Lymphocyte signaling: beyond knockouts.

Science.gov (United States)

Saveliev, Alexander; Tybulewicz, Victor L J

2009-04-01

The analysis of lymphocyte signaling was greatly enhanced by the advent of gene targeting, which allows the selective inactivation of a single gene. Although this gene 'knockout' approach is often informative, in many cases, the phenotype resulting from gene ablation might not provide a complete picture of the function of the corresponding protein. If a protein has multiple functions within a single or several signaling pathways, or stabilizes other proteins in a complex, the phenotypic consequences of a gene knockout may manifest as a combination of several different perturbations. In these cases, gene targeting to 'knock in' subtle point mutations might provide more accurate insight into protein function. However, to be informative, such mutations must be carefully based on structural and biophysical data.

Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

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Sakudo, Akikazu; Onodera, Takashi

2015-01-01

Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout (Prnp−/−) mice. So far, six types of Prnp−/− mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not fully reveal PrP functions. Recently, varieties of Prnp−/− cell lines established from Prnp−/− mice have contributed to the analysis of PrP functions. In this mini-review, we focus on Prnp−/− cell lines and summarize currently available Prnp−/− cell lines and their characterizations. In addition, we introduce the recent advances in the methodology of cell line generation with knockout or knockdown of the PrP gene. We also discuss how these cell lines have provided valuable insights into PrP functions and show future perspectives. PMID:25642423

An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.

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Xie, Yifang; Wang, Daqi; Lan, Feng; Wei, Gang; Ni, Ting; Chai, Renjie; Liu, Dong; Hu, Shijun; Li, Mingqing; Li, Dajin; Wang, Hongyan; Wang, Yongming

2017-05-24

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

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Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation.

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Yani Zhang

Full Text Available An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs into spermatogonial stem cells(SSCs. Three guides RNA (gRNAs were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1 and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40 in DF-1 cells, the knockdown efficiency was 23% (9/40 in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25. Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken's cells and inhibit ECSs differentiation into SSCs.

Adeno-associated virus LPL(S447X) gene therapy in LDL receptor knockout mice

NARCIS (Netherlands)

Rip, Jaap; Sierts, Jeroen A.; Vaessen, Stefan F. C.; Kastelein, John J. P.; Twisk, Jaap; Kuivenhoven, Jan Albert

2007-01-01

BACKGROUND: Overexpression of lipoprotein lipase (LPL) protects against atherosclerosis in genetically engineered mice. We tested whether a gene therapy vector that delivers human (h) LPL(S447X) cDNA to skeletal muscle could induce similar effects. METHODS: LDL receptor knockout (LDLr-/-) mice were

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

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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.

Prion protein (PrP) gene-knockout cell lines: insight into functions of the PrP

OpenAIRE

Sakudo, Akikazu; Onodera, Takashi

2015-01-01

Elucidation of prion protein (PrP) functions is crucial to fully understand prion diseases. A major approach to studying PrP functions is the use of PrP gene-knockout (Prnp ?/?) mice. So far, six types of Prnp ?/? mice have been generated, demonstrating the promiscuous functions of PrP. Recently, other PrP family members, such as Doppel and Shadoo, have been found. However, information obtained from comparative studies of structural and functional analyses of these PrP family proteins do not ...

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

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Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

DEFF Research Database (Denmark)

Jørgensen, Sebastian Beck; Wojtaszewski, Jørgen; Viollet, Benoit

2005-01-01

We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body a2- and a1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmi...

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

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Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

2017-11-16

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

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

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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.

Random phenotypic variation of yeast (Saccharomyces cerevisiae) single-gene knockouts fits a double pareto-lognormal distribution.

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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

Functional categorization of gene expression changes in the cerebellum of a Cln3-knockout mouse model for Batten disease.

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Brooks, Andrew I; Chattopadhyay, Subrata; Mitchison, Hannah M; Nussbaum, Robert L; Pearce, David A

2003-01-01

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is the most common progressive neurodegenerative disorder of childhood. The disease is inherited in an autosomal recessive manner and is the result of mutations in the CLN3 gene. One brain region severely affected in Batten disease is the cerebellum. Using a mouse model for Batten disease which shares pathological similarities to the disease in humans we have used oligonucleotide arrays to profile approximately 19000 mRNAs in the cerebellum. We have identified reproducible changes of twofold or more in the expression of 756 gene products in the cerebellum of 10-week-old Cln3-knockout mice as compared to wild-type controls. We have subsequently divided these genes with altered expression into 14 functional categories. We report a significant alteration in expression of genes associated with neurotransmission, neuronal cell structure and development, immune response and inflammation, and lipid metabolism. An apparent shift in metabolism toward gluconeogenesis is also evident in Cln3-knockout mice. Further experimentation will be necessary to understand the contribution of these changes in expression to a disease state. Detailed analysis of the functional consequences of altered expression of genes in the cerebellum of the Cln3-knockout mice may provide valuable clues in understanding the molecular basis of the pathological mechanisms underlying Batten disease.

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

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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.

Claudin-2 knockout by TALEN-mediated gene targeting in MDCK cells: claudin-2 independently determines the leaky property of tight junctions in MDCK cells.

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Shinsaku Tokuda

Full Text Available Tight junctions (TJs regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK II cells (a low-resistance strain of MDCK cells was shown to induce a three-fold increase in transepithelial electrical resistance (TER, which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs, a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000-4000 Ω·cm2 were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

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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.

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.

Deconstructing mammalian reproduction: using knockouts to define fertility pathways.

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Roy, Angshumoy; Matzuk, Martin M

2006-02-01

Reproduction is the sine qua non for the propagation of species and continuation of life. It is a complex biological process that is regulated by multiple factors during the reproductive life of an organism. Over the past decade, the molecular mechanisms regulating reproduction in mammals have been rapidly unraveled by the study of a vast number of mouse gene knockouts with impaired fertility. The use of reverse genetics to generate null mutants in mice through targeted disruption of specific genes has enabled researchers to identify essential regulators of spermatogenesis and oogenesis in vivo and model human disorders affecting reproduction. This review focuses on the merits, utility, and the variations of the knockout technology in studies of reproduction in mammals.

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

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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.

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

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.

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.

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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

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.

Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.

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Liu, Zhongliang; Hui, Yi; Shi, Lei; Chen, Zhenyu; Xu, Xiangjie; Chi, Liankai; Fan, Beibei; Fang, Yujiang; Liu, Yang; Ma, Lin; Wang, Yiran; Xiao, Lei; Zhang, Quanbin; Jin, Guohua; Liu, Ling; Zhang, Xiaoqing

2016-09-13

Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

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Zhenshan Wang

2015-11-01

Full Text Available Adenylyl Cyclase 3 (AC3 plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE. In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/− and wild-type (AC3+/+ mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

Disease Model Discovery from 3,328 Gene Knockouts by The International Mouse Phenotyping Consortium

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Meehan, Terrence F.; Conte, Nathalie; West, David B.; Jacobsen, Julius O.; Mason, Jeremy; Warren, Jonathan; Chen, Chao-Kung; Tudose, Ilinca; Relac, Mike; Matthews, Peter; Karp, Natasha; Santos, Luis; Fiegel, Tanja; Ring, Natalie; Westerberg, Henrik; Greenaway, Simon; Sneddon, Duncan; Morgan, Hugh; Codner, Gemma F; Stewart, Michelle E; Brown, James; Horner, Neil; Haendel, Melissa; Washington, Nicole; Mungall, Christopher J.; Reynolds, Corey L; Gallegos, Juan; Gailus-Durner, Valerie; Sorg, Tania; Pavlovic, Guillaume; Bower, Lynette R; Moore, Mark; Morse, Iva; Gao, Xiang; Tocchini-Valentini, Glauco P; Obata, Yuichi; Cho, Soo Young; Seong, Je Kyung; Seavitt, John; Beaudet, Arthur L.; Dickinson, Mary E.; Herault, Yann; Wurst, Wolfgang; de Angelis, Martin Hrabe; Lloyd, K.C. Kent; Flenniken, Ann M; Nutter, Lauryl MJ; Newbigging, Susan; McKerlie, Colin; Justice, Monica J.; Murray, Stephen A.; Svenson, Karen L.; Braun, Robert E.; White, Jacqueline K.; Bradley, Allan; Flicek, Paul; Wells, Sara; Skarnes, William C.; Adams, David J.; Parkinson, Helen; Mallon, Ann-Marie; Brown, Steve D.M.; Smedley, Damian

2017-01-01

Although next generation sequencing has revolutionised the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by our lack of knowledge of function and pathobiological mechanism for most genes. To address this challenge, the International Mouse Phenotyping Consortium (IMPC) is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout mouse strains across diverse biological systems through a broad set of standardised phenotyping tests, with all mice made readily available to the biomedical community. Analysing the first 3328 genes reveals models for 360 diseases including the first for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations are novel, providing the first functional evidence for 1092 genes and candidates in unsolved diseases such as Arrhythmogenic Right Ventricular Dysplasia 3. Finally, we describe our role in variant functional validation with the 100,000 Genomes and other projects. PMID:28650483

Generation of Newly Discovered Resistance Gene mcr-1 Knockout in Escherichia coli Using the CRISPR/Cas9 System.

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Sun, Lichang; He, Tao; Zhang, Lili; Pang, Maoda; Zhang, Qiaoyan; Zhou, Yan; Bao, Hongduo; Wang, Ran

2017-07-28

The mcr-1 gene is a new "superbug" gene discoverd in China in 2016 that makes bacteria highly resistant to the last-resort class of antibiotics. The mcr-1 gene raised serious concern about its possible global dissemination and spread. Here, we report a potential anti-resistant strategy using the CRISPR/Cas9-mediated approach that can efficiently induce mcr-1 gene knockout in Escherichia coli . Our findings suggested that using the CRISPR/Cas9 system to knock out the resistance gene mcr-1 might be a potential anti-resistant strategy. Bovine myeloid antimicrobial peptide-27 could help deliver plasmid pCas::mcr targeting specific DNA sequences of the mcr-1 gene into microbial populations.

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

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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.

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

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Song, Jun; Wang, Guoshun; Hoenerhoff, Mark J.; Ruan, Jinxue; Yang, Dongshan; Zhang, Jifeng; Yang, Jibing; Lester, Patrick A.; Sigler, Robert; Bradley, Michael; Eckley, Samantha; Cornelius, Kelsey; Chen, Kong; Kolls, Jay K.; Peng, Li; Ma, Liang; Chen, Yuqing Eugene; Sun, Fei; Xu, Jie

2018-01-01

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. PMID:29593714

Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

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Hosomi, Koji; Kuwana, Ritsuko; Takamatsu, Hiromu; Kohda, Tomoko; Kozaki, Shunji; Mukamoto, Masafumi

2015-06-01

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

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.

Enhanced genome editing tools for multi-gene deletion knock-out approaches using paired CRISPR sgRNAs in CHO cells

DEFF Research Database (Denmark)

Schmieder, Valerie; Bydlinski, Nina; Strasser, Richard

2017-01-01

(sgRNAs) for full gene deletions. This strategy also enables the targeting of regulatory regions, which would not respond to the conventional frameshift mutations, as shown by deleting the α-1,6-Fucosyltransferase 8 (FUT8) promoter resulting in a functional knock-out. Fut8 also served as model...

Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua.

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Zuo, Y-Y; Huang, J-L; Wang, J; Feng, Y; Han, T-T; Wu, Y-D; Yang, Y-H

2018-02-01

P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta-cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua. © 2017 The Royal Entomological Society.

CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.

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Minyan Li

Full Text Available Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2 is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo.

Gene knockout of the KCNJ8-encoded Kir6.1 K(ATP) channel imparts fatal susceptibility to endotoxemia.

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Kane, Garvan C; Lam, Chen-Fuh; O'Cochlain, Fearghas; Hodgson, Denice M; Reyes, Santiago; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Katusic, Zvonimir S; Terzic, Andre

2006-11-01

Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K(ATP) channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress-responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine- or metabolic-mediated vasodilation necessary to support myocardial perfusion in the wild-type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K(ATP) channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1-containing K(ATP) channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.

Generation of knockout rabbits using transcription activator-like effector nucleases

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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.

Efficient gene knock-out and knock-in with transgenic Cas9 in Drosophila.

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Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S; Gao, Guanjun

2014-03-21

Bacterial Cas9 nuclease induces site-specific DNA breaks using small gRNA as guides. Cas9 has been successfully introduced into Drosophila for genome editing. Here, we improve the versatility of this method by developing a transgenic system that expresses Cas9 in the Drosophila germline. Using this system, we induced inheritable knock-out mutations by injecting only the gRNA into embryos, achieved highly efficient mutagenesis by expressing gRNA from the promoter of a novel non-coding RNA gene, and recovered homologous recombination-based knock-in of a fluorescent marker at a rate of 4.5% by co-injecting gRNA with a circular DNA donor. Copyright © 2014 Xue et al.

Survey of quasi-free cluster knockout

International Nuclear Information System (INIS)

Roos, P.G.; Chant, N.S.

1975-01-01

The investigation of quasi-free knockout reactions has been proceeding for many years now, since the first experiments studying (p,2p) reactions on light nuclei. These experiments clearly showed the dominance of quasi-free proton knockout, and have provided information on the proton holes states in nuclei. From very early in the game people extended these studies to the knock-out of clusters, in an attempt to obtain nuclear structure information about clustering in nuclei. These cluster knockout reactions, excluding the nucleon knockout work, are examined. 20 figures, 16 references

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting

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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.

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.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice

International Nuclear Information System (INIS)

Chen, Mo; Wang, Qin; Zhu, Gang-Hua; Hu, Peng; Zhou, Yuan; Wang, Tian; Lai, Ruo-Sha; Xiao, Zi-An; Xie, Ding-Hua

2016-01-01

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN +/− mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN −/− mice exhibited progressive sensorineural hearing loss as reflected by auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN −/− mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene. - Highlights: • TPRN −/− mice were generated using TALEN technique. • TPRN −/− mice presented progressive hearing loss. • WT and TPRN −/− mice showed no difference in hair cell numbers. • TPRN −/− mice showed progressive degeneration of hair cell stereocilia.

The Impact of Oxytocin Gene Knockout on Sexual Behavior and Gene Expression Related to Neuroendocrine Systems in the Brain of Female Mice.

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Zimmermann-Peruzatto, Josi Maria; Lazzari, Virgínia Meneghini; Agnes, Grasiela; Becker, Roberta Oriques; de Moura, Ana Carolina; Guedes, Renata Padilha; Lucion, Aldo Bolten; Almeida, Silvana; Giovenardi, Márcia

2017-07-01

Social relations are built and maintained from the interaction among individuals. The oxytocin (OT), vasopressin (VP), estrogen, dopamine, and their receptors are involved in the modulation of sexual behavior in females. This study aimed to analyze the impact of OT gene knockout (OTKO) on sexual behavior and the gene expression of oxytocin (OTR), estrogen alpha (ERα), estrogen beta (ERβ), vasopressin (V 1a R), and dopamine (D 2 R) receptors in the olfactory bulb (OB), prefrontal cortex (PFC), hippocampus (HPC), and hypothalamus (HPT), as well as in the synthesis of VP in the HPT of female mice. Wild-type (WT) littermates were used for comparisons. The C DNAs were synthesized by polymerase chain reaction and the gene expression was calculated with the 2 -ΔΔCt formula. Our results showed that the absence of OT caused an increase in the frequency and duration of non-receptive postures and a decrease in receptive postures in the OTKO. OTKO females showed a significant decrease in the gene expression of OTR in the HPC, V 1a R in the HPT, and ERα and ERβ in the PFC. There was no significant difference in the gene expression of D 2 R of OTKO. However, OTKO showed an increased gene expression of V 1a R in the HPC. There is no significant difference in VP mRNA synthesis in the HPT between OTKO and WT. Our findings demonstrate that the absence of OT leads to significant changes in the expression of the studied genes (OTR, ERα, ERβ, V 1a R), and these changes may contribute to the decreased sexual behavior observed in OTKO females.

Sarcocystis pantherophis, n. sp. from eastern rat snakes (Pantherophis alleghaniensis) definitive hosts and interferongamma gene knockout mice as experimental intermediate hosts

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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....

CD44 and Bak expression in IL-6 or TNF-alpha gene knockout mice after whole lung irradiation

International Nuclear Information System (INIS)

Sakai, Minako; Iwakawa, Mayumi; Ohta, Toshie; Tsujii, Hirohiko; Imai, Takashi; Iwakura, Yoichiro

2008-01-01

To understand the molecular mechanisms that underlie radiation pneumonitis, we examined whether knockout of the tumor necrosis factor (TNF) or the interleukin (IL)-6 gene could give mice an inherent resistance to radiation in the acute phase of alveolar damage after thoracic irradiation. The temporal expression of inflammation (CD44) and apoptosis (Bak) markers in lung after thoracic irradiation was measured to determine the degree of alveolar damage. At 4 weeks post-irradiation (10 Gy), small inflammatory foci were observed in all mice, but there were no obvious histological differences between control (C57BL/6JSlc), TNF-alpha knockout (TNF KO), and IL-6 knockout (IL-6 KO) mice. However, immunohistochemical analysis of CD44 and Bak expression over a time course of 2 weeks highlighted significant differences between the three groups. C57BL/6JSlc and TNF KO mice had increased numbers of both CD44-positive and Bak-positive cells after irradiation, while the IL-6 KO mice showed stable levels of CD44 and Bak. In conclusion, the radioresistant status of IL-6 KO mice in the acute phase of alveolar damage after irradiation suggested an important role for IL-6 in radiation pneumonitis. (author)

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

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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

Hippocampal gene expression patterns in oxytocin male knockout mice are related to impaired social interaction.

