggesting a reduced capacity to combat systemic oxidative stress. Inflammatory serum proteins interleukin 1 beta (IL-1β) and monocyte chemoattractant protein 3 (MCP-3) were increased two weeks after GMA-SS exposure. Analysis of atherosclerotic plaques showed an increase in lesion area as the result of GMA-SS exposure. In conclusion, GMA-SS exposure showed evidence of systemic inflammation and increased plaque progression in apoE(-/-) mice. These results complement epidemiological and functional human studies that suggest welding may result in adverse cardiovascular effects. Published by Elsevier Ireland Ltd.

  17. Studies on functional roles of the histaminergic neuron system by using pharmacological agents, knockout mice and positron emission tomography

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    Watanabe, Takehiko; Yanai, Kazuhiko [Tohoku Univ., Sendai (Japan). Graduate School of Medicine

    2001-12-01

    Since one of us, Takehiko Watanabe (TW), elucidated the location and distribution of the histaminergic neuron system in the brain with antibody raised against L-histidine decarboxylase (a histamine-forming enzyme, HDC) as a marker in 1984 and came to Tohoku University School of Medicine in Sendai, we have been collaborating on the functions of this neuron system by using pharmacological agents, knockout mice of the histamine-related genes, and, in some cases, positron emission tomography (PET). Many of our graduate students and colleagues have been actively involved in histamine research since 1985. Our extensive studies have clarified some of the functions of histamine neurons using methods from molecular techniques to non-invasive human PET imaging. Histamine neurons are involved in many brain functions, such as spontaneous locomotion, arousal in wake-sleep cycle, appetite control, seizures, learning and memory, aggressive behavior and emotion. Particularly, the histaminergic neuron system is one of the most important neuron systems to maintain and stimulate wakefulness. Histamine also functions as a biprotection system against various noxious and unfavorable stimuli (for examples, convulsion, nociception, drug sensitization, ischemic lesions, and stress). Although activators of histamine neurons have not been clinically available until now, we would like to point out that the activation of the histaminergic neuron system is important to maintain mental health. Here, we summarize the newly-discovered functions of histamine neurons mainly on the basis of results from our research groups. (author)

  18. Knock-Out Serum Replacement and Melatonin Effects on Germ Cell Differentiation in Murine Testicular Explant Cultures.

    Science.gov (United States)

    Reda, Ahmed; Albalushi, Halima; Montalvo, Sheyla Cisneros; Nurmio, Mirja; Sahin, Zeliha; Hou, Mi; Geijsen, Niels; Toppari, Jorma; Söder, Olle; Stukenborg, Jan-Bernd

    2017-07-01

    Finding robust culture conditions for in vitro maturation (IVM) of male germ cells is still a challenge. Recently, a testis organ culture method, using Knockout Serum Replacement (KSR), was suggested as a promising approach. However, the efficiency of that model is still not optimal. Hence, we have tried to establish the culture conditions in two laboratories, and to improve the reliability of the culture system to generate mature germ cells. Male mice at three days of age were sacrificed. Testes were cut into small pieces which were cultured atop agarose stands, using Minimum Essential Medium alpha supplemented with different supplements; melatonin, Glutamax, and different concentrations of KSR. The results showed that the duration of culture beyond 18 days had an impact on the number of differentiated germ cells. Supplementation with melatonin and Glutamax revealed a positive influence on the efficiency of male germ cell differentiation in vitro. Furthermore, the results confirmed that KSR had a positive effect on germ cell maturation and testosterone production, with a concentration of at least 10%. In conclusion, this study emphasizes the beneficial role of at least 10% KSR in the IVM of germ cells.

  19. Knockout of Murine Mamld1 Impairs Testicular Growth and Daily Sperm Production but Permits Normal Postnatal Androgen Production and Fertility

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    Mami Miyado

    2017-06-01

    Full Text Available MAMLD1 has been implicated in testicular function in both human and mouse fetuses. Although three patients with MAMLD1 mutations were reported to have hypergonadotropic hypogonadism in their teens, the functional significance of MAMLD1 in the postnatal testis remains unclear. Here, we analyzed the phenotype of Mamld1 knockout (KO male mice at reproductive ages. The reproductive organs of KO male mice were morphologically unremarkable, except for relatively small testes. Seminiferous tubule size and number of proliferating spermatogonia/spermatocytes were reduced in the KO testis. Daily sperm production of KO mice was mildly attenuated, whereas total sperm counts in epididymal semen remained normal. Sperm motility and morphology, as well as androgen levels in serum and testicular tissues and the number of pups born from cross-mated wildtype (WT female mice, were comparable between WT and KO male mice. These results indicate that MAMLD1 contributes to the maintenance of postnatal testicular growth and daily sperm production but is dispensable for androgen biosynthesis and fertility. MAMLD1 likely plays supporting roles in multiple and continuous steps of male reproduction.

  20. Knockout of Murine Mamld1 Impairs Testicular Growth and Daily Sperm Production but Permits Normal Postnatal Androgen Production and Fertility.

    Science.gov (United States)

    Miyado, Mami; Yoshida, Kaoru; Miyado, Kenji; Katsumi, Momori; Saito, Kazuki; Nakamura, Shigeru; Ogata, Tsutomu; Fukami, Maki

    2017-06-19

    MAMLD1 has been implicated in testicular function in both human and mouse fetuses. Although three patients with MAMLD1 mutations were reported to have hypergonadotropic hypogonadism in their teens, the functional significance of MAMLD1 in the postnatal testis remains unclear. Here, we analyzed the phenotype of Mamld1 knockout (KO) male mice at reproductive ages. The reproductive organs of KO male mice were morphologically unremarkable, except for relatively small testes. Seminiferous tubule size and number of proliferating spermatogonia/spermatocytes were reduced in the KO testis. Daily sperm production of KO mice was mildly attenuated, whereas total sperm counts in epididymal semen remained normal. Sperm motility and morphology, as well as androgen levels in serum and testicular tissues and the number of pups born from cross-mated wildtype (WT) female mice, were comparable between WT and KO male mice. These results indicate that MAMLD1 contributes to the maintenance of postnatal testicular growth and daily sperm production but is dispensable for androgen biosynthesis and fertility. MAMLD1 likely plays supporting roles in multiple and continuous steps of male reproduction.