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Lazzari, Virginia Meneghini; Zimmermann-Peruzatto, Josi Maria; Agnes, Grasiela; Becker, Roberta Oriques; de Moura, Ana Carolina; Almeida, Silvana; Guedes, Renata Padilha; Giovenardi, Marcia

2017-11-02

Social interaction between animals is crucial for the survival and life in groups. It is well demonstrated that oxytocin (OT) and vasopressin (AVP) play critical roles in the regulation of social behaviors in mammals, however, other neurotransmitters and hormones are involved in the brain circuitry related to these behaviors. The present study aimed to investigate the gene expression of neurotransmitter receptors in the brain of OT knockout (OTKO) male mice. In this study, we evaluated the expression levels of the OT receptor (Oxtr), AVP receptors 1a and 1b (Avpr1a; Avpr1b), dopamine receptor 2 (Drd2), and the estrogen receptors alpha and beta (Esr1; Esr2) genes in the hippocampus (HPC), olfactory bulb (OB), hypothalamus (HPT) and prefrontal cortex (PFC). AVP gene (Avp) expression was analyzed in the HPT. Gene expression results were discussed regarding to social interaction and sexual behavior findings. Additionally, we analyzed the influence of OT absence on the Avp mRNA expression levels in the HPT. RNA extraction and cDNAs synthesis followed by quantitative polymerase chain reaction were performed for gene expression determination. Results were calculated with the 2 -ΔΔCt method. Our main finding was that HPC is more susceptible to gene expression changes due to the lack of OT. OTKOs exhibited decreased expression of Drd2 and Avpr1b, but increased expression of Oxtr in the HPC. In the PFC, Esr2 was increased. In the HPT, there was a reduced Avp expression in the OTKO group. No differences were detected in the OB and HPT. Despite these changes in gene expression, sexual behavior was not affected. However, OTKO showed higher social investigation and lower aggressive performance than wild-type mice. Our data highlight the importance of OT for proper gene expression of neurotransmitter receptors related to the regulation of social interaction in male mice. Copyright © 2017. Published by Elsevier B.V.

Development of a one-step gene knock-out and knock-in method for metabolic engineering of Aureobasidium pullulans.

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Guo, Jian; Wang, Yuanhua; Li, Baozhong; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2017-06-10

Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40bp length and 10μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic analysis of tissue from α1,3-galactosyltransferase knockout mice reveals that a wide variety of proteins and protein fragments change expression level.

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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.

Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

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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.

Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

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Li, Xiaoli; Li, Yaqing; Han, Gaoyang; Li, Xiaoran; Ji, Yasai; Fan, Zhirui; Zhong, Yali; Cao, Jing; Zhao, Jing; Mariusz, Goscinski; Zhang, Mingzhi; Wen, Jianguo; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice. PMID:27835892

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

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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 ...

[Effects of aquaporin-4 gene knockout on behavior changes and cerebral morphology during aging in mice].

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Su, Shengan; Lu, Yunbi; Zhang, Weiping

2013-05-01

To investigate the effects of aquaporin-4 (AQP4) gene knockout on the behavior changes and cerebral morphology during aging in mice,and to compare that of young and aged mice between AQP4 knockout mice (AQP4(-/-)) and wild type mice (AQP4(+/+)). Fifty-eight CD-1 mice were divided into four groups: young (2-3 months old) AQP4(-/-), aged (17-19 months old) AQP4(-/-), young AQP4(+/+) and aged AQP4(+/+). The activity levels and exploring behavior of mice were tested in open field. The neurons were stained with toluidine blue and NeuN, the astrocytes and microglia were stained with GFAP and Iba-1, respectively. The morphological changes of neuron, astrocyte and microglia were then analyzed. Compared with young mice, the total walking distance in open field of aged AQP4(+/+) mice and aged AQP4(-/-) mice decreased 41.2% and 44.1%, respectively (Ptime in the central area of open field. The density of neuron in cortex of aged AQP4(+/+) mice and aged AQP4(-/-) mice decreased 19.6% and 15.8%, respectively (P<0.05), while there was no difference in the thickness of neuron cell body in hippocampus CA1 region. The density of astrocyte in hippocampus CA3 region of aged AQP4(+/+) mice and aged AQP4(-/-) mice increased 57.7% and 64.3%, respectively (P<0.001), while there was no difference in the area of astrocyte. The area of microglia in hippocampus CA3 region of aged AQP4(+/+) mice and aged AQP4(-/-) mice increased 46.9% and 52.0%, respectively (P<0.01), while there was no difference in the density of microglia. Compared with AQP4(+/+) mice, the young and aged AQP4(-/-) mice showed smaller area of astrocyte in hippocampus CA3 region, reduced 18.0% in young mice and 23.6% in aged mice. There was no difference between AQP4(+/+) mice and AQP4(-/-) mice for other observed indexes. AQP4 may be involved in change of astrocyte and astrocyte-related behaviors during aging. AQP4 gene knockout may have limited effects on the change of neuron, microglia and most neuronal behaviors in aging

The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

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Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

2015-01-01

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.

Gamma aminobutyric acid transporter subtype 1 gene knockout mice: a new model for attention deficit/hyperactivity disorder

Institute of Scientific and Technical Information of China (English)

Ping Yang; Guoqiang Cai; Youqing Cai; Jian Fei; Guoxiang Liu

2013-01-01

Attention deficit/hyperactivity disorder (ADHD) is characterized by hyperactivity,impaired sustained attention,impulsivity,and is usually accompanied by varying degrees of learning difficulties and lack of motor coordination.However,the pathophysiology and etiology of ADHD remain inconclusive so far.Our previous studies have demonstrated that the gamma aminobutyric acid transporter subtype 1 (GAT1) gene knockout (ko) mouse (gat1-/-)is hyperactive and exhibited impaired memory performance in the Morris water maze.In the current study,we found that the gat1-/-mice showed low levels of attentional focusing and increased impulsivity.In addition,the gat1-/-mice displayed ataxia characterized by defects in motor coordination and balance skills.The hyperactivity in the ko mice was reduced by both methylphenidate and amphetamine.Collectively,these results suggest that GAT1 ko mouse is a new animal model for ADHD studying and GAT1 may be a new target to treat ADHD.

Reconstructing gene regulatory networks from knock-out data using Gaussian Noise Model and Pearson Correlation Coefficient.

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Mohamed Salleh, Faridah Hani; Arif, Shereena Mohd; Zainudin, Suhaila; Firdaus-Raih, Mohd

2015-12-01

A gene regulatory network (GRN) is a large and complex network consisting of interacting elements that, over time, affect each other's state. The dynamics of complex gene regulatory processes are difficult to understand using intuitive approaches alone. To overcome this problem, we propose an algorithm for inferring the regulatory interactions from knock-out data using a Gaussian model combines with Pearson Correlation Coefficient (PCC). There are several problems relating to GRN construction that have been outlined in this paper. We demonstrated the ability of our proposed method to (1) predict the presence of regulatory interactions between genes, (2) their directionality and (3) their states (activation or suppression). The algorithm was applied to network sizes of 10 and 50 genes from DREAM3 datasets and network sizes of 10 from DREAM4 datasets. The predicted networks were evaluated based on AUROC and AUPR. We discovered that high false positive values were generated by our GRN prediction methods because the indirect regulations have been wrongly predicted as true relationships. We achieved satisfactory results as the majority of sub-networks achieved AUROC values above 0.5. Copyright © 2015 Elsevier Ltd. All rights reserved.

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

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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.

Study of 19C by One-Neutron Knockout

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Hwang Jongwon

2016-01-01

Full Text Available The spectroscopic structure of 19C, a prominent one-neutron halo nucleus, has been studied with a 20C secondary beam at 290 MeV/nucleon and a carbon target. Neutron-unbound states populated by the one-neutron knockout reaction were investigated by means of the invariant mass method. The preliminary relative energy spectrum and parallel momentum distribution of the knockout residue, 19C*, were reconstructed from the measured four momenta of the 18C fragment, neutron, and beam. Three resonances were observed in the spectrum, which correspond to the states at Ex = 0.62(9, 1.42(10, and 2.89(10 MeV. The parallel momentum distributions for the 0.62-MeV and 2.89-MeV states suggest spin-parity assignments of 5/2+ and 1/2−, respectively. The 1.42-MeV state is in line with the reported 5/22+ state.

Split dose recovery studies using homologous recombination deficient gene knockout chicken B lymphocyte cells

International Nuclear Information System (INIS)

Rao, B.S.S.; Tano, Kaori; Utsumi, Hiroshi; Takeda, Shunichi

2007-01-01

To understand the role of proteins involved in double strand breaks (DSB) repair modulating sublethal damage (SLD) recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 -/- cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C -/- cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54 -/- cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52 -/- , XRCC2 -/- XRCC3 -/- RAD51C -/- and RAD51D -/- ) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52 -/- was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5 h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 -/- , XRCC3 -/- , RAD51C -/- and RAD51D -/- knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D -/- and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2 -/- , XRCC3 -/- , RAD51C -/- nd RAD51D -/- and further delayed when compared with that of RAD52 -/- and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery

Knockout mutations of insulin-like peptide genes enhance sexual receptivity in Drosophila virgin females.

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Watanabe, Kazuki; Sakai, Takaomi

2016-01-01

In the fruitfly Drosophila melanogaster, females take the initiative to mate successfully because they decide whether to mate or not. However, little is known about the molecular and neuronal mechanisms regulating sexual receptivity in virgin females. Genetic tools available in Drosophila are useful for identifying molecules and neural circuits involved in the regulation of sexual receptivity. We previously demonstrated that insulin-producing cells (IPCs) in the female brain are critical to the regulation of female sexual receptivity. Ablation and inactivation of IPCs enhance female sexual receptivity, suggesting that neurosecretion from IPCs inhibits female sexual receptivity. IPCs produce and release insulin-like peptides (Ilps) that modulate various biological processes such as metabolism, growth, lifespan and behaviors. Here, we report a novel role of the Ilps in sexual behavior in Drosophila virgin females. Compared with wild-type females, females with knockout mutations of Ilps showed a high mating success rate toward wild-type males, whereas wild-type males courted wild-type and Ilp-knockout females to the same extent. Wild-type receptive females retard their movement during male courtship and this reduced female mobility allows males to copulate. Thus, it was anticipated that knockout mutations of Ilps would reduce general locomotion. However, the locomotor activity in Ilp-knockout females was significantly higher than that in wild-type females. Thus, our findings indicate that the high mating success rate in Ilp-knockout females is caused by their enhanced sexual receptivity, but not by improvement of their sex appeal or by general sluggishness.

Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells

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Matyas Flemr

2015-07-01

Full Text Available Induction of double-strand DNA breaks (DSBs by engineered nucleases, such as CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs, stimulates knockin of exogenous DNA fragments via homologous recombination (HR. However, the knockin efficiencies reported so far have not allowed more complex in vitro genome modifications such as, for instance, simultaneous integration of a DNA fragment at two distinct genomic sites. We developed a reporter system to enrich for cells with engineered nuclease-assisted HR events. Using this system in mouse embryonic stem cells (mESCs, we achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. Our approach reduces the time and resources required for conditional knockout mESC generation dramatically.

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.

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

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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.

Knockout and transgenic mice of Trp53: what have we learned about p53 in breast cancer?

International Nuclear Information System (INIS)

Blackburn, Anneke C; Jerry, D Joseph

2002-01-01

The human p53 tumor suppressor gene TP53 is mutated at a high frequency in sporadic breast cancer, and Li-Fraumeni syndrome patients who carry germline mutations in one TP53 allele have a high incidence of breast cancer. In the 10 years since the first knockout of the mouse p53 tumor suppressor gene (designated Trp53) was published, much has been learned about the contribution of p53 to biology and tumor suppression in the breast through the use of p53 transgenic and knockout mice. The original mice deficient in p53 showed no mammary gland phenotype. However, studies using BALB/c-Trp53-deficient mice have demonstrated a delayed involution phenotype and a mammary tumor phenotype. Together with other studies of mutant p53 transgenes and p53 bitransgenics, a greater understanding has been gained of the role of p53 in involution, of the regulation of p53 activity by hormones, of the effect of mouse strain and modifier genes on tumor phenotype, and of the cooperation between p53 and other oncogenic pathways, chemical carcinogens and hormonal stimulation in mammary tumorigenesis. Both p53 transgenic and knockout mice are important in vivo tools for understanding breast cancer, and are yet to be exploited for developing therapeutic strategies in breast cancer

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

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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.

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.

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

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Chung, Amanda G; Belone, Phillip M; Bímová, Barbora Vošlajerová; Karn, Robert C; Laukaitis, Christina M

2017-04-01

The house mouse Androgen-binding protein ( Abp ) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg , encoding the alpha (ABPA) and beta-gamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27 , by replacing them with the neomycin resistance gene. The knockout genotype (-/-) showed no Abpa27 or Abpbg27 transcripts in submandibular gland complementary DNA (cDNA) libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the -/- genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the -/- animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the -/- genotype, compared with their +/+ and +/- siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity, and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP. Copyright © 2017 by the Genetics Society of America.

Post-irradiation studies on knock-out and pseudo-recoil releases of fission products from fissioning UO2

International Nuclear Information System (INIS)

Yamagishi, S.; Tanifuji, T.

1976-01-01

By using post-irradiation techniques, in-pile releases of 133 Xe, sup(85m)Kr, 88 Kr, 87 Kr and 138 Xe from UO 2 fissioning at low temperatures below about 200 0 C are studied: these are analyzed into a time-dependent knock-out and time-independent pseudo-recoil releases. For the latter, a 'self knock-out' mechanism is proposed: when a fission fragment loses thoroughly its energy near the UO 2 surface and stops there, it will knock out the surface substances and accordingly the fragment (i.e. the fission product) will be released. The effective thickness of the layer where the self knock-out occurs is found to be approximately 7A. As for the knock-out release, the following is estimated from its dependence on various factors: the knock-out release of fission products occurs from the surface layer with the effective thickness of approximately 20A: the shape of UO 2 matrix knocked out by one fission fragment passing through the surface is equivalent to a cylinder approximately 32A diameter by approximately 27A thick, (i.e. the knock-out coefficient for UO 2 is approximately 660 uranium atoms per knock-out event). On the basis of the above estimations, the conclusions derived from the past in-pile studies of fission gas releases are evaluated. (Auth.)

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

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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.

In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

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Andreas Holmgaard

2017-12-01

Full Text Available 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 in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs induce in vitro indel formation at high frequencies (44%–93%. A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP+ RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration.

Global gene expression profiles of MT knockout and wild-type mice in the condition of doxorubicin-induced cardiomyopathy.

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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.

Generation of knockout rabbits using transcription activator-like effector nucleases

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Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

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 ...

ANTXR2 Knock-Out Does Not Result in the Development of Hypertension in Rats.

Science.gov (United States)

Liu, Xiaoyan; Yuan, Wen; Li, Jing; Yang, Lei; Cai, Jun

2017-02-01

Our recent genetic study as well as robust evidences reported by previous genome-wide association studies (GWASs) have indicated that the single nucleotide polymorphism rs16998073, located near gene anthrax toxin receptor 2 (ANTXR2), was significantly associated with hypertension in Asians and Europeans. The aim of the present study was to determine whether ANTXR2 is the causal gene of hypertension at the 4q21 locus using an ANTXR2 knock-out model. Relative expression of ANTXR2 in Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were determined by real-time quantitative polymerase chain reaction and western blot analysis. ANTXR2 knock-out rats were created using CRISPR/Cas9-mediated genome editing and blood pressure values were measured in ANTXR2 -/- and wild type (WT) rats by tail-cuff method and carotid arterial catheterization method. Neither the mRNA nor protein levels of ANTXR2 were significantly different between tissues from SHRs and WKYs. To create ANTXR2 -/- rats, 67 base pairs were deleted in exon 1 of ANTXR2 using CRISPR/Cas9-mediated genome editing. ANTXR2 protein decreased significantly in aortas of ANTXR2 -/- rats, suggesting sufficient efficiency of ANTXR2 knock-out in this model. However, ANTXR2 -/- rats exhibited nearly the same blood pressure as WT rats at baseline conditions as well as during Angiotensin II (400ng/kg/min) infusion or high-salt diet treatment. These findings suggest that ANTXR2 might not be associated with hypertension and thus further functional analysis is warranted to identify the causal gene at this locus. © American Journal of Hypertension, Ltd 2016. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

Science.gov (United States)

Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

2016-10-01

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1 +/+ control group (group A, n=6); SIRT1 +/+ osteoarthritis group (group B, n=6); SIRT1 -/- control group (group C, n=6); SIRT1 -/- osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1 -/- osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1 +/+ osteoarthritis group and SIRT1 -/- control group, SIRT1 protein expression was not obviously changed in the SIRT1 -/- osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (Pknock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

Science.gov (United States)

Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

2017-10-01

RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

Directory of Open Access Journals (Sweden)

Christina Spilker

2016-03-01

Full Text Available Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB. Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS, a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle.

Science.gov (United States)

Modrzynska, Katarzyna; Pfander, Claudia; Chappell, Lia; Yu, Lu; Suarez, Catherine; Dundas, Kirsten; Gomes, Ana Rita; Goulding, David; Rayner, Julian C; Choudhary, Jyoti; Billker, Oliver

2017-01-11

A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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.

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.

Proton knock-out in Hall A

International Nuclear Information System (INIS)

Jager, K. de

2003-01-01

Proton knock-out is studied in a broad program in Hall A at Jefferson Lab. The first experiment performed in Hall A studied the 16 O(e,e'p) reaction. Since then proton knock-out experiments have studied a variety of aspects of that reaction, from single-nucleon properties to its mechanism, such as final-state interactions and two-body currents, in nuclei from 2 H to 16 O. In this review the accomplishments of this program will be summarized and an outlook given of expected future results. (orig.)

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

Cloning and knockout of formate hydrogen lyase and H{sub 2}-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Zhao, Hongxin; Ma, Kun; Lu, Yuan; Zhang, Chong; Wang, Liyan; Xing, Xin-Hui [Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084 (China)

2009-01-15

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A ({delta} hycA), IAM1183-O ({delta} hybO), and IAM1183-AO ({delta} hycA/ {delta} hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 {+-} 38.59, 2747.203 {+-} 13.25 and 3372.019 {+-} 4.39 (ml h{sup -1} g{sup -1}dry cell weight), respectively, higher than that of the wild strain (2321.861 {+-} 15.34 ml h{sup -1} g{sup -1}dry cell weight). The total H{sub 2} yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H{sub 2}/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H{sub 2}/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183 {delta} hycA/ {delta} hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L. (author)

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.

Knock-out reactions

International Nuclear Information System (INIS)

de Forest, T. Jr.

1977-01-01

It is pointed out that the primary motivation for performing high energy single nucleon knock-out reactions is based on the concept of quasi-elastic scattering. The validity of and corrections to the partial wave impulse approximation and kinematical invariance of knock-out reactions and tests of the reaction mechanism are treated. The effect of distortions on the momentum distribution in the effective momentum approximation for given parameters are plotted. 12 references

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17

OpenAIRE

Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

2012-01-01

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of ...

PDHA1 gene knockout in prostate cancer cells results in metabolic reprogramming towards greater glutamine dependence

Science.gov (United States)

Li, Yaqing; Li, Xiaoran; Li, Xiaoli; Zhong, Yali; Ji, Yasai; Yu, Dandan; Zhang, Mingzhi; Wen, Jian-Guo; Zhang, Hongquan; Goscinski, Mariusz Adam; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Alternative pathways of metabolism endowed cancer cells with metabolic stress. Inhibiting the related compensatory pathways might achieve synergistic anticancer results. This study demonstrated that pyruvate dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor cell metabolism by rendering the cells with increased expression of glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an increase in glutamine-dependent cell survival. Deprivation of glutamine induced cell growth inhibition, increased reactive oxygen species and decreased ATP production. Pharmacological blockade of the glutaminolysis pathway resulted in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1 KO cells. Further examination of the key glutaminolysis enzymes in human prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression were significantly associated with aggressive clinicopathological features and poor clinical outcome. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon alternative energy metabolism and targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in cancer cells may offer a potential novel therapeutic strategy. PMID:27462778

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

Science.gov (United States)

Kanda, Naho; Ichikawa, Machiko; Ono, Ayaka; Toyoda, Atsushi; Fujiyama, Asao; Abe, Jun; Tsuchikane, Yuki; Nishiyama, Tomoaki; Sekimoto, Hiroyuki

2017-12-19

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt + ) and mating-type minus (mt - ). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt + and mt - cells, respectively. PR-IP mediates the release of protoplasts from mt - cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt - cells of the C. psl. complex. When the knockout mt - cells were mixed with wild-type mt + cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

The vasopressin deficient Brattleboro rats: A natural knockout model used in the search for CNS effects of vasopressin

NARCIS (Netherlands)

Bohus, B; de Wied, David; Urban, I.J.A.; Burbach, J.P.H.; De Wied, D.

1999-01-01

Behavioral neuroscience is using mon and more gene knockout techniques to produce animals with a specific deletion. These studies have their precedent in nature. A mutation may result in a limited genetic defect, as seen in the vasopressin (VP) deficiency in the Brattleboro rat. The mutation is in a

A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

Directory of Open Access Journals (Sweden)

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.

Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

Science.gov (United States)

Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G

2001-01-01

One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and

A norm knockout method on indirect reciprocity to reveal indispensable norms

Science.gov (United States)

Yamamoto, Hitoshi; Okada, Isamu; Uchida, Satoshi; Sasaki, Tatsuya

2017-03-01

Although various norms for reciprocity-based cooperation have been suggested that are evolutionarily stable against invasion from free riders, the process of alternation of norms and the role of diversified norms remain unclear in the evolution of cooperation. We clarify the co-evolutionary dynamics of norms and cooperation in indirect reciprocity and also identify the indispensable norms for the evolution of cooperation. Inspired by the gene knockout method, a genetic engineering technique, we developed the norm knockout method and clarified the norms necessary for the establishment of cooperation. The results of numerical investigations revealed that the majority of norms gradually transitioned to tolerant norms after defectors are eliminated by strict norms. Furthermore, no cooperation emerges when specific norms that are intolerant to defectors are knocked out.

Single and double metallothionein knockout in the nematode C. elegans reveals cadmium dependent and independent toxic effects on life history traits

Energy Technology Data Exchange (ETDEWEB)

Hughes, Sam [School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3TL (United Kingdom); School of Biomedical and Health Sciences, Pharmaceutical Sciences Research Division, King' s College London, 150 Stamford Street, London SE1 9NH (United Kingdom); Stuerzenbaum, Stephen R. [School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3TL (United Kingdom) and School of Biomedical and Health Sciences, Pharmaceutical Sciences Research Division, King' s College London, 150 Stamford Street, London SE1 9NH (United Kingdom)]. E-mail: stephen.sturzenbaum@kcl.ac.uk

2007-01-15

The genome of the nematode Caenorhabditis elegans contains two metallothionein genes, both involved in metal homeostasis and/or detoxification. Single metallothionein knockout mutants have been created and now, for the first time, a double mutant has been isolated. Life history studies in the presence or absence of cadmium showed that all metallothionein mutants are viable. Although cadmium did not influence longevity, a dose dependent reduction in total brood size and volumetric growth was observed in wild type animals, which was magnified in single knockouts and further exacerbated in the double knockout. However, the metallothionein deletion caused two effects that are independent of cadmium exposure, namely all knockout strains displayed a reduced total brood size and the deletion of both metallothionein loci caused a significant reduction in volumetric growth. In summary, metallothionein is undoubtedly an important player in cadmium detoxification, but evidently also an important factor in cadmium independent pathways. - Metallothionein is a modifier of life-history parameters.

Single and double metallothionein knockout in the nematode C. elegans reveals cadmium dependent and independent toxic effects on life history traits

International Nuclear Information System (INIS)

Hughes, Sam; Stuerzenbaum, Stephen R.

2007-01-01

The genome of the nematode Caenorhabditis elegans contains two metallothionein genes, both involved in metal homeostasis and/or detoxification. Single metallothionein knockout mutants have been created and now, for the first time, a double mutant has been isolated. Life history studies in the presence or absence of cadmium showed that all metallothionein mutants are viable. Although cadmium did not influence longevity, a dose dependent reduction in total brood size and volumetric growth was observed in wild type animals, which was magnified in single knockouts and further exacerbated in the double knockout. However, the metallothionein deletion caused two effects that are independent of cadmium exposure, namely all knockout strains displayed a reduced total brood size and the deletion of both metallothionein loci caused a significant reduction in volumetric growth. In summary, metallothionein is undoubtedly an important player in cadmium detoxification, but evidently also an important factor in cadmium independent pathways. - Metallothionein is a modifier of life-history parameters

Metabonomic study of the biochemical profiles of heterozygous myostatin knockout swine

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Jianxiang XU,Dengke PAN,Jie ZHAO,Jianwu WANG,Xiaohong HE,Yuehui MA,Ning LI

2015-03-01

Full Text Available Myostatin is a transforming growth factor-β family member that normally acts to limit skeletal muscle growth. Myostatin gene (MSTN knockout (KO mice show possible effects for the prevention or treatment of metabolic disorders such as obesity and type 2 diabetes. We applied chromatography and mass spectrometry based metabonomics to assess system-wide metabolic response of heterozygous MSTN KO (MSTN+/- swine. Most of the metabolic data for MSTN+/- swine were similar to the data for wild type (WT control swine. There were, however, metabolic changes related to fatty acid metabolism, glucose utilization, lipid metabolism, as well as BCAA catabolism caused by monoallelic MSTN depletion.The statistical analyses suggested that: (1 most metabolic changes were not significant in MSTN+/- swine compared to WT swine; (2 only a few metabolic properties were significantly different between KO and WT swine, especially for lipid metabolism. Significantly, these minor changes were most evident in female KO swine and suggested differences in gender sensitivity to myostatin.

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

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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.

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)

Mice lacking pituitary tumor transforming gene show elevated exposure of DGalNAc carbohydrate determinants

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Lutsyk A. D.

2012-04-01

Full Text Available Aim. To investigate the influence of pituitary tumor transforming gene (pttg-1 knockout on glycome of parenchimal organs by means of lectin histochemistry. Methods. DGalNAc, DGlcNAc, NeuNAc carbohydrate determinants were labelled with soybean agglutinin (SBA and wheat germ agglutinin (WGA, conjugated to peroxidase, with subsequent visualization of the lectin-binding sites with diaminobenzidine. The testes and kidneys of murine strain BL6/C57 with the pttg-1 gene knockout (PTTG-KO were compared to the wild type (PTTG-WT animals, both groups 1 month of age. Results. Knockout of the pttg-1 gene was accompanied by enhanced exposure of the DGalNAc sugar residues within the Golgi complex of secondary spermatocytes, in a brush border of renal tubules and on the lumenal surface of collecting ducts. Conclusions. This study suggests that knockout of the pttg-1 gene may lead to the changes in carbohydrate processing in mammalian organism.

One-neutron knockout from Ne24-28 isotopes

CERN Document Server

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

2010-01-01

One-neutron knockout reactions of Ne24-28 in a beryllium target have been studied in the Fragment Separator (FRS), at GSI. The results include inclusive one-neutron knockout cross-sections as well as longitudinal-momentum distributions of the knockout fragments. The ground-state structure of the neutron-rich neon isotopes was obtained from an analysis of the measured momentum distributions. The results indicate that the two heaviest isotopes, Ne-27 and Ne-28, are dominated by a configuration in which a s(1/2) neutron is coupled to an excited state of the Ne-26 and Ne-27 core, respectively. (C) 2010 Elsevier B.V. All rights reserved.

Theoretical analysis of knock-out release of fission products from nuclear fuels

International Nuclear Information System (INIS)

Yamagishi, S.

1975-01-01

The knock-out release of fission products is studied theoretically. The general equations of knock-out release are derived, assuming that a fission fragment passing through the surface of nuclear fuels knocks out a local region of the surface with an effective thickness and an effective cross-sectional area. Using these equations, the knock-out release of fission gases is calculated for various cases. The conditions under which the knock-out coefficients (the average number of uranium atoms knocked out by one fission fragment) is obtainable are clarified by experiments on the knock-out release of fission gases. A method of determining the effective thickness and the effective cross-sectional area of a knock-out region is proposed. (Auth.)

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

Science.gov (United States)

Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Tailoring the Immune Response via Customization of Pathogen Gene Expression.

Science.gov (United States)

Runco, Lisa M; Stauft, Charles B; Coleman, J Robert

2014-01-01

The majority of studies focused on the construction and reengineering of bacterial pathogens have mainly relied on the knocking out of virulence factors or deletion/mutation of amino acid residues to then observe the microbe's phenotype and the resulting effect on the host immune response. These knockout bacterial strains have also been proposed as vaccines to combat bacterial disease. Theoretically, knockout strains would be unable to cause disease since their virulence factors have been removed, yet they could induce a protective memory response. While knockout strains have been valuable tools to discern the role of virulence factors in host immunity and bacterial pathogenesis, they have been unable to yield clinically relevant vaccines. The advent of synthetic biology and enhanced user-directed gene customization has altered this binary process of knockout, followed by observation. Recent studies have shown that a researcher can now tailor and customize a given microbe's gene expression to produce a desired immune response. In this commentary, we highlight these studies as a new avenue for controlling the inflammatory response as well as vaccine development.

Production of a mouse strain with impaired glucose tolerance by systemic heterozygous knockout of the glucokinase gene and its feasibility as a prediabetes model

Science.gov (United States)

SAITO, Mikako; KANEDA, Asako; SUGIYAMA, Tae; IIDA, Ryousuke; OTOKUNI, Keiko; KABURAGI, Misako; MATSUOKA, Hideaki

2015-01-01

Exon II of glucokinase (Gk) was deleted to produce a systemic heterozygous Gk knockout (Gk+/−) mouse. The relative expression levels of Gk in the heart, lung, liver, stomach, and pancreas in Gk+/− mice ranged from 0.41–0.68 versus that in wild (Gk+/+) mice. On the other hand, its expression levels in the brain, adipose tissue, and muscle ranged from 0.95–1.03, and its expression levels in the spleen and kidney were nearly zero. Gk knockout caused no remarkable off-target effect on the expression of 7 diabetes causing genes (Shp, Hnf1a, Hnf1b, Irs1, Irs2, Kir6.2, and Pdx1) in 10 organs. The glucose tolerance test was conducted to determine the blood glucose concentrations just after fasting for 24 h (FBG) and at 2 h after high-glucose application (GTT2h). The FBG-GTT2h plots obtained with the wild strain fed the control diet (CD), Gk+/− strain fed the CD, and Gk+/− strain fed the HFD were distributed in separate areas in the FBG-GTT2h diagram. The respective areas could be defined as the normal state, prediabetes state, and diabetes state, respectively. Based on the results, the criteria for prediabetes could be defined for the Gk+/− strain developed in this study. PMID:25765873

Brief Report: Altered Social Behavior in Isolation-Reared "Fmr1" Knockout Mice

Science.gov (United States)

Heitzer, Andrew M.; Roth, Alexandra K.; Nawrocki, Lauren; Wrenn, Craige C.; Valdovinos, Maria G.

2013-01-01

Social behavior abnormalities in Fragile X syndrome (FXS) are characterized by social withdrawal, anxiety, and deficits in social cognition. To assess these deficits, a model of FXS, the "Fmr1" knockout mouse ("Fmr1" KO), has been utilized. This mouse model has a null mutation in the fragile X mental retardation 1 gene ("Fmr1") and displays…

Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

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So-Jung Kim

2017-03-01

Full Text Available Nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2 is a well-known transcription factor that regulates the expression of a large number of anti-oxidant genes in mammalian cells (J.H. Kim et al., 2014. Here, we generated a homozygous Nrf2 knockout human embryonic stem cell (hESC line, H9Nrf2KO-A13, using the CRISPR/Cas9 genome editing method. The Nrf2 homozygous knockout H9 cell line maintains pluripotency, differentiation potential into three germ layers, and a normal karyotype.

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

Science.gov (United States)

Liao, Shi-Wei; Lee, Jen-Jie; Ptak, Christopher P; Wu, Ying-Chen; Hsuan, Shih-Ling; Kuo, Chih-Jung; Chen, Ter-Hsin

2018-03-01

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

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

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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

Knockout reactions: experimental aspects

Energy Technology Data Exchange (ETDEWEB)

Cortina Gil, D. [Santiago de Compostela Univ. (Spain)

2007-07-01

The availability of radioactive beams has given rise to intense activity in the field of direct reactions. The removal of one(two)-nucleon (referred to as nucleon knockout in this text) from a fast exotic projectile has been extensively investigated. This lecture provides a general overview of the experimental results achieved using this technique. The sensitivity of the method to different experimental aspects is illustrated with a few examples. Special attention is given to the application of nucleon-knockout reactions as a general purpose spectroscopic tool. (author)

Knockout reactions: experimental aspects

International Nuclear Information System (INIS)

Cortina Gil, D.

2007-01-01

The availability of radioactive beams has given rise to intense activity in the field of direct reactions. The removal of one(two)-nucleon (referred to as nucleon knockout in this text) from a fast exotic projectile has been extensively investigated. This lecture provides a general overview of the experimental results achieved using this technique. The sensitivity of the method to different experimental aspects is illustrated with a few examples. Special attention is given to the application of nucleon-knockout reactions as a general purpose spectroscopic tool. (author)

One-neutron knockout from {sup 24-28}Ne isotopes

Energy Technology Data Exchange (ETDEWEB)

Rodriguez-Tajes, C., E-mail: carme.rodriguez@usc.e [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Cortina-Gil, D.; Alvarez-Pol, H. [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Aumann, T. [GSI Helmholtzzentrum fuer Schwerionenforschung, 64291 Darmstadt (Germany); Benjamim, E.; Benlliure, J. [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Borge, M.J.G. [Instituto de Estructura de la Materia, CSIC, 28006 Madrid (Spain); Caamano, M.; Casarejos, E. [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Chatillon, A. [GSI Helmholtzzentrum fuer Schwerionenforschung, 64291 Darmstadt (Germany); Eppinger, K.; Faestermann, T. [Physik Department E12, Technische Universitaet Muenchen, 85748 Garching (Germany); Gascon, M. [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Geissel, H. [GSI Helmholtzzentrum fuer Schwerionenforschung, 64291 Darmstadt (Germany); Gernhaeuser, R. [Physik Department E12, Technische Universitaet Muenchen, 85748 Garching (Germany); Jonson, B. [Fundamental Fysik, Chalmers Tekniska Hoegskola, 412 96 Goeteborg (Sweden); PH Department, CERN, 1211 Geneve 23 (Switzerland); Kanungo, R. [Astronomy and Physics Department, Saint Mary' s University, Halifax, NS B3H 3C3 (Canada); Kruecken, R. [Physik Department E12, Technische Universitaet Muenchen, 85748 Garching (Germany); Kurtukian, T. [Departamento de Fisica de Particulas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela (Spain); Larsson, K. [Fundamental Fysik, Chalmers Tekniska Hoegskola, 412 96 Goeteborg (Sweden)

2010-04-05

One-neutron knockout reactions of {sup 24-28}Ne in a beryllium target have been studied in the Fragment Separator (FRS), at GSI. The results include inclusive one-neutron knockout cross-sections as well as longitudinal-momentum distributions of the knockout fragments. The ground-state structure of the neutron-rich neon isotopes was obtained from an analysis of the measured momentum distributions. The results indicate that the two heaviest isotopes, {sup 27}Ne and {sup 28}Ne, are dominated by a configuration in which a s{sub 1/2} neutron is coupled to an excited state of the {sup 26}Ne and {sup 27}Ne core, respectively.

Co-ordinate regulation of the cystic fibrosis and multidrug resistance genes in cystic fibrosis knockout mice.

Science.gov (United States)

Trezise, A E; Ratcliff, R; Hawkins, T E; Evans, M J; Freeman, T C; Romano, P R; Higgins, C F; Colledge, W H

1997-04-01

The cystic fibrosis (Cftr and multidrug resistance (Mdr1) genes encode structurally similar proteins which are members of the ABC transporter superfamily. These genes exhibit complementary patterns of expression in vivo, suggesting that the regulation of their expression may be co-ordinated. We have tested this hypothesis in vivo by examining Cftr and Mdr1 expression in cystic fibrosis knockout transgenic mice (Cftr(tm1CAM)). Cftr mRNA expression in Cftr(tm1CAM)/Cftr(tm1CAM) mice was 4-fold reduced in the intestine, as compared with littermate wild-type mice. All other Cftr(tm1CAM)/Cftr(tm1CAM) mouse tissues examined showed similar reductions in Cftr expression. In contrast, we observed a 4-fold increase in Mdr1 mRNA expression in the intestines of neonatal and 3- to 4-week-old Cftr(tm1CAM)/Cftr(tm1CAM) mice, as compared with age-matched +/+ mice, and an intermediate level of Mdr1 mRNA in heterozygous Cftr(tm1CAM) mice. In 10-week-old, Cftr(tm1CAM)/Cftr(tm1CAM) mice and in contrast to the younger mice, Mdr1 mRNA expression was reduced, by 3-fold. The expression of two control genes, Pgk-1 and Mdr2, was similar in all genotypes, suggesting that the changes in Mdr1 mRNA levels observed in the Cftr(tm1CAM)/Cftr(tm1CAM) mice are specific to the loss of Cftr expression and/or function. These data provide further evidence supporting the hypothesis that the regulation Cftr and Mdr1 expression is co-ordinated in vivo, and that this co-ordinate regulation is influenced by temporal factors.

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.

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.

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

Science.gov (United States)

Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

2016-05-20

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

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Karoly Toth

2015-08-01

Full Text Available Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as

Physiological roles of CNS muscarinic receptors gained from knockout mice

DEFF Research Database (Denmark)

Thomsen, Morgane; Sørensen, Gunnar; Dencker, Ditte

2017-01-01

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......, 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...

RNaseT2 knockout rats exhibit hippocampal neuropathology and deficits in memory.