  1. Rapid-throughput skeletal phenotyping of 100 knockout mice identifies 9 new genes that determine bone strength.

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    J H Duncan Bassett

    Full Text Available Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.

  2. A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

    Science.gov (United States)

    Jin, Jun-Xue; Lee, Sanghoon; Setyawan, Erif Maha Nugraha; Taweechaipaisankul, Anukul; Kim, Geon A; Han, Ho Jae; Ahn, Curie; Lee, Byeong Chun

    2018-01-18

    The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species. © 2018 Wiley Periodicals, Inc.

  3. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID using zinc-finger nucleases.

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    Tomoji Mashimo

    Full Text Available BACKGROUND: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. METHODOLOGY/PRINCIPAL FINDINGS: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID. Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. CONCLUSIONS AND SIGNIFICANCE: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  4. Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice.

    Science.gov (United States)

    Amano, Akiko; Kondo, Yoshitaka; Noda, Yoshihiro; Ohta, Mitsuhiro; Kawanishi, Noriaki; Machida, Shuichi; Mitsuhashi, Kazuteru; Senmaru, Takafumi; Fukui, Michiaki; Takaoka, Osamu; Mori, Taisuke; Kitawaki, Jo; Ono, Masafumi; Saibara, Toshiji; Obayashi, Hiroshi; Ishigami, Akihito

    2017-05-15

    Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. [New method for analyzing pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies].

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    Zhao, Yan; Qu, Hui-Hua; Wang, Qing-Guo

    2013-09-01

    Study on pharmacodynamic material basis of traditional Chinese medicines is one of the key issues for the modernization of traditional Chinese medicine. Having introduced the monoclonal antibody technology into the study on pharmacodynamic material basis of traditional Chinese medicines, the author prepared the immunoaffinity chromatography column by using monoclonal antibodies in active components of traditional Chinese medicines, so as to selectively knock out the component from herbs or traditional Chinese medicine compounds, while preserving all of the other components and keeping their amount and ratio unchanged. A comparative study on pharmacokinetics and pharmacodynamics was made to explicitly reveal the correlation between the component and the main purpose of traditional Chinese medicines and compounds. The analysis on pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies is a new method for study pharmacodynamic material basis in line with the characteristics of traditional Chinese medicines. Its results can not only help study material basis from a new perspective, but also help find the modern scientific significance in single herb or among compounds of traditional Chinese medicines.

  6. Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays.

    Science.gov (United States)

    Bezrutczyk, Margaret; Hartwig, Thomas; Horschman, Marc; Char, Si Nian; Yang, Jinliang; Yang, Bing; Frommer, Wolf B; Sosso, Davide

    2018-04-01

    Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H + symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  7. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice

    Science.gov (United States)

    van Putten, Maaike; Hulsker, Margriet; Young, Courtney; Nadarajah, Vishna D.; Heemskerk, Hans; van der Weerd, Louise; 't Hoen, Peter A. C.; van Ommen, Gert-Jan B.; Aartsma-Rus, Annemieke M.

    2013-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to restore the synthesis of functional dystrophin are underway. The dystrophin levels needed for improvement of muscle pathology, function, and overall vitality are not known. Here, we describe the mdx/utrn−/−/XistΔhs mouse model, which expresses a range of low dystrophin levels, depending on the degree of skewing of X inactivation in a utrophin-negative background. Mdx/utrn−/− mice develop severe muscle weakness, kyphosis, respiratory and heart failure, and premature death closely resembling DMD pathology. We show that at dystrophin levels 4% dystrophin, histopathology is ameliorated, as well. These findings suggest that the dystrophin levels needed to benefit vitality and functioning of patients with DMD might be lower than those needed for full protection against muscle damage.—Van Putten, M., Hulsker, M., Young, C., Nadarajah, V. D., Heemskerk, H., van der Weerd, L., 't Hoen, P. A. C., van Ommen, G. J. B., Aartsma-Rus, A. M. Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice. PMID:23460734

  8. Imbalanced lipid homeostasis in the conditional Dicer1 knockout mouse epididymis causes instability of the sperm membrane.

    Science.gov (United States)

    Björkgren, Ida; Gylling, Helena; Turunen, Heikki; Huhtaniemi, Ilpo; Strauss, Leena; Poutanen, Matti; Sipilä, Petra

    2015-02-01

    During epididymal sperm maturation, the lipid content of the sperm membrane is modified, which facilitates sperm motility and fertility. However, little is known about the mechanisms regulating the maturation process. By generating a conditional knockout (cKO) of Dicer1 in the proximal part of the mouse epididymis, we studied the role of RNA interference in epididymal functions. The Dicer1 cKO epididymis displayed an altered lipid homeostasis associated with a 0.6-fold reduction in the expression of the gene elongation of very long chain fatty acids-like 2, an enzyme needed for production of long-chain polyunsaturated fatty acids (PUFAs). Furthermore, the expression of several factors involved in cholesterol synthesis was up-regulated. Accordingly, the Dicer1 cKO sperm membrane showed a 0.7-fold decrease in long-chain PUFAs, whereas the amount of cholesterol in acrosome-reacted sperm displayed a 1.7-fold increase. The increased cholesterol:PUFA ratio of the sperm membrane caused breakage of the neck and acrosome region and immotility of sperm. Dicer1 cKO mice sperm also displayed reduced ability to bind to and fertilize the oocyte in vitro. This study thus shows that Dicer1 is critical for lipid synthesis in the epididymis, which directly affects sperm membrane integrity and male fertility. © FASEB.

  9. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

    Science.gov (United States)

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-12-15

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration.

  10. Rescue of heart lipoprotein lipase-knockout mice confirms a role for triglyceride in optimal heart metabolism and function.

    Science.gov (United States)

    Khan, Raffay S; Lin, Yan; Hu, Yunying; Son, Ni-Huiping; Bharadwaj, Kalyani G; Palacios, Carla; Chokshi, Aalap; Ji, Ruiping; Yu, Shuiqing; Homma, Sunichi; Schulze, P Christian; Tian, Rong; Goldberg, Ira J

    2013-12-01

    Hearts utilize fatty acids as a primary source of energy. The sources of those lipids include free fatty acids and lipoprotein triglycerides. Deletion of the primary triglyceride-hydrolyzing enzyme lipoprotein lipase (LPL) leads to cardiac dysfunction. Whether heart LPL-knockout (hLPL0) mice are compromised due a deficiency in energetic substrates is unknown. To test whether alternative sources of energy will prevent cardiac dysfunction in hLPL0 mice, two different models were used to supply nonlipid energy. 1) hLPL0 mice were crossed with mice transgenically expressing GLUT1 in cardiomyocytes to increase glucose uptake into the heart; this cross-corrected cardiac dysfunction, reduced cardiac hypertrophy, and increased myocardial ATP. 2) Mice were randomly assigned to a sedentary or training group (swimming) at 3 mo of age, which leads to increased skeletal muscle production of lactate. hLPL0 mice had greater expression of the lactate transporter monocarboxylate transporter-1 (MCT-1) and increased cardiac lactate uptake. Compared with hearts from sedentary hLPL0 mice, hearts from trained hLPL0 mice had adaptive hypertrophy and improved cardiac function. We conclude that defective energy intake and not the reduced uptake of fat-soluble vitamins or cholesterol is responsible for cardiac dysfunction in hLPL0 mice. In addition, our studies suggest that adaptations in cardiac metabolism contribute to the beneficial effects of exercise on the myocardium of patients with heart failure.

  11. The time point of β-catenin knockout in hepatocytes determines their response to xenobiotic activation of the constitutive androstane receptor

    International Nuclear Information System (INIS)

    Ganzenberg, Katrin; Singh, Yasmin; Braeuning, Albert

    2013-01-01

    The constitutive androstane receptor (CAR) controls the expression of drug-metabolizing enzymes and regulates hepatocyte proliferation. Studies with transgenic mice with an early postnatal conditional hepatocyte-specific knockout of the β-catenin gene Ctnnb1 revealed that β-catenin deficiency decreases the magnitude of induction of drug-metabolizing enzymes by CAR activators, abrogates zonal differences in the hepatocytes’ susceptibility to these compounds, and impacts on hepatocyte proliferation. These data, however, do not allow distinguishing between effects caused by β-catenin deficiency during postnatal liver development and acute effects of β-catenin deficiency in the adult animal at the time point of CAR activation. Therefore, CAR activation was now studied in a different mouse model allowing for the hepatocyte-specific knockout of β-catenin in adult mice. Treatment of these mice with 3 mg/kg body weight of the model CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) confirmed previous findings related to the coordinate regulation of drug metabolism by β-catenin and CAR. More importantly, the present study clarified that the impact of β-catenin signaling on CAR-mediated enzyme induction in the liver is not merely due to developmental defects caused by a postnatal lack of β-catenin, but depends on the presence of β-catenin at the time point of xenobiotic treatment. The study also revealed interesting differences between the two mouse models: hepatic zonation of TCPOBOP-dependent induction of drug-metabolizing enzymes was restored in mice with late knockout of β-catenin, and the strong proliferative response of female mice was exclusively abolished when using animals with a late β-catenin knockout. This suggests a β-catenin-dependent postnatal priming of hepatocytes during postnatal liver development, later affecting the proliferative response of adult animals to CAR-activating xenobiotics

  12. Green tea polyphenol treatment attenuates atherosclerosis in high-fat diet-fed apolipoprotein E-knockout mice via alleviating dyslipidemia and up-regulating autophagy.

    Directory of Open Access Journals (Sweden)

    Shibin Ding

    Full Text Available Green tea polyphenol (GTP is a polyphenol source from green tea that has drawn wide attention owing to epidemiological evidence of its beneficial effects in the prevention of cardiovascular disease; the underlying molecular mechanisms of these effects are not well understood. This study aimed to investigate the effects of GTP treatment on autophagy regulation in the vessel wall and lipid metabolism of HFD-fed male ApoE-knockout mice.Adult male ApoE-knockout mice (n = 30 fed with a high-fat diet (HFD were treated with either vehicle or GTP (3.2 or 6.4 g/L administered via drinking water for 15 weeks, and C57BL/6J mice fed with standard chow diet (STD were used as the control group. Metabolic parameters, expression of key mRNAs and proteins of hepatic lipid metabolism and autophagy in the vessel wall of mice were determined after the 15-week treatment.A HFD induced atherosclerosis formation and lipid metabolism disorders as well as reduced autophagy expression in the vessel wall of ApoE-knockout mice, but GTP treatment alleviated the lipid metabolism disorders, decreased the oxLDL levels in serum, and increased the mRNA and protein expressions of hepatic PPARα and autophagy markers (LC3, Beclin1 and p62 in the vessel wall of ApoE-knockout mice.Our findings suggest that GTP supplementation showed marked suppression of atherogenesis through improved lipid metabolism as well as through a direct impact on oxLDL and autophagy flux in the vessel wall.

  13. Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail to Display Hallmarks of Adult-Onset Type II Citrullinemia

    OpenAIRE

    Tsui, LC; Jalil, MA; Saheki, T; Kobayashi, K; Robinson, BH; Horiuchi, M; Iijima, M; Li, MX; Sinasac, DS; Begum, L; Moriyama, M

    2004-01-01

    Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resul...

  14. CLIP-GENE: a web service of the condition specific context-laid integrative analysis for gene prioritization in mouse TF knockout experiments.

    Science.gov (United States)

    Hur, Benjamin; Lim, Sangsoo; Chae, Heejoon; Seo, Seokjun; Lee, Sunwon; Kang, Jaewoo; Kim, Sun

    2016-10-24

    Transcriptome data from the gene knockout experiment in mouse is widely used to investigate functions of genes and relationship to phenotypes. When a gene is knocked out, it is important to identify which genes are affected by the knockout gene. Existing methods, including differentially expressed gene (DEG) methods, can be used for the analysis. However, existing methods require cutoff values to select candidate genes, which can produce either too many false positives or false negatives. This hurdle can be addressed either by improving the accuracy of gene selection or by providing a method to rank candidate genes effectively, or both. Prioritization of candidate genes should consider the goals or context of the knockout experiment. As of now, there are no tools designed for both selecting and prioritizing genes from the mouse knockout data. Hence, the necessity of a new tool arises. In this study, we present CLIP-GENE, a web service that selects gene markers by utilizing differentially expressed genes, mouse transcription factor (TF) network, and single nucleotide variant information. Then, protein-protein interaction network and literature information are utilized to find genes that are relevant to the phenotypic differences. One of the novel features is to allow researchers to specify their contexts or hypotheses in a set of keywords to rank genes according to the contexts that the user specify. We believe that CLIP-GENE will be useful in characterizing functions of TFs in mouse experiments. http://epigenomics.snu.ac.kr/CLIP-GENE REVIEWERS: This article was reviewed by Dr. Lee and Dr. Pongor.