Science.gov (United States)

Sinkevicius, Kerstin W; Morrison, Thomas R; Kulkarni, Praveen; Cagliostro, Martha K Caffrey; Iriah, Sade; Malmberg, Samantha; Sabrick, Julia; Honeycutt, Jennifer A; Askew, Kim L; Trivedi, Malav; Ferris, Craig F

2018-05-10

RNASET2 deficiency in humans is associated with infant cystic leukoencephalopathy, which causes psychomotor impairment, spasticity, and epilepsy. A zebrafish mutant model suggests that loss of RNASET2 function leads to neurodegeneration due to the accumulation of non-degraded RNA in the lysosomes. The goal of this study was to characterize the first rodent model of RNASET2 deficiency. The brains of 3- and 12-month-old RNaseT2 knockout rats were studied using multiple magnetic resonance imaging modalities and behavioral tests. While T1 and T2 weighted images of RNaseT2 knockout rats exhibited no evidence of cystic lesions, the prefrontal cortex and hippocampal complex were enlarged in knockout animals. Diffusion weighted imaging showed altered anisotropy and putative gray matter changes in the hippocampal complex of the RNaseT2 knockout rats. Immunohistochemistry for glial fibrillary acidic protein (GFAP) showed the presence of hippocampal neuroinflammation. Decreased levels of lysosome-associated membrane protein 2 (LAMP2) and elevated acid phosphatase and β-N-Acetylglucosaminidase (NAG) activities indicated that the RNASET2 knockout rats likely had altered lysosomal function and potential defects in autophagy. Object recognition tests confirmed the RNaseT2 knockout rats exhibited memory deficits. However, the Barnes maze, and balance beam and rotarod tests, indicated there were no differences in spatial memory or motor impairments, respectively. Overall, patients with RNASET2 deficiency exhibited a more severe neurodegeneration phenotype than was observed in the RNaseT2 knockout rats. However, the vulnerability of the knockout rat hippocampus as evidenced by neuroinflammation, altered lysosomal function, and cognitive defects indicates this is still a useful in vivo model to study RNASET2 function. © 2018. Published by The Company of Biologists Ltd.

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.

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

Haloperidol inhibits the development of atherosclerotic lesions in LDL receptor knockout mice.

Science.gov (United States)

van der Sluis, Ronald J; Nahon, Joya E; Reuwer, Anne Q; Van Eck, Miranda; Hoekstra, Menno

2015-05-01

Antipsychotic drugs have been shown to modulate the expression of ATP-binding cassette transporter A1 (ABCA1), a key factor in the anti-atherogenic reverse cholesterol transport process, in vitro. Here we evaluated the potential of the typical antipsychotic drug haloperidol to modulate the cholesterol efflux function of macrophages in vitro and their susceptibility to atherosclerosis in vivo. Thioglycollate-elicited peritoneal macrophages were used for in vitro studies. Hyperlipidaemic low-density lipoprotein (LDL) receptor knockout mice were implanted with a haloperidol-containing pellet and subsequently fed a Western-type diet for 5 weeks to induce the development of atherosclerotic lesions in vivo. Haloperidol induced a 54% decrease in the mRNA expression of ABCA1 in peritoneal macrophages. This coincided with a 30% decrease in the capacity of macrophages to efflux cholesterol to apolipoprotein A1. Haloperidol treatment stimulated the expression of ABCA1 (+51%) and other genes involved in reverse cholesterol transport, that is, CYP7A1 (+98%) in livers of LDL receptor knockout mice. No change in splenic ABCA1 expression was noted. However, the average size of the atherosclerotic size was significantly smaller (-31%) in the context of a mildly more atherogenic metabolic phenotype upon haloperidol treatment. More importantly, haloperidol markedly lowered MCP-1 expression (-70%) and secretion (-28%) by peritoneal macrophages. Haloperidol treatment lowered the susceptibility of hyperlipidaemic LDL receptor knockout mice to develop atherosclerotic lesions. Our findings suggest that the beneficial effect of haloperidol on atherosclerosis susceptibility can be attributed to its ability to inhibit macrophage chemotaxis. © 2015 The British Pharmacological Society.

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....

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Science.gov (United States)

Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

2015-08-04

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

Synthetic promoter libraries- tuning of gene expression

DEFF Research Database (Denmark)

Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

2006-01-01

knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

Directory of Open Access Journals (Sweden)

Susan Richter

Full Text Available Zinc finger nucleases (ZFN are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK, in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116. All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002 and 4.3±0.8% (p = 0.001 for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

Science.gov (United States)

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G; Ronimus, Ron S; McGee, Sean L; Wilson, William R

2013-01-01

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

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 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...... 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...

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

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

Science.gov (United States)

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.

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

Directory of Open Access Journals (Sweden)

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.

Gene knockout of tau expression does not contribute to the pathogenesis of prion disease.

Science.gov (United States)

Lawson, Victoria A; Klemm, Helen M; Welton, Jeremy M; Masters, Colin L; Crouch, Peter; Cappai, Roberto; Ciccotosto, Giuseppe D

2011-11-01

Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.

Characterization of Bombyx mori nucleopolyhedrovirus with a knockout of Bm17.

Science.gov (United States)

Shen, Hongxing; Zhou, Yang; Zhang, Wen; Nin, Bin; Wang, Hua; Wang, Xiaochun; Shao, Shihe; Chen, Huiqing; Guo, Zhongjian; Liu, Xiaoyong; Yao, Qin; Chen, Keping

2012-12-01

Open reading frame 17 (Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells.

Age- and region-specific imbalances of basal amino acids and monoamine metabolism in limbic regions of female Fmr1 knock-out mice.

Science.gov (United States)

Gruss, Michael; Braun, Katharina

2004-07-01

The Fragile X syndrome, a common form of mental retardation in humans, originates from the loss of expression of the Fragile X mental retardation gene leading to the absence of the encoded Fragile X mental retardation protein 1 (FMRP). A broad pattern of morphological and behavioral abnormalities is well described for affected humans as well as Fmr1 knock-out mice, a transgenic animal model for the human Fragile X syndrome. In the present study, we examined neurochemical differences between female Fmr1 knock-out and wildtype mice with particular focus on neurotransmission. Significant age- and region-specific differences of basal tissue neurotransmitter and metabolite levels measured by high performance liquid chromatography were found. Those differences were more numerous in juvenile animals (postnatal day (PND) 28-31) compared to adults (postnatal day 209-221). In juvenile female knock-out mice, especially aspartate and taurine were increased in cortical regions, striatum, cerebellum, and brainstem. Furthermore, compared to the wildtype animals, the juvenile knock-out mice displayed an increased level of neuronal inhibition in the hippocampus and brainstem reflected by decreased ratios of (aspartate + glutamate)/(taurine + GABA), as well as an increased dopamine (DA) turnover in cortical regions, striatum, and hippocampus. These results provide the first evidence that the lack of FMRP expression in female Fmr1 knock-out mice is accompanied by age-dependent, region-specific alterations in brain amino acids, and monoamine turnover, which might be related to the reported synaptical and behavioural alterations in these animals.

The normal function of a speciation gene, Odysseus, and its hybrid sterility effect.

Science.gov (United States)

Sun, Sha; Ting, Chau-Ti; Wu, Chung-I

2004-07-02

To understand how postmating isolation is connected to the normal process of species divergence and why hybrid male sterility is often the first sign of speciation, we analyzed the Odysseus (OdsH) gene of hybrid male sterility in Drosophila. We carried out expression analysis, transgenic study, and gene knockout. The combined evidence suggests that the sterility phenotype represents a novel manifestation of the gene function rather than the reduction or loss of the normal one. The gene knockout experiment identified the normal function of OdsH as a modest enhancement of sperm production in young males. The implication of a weak effect of OdsH on the normal phenotype but a strong influence on hybrid male sterility is discussed in light of Haldane's rule of postmating isolation.

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

Directory of Open Access Journals (Sweden)

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.

Activation of PPARγ Ameliorates Spatial Cognitive Deficits through Restoring Expression of AMPA Receptors in Seipin Knock-Out Mice.

Science.gov (United States)

Zhou, Libin; Chen, Tingting; Li, Guoxi; Wu, Chaoming; Wang, Conghui; Li, Lin; Sha, Sha; Chen, Lei; Liu, George; Chen, Ling

2016-01-27

A characteristic phenotype of congenital generalized lipodystrophy 2 (CGL2) that is caused by loss-of-function of seipin gene is mental retardation. Here, we show that seipin deficiency in hippocampal CA1 pyramidal cells caused the reduction of peroxisome proliferator-activated receptor gamma (PPARγ). Twelve-week-old systemic seipin knock-out mice and neuronal seipin knock-out (seipin-nKO) mice, but not adipose seipin knock-out mice, exhibited spatial cognitive deficits as assessed by the Morris water maze and Y-maze, which were ameliorated by the treatment with the PPARγ agonist rosiglitazone (rosi). In addition, seipin-nKO mice showed the synaptic dysfunction and the impairment of NMDA receptor-dependent LTP in hippocampal CA1 regions. The density of AMPA-induced current (IAMPA) in CA1 pyramidal cells and GluR1/GluR2 expression were significantly reduced in seipin-nKO mice, whereas the NMDA-induced current (INMDA) and NR1/NR2 expression were not altered. Rosi treatment in seipin-nKO mice could correct the decrease in expression and activity of AMPA receptor (AMPAR) and was accompanied by recovered synaptic function and LTP induction. Furthermore, hippocampal ERK2 and CREB phosphorylation in seipin-nKO mice were reduced and this could be rescued by rosi treatment. Rosi treatment in seipin-nKO mice elevated BDNF concentration. The MEK inhibitor U0126 blocked rosi-restored AMPAR expression and LTP induction in seipin-nKO mice, but the Trk family inhibitor K252a did not. These findings indicate that the neuronal seipin deficiency selectively suppresses AMPAR expression through reducing ERK-CREB activities, leading to the impairment of LTP and spatial memory, which can be rescued by PPARγ activation. Congenital generalized lipodystrophy 2 (CGL2), caused by loss-of-function mutation of seipin gene, is characterized by mental retardation. By the generation of systemic or neuronal seipin knock-out mice, the present study provides in vivo evidence that neuronal seipin

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.

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

Science.gov (United States)

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Science.gov (United States)

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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

VIP Gene Deletion in Mice Causes Cardiomyopathy Associated with Upregulation of Heart Failure Genes

Energy Technology Data Exchange (ETDEWEB)

Szema, Anthony M.; Hamidi, Sayyed A.; Smith, S. David; Benveniste, Helene; Katare, Rajesh Gopalrao

2013-05-20

Vasoactive Intestinal Peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, is absent in pulmonary arteries of patients with idiopathic pulmonary arterial hypertension (PAH). We previously determined that targeted deletion of the VIP gene in mice leads to PAH with pulmonary vascular remodeling and right ventricular (RV) dilatation. Whether the left ventricle is also affected by VIP gene deletion is unknown. In the current study, we examined if VIP knockout mice (VIP-/-) develop both right (RV) and left ventricular (LV) cardiomyopathy, manifested by LV dilatation and systolic dysfunction, as well as overexpression of genes conducive to heart failure.

Effect of Duplicate Genes on Mouse Genetic Robustness: An Update

Directory of Open Access Journals (Sweden)

Zhixi Su

2014-01-01

Full Text Available In contrast to S. cerevisiae and C. elegans, analyses based on the current knockout (KO mouse phenotypes led to the conclusion that duplicate genes had almost no role in mouse genetic robustness. It has been suggested that the bias of mouse KO database toward ancient duplicates may possibly cause this knockout duplicate puzzle, that is, a very similar proportion of essential genes (PE between duplicate genes and singletons. In this paper, we conducted an extensive and careful analysis for the mouse KO phenotype data and corroborated a strong effect of duplicate genes on mouse genetics robustness. Moreover, the effect of duplicate genes on mouse genetic robustness is duplication-age dependent, which holds after ruling out the potential confounding effect from coding-sequence conservation, protein-protein connectivity, functional bias, or the bias of duplicates generated by whole genome duplication (WGD. Our findings suggest that two factors, the sampling bias toward ancient duplicates and very ancient duplicates with a proportion of essential genes higher than that of singletons, have caused the mouse knockout duplicate puzzle; meanwhile, the effect of genetic buffering may be correlated with sequence conservation as well as protein-protein interactivity.

Msx homeobox gene family and craniofacial development.

Science.gov (United States)

Alappat, Sylvia; Zhang, Zun Yi; Chen, Yi Ping

2003-12-01

Vertebrate Msx genes are unlinked, homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development. Inductive interactions mediated by the Msx genes are essential for normal craniofacial, limb and ectodermal organ morphogenesis, and are also essential to survival in mice, as manifested by the phenotypic abnormalities shown in knockout mice and in humans. This review summarizes studies on the expression, regulation, and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice. Key words: Msx genes, craniofacial, tooth, cleft palate, suture, development, transcription factor, signaling molecule.

Phytosterol Feeding Causes Toxicity in ABCG5/G8 Knockout Mice

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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

Comprehensive behavioral study of mGluR3 knockout mice: implication in schizophrenia related endophenotypes

Science.gov (United States)

2014-01-01

Background We previously performed systematic association studies of glutamate receptor gene family members with schizophrenia, and found positive associations of polymorphisms in the GRM3 (a gene of metabotropic glutamate receptor 3: mGluR3) with the disorder. Physiological roles of GRM3 in brain functions and its functional roles in the pathogenesis of schizophrenia remain to be resolved. Results We generated mGluR3 knockout (KO) mice and conducted comprehensive behavioral analyses. KO mice showed hyperactivity in the open field, light/dark transition, and 24-hour home cage monitoring tests, impaired reference memory for stressful events in the Porsolt forced swim test, impaired contextual memory in cued and contextual fear conditioning test, and impaired working memory in the T-Maze forced alternation task test. Hyperactivity and impaired working memory are known as endophenotypes of schizophrenia. We examined long-term synaptic plasticity by assessing long-term potentiation (LTP) in the CA1 region in the hippocampi of KO and wild-type (WT) mice. We observed no differences in the amplitude of LTP between the two genotypes, suggesting that mGluR3 is not essential for LTP in the CA1 region of the mouse hippocampus. As hyperactivity is typically associated with increased dopaminergic transmission, we performed in vivo microdialysis measurements of extracellular dopamine in the nucleus accumbens of KO and WT mice. We observed enhancements in the methamphetamine (MAP)-induced release of dopamine in KO mice. Conclusions These results demonstrate that a disturbance in the glutamate-dopamine interaction may be involved in the pathophysiology of schizophrenia-like behavior, such as hyperactivity in mGluR3 KO mice. PMID:24758191

Age-dependent changes of cerebral copper metabolism in Atp7b -/- knockout mouse model of Wilson's disease by [64Cu]CuCl2-PET/CT.

Science.gov (United States)

Xie, Fang; Xi, Yin; Pascual, Juan M; Muzik, Otto; Peng, Fangyu

2017-06-01

Copper is a nutritional metal required for brain development and function. Wilson's disease (WD), or hepatolenticular degeneration, is an inherited human copper metabolism disorder caused by a mutation of the ATP7B gene. Many WD patients present with variable neurological and psychiatric symptoms, which may be related to neurodegeneration secondary to copper metabolism imbalance. The objective of this study was to explore the feasibility and use of copper-64 chloride ([ 64 C]CuCl 2 ) as a tracer for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD using an Atp7b -/- knockout mouse model of WD and positron emission tomography/computed tomography (PET/CT) imaging. Continuing from our recent study of biodistribution and radiation dosimetry of [ 64 C]CuCl 2 in Atp7b -/- knockout mice, PET quantitative analysis revealed low 64 Cu radioactivity in the brains of Atp7b -/- knockout mice at 7th weeks of age, compared with 64 Cu radioactivity in the brains of age- and gender-matched wild type C57BL/6 mice, at 24 h (h) post intravenous injection of [ 64 C]CuCl 2 as a tracer. Furthermore, age-dependent increase of 64 Cu radioactivity was detected in the brains of Atp7b -/- knockout mice from the 13th to 21th weeks of age, based on the data derived from a longitudinal [ 64 C]CuCl 2 -PET/CT study of Atp7b -/- knockout mice with orally administered [ 64 Cu]CuCl 2 as a tracer. The findings of this study support clinical use of [ 64 Cu]CuCl 2 -PET/CT imaging as a tool for noninvasive assessment of age-dependent changes of cerebral copper metabolism in WD patients presenting with variable neurological and psychiatric symptoms.

Systematic screening for skin, hair, and nail abnormalities in a large-scale knockout mouse program.

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John P Sundberg

Full Text Available The International Knockout Mouse Consortium was formed in 2007 to inactivate ("knockout" all protein-coding genes in the mouse genome in embryonic stem cells. Production and characterization of these mice, now underway, has generated and phenotyped 3,100 strains with knockout alleles. Skin and adnexa diseases are best defined at the gross clinical level and by histopathology. Representative retired breeders had skin collected from the back, abdomen, eyelids, muzzle, ears, tail, and lower limbs including the nails. To date, 169 novel mutant lines were reviewed and of these, only one was found to have a relatively minor sebaceous gland abnormality associated with follicular dystrophy. The B6N(Cg-Far2tm2b(KOMPWtsi/2J strain, had lesions affecting sebaceous glands with what appeared to be a secondary follicular dystrophy. A second line, B6N(Cg-Ppp1r9btm1.1(KOMPVlcg/J, had follicular dystrophy limited to many but not all mystacial vibrissae in heterozygous but not homozygous mutant mice, suggesting that this was a nonspecific background lesion. We discuss potential reasons for the low frequency of skin and adnexal phenotypes in mice from this project in comparison to those seen in human Mendelian diseases, and suggest alternative approaches to identification of human disease-relevant models.

Global gene expression analysis in a mouse model for Norrie disease: late involvement of photoreceptor cells.

Science.gov (United States)

Lenzner, Steffen; Prietz, Sandra; Feil, Silke; Nuber, Ulrike A; Ropers, H-Hilger; Berger, Wolfgang

2002-09-01

Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exudative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot-containing extracellular signaling molecule of unknown function. In the current study, gene expression was determined in a mouse model of ND, to unravel disease-associated mechanisms at the molecular level. Gene transcription in the eyes of 2-year-old Ndp knockout mice was compared with that in the eyes of age-matched wild-type control animals, by means of cDNA subtraction and microarrays. Clones (n = 3072) from the cDNA subtraction libraries were spotted onto glass slides and hybridized with fluorescently labeled RNA-derived targets. More than 230 differentially expressed clones were sequenced, and their expression patterns were verified by virtual Northern blot analysis. Numerous gene transcripts that are absent or downregulated in the eye of Ndp knockout mice are photoreceptor cell specific. In younger Ndp knockout mice (up to 1 year old), however, all these transcripts were found to be expressed at normal levels. The identification of numerous photoreceptor cell-specific transcripts with a reduced expression in 2-year-old, but not in young, Ndp knockout mice indicates that normal gene expression in these light-sensitive cells of mutant mice is established and maintained over a long period and that rods and cones are affected relatively late in the mouse model of ND. Obviously, the absence of the Ndp gene product is not compatible with long-term survival of photoreceptor cells in the mouse.