  15. Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion.

    Science.gov (United States)

    Wang, Tzi-Yuan; Huang, Chih-Jen; Chen, Hsin-Liang; Ho, Po-Chun; Ke, Huei-Mien; Cho, Hsing-Yi; Ruan, Sz-Kai; Hung, Kuo-Yen; Wang, I-Li; Cai, Ya-Wun; Sung, Huang-Mo; Li, Wen-Hsiung; Shih, Ming-Che

    2013-09-03

    As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular

  16. Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

    OpenAIRE

    Singh, Jaspreet; Suhail, Hamid; Giri, Shailendra

    2015-01-01

    X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice)...

  17. Effect of Diet High in Coconut Oil on Cardiovascular Disease Risk in ApoE Knockout and Wild Type Mice (Mus musculus)

    Science.gov (United States)

    2016-04-07

    Objective: We evaluated the risk of cardiovascular disease in both control and proatherosclerotic mice consuming diets high in coconut oil. Methods...The mice were weighed and randomly assigned to receive a custom diet with either coconut oil or milk fat. Both diets were formulated to have the...significant differences were seen between knockout and wildtype mice in aorta score regardless of diet, and in liver score with coconut oil diet

  18. Similar Intracellular Peptide Profile of TAP1/beta 2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

    OpenAIRE

    Castro, Leandro Mantovani de [UNIFESP; Berti, Denise A.; Russo, Lilian C.; Coelho, Veronica; Gozzo, Fabio C.; Oliveira, Vitor [UNIFESP; Ferro, Emer Suavinho [UNIFESP

    2010-01-01

    Cells produce and use peptides in distinctive ways. in the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/beta 2m(-/-) (transporter associated with antigen-processing 1/beta 2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some p...

  19. Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice

    DEFF Research Database (Denmark)

    Bentzon, Jacob Fog; Weile, Charlotte; Sondergaard, Claus S

    2006-01-01

    Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE-/-) mice have concluded that a substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of hematopoietic origin. This pathway, however, remains controversial. In the present...... study, we reexamined the origin of plaque SMCs in apoE-/- mice by a series of BM transplantations and in a novel model of atherosclerosis induced in surgically transferred arterial segments....

  20. The Anti-Non-Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells is Encoded by a Restricted Number of Germline Progenitors1

    OpenAIRE

    Kiernan, Kathleen; Harnden, Ivan; Gunthart, Mirja; Gregory, Clare; Meisner, Jessica; Kearns-Jonker, Mary

    2008-01-01

    Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into non-human primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes der...

  1. Comparison of the hepatic and thyroid gland effects of sodium phenobarbital in wild type and constitutive androstane receptor (CAR) knockout rats and pregnenolone-16α-carbonitrile in wild type and pregnane X receptor (PXR) knockout rats.

    Science.gov (United States)

    Haines, Corinne; Chatham, Lynsey R; Vardy, Audrey; Elcombe, Clifford R; Foster, John R; Lake, Brian G

    2018-03-13

    A number of chemicals produce liver and thyroid gland tumours in rodents by nongenotoxic modes of action (MOAs). In this study the hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were examined in male Sprague-Dawley wild type (WT) rats and in CAR knockout (CAR KO) rats and the effects of the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in WT and PXR knockout (PXR KO) rats. Rats were either fed diets containing 0 (control) or 500 ppm NaPB or were dosed with 0 (control) or 100 mg/kg/day PCN orally for 7 days. The treatment of WT rats with NaPB and PCN for 7 days resulted in increased relative liver weight, increased hepatocyte replicative DNA synthesis (RDS) and the induction of cytochrome P450 CYP2B and CYP3A subfamily enzyme, mRNA and protein levels. In marked contrast, the treatment of CAR KO rats with NaPB and PXR KO rats with PCN did not result in any increases in liver weight and induction of CYP2B and CYP3A enzymes. The treatment of CAR KO rats with NaPB had no effect on hepatocyte RDS, while PCN produced only a small increase in hepatocyte RDS in PXR KO rats. Treatment with NaPB had no effect on thyroid gland weight in WT and CAR KO rats, whereas treatment with PCN resulted in an increase in relative thyroid gland weight in WT, but not in PXR KO, rats. Thyroid gland follicular cell RDS was increased by the treatment of WT rats with NaPB and PCN, with NaPB also producing a small increase in thyroid gland follicular cell RDS in CAR KO rats. Overall, the present study with CAR KO rats demonstrates that a functional CAR is required for NaPB-mediated increases in liver weight, stimulation of hepatocyte RDS and induction of hepatic CYP enzymes. The studies with PXR KO rats demonstrate that a functional PXR is required for PCN-mediated increases in liver weight and induction of hepatic CYP enzymes; with induction of hepatocyte RDS also being

  2. Comparison of the Tastes of L-Alanine and Monosodium Glutamate in C57BL/6J Wild Type and T1r3 Knockout Mice.

    Science.gov (United States)

    Eddy, Meghan C; Eschle, Benjamin K; Delay, Eugene R

    2017-09-01

    Previous research showed that L-alanine and monosodium L-glutamate elicit similar taste sensations in rats. This study reports the results of behavioral experiments designed to compare the taste capacity of C57BL/6J wild type and T1r3- mice for these 2 amino acids. In conditioned taste aversion (CTA) experiments, wild-type mice exhibited greater sensitivity than knockout mice for both L-amino acids, although knockout mice were clearly able to detect both amino acids at 50 mM and higher concentrations. Generalization of CTA between L-alanine and L-glutamate was bidirectionally equivalent for both mouse genotypes, indicating that both substances elicited similar tastes in both genotypes. This was verified by the discrimination experiments in which both mouse genotypes performed at or near chance levels at 75 and 150 mM. Above 150 mM, discrimination performance improved, suggesting the taste qualities of the 2 L-amino acids are not identical. No differences between knockout and wild-type mice in discrimination ability were detected. These results indicate that while the T1r3 receptor is important for tasting L-alanine and L-glutamate, other receptors are also important for tasting these amino acids. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Identification of gene knockout strategies using a hybrid of an ant colony optimization algorithm and flux balance analysis to optimize microbial strains.