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

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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.

Protease activity of legumain is inhibited by an increase of cystatin E/M in the DJ-1-knockout mouse spleen, cerebrum and heart

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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.

Skeletal muscle gene expression after myostatin knockout in mature mice Address for reprint requests and other correspondence: S. Welle, Univ. of Rochester Medical Center, 601 Elmwood Ave., Box 693, Rochester, NY 14642 (e-mail: ).

OpenAIRE

Welle, Stephen; Cardillo, Andrew; Zanche, Michelle; Tawil, Rabi

2009-01-01

There is much interest in developing anti-myostatin agents to reverse or prevent muscle atrophy in adults, so it is important to characterize the effects of reducing myostatin activity after normal muscle development. For assessment of the effect of loss of myostatin signaling on gene expression in muscle, RNA from mice with postdevelopmental myostatin knockout was analyzed with oligonucleotide microarrays. Myostatin was undetectable in muscle within 2 wk after Cre recombinase activation in 4...

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.

ANTXR2 is a potential causative gene in the genome-wide association study of the blood pressure locus 4q21.

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Park, So Yon; Lee, Hyeon-Ju; Ji, Su-Min; Kim, Marina E; Jigden, Baigalmaa; Lim, Ji Eun; Oh, Bermseok

2014-09-01

Hypertension is the most prevalent cardiovascular disease worldwide, but its genetic basis is poorly understood. Recently, genome-wide association studies identified 33 genetic loci that are associated with blood pressure. However, it has been difficult to determine whether these loci are causative owing to the lack of functional analyses. Of these 33 genome-wide association studies (GWAS) loci, the 4q21 locus, known as the fibroblast growth factor 5 (FGF5) locus, has been linked to blood pressure in Asians and Europeans. Using a mouse model, we aimed to identify a causative gene in the 4q21 locus, in which four genes (anthrax toxin receptor 2 (ANTXR2), PR domain-containing 8 (PRDM8), FGF5 and chromosome 4 open reading frame 22 (C4orf22)) were near the lead single-nucleotide polymorphism (rs16998073). Initially, we examined Fgf5 gene by measuring blood pressure in Fgf5-knockout mice. However, blood pressure did not differ between Fgf5 knockout and wild-type mice. Therefore, the other candidate genes were studied by in vivo small interfering RNA (siRNA) silencing in mice. Antxr2 siRNA was pretreated with polyethylenimine and injected into mouse tail veins, causing a significant decrease in Antxr2 mRNA by 22% in the heart. Moreover, blood pressure measured under anesthesia in Antxr2 siRNA-injected mice rose significantly compared with that of the controls. These results suggest that ANTXR2 is a causative gene in the human 4q21 GWAS-blood pressure locus. Additional functional studies of ANTXR2 in blood pressure may identify a novel genetic pathway, thus increasing our understanding of the etiology of essential hypertension.

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.

Problem-Solving Test: Targeted Gene Disruption

Science.gov (United States)

Szeberenyi, Jozsef

2008-01-01

Mutational inactivation of a specific gene is the most powerful technique to analyze the biological function of the gene. This approach has been used for a long time in viruses, bacteria, yeast, and fruit fly, but looked quite hopeless in more complex organisms. Targeted inactivation of specific genes (also known as knock-out mutation) in mice is…

Eliminating graphs by means of parallel knock-out schemes

NARCIS (Netherlands)

Broersma, H.J.; Fomin, F.V.; Královic, R.; Woeginger, G.J.

2007-01-01

In 1997 Lampert and Slater introduced parallel knock-out schemes, an iterative process on graphs that goes through several rounds. In each round of this process, every vertex eliminates exactly one of its neighbors. The parallel knock-out number of a graph is the minimum number of rounds after which

Eliminating graphs by means of parallel knock-out schemes

NARCIS (Netherlands)

Broersma, Haitze J.; Fomin, F.V.; Královič, R.; Woeginger, Gerhard

In 1997 Lampert and Slater introduced parallel knock-out schemes, an iterative process on graphs that goes through several rounds. In each round of this process, every vertex eliminates exactly one of its neighbors. The parallel knock-out number of a graph is the minimum number of rounds after which

Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos.

Science.gov (United States)

Jones, Elizabeth A V; Yuan, Li; Breant, Christine; Watts, Ryan J; Eichmann, Anne

2008-08-01

Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild-type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin 40 and ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.

Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish.

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Wanlada Klangnurak

Full Text Available We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm, were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

High-fat feeding in cardiomyocyte-restricted PPARdelta knockout mice leads to cardiac overexpression of lipid metabolic genes but fails to rescue cardiac phenotypes.

Science.gov (United States)

Li, Yuquan; Cheng, Lihong; Qin, Qianhong; Liu, Jian; Lo, Woo-kuen; Brako, Lowrence A; Yang, Qinglin

2009-10-01

Peroxisome proliferator-activated receptor delta (PPARdelta) is an essential determinant of basal myocardial fatty acid oxidation (FAO) and bioenergetics. We wished to determine whether increased lipid loading affects the PPARdelta deficient heart in transcriptional regulation of FAO and in the development of cardiac pathology. Cardiomyocyte-restricted PPARdelta knockout (CR-PPARdelta(-/-)) and control (alpha-MyHC-Cre) mice were subjected to 48 h of fasting and to a long-term maintenance on a (28 weeks) high-fat diet (HFD). The expression of key FAO proteins in heart was examined. Serum lipid profiles, cardiac pathology, and changes of various transduction signaling pathways were also examined. Mice subjected to fasting exhibited upregulated transcript expression of FAO genes in the CR-PPARdelta(-/-) hearts. Moreover, long-term HFD in CR-PPARdelta(-/-) mice induced a strikingly greater transcriptional response. After HFD, genes encoding key FAO enzymes were expressed remarkably more in CR-PPARdelta(-/-) hearts than in those of control mice. Despite the marked rise of FAO gene expression, corresponding protein expression remained low in the CR-PPARdelta(-/-) heart, accompanied by abnormalities in sarcomere structures and mitochondria that were similar to those of CR-PPARdelta(-/-) hearts with regular chow feeding. The CR-PPARdelta(-/-) mice displayed increased expression of PPARgamma co-activator-1alpha (PGC-1alpha) and PPARalpha in the heart with deactivated Akt and p42/44 MAPK signaling in response to HFD. We conclude that PPARdelta is an essential determinant of myocardial FAO. Increased lipid intake activates cardiac expression of FAO genes via PPARalpha/PGC-1alpha pathway, albeit it is not sufficient to improve cardiac pathology due to PPARdelta deficiency.

Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

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Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2014-02-23

Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder

OpenAIRE

Fuchs, Claudia; Gennaccaro, Laura; Trazzi, Stefania; Bastianini, Stefano; Bettini, Simone; Martire, Viviana Lo; Ren, Elisa; Medici, Giorgio; Zoccoli, Giovanna; Rimondini, Roberto; Ciani, Elisabetta

2018-01-01

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/−) mouse, has been little characterized. The lack of...

p21WAF1/Cip1/Sdi1 knockout mice respond to doxorubicin with reduced cardiotoxicity

International Nuclear Information System (INIS)

Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho; Williams, Stuart; Chen, Qin M.

2011-01-01

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21 WAF1/Cip1/Sdi1 (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: ► Doxorubicin induces p21 elevation in the myocardium. ► Doxorubicin causes dilated cardiomyopathy in wild type mice. ► p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. ► Lack of inflammatory response correlates with the resistance in p21 knockout mice.

Autism-related behavioral abnormalities in synapsin knockout mice.

Science.gov (United States)

Greco, Barbara; Managò, Francesca; Tucci, Valter; Kao, Hung-Teh; Valtorta, Flavia; Benfenati, Fabio

2013-08-15

Several synaptic genes predisposing to autism-spectrum disorder (ASD) have been identified. Nonsense and missense mutations in the SYN1 gene encoding for Synapsin I have been identified in families segregating for idiopathic epilepsy and ASD and genetic mapping analyses have identified variations in the SYN2 gene as significantly contributing to epilepsy predisposition. Synapsins (Syn I/II/III) are a multigene family of synaptic vesicle-associated phosphoproteins playing multiple roles in synaptic development, transmission and plasticity. Lack of SynI and/or SynII triggers a strong epileptic phenotype in mice associated with mild cognitive impairments that are also present in the non-epileptic SynIII(-/-) mice. SynII(-/-) and SynIII(-/-) mice also display schizophrenia-like traits, suggesting that Syns could be involved in the regulation of social behavior. Here, we studied social interaction and novelty, social recognition and social dominance, social transmission of food preference and social memory in groups of male SynI(-/-), SynII(-/-) and SynIII(-/-) mice before and after the appearance of the epileptic phenotype and compared their performances with control mice. We found that deletion of Syn isoforms widely impairs social behaviors and repetitive behaviors, resulting in ASD-related phenotypes. SynI or SynIII deletion altered social behavior, whereas SynII deletion extensively impaired various aspects of social behavior and memory, altered exploration of a novel environment and increased self-grooming. Social impairments of SynI(-/-) and SynII(-/-) mice were evident also before the onset of seizures. The results demonstrate an involvement of Syns in generation of the behavioral traits of ASD and identify Syn knockout mice as a useful experimental model of ASD and epilepsy. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

Science.gov (United States)

Zou, Yunlong; Li, Zhiyuan; Zou, Yunjing; Hao, Haiyang; Li, Ning; Li, Qiuyan

2018-04-15

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases. Copyright © 2018. Published by Elsevier Inc.

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.

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.

Cortical Gene Expression After a Conditional Knockout of 67 kDa Glutamic Acid Decarboxylase in Parvalbumin Neurons.

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Georgiev, Danko; Yoshihara, Toru; Kawabata, Rika; Matsubara, Takurou; Tsubomoto, Makoto; Minabe, Yoshio; Lewis, David A; Hashimoto, Takanori

2016-07-01

In the cortex of subjects with schizophrenia, expression of glutamic acid decarboxylase 67 (GAD67), the enzyme primarily responsible for cortical GABA synthesis, is reduced in the subset of GABA neurons that express parvalbumin (PV). This GAD67 deficit is accompanied by lower cortical levels of other GABA-associated transcripts, including GABA transporter-1, PV, brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B, somatostatin, GABAA receptor α1 subunit, and KCNS3 potassium channel subunit mRNAs. In contrast, messenger RNA (mRNA) levels for glutamic acid decarboxylase 65 (GAD65), another enzyme for GABA synthesis, are not altered. We tested the hypothesis that this pattern of GABA-associated transcript levels is secondary to the GAD67 deficit in PV neurons by analyzing cortical levels of these GABA-associated mRNAs in mice with a PV neuron-specific GAD67 knockout. Using in situ hybridization, we found that none of the examined GABA-associated transcripts had lower cortical expression in the knockout mice. In contrast, PV, BDNF, KCNS3, and GAD65 mRNA levels were higher in the homozygous mice. In addition, our behavioral test battery failed to detect a change in sensorimotor gating or working memory, although the homozygous mice exhibited increased spontaneous activities. These findings suggest that reduced GAD67 expression in PV neurons is not an upstream cause of the lower levels of GABA-associated transcripts, or of the characteristic behaviors, in schizophrenia. In PV neuron-specific GAD67 knockout mice, increased levels of PV, BDNF, and KCNS3 mRNAs might be the consequence of increased neuronal activity secondary to lower GABA synthesis, whereas increased GAD65 mRNA might represent a compensatory response to increase GABA synthesis. © The Author 2016. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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...

A new social gene in Dictyostelium discoideum, chtB

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Santorelli Lorenzo A

2013-01-01

Full Text Available Abstract Background Competitive social interactions are ubiquitous in nature, but their genetic basis is difficult to determine. Much can be learned from single gene knockouts in a eukaryote microbe. The mutants can be competed with the parent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior in the construction of a multicellular fruiting body. It is a good model organism to study the genetic basis of cooperation since it has a sequenced genome and it is amenable to genetic manipulation. When two strains of D. discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in the lab or found in the wild and genetic analyses have shown that cheating behavior can be achieved through many pathways. Results We have characterized the knockout mutant chtB, which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies on their own. When mixed in equal proportions with parental strain cells, chtB mutants contributed almost 60% of the total number of spores. To do so, chtB cells inhibit wild type cells from becoming spores, as indicated by counts and by the wild type cells’ reduced expression of the prespore gene, cotB. We found no obvious fitness costs (morphology, doubling time in liquid medium, spore production, and germination efficiency associated with the cheating ability of the chtB knockout. Conclusions In this study we describe a new gene in D. discoideum, chtB, which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, we did not detect any fitness costs associated with this behavior.

Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

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DasSarma Shiladitya

2007-06-01

Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

Female preproenkephalin-knockout mice display altered emotional responses

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Ragnauth, A.; Schuller, A.; Morgan, M.; Chan, J.; Ogawa, S.; Pintar, J.; Bodnar, R. J.; Pfaff, D. W.

2001-01-01

The endogenous opioid system has been implicated in sexual behavior, palatable intake, fear, and anxiety. The present study examined whether ovariectomized female transgenic preproenkephalin-knockout (PPEKO) mice and their wild-type and heterozygous controls displayed alterations in fear and anxiety paradigms, sucrose intake, and lordotic behavior. To examine stability of responding, three squads of the genotypes were tested across seasons over a 20-month period. In a fear-conditioning paradigm, PPEKO mice significantly increased freezing to both fear and fear + shock stimuli relative to controls. In the open field, PPEKO mice spent significantly less time and traversed significantly less distance in the center of an open field than wild-type controls. Further, PPEKO mice spent significantly less time and tended to be less active on the light side of a dark–light chamber than controls, indicating that deletion of the enkephalin gene resulted in exaggerated responses to fear or anxiety-provoking environments. These selective deficits were observed consistently across testing squads spanning 20 months and different seasons. In contrast, PPEKO mice failed to differ from corresponding controls in sucrose, chow, or water intake across a range (0.0001–20%) of sucrose concentrations and failed to differ in either lordotic or female approach to male behaviors when primed with estradiol and progesterone, thereby arguing strongly for the selectivity of a fear and anxiety deficit which was not caused by generalized and nonspecific debilitation. These transgenic data strongly suggest that opioids, and particularly enkephalin gene products, are acting naturally to inhibit fear and anxiety. PMID:11172058

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

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

Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

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Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

2015-01-21

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. Copyright © 2015 the authors 0270-6474/15/350943-17$15.00/0.

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

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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.

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

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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.

Role of plnB gene in the regulation of bacteriocin production in Lactobacillus paraplantarum L-XM1.

Science.gov (United States)

Zhang, Xiangmei; Shang, Nan; Zhang, Xu; Gui, Meng; Li, Pinglan

2013-06-12

Homologues of plnB gene have been shown to participate in regulation of bacteriocin production through quorum sensing system in other organisms, to investigate the possible role of plnB gene in Lactobacillus paraplantarum L-XM1, we cloned and insertionally inactivated the plnB gene. The plnB knockout mutant ΔplnB21 showed loss of bacteriocin production, its Bac⁺ phenotype could not be restored even after the addition of PlnA. Furthermore, reverse transcription-PCR analysis from total RNA preparations showed that the bacteriocin structural genes of the plnEF and plnJK were not transcribed in the plnB knockout mutant compared with the wild-type strain. It was therefore concluded that plnB is invovled in a quorum sensing based bacteriocin production. This is the first demonstration of a role for plnB by gene knockout in L. paraplantarum. Copyright © 2012 Elsevier GmbH. All rights reserved.

Differentially expressed genes in embryonic cardiac tissues of mice lacking Folr1 gene activity

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Schwartz Robert J

2007-11-01

Full Text Available Abstract Background Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs, it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects 123. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis. Results We have observed cardiovascular abnormalities including outflow tract and aortic arch arterial defects in genetically compromised Folr1 knockout mice. In order to investigate the molecular mechanisms underlying the failure to complete development of outflow tract and aortic arch arteries in the Folr1 knockout mouse model, we examined tissue-specific gene expression difference between Folr1 nullizygous embryos and morphologically normal heterozygous embryos during early cardiac development (14-somite stage, heart tube looping (28-somite stage, and outflow track septation (38-somite stage. Microarray analysis was performed as a primary screening, followed by investigation using quantitative real-time PCR assays. Gene ontology analysis highlighted the following ontology groups: cell migration, cell motility and localization of cells, structural constituent of cytoskeleton, cell-cell adhesion, oxidoreductase, protein folding and mRNA processing. This study provided preliminary data and suggested potential candidate genes for further description and investigation. Conclusion The results suggested that Folr1 gene ablation and abnormal folate homeostasis altered gene expression in developing heart and conotruncal tissues. These changes affected normal cytoskeleton structures, cell migration and

p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

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Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Williams, Stuart [Biomedical Engineering Program, College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Chen, Qin M., E-mail: qchen@email.arizona.edu [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States)

2011-11-15

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

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

Science.gov (United States)

2012-01-01

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. PMID:22647713

The International Mouse Phenotyping Consortium Web Portal, a unified point of access for knockout mice and related phenotyping data

Science.gov (United States)

Koscielny, Gautier; Yaikhom, Gagarine; Iyer, Vivek; Meehan, Terrence F.; Morgan, Hugh; Atienza-Herrero, Julian; Blake, Andrew; Chen, Chao-Kung; Easty, Richard; Di Fenza, Armida; Fiegel, Tanja; Grifiths, Mark; Horne, Alan; Karp, Natasha A.; Kurbatova, Natalja; Mason, Jeremy C.; Matthews, Peter; Oakley, Darren J.; Qazi, Asfand; Regnart, Jack; Retha, Ahmad; Santos, Luis A.; Sneddon, Duncan J.; Warren, Jonathan; Westerberg, Henrik; Wilson, Robert J.; Melvin, David G.; Smedley, Damian; Brown, Steve D. M.; Flicek, Paul; Skarnes, William C.; Mallon, Ann-Marie; Parkinson, Helen

2014-01-01

The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated ‘data wranglers’ work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases. PMID:24194600

Effects of PPARs agonists on cardiac metabolism in littermate and cardiomyocyte-specific PPAR-γ-knockout (CM-PGKO mice.