    Science.gov (United States)

    Lu, Shi Jing; Salleh, Abdul Hakim Mohamed; Mohamad, Mohd Saberi; Deris, Safaai; Omatu, Sigeru; Yoshioka, Michifumi

    2014-09-28

    Reconstructions of genome-scale metabolic networks from different organisms have become popular in recent years. Metabolic engineering can simulate the reconstruction process to obtain desirable phenotypes. In previous studies, optimization algorithms have been implemented to identify the near-optimal sets of knockout genes for improving metabolite production. However, previous works contained premature convergence and the stop criteria were not clear for each case. Therefore, this study proposes an algorithm that is a hybrid of the ant colony optimization algorithm and flux balance analysis (ACOFBA) to predict near optimal sets of gene knockouts in an effort to maximize growth rates and the production of certain metabolites. Here, we present a case study that uses Baker's yeast, also known as Saccharomyces cerevisiae, as the model organism and target the rate of vanillin production for optimization. The results of this study are the growth rate of the model organism after gene deletion and a list of knockout genes. The ACOFBA algorithm was found to improve the yield of vanillin in terms of growth rate and production compared with the previous algorithms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Serotonin and urocortin 1 in the dorsal raphe and Edinger-Westphal nuclei after early life stress in serotonin transporter knockout rats.

    Science.gov (United States)

    van der Doelen, Rick H A; Robroch, Berit; Arnoldussen, Ilse A; Schulpen, Maya; Homberg, Judith R; Kozicz, Tamás

    2017-01-06

    The interaction of early life stress (ELS) and the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) has been associated with increased risk to develop depression in later life. We have used the maternal separation paradigm as a model for ELS exposure in homozygous and heterozygous 5-HTT knockout rats and measured urocortin 1 (Ucn1) mRNA and/or protein levels, Ucn1 DNA methylation, as well as 5-HT innervation in the centrally projecting Edinger-Westphal (EWcp) and dorsal raphe (DR) nuclei, both implicated in the regulation of stress response. We found that ELS and 5-HTT genotype increased the number of 5-HT neurons in specific DR subdivisions, and that 5-HTT knockout rats showed decreased 5-HT innervation of EWcp-Ucn1 neurons. Furthermore, ELS was associated with increased DNA methylation of the promoter region of the Ucn1 gene and increased expression of 5-HT receptor 1A in the EWcp. In contrast, 5-HTT deficiency was associated with site-specific alterations in DNA methylation of the Ucn1 promoter, and heterozygous 5-HTT knockout rats showed decreased expression of CRF receptor 1 in the EWcp. Together, our findings extend the existing literature on the relationship between EWcp-Ucn1 and DR-5-HT neurons. These observations will further our understanding on their potential contribution to mediate affect as a function of ELS interacting with 5-HTTLPR. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

    Science.gov (United States)

    Pang, Xiao-Yang; Cui, Wen-Ming; Liu, Lu; Zhang, Shu-Wen; Lv, Jia-Ping

    2014-01-01

    Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  6. Cardiomyocyte-specific conditional knockout of the histone chaperone HIRA in mice results in hypertrophy, sarcolemmal damage and focal replacement fibrosis

    Directory of Open Access Journals (Sweden)

    Nicolas Valenzuela

    2016-03-01

    Full Text Available HIRA is the histone chaperone responsible for replication-independent incorporation of histone variant H3.3 within gene bodies and regulatory regions of actively transcribed genes, and within the bivalent promoter regions of developmentally regulated genes. The HIRA gene lies within the 22q11.2 deletion syndrome critical region; individuals with this syndrome have multiple congenital heart defects. Because terminally differentiated cardiomyocytes have exited the cell cycle, histone variants should be utilized for the bulk of chromatin remodeling. Thus, HIRA is likely to play an important role in epigenetically defining the cardiac gene expression program. In this study, we determined the consequence of HIRA deficiency in cardiomyocytes in vivo by studying the phenotype of cardiomyocyte-specific Hira conditional-knockout mice. Loss of HIRA did not perturb heart development, but instead resulted in cardiomyocyte hypertrophy and susceptibility to sarcolemmal damage. Cardiomyocyte degeneration gave way to focal replacement fibrosis and impaired cardiac function. Gene expression was widely altered in Hira conditional-knockout hearts. Significantly affected pathways included responses to cellular stress, DNA repair and transcription. Consistent with heart failure, fetal cardiac genes were re-expressed in the Hira conditional knockout. Our results suggest that transcriptional regulation by HIRA is crucial for cardiomyocyte homeostasis.

  7. Knockout of the Arp2/3 complex in epidermis causes a psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2.

    Science.gov (United States)

    van der Kammen, Rob; Song, Ji-Ying; de Rink, Iris; Janssen, Hans; Madonna, Stefania; Scarponi, Claudia; Albanesi, Cristina; Brugman, Wim; Innocenti, Metello

    2017-12-15

    The Arp2/3 complex assembles branched actin filaments, which are key to many cellular processes, but its organismal roles remain poorly understood. Here, we employed conditional A rpc4 knockout mice to study the function of the Arp2/3 complex in the epidermis. We found that depletion of the Arp2/3 complex by knockout of Arpc4 results in skin abnormalities at birth that evolve into a severe psoriasis-like disease hallmarked by hyperactivation of transcription factor Nrf2. Knockout of Arpc4 in cultured keratinocytes was sufficient to induce nuclear accumulation of Nrf2, upregulation of Nrf2 target genes and decreased filamentous actin levels. Furthermore, pharmacological inhibition of the Arp2/3 complex unmasked the role of branched actin filaments in Nrf2 regulation. Consistent with this, we revealed that Nrf2 associates with the actin cytoskeleton in cells and binds to filamentous actin in vitro Finally, we discovered that Arpc4 is downregulated in both human and mouse psoriatic epidermis. Thus, the Arp2/3 complex affects keratinocyte shape and transcriptome through an actin-based cell-autonomous mechanism that influences epidermal morphogenesis and homeostasis. © 2017. Published by The Company of Biologists Ltd.