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Michelangela Barbieri

Full Text Available Understanding the molecular regulatory mechanisms controlling for myocardial lipid metabolism is of critical importance for the development of new therapeutic strategies for heart diseases. The role of PPARγ and thiazolidinediones in regulation of myocardial lipid metabolism is controversial. The aim of our study was to assess the role of PPARγ on myocardial lipid metabolism and function and differentiate local/from systemic actions of PPARs agonists using cardiomyocyte-specific PPARγ -knockout (CM-PGKO mice. To this aim, the effect of PPARγ, PPARγ/PPARα and PPARα agonists on cardiac function, intra-myocyte lipid accumulation and myocardial expression profile of genes and proteins, affecting lipid oxidation, uptake, synthesis, and storage (CD36, CPT1MIIA, AOX, FAS, SREBP1-c and ADPR was evaluated in cardiomyocyte-specific PPARγ-knockout (CM-PGKO and littermate control mice undergoing standard and high fat diet (HFD. At baseline, protein levels and mRNA expression of genes involved in lipid uptake, oxidation, synthesis, and accumulation of CM-PGKO mice were not significantly different from those of their littermate controls. At baseline, no difference in myocardial lipid content was found between CM-PGKO and littermate controls. In standard condition, pioglitazone and rosiglitazone do not affect myocardial metabolism while, fenofibrate treatment significantly increased CD36 and CPT1MIIA gene expression. In both CM-PGKO and control mice submitted to HFD, six weeks of treatment with rosiglitazone, fenofibrate and pioglitazone lowered myocardial lipid accumulation shifting myocardial substrate utilization towards greater contribution of glucose. In conclusion, at baseline, PPARγ does not play a crucial role in regulating cardiac metabolism in mice, probably due to its low myocardial expression. PPARs agonists, indirectly protect myocardium from lipotoxic damage likely reducing fatty acids delivery to the heart through the actions on adipose

[Influence of tissue-specific superoxide dismutase genes expression in brain cells on Drosophila melanogaster sensitivity to oxidative stress and viability].

Science.gov (United States)

Vitushynska, M V; Matiytsiv, N P; Chernyk, Y

2015-01-01

The study has shown that both functional gene knockout Sodl and Sod2 and their overexpression in neurons and glial tissue increase the sensitivity of Drosophila melanogaster to oxidative stress (OS) conditions. The lowest survival rate was only 20.5% in insects with Sod2 knockout in neurons. Comparative analysis of the survival curves showed that adults with altered tissue-specific expression of the studied genes had reduced average and maximum life span. Under OS conditions induced by 5% hydrogen peroxide the life spans of wild type Oregon R and transgenic insects were significantly reduced. Altered Sod gene expression in glial tissue leads to degenerative changes in Drosophila brain at the young age. During the aging of insects and the action of pro-oxidants increasing of neurodegenerative phenotype is observed.

One-neutron knockout from {sup 51-55}Sc

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Schwertel, S.; Maierbeck, P.; Gernhaeuser, R.; Bildstein, V.; Boehmer, M.; Eppinger, K.; Faestermann, T.; Friese, J.; Fabbietti, L.; Maier, L.; Winkler, S. [Technische Universitaet Muenchen, Physik Department E12, Garching (Germany); Kruecken, R. [Technische Universitaet Muenchen, Physik Department E12, Garching (Germany); TRIUMF, Vancouver (Canada); University of British Columbia, Department of Physics and Astronomy, Vancouver (Canada); Kroell, T. [Technische Universitaet Muenchen, Physik Department E12, Garching (Germany); Technische Universitaet Darmstadt, Institut fuer Kernphysik, Darmstadt (Germany); Alvarez-Pol, H.; Benjamim, E.A.; Benlliure, J.; Caamano, M.; Cortina-Gil, D.; Gascon, M.; Kurtukian, T.; Perez, D.; Rodriguez-Tajes, C. [Universidade de Santiago de Compostela, Departamento de Fisica de Particulas, Santiago de Compostela (Spain); 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. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Borge, M.J.G.; Pascual-Izarra, C.; Perea, A.; Tengblad, O. [CSIC, Instituto de Estructura de la Materia, Madrid (Spain); Buerger, A. [University of Oslo, SAFE/OCL, Oslo (Norway); CEA, Gif-sur-Yvette (France); Casarejos, E.; Brown, B.A. [University of Vigo, Vigo (Spain); Enders, J.; Schrieder, G. [Technische Universitaet Darmstadt, Institut fuer Kernphysik, Darmstadt (Germany); Hansen, P.G. [Michigan State University, NSCL, East Lansing, Michigan (United States); Jonson, B.; Nyman, G. [Chalmers Tekniska Hoegskola och Goeteborgs Universitet, Experimentell Fysik, Goeteborg (Sweden); Kanungo, R. [TRIUMF, Vancouver (Canada); GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Saint Mary' s University, Halifax (Canada); Kiselev, O. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Johannes Gutenberg Universitaet, Mainz (Germany); Paul Scherrer Institut, Villigen (Switzerland); Larsson, K. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Chalmers Tekniska Hoegskola och Goeteborgs Universitet, Experimentell Fysik, Goeteborg (Sweden); Le Bleis, T. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); IN2P3-CNRS/Universite Louis Pasteur, Institut Pluridisciplinaire Hubert Curien, Strasbourg Cedex 2 (France); Mahata, K. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Paul Scherrer Institut, Villigen (Switzerland); Nilsson, T. [Technische Universitaet Darmstadt, Institut fuer Kernphysik, Darmstadt (Germany); Chalmers Tekniska Hoegskola och Goeteborgs Universitet, Experimentell Fysik, Goeteborg (Sweden); Prochazka, A. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany); Comenius University, Faculty of Mathematics and Physics, Bratislava (Slovakia); Rossi, D. [Johannes Gutenberg Universitaet, Mainz (Germany); Sitar, B. [Comenius University, Faculty of Mathematics and Physics, Bratislava (Slovakia); Otsuka, T. [University of Tokyo, Hongo, Bunkyo-ku, Department of Physics, Tokyo (Japan); Tostevin, J.A. [University of Surrey, Department of Physics, Faculty of Engineering and Physical Sciences, Guildford (United Kingdom); Rae, W.D.M. [Garsington, Oxfordshire (United Kingdom)

2012-12-15

Results are presented from a one-neutron knockout experiment at relativistic energies of {approx} 420 A MeV on {sup 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 {nu}p{sub 1/2}, {nu}p{sub 3/2}, (L = 1) and {nu}f{sub 7/2}, {nu}f{sub 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 {nu}f{sub 7/2} to the {nu}p{sub 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{sub low} {sub k} obtained by evolving a chiral N3LO nucleon-nucleon potential. (orig.)

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.

Tempol improves lipid profile and prevents left ventricular hypertrophy in LDL receptor gene knockout (LDLr-/-) mice on a high-fat diet.

Science.gov (United States)

Viana Gonçalves, Igor Cândido; Cerdeira, Cláudio Daniel; Poletti Camara, Eduardo; Dias Garcia, José Antônio; Ribeiro Pereira Lima Brigagão, Maísa; Bessa Veloso Silva, Roberta; Bitencourt Dos Santos, Gérsika

2017-09-01

Dyslipidemia is associated with increased risk of cardiovascular disease and atherosclerosis, and hence with high morbidity and mortality. This study investigated the effects of the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) on lipid profile and cardiac morphology in low-density lipoprotein (LDL) receptor gene knockout (LDLr-/-) mice. Male LDLr-/- mice (three months old, approximately 22 g weight) were divided into the following groups: controls, including (1) standard chow (SC, n=8) and (2) high-fat diet (HFD, n=8); and treatment, including (3) standard chow + Tempol (SC+T, n=8) (30 mg/kg administered by gavage, once daily) and (4) high-fat diet + Tempol (HFD+T, n=8) (30 mg/kg). After 30 days of the diet/treatment, whole blood was collected for analysis of biochemical parameters (total cholesterol, triglycerides [TG], high-density lipoprotein [HDL], LDL, and very low-density lipoprotein [VLDL]). The heart was removed through thoracotomy and histological analysis of the left ventricle was performed. A significant increase in TG, LDL, and VLDL and marked left ventricular hypertrophy (LVH) were demonstrated in the HFD group relative to the SC group (p<0.05), while Tempol treatment (HFD+T group) significantly (p<0.05) prevented increases in the levels of these lipid profile markers and attenuated LVH compared with the HFD group. In this study, Tempol showed potential for the prevention of events related to serious diseases of the cardiovascular system. Copyright © 2017 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.

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

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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.

Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure

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Chakraborty Tandra

2009-10-01

Full Text Available Abstract Background Previous studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s and site(s of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds. Results Unlike hyperactive VGF knockout mice on a mixed C57Bl6-129/SvJ background, homozygous mutant mice on a C57Bl6 background were hypermetabolic with similar locomotor activity levels to Vgf+/Vgf+ mice, during day and night cycles, indicating that mechanism(s other than hyperactivity were responsible for their increased energy expenditure. In Vgf-/Vgf- knockout mice, morphological analysis of brown and white adipose tissues (BAT and WAT indicated decreased fat storage in both tissues, and decreased adipocyte perimeter and area in WAT. Changes in gene expression measured by real-time RT-PCR were consistent with increased fatty acid oxidation and uptake in BAT, and increased lipolysis, decreased lipogenesis, and brown adipocyte differentiation in WAT, suggesting that increased sympathetic nervous system activity in Vgf-/Vgf- mice may be associated with or responsible for alterations in energy expenditure and fat storage. In addition, uncoupling protein 1 (UCP1 and UCP2 protein levels, mitochondrial number, and mitochondrial cristae density were upregulated in Vgf-/Vgf- BAT. Using immunohistochemical and histochemical techniques, we detected VGF in nerve fibers innervating BAT and Vgf promoter-driven reporter expression in cervical and thoracic spinal ganglia that project to and innervate the chest wall and tissues including BAT. Moreover, VGF peptide levels were quantified by radioimmunoassay in BAT, and were found to be down-regulated by a high fat diet. Lastly, despite being hypermetabolic

Gene function analysis by artificial microRNAs in Physcomitrella patens.

KAUST Repository

Khraiwesh, Basel

2011-01-01

MicroRNAs (miRNAs) are ~21 nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest. During the last decade the moss Physcomitrella patens emerged as a model plant for functional gene analysis based on its unique ability to integrate DNA into the nuclear genome by homologous recombination which allows for the generation of targeted gene knockout mutants. In addition to this, we developed a protocol to express amiRNAs in P. patens that has particular advantages over the generation of knockout mutants and might be used to speed up reverse genetics approaches in this model species.

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.

Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

Science.gov (United States)

Varshney, Gaurav Kumar

2014-01-01

The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

NCKX3 was compensated by calcium transporting genes and bone resorption in a NCKX3 KO mouse model.

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Yang, Hyun; Ahn, Changhwan; Shin, Eun-Kyeong; Lee, Ji-Sun; An, Beum-Soo; Jeung, Eui-Bae

2017-10-15

Gene knockout is the most powerful tool for determination of gene function or permanent modification of the phenotypic characteristics of an animal. Existing methods for gene disruption are limited by their efficiency, time required for completion and potential for confounding off-target effects. In this study, a rapid single-step approach to knockout of a targeted gene in mice using zinc-finger nucleases (ZFNs) was demonstrated for generation of mutant (knockout; KO) alleles. Specifically, ZFNs to target the sodium/calcium/potassium exchanger3 (NCKX3) gene in C57bl/6j were designed using the concept of this approach. NCKX3 KO mice were generated and the phenotypic characterization and molecular regulation of active calcium transporting genes was assessed when mice were fed different calcium diets during growth. General phenotypes such as body weight and plasma ion level showed no distinct abnormalities. Thus, the potassium/sodium/calcium exchanger of NCKX3 KO mice proceeded normally in this study. As a result, the compensatory molecular regulation of this mechanism was elucidated. Renal TRPV5 mRNA of NCKX3 KO mice increased in both male and female mice. Expression of TRPV6 mRNA was only down-regulated in the duodenum of male KO mice. Renal- and duodenal expression of PTHR and VDR were not changed; however, GR mRNA expression was increased in the kidney of NCKX3 KO mice. Depletion of the NCKX3 gene in a KO mouse model showed loss of bone mineral contents and increased plasma parathyroid hormone, suggesting that NCKX3 may play a role in regulating calcium homeostasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence of Neurobiological Changes in the Presymptomatic PINK1 Knockout Rat.

Science.gov (United States)

Ferris, Craig F; Morrison, Thomas R; Iriah, Sade; Malmberg, Samantha; Kulkarni, Praveen; Hartner, Jochen C; Trivedi, Malav

2018-01-01

Genetic models of Parkinson's disease (PD) coupled with advanced imaging techniques can elucidate neurobiological disease progression, and can help identify early biomarkers before clinical signs emerge. PTEN-induced putative kinase 1 (PINK1) helps protect neurons from mitochondrial dysfunction, and a mutation in the associated gene is a risk factor for recessive familial PD. The PINK1 knockout (KO) rat is a novel model for familial PD that has not been neuroradiologically characterized for alterations in brain structure/function, alongside behavior, prior to 4 months of age. To identify biomarkers of presymptomatic PD in the PINK1 -/- rat at 3 months using magnetic resonance imaging techniques. At postnatal weeks 12-13; one month earlier than previously reported signs of motor and cognitive dysfunction, this study combined imaging modalities, including assessment of quantitative anisotropy across 171 individual brain areas using an annotated MRI rat brain atlas to identify sites of gray matter alteration between wild-type and PINK1 -/- rats. The olfactory system, hypothalamus, thalamus, nucleus accumbens, and cerebellum showed differences in anisotropy between experimental groups. Molecular analyses revealed reduced levels of glutathione, ATP, and elevated oxidative stress in the substantia nigra, striatum and deep cerebellar nuclei. Mitochondrial genes encoding proteins in Complex IV, along with mRNA levels associated with mitochondrial function and genes involved in glutathione synthesis were reduced. Differences in brain structure did not align with any cognitive or motor impairment. These data reveal early markers, and highlight novel brain regions involved in the pathology of PD in the PINK1 -/- rat before behavioral dysfunction occurs.

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.

Two-proton knockout on neutron-rich nuclei

International Nuclear Information System (INIS)

Bazin, D.; Brown, B.A.; Campbell, C.M.; Church, J.A.; Dinca, D.C.; Enders, J.; Gade, A.; Glasmacher, T.; Hansen, P.G.; Mueller, W.F.; Olliver, H.; Perry, B.C.; Sherrill, B.M.; Terry, J.R.; Tostevin, J.A.

2004-01-01

Two-proton knockout reactions on neutron-rich nuclei [Phys. Rev. Lett. 91 (2003) 012501] have been studied in inverse kinematics at intermediate energy. Strong evidence that the two-proton removal from a neutron-rich system proceeds as a direct reaction is presented, together with a preliminary theoretical discussion of the partial cross sections based on eikonal reaction theory and the many-body shell model. They show that this reaction can be used to characterize the wave functions of the projectiles and holds great promise for the study of neutron-rich nuclei

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

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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.

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

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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.

Inflammation in Lafora Disease: Evolution with Disease Progression in Laforin and Malin Knock-out Mouse Models.

Science.gov (United States)

López-González, Irene; Viana, Rosa; Sanz, Pascual; Ferrer, Isidre

2017-07-01

Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a -/- (laforin knock-out) and Epm2b -/- (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 messenger RNAs (mRNAs) are significantly increased in Epm2a -/- mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα, and Il10ra mRNAs are significantly upregulated in Epm2b -/- at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα, and Cox2 particularly in Epm2b -/- mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b -/- mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD.

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.

Science.gov (United States)

Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki; Takai, Yuki; Okumura, Yoshiko; Baba, Miki; Datsenko, Kirill A; Tomita, Masaru; Wanner, Barry L; Mori, Hirotada

2006-01-01

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Unimpaired dendritic cell functions in MVP/LRP knockout mice.

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Mossink, Marieke H; de Groot, Jan; van Zon, Arend; Fränzel-Luiten, Erna; Schoester, Martijn; Scheffer, George L; Sonneveld, Pieter; Scheper, Rik J; Wiemer, Erik A C

2003-09-01

Dendritic cells (DCs) act as mobile sentinels of the immune system. By stimulating T lymphocytes, DCs are pivotal for the initiation of both T- and B-cell-mediated immune responses. Recently, ribonucleoprotein particles (vaults) were found to be involved in the development and/or function of human DCs. To further investigate the role of vaults in DCs, we examined the effects of disruption of the major vault protein (MVP/LRP) on the development and antigen-presenting capacity of DCs, using our MVP/LRP knockout mouse model. Mononuclear bone marrow cells were isolated from wild-type and knockout mice and stimulated to differentiate to DCs. Like human DCs, the wild-type murine DC cultures strongly expressed MVP/LRP. Nevertheless, the MVP/LRP-deficient DCs developed normally and showed similar expression levels of several DC surface markers. No differences were observed in in vitro studies on the antigen uptake and presenting capacities of the wild-type and MVP/LRP knockout DCs. Moreover, immunization of the MVP/LRP-deficient mice with several T-cell antigens led to responses similar to those observed in the wild-type mice, indicating that the in vivo DC migration and antigen-presentation capacities are intact. Moreover, no differences were observed in the induction of the T cell-dependent humoral responses and orally induced peripheral T-cell tolerance. In conclusion, vaults are not required for primary DC functions. Their abundance in DCs may, however, still reflect basic roles in myeloid cell proliferation and DC development.

Requirement of NMDA receptor reactivation for consolidation and storage of nondeclarative taste memory revealed by inducible NR1 knockout.

Science.gov (United States)

Cui, Zhenzhong; Lindl, Kathryn A; Mei, Bing; Zhang, Shuqing; Tsien, Joe Z

2005-08-01

We employed an inducible, reversible and region-specific gene knockout technique to investigate the requirements for cortical NMDA receptors (NMDAR) during the various stages (acquisition, consolidation and storage, and retrieval) of nondeclarative, hippocampal-independent memory in mice using a conditioned taste aversion memory paradigm. Here we show that temporary knockout of the cortical NMDAR during either the learning or postlearning consolidation stage, but not during the retrieval stage, causes severe performance deficits in the 1-month taste memory retention tests. More importantly, we found that the consolidation and storage of the long-term nondeclarative taste memories requires cortical NMDAR reactivation. Thus, the dynamic engagement of the NMDAR during the postlearning stage leads us to postulate that NMDAR reactivation-mediated synaptic re-entry reinforcement is crucial for overcoming the destabilizing effects intrinsic to synaptic protein turnover and for achieving consolidation and storage of nondeclarative memories in the brain.

Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory.