  8. A selective histone deacetylase-6 inhibitor improves BDNF trafficking in hippocampal neurons from Mecp2 knockout mice:implications for Rett syndrome

    Directory of Open Access Journals (Sweden)

    Xin eXu

    2014-03-01

    Full Text Available Rett syndrome (RTT is a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2. One of the most prominent gene targets of MeCP2 is brain-derived neurotrophic factor (Bdnf, a potent modulator of activity-dependent synaptic development, function and plasticity. Dysfunctional BDNF signaling has been demonstrated in several pathophysiological mechanisms of RTT disease progression. To evaluate whether the dynamics of BDNF trafficking is affected by Mecp2 deletion, we analyzed movements of BDNF tagged with yellow fluorescent protein (YFP in cultured hippocampal neurons by time-lapse fluorescence imaging. We found that both anterograde and retrograde vesicular trafficking of BDNF-YFP are significantly impaired in Mecp2 knockout hippocampal neurons. Selective inhibitors of histone deacetylase 6 (HDAC6 show neuroprotective effects in neurodegenerative diseases and stimulate microtubule-dependent vesicular trafficking of BDNF-containing dense core vesicles. Here, we show that the selective HDAC6 inhibitor Tubastatin-A increased the velocity of BDNF-YFP vesicles in Mecp2 knockout neurons in both directions by increasing αtubulin acetylation. Tubastatin-A also restored activity-dependent BDNF release from Mecp2 knockout neurons to levels comparable to those shown by wildtype neurons. These findings demonstrate that a selective HDAC6 inhibitor is a potential pharmacological strategy to reverse cellular and synaptic impairments in RTT resulting from impaired BDNF signaling.

  9. Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Pang

    Full Text Available Autolysis of lactic acid bacteria (LAB plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur, was cloned and sequenced (GenBank accession number: KF157911. Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

  10. Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects.

    Science.gov (United States)

    McKinley, Kara L; Cheeseman, Iain M

    2017-02-27

    Defining the genes that are essential for cellular proliferation is critical for understanding organismal development and identifying high-value targets for disease therapies. However, the requirements for cell-cycle progression in human cells remain incompletely understood. To elucidate the consequences of acute and chronic elimination of cell-cycle proteins, we generated and characterized inducible CRISPR/Cas9 knockout human cell lines targeting 209 genes involved in diverse cell-cycle processes. We performed single-cell microscopic analyses to systematically establish the effects of the knockouts on subcellular architecture. To define variations in cell-cycle requirements between cultured cell lines, we generated knockouts across cell lines of diverse origins. We demonstrate that p53 modulates the phenotype of specific cell-cycle defects through distinct mechanisms, depending on the defect. This work provides a resource to broadly facilitate robust and long-term depletion of cell-cycle proteins and reveals insights into the requirements for cell-cycle progression. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Modeling and simulation of the main metabolism in Escherichia coli and its several single-gene knockout mutants with experimental verification

    Directory of Open Access Journals (Sweden)

    McFadden Johnjoe

    2010-11-01

    Full Text Available Abstract Background It is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth. Results In the present study, we considered the simulation of the main metabolic pathways such as glycolysis, TCA cycle, pentose phosphate (PP pathway, and the anapleorotic pathways using enzymatic reaction models of E. coli. Once intracellular fluxes were computed by this model, the specific ATP production rate, the specific CO2 production rate, and the specific NADPH production rate could be estimated. The specific ATP production rate thus computed was used for the estimation of the specific growth rate. The CO2 production rate could be used to estimate cell yield, and the specific NADPH production rate could be used to determine the flux of the oxidative PP pathway. The batch and continuous cultivations were simulated where the changing patterns of extracellular and intra-cellular metabolite concentrations were compared with experimental data. Moreover, the effects of the knockout of such pathways as Ppc, Pck and Pyk on the metabolism were simulated. It was shown to be difficult for the cell to grow in Ppc mutant due to low concentration of OAA, while Pck mutant does not necessarily show this phenomenon. The slower growth rate of the Ppc mutant was properly

  12. Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse

    Directory of Open Access Journals (Sweden)

    Soper Jessica

    2007-07-01

    Full Text Available Abstract Background Preterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition. Methods Gestational d18.0 uteri (n = 4 were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization. Results We identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri. Conclusion To our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and

  13. Evaluation of organ-specific glucose metabolism by 18F-FDG in insulin receptor substrate-1 (IRS-1) knockout mice as a model of insulin resistance

    International Nuclear Information System (INIS)

    Cheng, Chao; Nakamura, Akinobu; Minamimoto, Ryogo; Shinoda, Kazuaki; Tateishi, Ukihide; Terauchi, Yasuo; Inoue, Tomio; Goto, Atsuhi; Kadowaki, Takashi

    2011-01-01

    Insulin resistance (IR) is a physiological condition in which the body produces insulin but does not result in a sufficient biological effect. Insulin resistance is usually asymptomatic but is associated with health problems and is a factor in the metabolic syndrome. The aim of the present study is to clarify organ-specific insulin resistance in normal daily conditions using [ 18 F]-2-fluoro-2-deoxy-D-glucose ([ 18 F]-FDG). The biodistribution of [ 18 F]-FDG was examined in insulin receptor substrate-1 (IRS-1) knockout mice, an animal model of skeletal muscle insulin resistance, and C57BL/6J (wild-type) mice with and without insulin loading. Mice received 0.5 MBq of [ 18 F]-FDG injected into the tail vein, immediately followed by nothing (control cohorts) or an intraperitoneal injection of 1.5 mU/g body weight of human insulin as an insulin loading test. Blood glucose concentrations for all of the experimental animals were assessed at 0, 20, 40, and 60 min post-injection. The mice were subsequently killed, and tissue was collected for evaluation of [ 18 F]-FDG biodistribution. The radioactivity of each organ was measured using a gamma counter. In the absence of insulin, the blood glucose concentrations of wild-type mice (132±26 mg/dl) and IRS-1 knockout mice (134±18 mg/dl) were not significantly different. Blood glucose concentrations decreased following insulin administration, with lower concentrations in wild-type mice than in knockout mice at 20, 40, and 60 min. A statistically significant difference in [ 18 F]-FDG uptake between wild-type mice and IRS-1 knockout mice was confirmed in the heart, abdominal muscle, and femoral muscle. With insulin loading, [ 18 F]-FDG uptake in the heart, back muscle, and abdominal muscle was significantly increased compared to without insulin loading in both wild-type mice and knockout mice. Our results showed that IR significantly affected [ 18 F]-FDG uptake in the heart in normal daily conditions. IR was associated with

  14. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    Science.gov (United States)

    Cubells, Joseph F; Schroeder, Jason P; Barrie, Elizabeth S; Manvich, Daniel F; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A; Liles, L Cameron; Squires, Katherine E; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency

  15. Human Bacterial Artificial Chromosome (BAC Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Joseph F Cubells

    Full Text Available Dopamine β-hydroxylase (DBH converts dopamine (DA to norepinephrine (NE in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/- mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP in the human DBH gene promoter (-970C>T; rs1611115 is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS, which can be converted to NE by aromatic acid decarboxylase (AADC in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh

  16. Mice with conditional NeuroD1 knockout display reduced aberrant hippocampal neurogenesis but no change in epileptic seizures.

    Science.gov (United States)

    Brulet, Rebecca; Zhu, Jingfei; Aktar, Mahafuza; Hsieh, Jenny; Cho, Kyung-Ok

    2017-07-01

    Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroD1 (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroD1+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroD1 in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroD1+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroD1 cKO mice, suggesting that NeuroD1 cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Prox1+ hilar ectopic granule cells were unchanged after NeuroD1 cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons. Published by Elsevier Inc.

  17. Spatial learning in the 5-HT1B receptor knockout mouse: selective facilitation/impairment depending on the cognitive demand.

    Science.gov (United States)

    Buhot, Marie-Christine; Wolff, Mathieu; Benhassine, Narimane; Costet, Pierre; Hen, René; Segu, Louis

    2003-01-01

    Age-related memory decline is associated with a combined dysfunction of the cholinergic and serotonergic systems in the hippocampus and frontal cortex, in particular. The 5-HT1B receptor occupies strategic cellular and subcellular locations in these structures, where it plays a role in the modulation of ACh release. In an attempt to characterize the contribution of this receptor to memory functions, 5-HT1B receptor knockout (KO) mice were submitted to various behavioral paradigms carried out in the same experimental context (water maze), which were aimed at exposing mice to various levels of memory demand. 5-HT1BKO mice exhibited a facilitation in the acquisition of a hippocampal-dependent spatial reference memory task in the Morris water maze. This facilitation was selective of task difficulty, showing thus that the genetic inactivation of the 5-HT1B receptor is associated with facilitation when the complexity of the task is increased, and reveals a protective effect on age-related hippocampal-dependent memory decline. Young-adult and aged KO and wild-type (WT) mice were equally able to learn a delayed spatial matching-to-sample working memory task in a radial-arm water maze with short (0 or 5 min) delays. However, 5-HT1BKO mice, only, exhibited a selective memory impairment at intermediate and long (15, 30, and 60 min) delays. Treatment by scopolamine induced the same pattern of performance in wild type as did the mutation for short (5 min, no impairment) and long (60 min, impairment) delays. Taken together, these studies revealed a beneficial effect of the mutation on the acquisition of a spatial reference memory task, but a deleterious effect on a working memory task for long delays. This 5-HT1BKO mouse story highlights the problem of the potential existence of "global memory enhancers."

  18. Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level

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    Wang Jia

    2009-11-01

    Full Text Available Abstract Background Protein kinase C interacting protein (PKCI/HINT1 is a small protein belonging to the histidine triad (HIT family proteins. Its brain immunoreactivity is located in neurons and neuronal processes. PKCI/HINT1 gene knockout (KO mice display hyper-locomotion in response to D-amphetamine which is considered a positive symptom of schizophrenia in animal models. Postmortem studies identified PKCI/HINT1 as a candidate molecule for schizophrenia and bipolar disorder. We investigated the hypothesis that the PKCI/HINT1 gene may play an important role in regulating mood function in the CNS. We submitted PKCI/HINT1 KO mice and their wild type (WT littermates to behavioral tests used to study anti-depressant, anxiety like behaviors, and goal-oriented behavior. Additionally, as many mood disorders coincide with modifications of hypothalamic-pituitary-adrenal (HPA axis function, we assessed the HPA activity through measurement of plasma corticosterone levels. Results Compared to the WT controls, KO mice exhibited less immobility in the forced swim (FST and the tail suspension (TST tests. Activity in the TST tended to be attenuated by acute treatment with valproate at 300 mg/kg in KO mice. The PKCI/HINT1 KO mice presented less thigmotaxis in the Morris water maze and spent progressively more time in the lit compartment in the light/dark test. In a place navigation task, KO mice exhibited enhanced acquisition and retention. Furthermore, the afternoon basal plasma corticosterone level in PKCI/HINT1 KO mice was significantly higher than in the WT. Conclusion PKCI/HINT1 KO mice displayed a phenotype of behavioral and endocrine features which indicate changes of mood function, including anxiolytic-like and anti-depressant like behaviors, in conjunction with an elevated corticosterone level in plasma. These results suggest that the PKCI/HINT 1 gene could be important for the mood regulation function in the CNS.

  19. Catalase can protect spermatozoa of FSH receptor knock-out mice against oxidant-induced DNA damage in vitro.

    Science.gov (United States)

    Libman, J; Gabriel, M S; Sairam, M R; Zini, A

    2010-12-01

    The aetiology of sperm DNA damage is likely multi-factorial with abnormal compaction of nuclear DNA, abortive apoptosis and oxidative stress implicated as potential causes of DNA damage. The objective of this study was to evaluate DNA damage in spermatozoa from wild-type (WT) and FSH receptor knock-out (FORKO) mice, compare the relative susceptibility of spermatozoa from these animals to oxidative DNA damage, and examine the protective effect of the antioxidant catalase on sperm DNA damage. Epididymal spermatozoa from FORKO mice (n = 5) and WT controls (n = 5) were extracted and incubated with or without catalase. Sperm DNA damage was assessed immediately after epididymal extraction (time 0 control) and following 2-h incubation at 37 °C. DNA damage was measured by the sperm chromatin structure assay and the results expressed as the %DNA fragmentation index or %DFI. Freshly retrieved epididymal spermatozoa from WT mice had a significantly lower mean (±SD) %DFI than that of FORKO mice (2.7 ± 1.8 vs. 6.4 ± 2.9%, p catalase protected these spermatozoa from DNA damage (9.8 ± 4.1 vs. 17.9 ± 9.2%, respectively, p 0.05) and the addition of catalase (vs. no catalase) did not result in a significant reduction in %DFI (5.8 ± 5.0 vs. 7.7 ± 6.5%, respectively, p > 0.05). These data indicate that catalase may protect sperm nuclear DNA from oxidative stress in vitro. The data also demonstrate the differential susceptibility of WT and FORKO mice spermatozoa to oxidative stress. © 2010 The Authors. International Journal of Andrology © 2010 European Academy of Andrology.