Science.gov (United States)

Chen, Adele P; Ohno, Masuo; Giese, K Peter; Kühn, Ralf; Chen, Rachel L; Silva, Alcino J

2006-01-01

Raf kinases are downstream effectors of Ras and upstream activators of the MEK-ERK cascade. Ras and MEK-ERK signaling play roles in learning and memory (L&M) and neural plasticity, but the roles of Raf kinases in L&M and plasticity are unclear. Among Raf isoforms, B-raf is preferentially expressed in the brain. To determine whether B-raf has a role in synaptic plasticity and L&M, we used the Cre-LoxP gene targeting system to derive forebrain excitatory neuron B-raf knockout mice. This conditional knockout resulted in deficits in ERK activation and hippocampal long-term potentiation (LTP) and impairments in hippocampus-dependent L&M, including spatial learning and contextual discrimination. Despite the widespread expression of B-raf, this mutation did not disrupt other forms of L&M, such as cued fear conditioning and conditioned taste aversion. Our findings demonstrate that B-raf plays a role in hippocampal ERK activation, synaptic plasticity, and L&M.

Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

Energy Technology Data Exchange (ETDEWEB)

Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio, E-mail: y_mukai@nagahama-i-bio.ac.jp

2015-07-31

Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism

OpenAIRE

Deng, Jiao; Li, Jiejie; Li, Liaoliao; Feng, Chenzhuo; Xiong, Lize; Zuo, Zhiyi

2013-01-01

Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young a...

Chronic minocycline treatment improves social recognition memory in adult male Fmr1 knockout mice.

Science.gov (United States)

Yau, Suk Yu; Chiu, Christine; Vetrici, Mariana; Christie, Brian R

2016-10-01

Fragile X syndrome (FXS) is caused by a mutation in the Fmr1 gene that leads to silencing of the gene and a loss of its gene product, Fragile X mental retardation protein (FMRP). Some of the key behavioral phenotypes for FXS include abnormal social anxiety and sociability. Here we show that Fmr1 knock-out (KO) mice exhibit impaired social recognition when presented with a novel mouse, and they display normal social interactions in other sociability tests. Administering minocycline to Fmr1 KO mice throughout critical stages of neural development improved social recognition memory in the novel mouse recognition task. To determine if synaptic changes in the prefrontal cortex (PFC) could have played a role in this improvement, we examined PSD-95, a member of the membrane-associated guanylate kinase family, and signaling molecules (ERK1/2, and Akt) linked to synaptic plasticity in the PFC. Our analyses indicated that while minocycline treatment can enhance behavioral performance, it does not enhance expression of PSD-95, ERK1/2 or Akt in the PFC. Copyright © 2016 Elsevier B.V. All rights reserved.

Quasi-free knockout reactions with the proton-dripline nucleus {sup 17}Ne

Energy Technology Data Exchange (ETDEWEB)

Wamers, Felix; Aumann, Thomas [Institut fuer Kernphysik, TU, Darmstadt (Germany); Heil, Michael [Kernreaktionen und Nukleare Astrophysik, GSI, Darmstadt (Germany); Marganiec, Justyna [ExtreMe Matter Institute EMMI, GSI, Darmstadt (Germany); Plag, Ralf [Kernreaktionen und Nukleare Astrophysik, GSI, Darmstadt (Germany); Goethe Universitaet, Frankfurt a.M. (Germany); Collaboration: R3B-Collaboration

2011-07-01

{sup 17}Ne is a proton-dripline nucleus that has raised special interest in nuclear-structure physics in recent years. As a ({sup 15}O+2p) Borromean 3-body system, it is often considered to be a 2-proton-halo nucleus, yet lacking concluding experimental evidence about its structure. We have studied breakup reactions of 500 AMeV {sup 17}Ne secondary beams using the R{sup 3}B-LAND setup at GSI. One focus was on the quasi-free one-proton knockout in a proton-rich paraffin (CH{sub 2}) target in inverse kinematics, i.e., {sup 17}Ne(p,2p){sup 16}F{yields}{sup 15}O+p, in comparison to the one-proton knockout with a carbon target. Recoil protons have been detected with Si-Strip detectors and the surrounding 4{pi} NaI spectrometer ''Crystal Ball'', thus providing a clean signature for quasi-free knockout. First results on two-proton removal cross sections with CH{sub 2} and C targets will be presented, as well as transverse momentum distributions of the {sup 15}O core in {sup 17}Ne. Projectile-like forward protons after one-proton knockout from {sup 17}Ne have been measured in coincidence with the {sup 15}O residual core, leading to the relative-energy spectrum of the unbound {sup 16}F. Possible interpretations and implications regarding the structure of {sup 17}Ne are discussed.

Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes.

Science.gov (United States)

Upton, Dana C; Unniraman, Shyam

2011-11-01

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence

[Genes in the development of female genital tract].

Science.gov (United States)

Chen, Na; Zhu, Lan; Lang, Jing-he

2013-12-01

Female genital tract, which includes oviduct, uterus, and vagina, is critical for female reproduction. In recent years, animal experiments using knockout mice and genetic studies on patients with female genital malformations have contributed substantially to our understanding of the molecular mechanisms in the female genital tract development. Here we review genes that are involved in various stages of female genital tract formation and development.

Voluntary exercise decreases atherosclerosis in nephrectomised ApoE knockout mice.

Directory of Open Access Journals (Sweden)

Cecilia M Shing

Full Text Available Cardiovascular disease is the main cause of morbidity and mortality in patients with kidney disease. The effectiveness of exercise for cardiovascular disease that is accelerated by the presence of chronic kidney disease remains unknown. The present study utilized apolipoprotein E knockout mice with 5/6 nephrectomy as a model of combined kidney disease and cardiovascular disease to investigate the effect of exercise on aortic plaque formation, vascular function and systemic inflammation. Animals were randomly assigned to nephrectomy or control and then to either voluntary wheel running exercise or sedentary. Following 12-weeks, aortic plaque area was significantly (p0.05. Nephrectomy increased IL-6 and TNF-α concentrations compared with control mice (p0.05. Exercise was an effective non-pharmacologic approach to slow cardiovascular disease in the presence of kidney disease in the apolipoprotein E knockout mouse.

Identifying essential genes in bacterial metabolic networks with machine learning methods

Science.gov (United States)

2010-01-01

Background Identifying essential genes in bacteria supports to identify potential drug targets and an understanding of minimal requirements for a synthetic cell. However, experimentally assaying the essentiality of their coding genes is resource intensive and not feasible for all bacterial organisms, in particular if they are infective. Results We developed a machine learning technique to identify essential genes using the experimental data of genome-wide knock-out screens from one bacterial organism to infer essential genes of another related bacterial organism. We used a broad variety of topological features, sequence characteristics and co-expression properties potentially associated with essentiality, such as flux deviations, centrality, codon frequencies of the sequences, co-regulation and phyletic retention. An organism-wise cross-validation on bacterial species yielded reliable results with good accuracies (area under the receiver-operator-curve of 75% - 81%). Finally, it was applied to drug target predictions for Salmonella typhimurium. We compared our predictions to the viability of experimental knock-outs of S. typhimurium and identified 35 enzymes, which are highly relevant to be considered as potential drug targets. Specifically, we detected promising drug targets in the non-mevalonate pathway. Conclusions Using elaborated features characterizing network topology, sequence information and microarray data enables to predict essential genes from a bacterial reference organism to a related query organism without any knowledge about the essentiality of genes of the query organism. In general, such a method is beneficial for inferring drug targets when experimental data about genome-wide knockout screens is not available for the investigated organism. PMID:20438628

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.

Synthesizing genome-wide association studies and expression microarray reveals novel genes that act in the human growth plate to modulate height.

Science.gov (United States)

Lui, Julian C; Nilsson, Ola; Chan, Yingleong; Palmer, Cameron D; Andrade, Anenisia C; Hirschhorn, Joel N; Baron, Jeffrey

2012-12-01

Previous meta-analysis of genome-wide association (GWA) studies has identified 180 loci that influence adult height. However, each GWA locus typically comprises a set of contiguous genes, only one of which presumably modulates height. We reasoned that many of the causative genes within these loci influence height because they are expressed in and function in the growth plate, a cartilaginous structure that causes bone elongation and thus determines stature. Therefore, we used expression microarray studies of mouse and rat growth plate, human disease databases and a mouse knockout phenotype database to identify genes within the GWAS loci that are likely required for normal growth plate function. Each of these approaches identified significantly more genes within the GWA height loci than at random genomic locations (P analysis strongly implicates 78 genes in growth plate function, including multiple genes that participate in PTHrP-IHH, BMP and CNP signaling, and many genes that have not previously been implicated in the growth plate. Thus, this analysis reveals a large number of novel genes that regulate human growth plate chondrogenesis and thereby contribute to the normal variations in human adult height. The analytic approach developed for this study may be applied to GWA studies for other common polygenic traits and diseases, thus providing a new general strategy to identify causative genes within GWA loci and to translate genetic associations into mechanistic biological insights.

Parallel knock-out schemes in networks

NARCIS (Netherlands)

Broersma, H.J.; Fomin, F.V.; Woeginger, G.J.

2004-01-01

We consider parallel knock-out schemes, a procedure on graphs introduced by Lampert and Slater in 1997 in which each vertex eliminates exactly one of its neighbors in each round. We are considering cases in which after a finite number of rounds, where the minimimum number is called the parallel

Proton-induced $\\alpha$-cluster knockout from $^{12}$C

CERN Document Server

Cowley, A A; Förtsch, S V; Buthelezi, E Z; Neveling, R; Smit, F D; Steyn, G F; van Zyl, J J

2010-01-01

Results of a study of the (p, p ) reaction on 12C with polarized incident protons of 100 MeV are reviewed. Experimental cross section and analyzing power distributions are compared with predictions of a distorted wave impulse approximation (DWIA) theory. The theory reproduces the data reasonably well, suggesting that a quasifree knockout mechanism dominates the reaction. Spectroscopic information extracted from the cross section data is in agreement with a shell model prediction.

Sodium homeostasis is preserved in a global 11β-hydroxysteroid dehydrogenase type 1 knockout mouse model

DEFF Research Database (Denmark)

Christensen, Thorbjørn H; Bailey, Matthew A; Kenyon, Christopher J

2015-01-01

hypothesized that loss of renal 11βHSD1 would result in salt wasting and tested this in a knockout mouse model in which 11βHSD1 was deleted in all body tissues. In balance studies, 11βHSD1 deletion had no effect on water, sodium or potassium metabolism; transition to a low-sodium diet did not reveal...... that global deletion of 11βHSD1 in the mouse would give rise to a salt-wasting renal phenotype. What is the main finding and its importance? We subjected a mouse model of global 11βHSD1 deletion to studies of water and electrolyte balance, renal clearance, urinary steroid excretion, renin-angiotensin system...... a natriuretic phenotype. Renal clearance studies demonstrated identical haemodynamic parameters (arterial blood pressure, renal blood flow and glomerular filtration rate) in knockout and wild-type mice, but revealed an augmented kaliuretic response to thiazides in 11βHSD1 knockout animals. There was no effect...

Properties of the {sup 7}He ground state from {sup 8}He neutron knockout

Energy Technology Data Exchange (ETDEWEB)

Aksyutina, Yu. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Fundamental Fysik, Chalmers Tekniska Hoegskola, S-412 96 Goeteborg (Sweden); Johansson, H.T. [Fundamental Fysik, Chalmers Tekniska Hoegskola, S-412 96 Goeteborg (Sweden); Aumann, T.; Boretzky, K. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Borge, M.J.G. [Instituto Estructura de la Materia, CSIC, E-28006 Madrid (Spain); Chatillon, A. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Chulkov, L.V. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Kurchatov Institute, RU-123182 Moscow (Russian Federation); Cortina-Gil, D. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); University of Santiago de Compostela, 15706 Santiago de Compostela (Spain); Pramanik, U. Datta [Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Emling, H. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Forssen, C. [Fundamental Fysik, Chalmers Tekniska Hoegskola, S-412 96 Goeteborg (Sweden); Fynbo, H.O.U. [Department of Physics and Astronomy, University of Aarhus, DK-8000 Aarhus C (Denmark); Geissel, H.; Ickert, G. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Jonson, B. [Fundamental Fysik, Chalmers Tekniska Hoegskola, S-412 96 Goeteborg (Sweden)], E-mail: bjn@fy.chalmers.se; Kulessa, R. [Instytut Fizyki, Universytet Jagiellonski, PL-30-059 Krakow (Poland); Langer, C. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Lantz, M. [Fundamental Fysik, Chalmers Tekniska Hoegskola, S-412 96 Goeteborg (Sweden); LeBleis, T. [GSI Helmholtzzentrum fuer Schwerionenforschung GmbH, D-64291 Darmstadt (Germany); Lindahl, A.O. [Institutionen foer Fysik, University of Gothenburg, S-412 96 Goeteborg (Sweden)] (and others)

2009-08-24

The unbound nucleus {sup 7}He, produced in neutron-knockout reactions with a 240 MeV/u {sup 8}He beam in a liquid-hydrogen target, has been studied in an experiment at the ALADIN-LAND setup at GSI. From an R-matrix analysis the resonance parameters for {sup 7}He as well as the spectroscopic factor for the {sup 6}He(0{sup +}) + n configuration in its ground-state have been obtained. The spectroscopic factor is 0.61 confirming that {sup 7}He is not a pure single-particle state. An analysis of {sup 5}He data from neutron-knockout reactions of {sup 6}He in a carbon target reveals the presence of an s-wave component at low energies in the {alpha}+n relative energy spectrum. A possible low-lying exited state in {sup 7}He observed in neutron knockout data from {sup 8}He in a carbon target and tentatively interpreted as a I{sup {pi}}=1/2{sup -} state, could not be observed in the present experiment. Possible explanations of the shape difference between the {sup 7}He resonance obtained in the two knockout reactions are discussed in terms of target-dependence or different reaction mechanisms at relativistic energies.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

Science.gov (United States)

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

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.

Studies of UCP2 transgenic and knockout mice reveal that liver UCP2 is not essential for the antiobesity effects of fish oil.

Science.gov (United States)

Tsuboyama-Kasaoka, Nobuyo; Sano, Kayo; Shozawa, Chikako; Osaka, Toshimasa; Ezaki, Osamu

2008-03-01

Uncoupling protein 2 (UCP2) is a possible target molecule for energy dissipation. Many dietary fats, including safflower oil and lard, induce obesity in C57BL/6 mice, whereas fish oil does not. Fish oil increases UCP2 expression in hepatocytes and may enhance UCP2 activity by activating the UCP2 molecule or altering the lipid bilayer environment. To examine the role of liver UCP2 in obesity, we created transgenic mice that overexpressed human UCP2 in hepatocytes and examined whether UCP2 transgenic mice showed less obesity when fed a high-fat diet (safflower oil or lard). In addition, we examined whether fish oil had antiobesity effects in UCP2 knockout mice. UCP2 transgenic and wild-type mice fed a high-fat diet (safflower oil or lard) developed obesity to a similar degree. UCP2 knockout and wild-type mice fed fish oil had lower rates of obesity than mice fed safflower oil. Remarkably, safflower oil did not induce obesity in female UCP2 knockout mice, an unexpected phenotype for which we presently have no explanation. However, this unexpected effect was not observed in male UCP2 knockout mice or in UCP2 knockout mice fed a high-lard diet. These data indicate that liver UCP2 is not essential for fish oil-induced decreases in body fat.

miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity.

Directory of Open Access Journals (Sweden)

Judit Remenyi

Full Text Available miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.

A Mutation in the Dmp1 Gene Alters Phosphate Responsiveness in Mice

Science.gov (United States)

Gerard-O'Riley, Rita L.; Acton, Dena; McQueen, Amie K.; Strobel, Isabel E.; Witcher, Phillip C.; Feng, Jian Q.; Econs, Michael J.

2017-01-01

Mutations in the dentin matrix protein 1 (DMP1) gene cause autosomal recessive hypophosphatemic rickets (ARHR). Hypophosphatemia in ARHR results from increased circulating levels of the phosphaturic hormone, fibroblast growth factor 23 (FGF23). Similarly, elevated FGF23, caused by mutations in the PHEX gene, is responsible for the hypophosphatemia in X-linked hypophosphatemic rickets (XLH). Previously, we demonstrated that a Phex mutation in mice creates a lower set point for extracellular phosphate, where an increment in phosphorus further stimulates Fgf23 production to maintain low serum phosphorus levels. To test the presence of the similar set point defect in ARHR, we generated 4- and 12-week-old Dmp1/Galnt3 double knockout mice and controls, including Dmp1 knockout mice (a murine model of ARHR), Galnt3 knockout mice (a murine model of familial tumoral calcinosis), and phenotypically normal double heterozygous mice. Galnt3 knockout mice had increased proteolytic cleavage of Fgf23, leading to low circulating intact Fgf23 levels with consequent hyperphosphatemia. In contrast, Dmp1 knockout mice had little Fgf23 cleavage and increased femoral Fgf23 expression, resulting in hypophosphatemia and low femoral bone mineral density (BMD). However, introduction of the Galnt3 null allele to Dmp1 knockout mice resulted in a significant increase in serum phosphorus and normalization of BMD. This increased serum phosphorus was accompanied by markedly elevated Fgf23 expression and circulating Fgf23 levels, an attempt to reduce serum phosphorus in the face of improving phosphorus levels. These data indicate that a Dmp1 mutation creates a lower set point for extracellular phosphate and maintains it through the regulation of Fgf23 cleavage and expression. PMID:28005411

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)

Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

Science.gov (United States)

Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

2016-02-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

Science.gov (United States)

Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

2017-08-01

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Skeletal Muscle Fibre-Specific Knockout of p53 Does Not Reduce Mitochondrial Content or Enzyme Activity

Directory of Open Access Journals (Sweden)

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.

Structure around the island of inversion with single-neutron knockout reactions at GANIL

CERN Document Server

Fernández-Domínguez, B; Patterson, N; Thomas, J S; Orr, N; Chartier, M; Catford, W; Achouri, N L; Angélique, J-C; Ashwood, N I; Banu, A; Bastin,B; Brown, J; Borcea, R; Franchoo, S; Freer, M; Gaudefroy, L; Laurent, B; Labiche, M; Lemmon, R C; Negoita, F; Paschalis, S; Paul, E S; Petri, M; Roussel-Chomaz, P; Staniou, M; Taylor, M J; Trache, L

2010-01-01

The nuclear structure of the 31Mg nucleus has been studied with the singleneutron knockout reaction. We report on the preliminary results of an experiment performed with the EXOGAM array coupled, for the first time, to the SPEG spectrometer at GANIL.We present a provisional result for the inclusive single-neutron knockout cross section of σinc= 90(5) mb. Preliminary exclusive cross sections for the measured bound states, including the ground state, are also presented. Finally, preliminary longitudinal momentum distributions for the ground state and first excited state are also shown. These results are compared to Monte Carlo Shell-Model calculations in the sd-pf region.