  20. Effects of High-Fat Diet on Stress Response in Male and Female Wildtype and Prolactin Knockout Mice.

    Science.gov (United States)

    Kalyani, Manu; Hasselfeld, Kathryn; Janik, James M; Callahan, Phyllis; Shi, Haifei

    2016-01-01

    Prolactin (PRL) is well characterized for its roles in initiation and maintenance of lactation, and it also suppresses stress-induced responses. Feeding a high-fat diet (HFD) disrupts activity of the hypothalamic-pituitary-adrenal (HPA) axis. Whether PRL regulates HPA axis activation under HFD feeding is not clear. Male and female wildtype (WT) and PRL knockout (KO) mice were fed either a standard low-fat diet (LFD) or HFD for 12 weeks. Circulating corticosterone (CORT) levels were measured before, during, and after mice were subjected to an acute restraint stress or remained in their home cages as no stress controls. HFD feeding increased leptin levels, but the increase was lower in KO than in WT mice. All stressed female groups and only LFD-fed stressed males had elevated CORT levels compared to their no stress same-sex counterparts regardless of genotype. These results indicated that HFD consumption blunted the HPA axis response to acute stress in males but not females. Additionally, basal hypothalamic CRH content was lower in HFD than LFD males, but was similar among female groups. Furthermore, although basal CORT levels were similar among KO and WT groups, CORT levels were higher in KO mice than their WT counterparts during stress, suggesting that loss of PRL led to greater HPA axis activation. Basal PRL receptor mRNA levels in the choroid plexus were higher in HFD than LFD same-sex counterparts, suggesting activation of central PRL's action by HFD feeding in both males and females. Current results confirmed PRL's roles in suppression of the stress-induced HPA axis activation. Although HFD feeding activated central PRL's action in both sexes, only the male HPA axis was dampened by HFD feeding.

  1. Orexin/Hypocretin and Histamine: Distinct Roles in the Control of Wakefulness Demonstrated Using Knock-Out Mouse Models

    Science.gov (United States)

    Anaclet, Christelle; Parmentier, Régis; Ouk, Koliane; Guidon, Gérard; Buda, Colette; Sastre, Jean-Pierre; Akaoka, Hidéo; Sergeeva, Olga A.; Yanagisawa, Masashi; Ohtsu, Hiroshi; Franco, Patricia; Haas, Helmut L.; Lin, Jian Sheng

    2009-01-01

    To determine the respective role played by orexin/hypocretin and histamine (HA) neurons in maintaining wakefulness (W), we characterized the behavioral and sleep-wake phenotypes of orexin(Ox) knockout(−/−) mice and compared them with those of histidine-decarboxylase(HDC, HA-synthesizing enzyme)−/−mice. While both mouse strains displayed sleep fragmentation and increased paradoxical sleep(PS), they presented a number of marked differences: 1) The PS-increase in HDC−/−mice was seen during lightness, whereas that in Ox−/−mice occurred during darkness; 2) Contrary to HDC−/−, Ox−/−mice had no W deficiency around lights-off, nor an abnormal EEG and responded to a new environment with increased W; 3) Only Ox−/−, but not HDC−/−mice, displayed narcolepsy and deficient W when faced with motor challenge. Thus, when placed on a wheel, WT, but not littermate Ox−/−mice, voluntarily spent their time in turning it and as a result, remained highly awake; this was accompanied by dense c-fos expression in many areas of their brains, including Ox-neurons in the dorsolateral hypothalamus. The W and motor deficiency of Ox−/−mice was due to the absence of Ox because intraventricular dosing of Ox-A restored their W amount and motor performance whereas SB-334867 (Ox1-receptor antagonist, i.p.) impaired W and locomotion of WT mice during the test. These data indicate that Ox, but not HA, promotes W through enhanced locomotion and suggest that HA and Ox neurons exert a distinct, but complementary and synergistic control of W: the neuropeptide being more involved in its behavioral aspects, whereas the amine is mainly responsible for its qualitative cognitive aspects and cortical-EEG activation. PMID:19923277

  2. Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs as a model of PTEN deficiency disease.

    Directory of Open Access Journals (Sweden)

    Yuriko Matsuzaki

    Full Text Available Phosphatase and tensin homolog (PTEN is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K-AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator-like effector nuclease (TALEN technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

  3. The Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Protects against Dyslipidemia-Related Kidney Injury in Apolipoprotein E Knockout Mice

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    Jingjing Li

    2014-06-01

    Full Text Available The goal of this study was to investigate the possible protective effects of sitagliptin against dyslipidemia-related kidney injury in apolipoprotein E knockout (apoE−/− mice. Eight-week-old male apoE−/− mice were randomized to receive either a high fat diet (HFD, apoE−/− group or HFD mixed with sitagliptin (sita + apoE−/− group for 16 weeks. A control group of age- and gender-matched C57BL/6J mice were fed a HFD. The apoE−/− group exhibited increases in body weight and serum lipid levels in addition to high-density lipoprotein, and increases in 24-h urinary 8-hydroxy-2-deoxyguanosine and albuminuria excretion. Decreased insulin sensitivity was also observed in the apoE−/− group. These mice additionally contained enlargements of the glomerular mesangial matrix area, lipid deposition area, and renal interstitium collagen area. The apoE−/− group also demonstrated down-regulation of phosphorylated AMP-activated protein kinase (AMPK, increases in renal mRNA expression of transforming growth factor-beta 1 (TGF-β1 and fibronectin (FN, and increased protein expression of Akt, TGF-β1, FN and p38/ERK mitogen-activated protein kinase (MAPK. Sitagliptin treatment successfully ameliorated all the deleterious effects of dyslipidemia tested. To our knowledge, this is the first time that sitagliptin has been shown to reverse the renal dysfunction and structural damage induced by dyslipidemia in apoE−/− mice. Our results suggest that the renoprotective mechanism of sitagliptin may be due to a reduction in Akt levels, a restoration of AMPK activity, and inhibition of TGF-β1, FN, and p38/ERK MAPK signaling pathways.

  4. Aged neuronal nitric oxide knockout mice show preserved olfactory learning in bo