Methamphetamine-induced changes in the striatal dopamine pathway in μ-opioid receptor knockout mice

Directory of Open Access Journals (Sweden)

Park Sang Won

2011-11-01

Full Text Available Abstract Background Repeated exposure to methamphetamine (METH can cause not only neurotoxicity but also addiction. Behavioral sensitization is widely used as an animal model for the study of drug addiction. We previously reported that the μ-opioid receptor knockout mice were resistant to METH-induced behavioral sensitization but the mechanism is unknown. Methods The present study determined whether resistance of the μ-opioid receptor (μ-OR knockout mice to behavioral sensitization is due to differential expression of the stimulatory G protein α subunit (Gαs or regulators of G-protein signaling (RGS coupled to the dopamine D1 receptor. Mice received daily intraperitoneal injections of saline or METH (10 mg/kg for 7 consecutive days to induce sensitization. On day 11(following 4 abstinent days, mice were either given a test dose of METH (10 mg/kg for behavioral testing or sacrificed for neurochemical assays without additional METH treatment. Results METH challenge-induced stereotyped behaviors were significantly reduced in the μ-opioid receptor knockout mice when compared with those in wild-type mice. Neurochemical assays indicated that there is a decrease in dopamine D1 receptor ligand binding and an increase in the expression of RGS4 mRNA in the striatum of METH-treated μ-opioid receptor knockout mice but not of METH-treated wild-type mice. METH treatment had no effect on the expression of Gαs and RGS2 mRNA in the striatum of either strain of mice. Conclusions These results indicate that down-regulation of the expression of the dopamine D1 receptor and up-regulation of RGS4 mRNA expression in the striatum may contribute to the reduced response to METH-induced stereotypy behavior in μ-opioid receptor knockout mice. Our results highlight the interactions of the μ-opioid receptor system to METH-induced behavioral responses by influencing the expression of RGS of dopamine D1 receptors.

Association between the SPRY1 gene polymorphism and obesity-related traits and osteoporosis in Korean women.

Science.gov (United States)

Jin, Hyun-Seok; Kim, Bo-Young; Kim, Jeonghyun; Hong, Kyung-Won; Jung, Suk-Yul; Lee, Yun-Seok; Huh, Dam; Oh, Bermseok; Chung, Yoon-Sok; Jeong, Seon-Yong

2013-01-01

Emerging evidence has revealed a close relationship between obesity and osteoporosis. It was reported recently that conditional knockout of the Spry1 gene in mice adipocytes causes an increase in body fat and a decrease in bone mass, and that these phenotypes are rescued by Spry1 overexpression in adipose tissue. In this study, we investigated whether genetic variation in the human SPRY1 gene is associated with obesity-related phenotypes and/or osteoporosis in humans. We performed a candidate gene association analysis between the four single nucleotide polymorphisms (SNPs) and 14 imputed SNPs in the SPRY1 gene and obesity-related traits and osteoporosis in a Korean women cohort (3013 subjects). All four SPRY1 gene SNPs were significantly associated with either obesity-related traits or osteoporosis. The TGCC haplotype in the SRPY1 gene showed simultaneous association with an increased risk for obesity-related traits, percentage body fat (p=0.0087) and percentage abdominal fat (p=0.047), and osteoporosis (odds ratio=1.50; p=0.025) in the recessive genetic model. Our results support a previous finding in conditional Spry1 gene knockout mice and suggest that the SPRY1 gene is an important genetic factor for determining the risk of both obesity and osteoporosis in humans. Copyright © 2012 Elsevier Inc. All rights reserved.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

Science.gov (United States)

Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

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

Test of distorted wave kinematic coupling approximation calculations for knockout reactions

International Nuclear Information System (INIS)

Jain, A.K.

1990-01-01

A test has been devised to check the validity of conventional distorted-wave impulse approximation (DWIA) treatment of knockout reactions. The conventional DWIA formalism separates the three-body final state Schroedinger equation for a knockout reaction into two two-body Schroedinger equations by assuming an asymptotic constant value for the three-body coupling term commonly known as the kinematic coupling approximation (KCA). In the test case, which consists of an extreme asymmetric situation where one of the distorting optical potentials is assumed to vanish, the three-body final state Schroedinger equation can be solved exactly as a product of two two-body solutions using one particular set of relative coordinates. Large influence of the three-body coupling term is seen in the comparison of the exact and KCA results for (α,2α) and (p,pα) knockout reactions when the distorting optical potentials are weakly absorbing

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.

Increased Contextual Fear Conditioning in iNOS Knockout Mice: Additional Evidence for the Involvement of Nitric Oxide in Stress-Related Disorders and Contribution of the Endocannabinoid System

Science.gov (United States)

Gomes, Felipe V.; Silva, Andréia L.; Uliana, Daniela L.; Camargo, Laura H. A.; Guimarães, Francisco S.; Cunha, Fernando Q.; Joca, Sâmia R. L.; Resstel, Leonardo B. M.

2015-01-01

Background: Inducible or neuronal nitric oxide synthase gene deletion increases or decreases anxiety-like behavior in mice, respectively. Since nitric oxide and endocannabinoids interact to modulate defensive behavior, the former effect could involve a compensatory increase in basal brain nitric oxide synthase activity and/or changes in the endocannabinoid system. Thus, we investigated the expression and extinction of contextual fear conditioning of inducible nitric oxide knockout mice and possible involvement of endocannabinoids in these responses. Methods: We evaluated the effects of a preferential neuronal nitric oxide synthase inhibitor, 7-nitroindazol, nitric oxide synthase activity, and mRNA changes of nitrergic and endocannabinoid systems components in the medial prefrontal cortex and hippocampus of wild-type and knockout mice. The effects of URB597, an inhibitor of the fatty acid amide hydrolase enzyme, which metabolizes the endocannabinoid anandamide, WIN55,212-2, a nonselective cannabinoid agonist, and AM281, a selective CB1 antagonist, on contextual fear conditioning were also evaluated. Results: Contextual fear conditioning expression was similar in wild-type and knockout mice, but the latter presented extinction deficits and increased basal nitric oxide synthase activity in the medial prefrontal cortex. 7-Nitroindazol decreased fear expression and facilitated extinction in wild-type and knockout mice. URB597 decreased fear expression in wild-type and facilitated extinction in knockout mice, whereas WIN55,212-2 and AM281 increased it in wild-type mice. Nonconditioned knockout mice showed changes in the mRNA expression of nitrergic and endocannabinoid system components in the medial prefrontal cortex and hippocampus that were modified by fear conditioning. Conclusion: These data reinforce the involvement of the nitric oxide and endocannabinoids (anandamide) in stress-related disorders and point to a deregulation of the endocannabinoid system in

[BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs].

Science.gov (United States)

Song, Shaozheng; Zhu, Mengmin; Yuan, Yuguo; Rong, Yao; Xu, Sheng; Chen, Si; Mei, Junyan; Cheng, Yong

2016-03-01

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi Sordaria macrospora and Neurospora crassa.

Science.gov (United States)

Nowrousian, Minou

2009-04-01

During fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. Analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete Sordaria macrospora. Real time PCR analysis showed that the genes from the orthologous cluster in Neurospora crassa are also upregulated during development. The cluster encodes putative polyketide biosynthesis enzymes, including a reducing polyketide synthase. Analysis of knockout strains of a predicted dehydrogenase gene from the cluster showed that mutants in N. crassa and S. macrospora are delayed in fruiting body formation. In addition to the upregulated cluster, the N. crassa genome comprises another cluster containing a polyketide synthase gene, and five additional reducing polyketide synthase (rpks) genes that are not part of clusters. To study the role of these genes in sexual development, expression of the predicted rpks genes in S. macrospora (five genes) and N. crassa (six genes) was analyzed; all but one are upregulated during sexual development. Analysis of knockout strains for the N. crassa rpks genes showed that one of them is essential for fruiting body formation. These data indicate that polyketides produced by RPKSs are involved in sexual development in filamentous ascomycetes.

Histone deacetylase 6 inhibition reduces cysts by decreasing cAMP and Ca2+ in knock-out mouse models of polycystic kidney disease.

Science.gov (United States)

Yanda, Murali K; Liu, Qiangni; Cebotaru, Valeriu; Guggino, William B; Cebotaru, Liudmila

2017-10-27

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 ( pkd1 ), primarily a signaling molecule, and PC2 ( pkd2 ), a Ca 2+ channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca 2+ was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca 2+ and increased ATP-simulated Ca 2+ release. HDAC6 inhibition reduced the release of Ca 2+ from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ -ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca 2+ levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Postnatal Gene Therapy Improves Spatial Learning Despite the Presence of Neuronal Ectopia in a Model of Neuronal Migration Disorder

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Huaiyu Hu

2016-11-01

Full Text Available Patients with type II lissencephaly, a neuronal migration disorder with ectopic neurons, suffer from severe mental retardation, including learning deficits. There is no effective therapy to prevent or correct the formation of neuronal ectopia, which is presumed to cause cognitive deficits. We hypothesized that learning deficits were not solely caused by neuronal ectopia and that postnatal gene therapy could improve learning without correcting the neuronal ectopia formed during fetal development. To test this hypothesis, we evaluated spatial learning of cerebral cortex-specific protein O-mannosyltransferase 2 (POMT2, an enzyme required for O-mannosyl glycosylation knockout mice and compared to the knockout mice that were injected with an adeno-associated viral vector (AAV encoding POMT2 into the postnatal brains with Barnes maze. The data showed that the knockout mice exhibited reduced glycosylation in the cerebral cortex, reduced dendritic spine density on CA1 neurons, and increased latency to the target hole in the Barnes maze, indicating learning deficits. Postnatal gene therapy restored functional glycosylation, rescued dendritic spine defects, and improved performance on the Barnes maze by the knockout mice even though neuronal ectopia was not corrected. These results indicate that postnatal gene therapy improves spatial learning despite the presence of neuronal ectopia.

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

Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants

DEFF Research Database (Denmark)

Claesson, M H; Endel, B; Ulrik, J

1994-01-01

Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b...

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

Directory of Open Access Journals (Sweden)

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.

A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

Science.gov (United States)

Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

2015-07-28

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice

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Hao Wang

2017-02-01

Full Text Available We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA based on the DNA microarray data from GPER-knockout versus GPER-intact (intact cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article “Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling” (Wang et al., 2016 [1]. Data have been deposited to the Gene Expression Omnibus (GEO database repository with the dataset identifier GSE86843.

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

Science.gov (United States)

Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. 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 suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. 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 and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout 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 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

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

Gene targeting and cloning in pigs using fetal liver derived cells.

Science.gov (United States)

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Knockout beyond the dripline

Energy Technology Data Exchange (ETDEWEB)

Bonaccorso, A. [INFN, Sez. di Pisa, Largo B. Pontecorvo 3, 56127 Pisa (Italy); Charity, R. J. [Department of Chemistry, Washington University, St. Louis, Missouri 63130 (United States); Kumar, R. [Department of Physics, Deenbandhu Chhoturam University of Science and Technology, Murthal, Sonepat-131039 Haryana (India); Salvioni, G. [INFN, Sez. di Pisa, Largo B. Pontecorvo 3, 56127 Pisa, Italy and Dipartimento di Fisica, Università di Pisa, Largo B. Pontecorvo 3, 56127, Pisa (Italy)

2015-02-24

In this contribution, we will describe neutron and proton removal from {sup 9}C and {sup 7}Be which are two particularly interesting nuclei entering the nucleo-synthesis pp-chain [1, 2]. Neutron and proton removal reactions have been used in the past twenty years to probe the single-particle structure of exotic nuclei. The core parallel-momentum distribution can give information on the angular momentum and spin of the nucleon initial state while the total removal cross section is sensitive to the asymptotic part of the initial wave function and also to the reaction mechanism. Because knockout is a peripheral reaction from which the Asymptotic Normalization Constant (ANC) of the single-particle wave function can be extracted, it has been used as an indirect method to obtain the rate of reactions like {sup 8}B(p,γ){sup 9}C or {sup 7}Be(p,γ){sup 8}B. Nucleon removal has recently been applied by the HiRA collaboration [3] to situations in which the remaining “core” is beyond the drip line, such as {sup 8}C and {sup 6}Be, unbound by one or more protons, and whose excitation-energy spectrum can be obtained by the invariant-mass method. By gating on the ground-state peak, “core” parallel-momentum distributions and total knockout cross sections have been obtained similar to previous studies with well-bound “cores”. In addition for each projectile, knock out to final bound states has also been obtained in several cases. We will report on the theoretical description and comparison to this experimental data for a few cases for which advances in the accuracy of the transfer-to-the continuum model [4, 5] have been made [6]. These include the use, when available, of “ab-initio” overlaps for the initial state [7] and in particular their ANC values [8]. Also, the construction of a nucleus-target folding potential for the treatment of the core-target S-matrix [9] using for the cores “ab-initio” densities [10] and state-of-the-art n−{sup 9}Be optical

Sarcocystis jamaicensis n. sp., from Red-Tailed Hawks (Buteo jamaicensis) Definitive Host and IFN-γ Gene Knockout Mice as Experimental Intermediate Host.

Science.gov (United States)

Verma, S K; von Dohlen, A Rosypal; Mowery, J D; Scott, D; Rosenthal, B M; Dubey, J P; Lindsay, D S

2017-10-01

Here, we report a new species of Sarcocystis with red-tailed hawk (RTH, Buteo jamaicensis) as the natural definitive host and IFN-γ gene knockout (KO) mice as an experimental intermediate host in which sarcocysts form in muscle. Two RTHs submitted to the Carolina Raptor Center, Huntersville, North Carolina, were euthanized because they could not be rehabilitated and released. Fully sporulated 12.5 × 9.9-μm sized sporocysts were found in intestinal scrapings of both hawks. Sporocysts were orally fed to laboratory-reared outbred Swiss Webster mice (SW, Mus musculus) and also to KO mice. The sporocysts were infective for KO mice but not for SW mice. All SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in any SW mice euthanized on days 54, 77, 103 (n = 2) or 137 post-inoculation (PI). The KO mice developed neurological signs and were necropsied between 52 to 68 days PI. Schizonts/merozoites were found in all KO mice euthanized on days 52, 55 (n = 3), 59, 61 (n = 2), 66, and 68 PI and they were confined to the brain. The predominant lesion was meningoencephalitis characterized by perivascular cuffs, granulomas, and necrosis of the neural tissue. The schizonts/merozoites were located in neural tissue and were apparently extravascular. Brain homogenates from infected KO mice were infective to KO mice by subcutaneous inoculation and when seeded on to CV-1 cells. Microscopic sarcocysts were found in skeletal muscles of 5 of 8 KO mice euthanized between 55-61 days PI. Only a few sarcocysts were detected. Sarcocysts were microscopic, up to 3.5 mm long. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 μm thick) and smooth. By transmission electron microscopy, the sarcocyst wall classified as "type 1j" (new designation). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes revealed a close relationship with Sarcocystis microti and Sarcocystis glareoli; both species infect birds as definitive hosts

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.

Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production.

Science.gov (United States)

Masud, H M Abdullah Al; Watanabe, Takahiro; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

2017-12-01

Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious

Genotypic differences in intruder-evoked immediate early gene activation in male, but not female, vasopressin 1b receptor knockout mice.

Science.gov (United States)

Witchey, Shannah K; Stevenson, Erica L; Caldwell, Heather K

2016-11-24

The neuropeptide arginine vasopressin (Avp) modulates social behaviors via its two centrally expressed receptors, the Avp 1a receptor and the Avp 1b receptor (Avpr1b). Recent work suggests that, at least in mice, Avp signaling through Avpr1b within the CA2 region of the hippocampus is critical for normal aggressive behaviors and social recognition memory. However, this brain area is just one part of a larger neural circuit that is likely to be impacted in Avpr1b knockout (-/-) mice. To identify other brain areas that are affected by altered Avpr1b signaling, genotypic differences in immediate early gene activation, i.e. c-FOS and early growth response factor 1 (EGR-1), were quantified using immunocytochemistry following a single exposure to an intruder. In females, no genotypic differences in intruder-evoked c-FOS or EGR-1 immunoreactivity were observed in any of the brain areas measured. In males, while there were no intruder-evoked genotypic differences in c-FOS immunoreactivity, genotypic differences were observed in EGR-1 immunoreactivity within the ventral bed nucleus of the stria terminalis and the anterior hypothalamus; with Avpr1b -/- males having less EGR-1 immunoreactivity in these regions than controls. These data are the first to identify specific brain areas that may be a part of a neural circuit that includes Avpr1b-expressing cells in the CA2 region of the hippocampus. It is thought that this circuit, when working properly, plays a role in how an animal evaluates its social context.

Differential Gene Expression by Lactobacillus plantarum WCFS1 in Response to Phenolic Compounds Reveals New Genes Involved in Tannin Degradation.

Science.gov (United States)

Reverón, Inés; Jiménez, Natalia; Curiel, José Antonio; Peñas, Elena; López de Felipe, Félix; de Las Rivas, Blanca; Muñoz, Rosario

2017-04-01

Lactobacillus plantarum is a lactic acid bacterium that can degrade food tannins by the successive action of tannase and gallate decarboxylase enzymes. In the L. plantarum genome, the gene encoding the catalytic subunit of gallate decarboxylase ( lpdC , or lp_2945 ) is only 6.5 kb distant from the gene encoding inducible tannase ( L. plantarum tanB [ tanB Lp ], or lp_2956 ). This genomic context suggests concomitant activity and regulation of both enzymatic activities. Reverse transcription analysis revealed that subunits B ( lpdB , or lp_0271 ) and D ( lpdD , or lp_0272 ) of the gallate decarboxylase are cotranscribed, whereas subunit C ( lpdC , or lp_2945 ) is cotranscribed with a gene encoding a transport protein ( gacP , or lp_2943 ). In contrast, the tannase gene is transcribed as a monocistronic mRNA. Investigation of knockout mutations of genes located in this chromosomal region indicated that only mutants of the gallate decarboxylase (subunits B and C), tannase, GacP transport protein, and TanR transcriptional regulator ( lp_2942 ) genes exhibited altered tannin metabolism. The expression profile of genes involved in tannin metabolism was also analyzed in these mutants in the presence of methyl gallate and gallic acid. It is noteworthy that inactivation of tanR suppresses the induction of all genes overexpressed in the presence of methyl gallate and gallic acid. This transcriptional regulator was also induced in the presence of other phenolic compounds, such as kaempferol and myricetin. This study complements the catalog of L. plantarum expression profiles responsive to phenolic compounds, which enable this bacterium to adapt to a plant food environment. IMPORTANCE Lactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables when tannins are present. L. plantarum strains degrade tannins to the less-toxic pyrogallol by the successive action of tannase and gallate decarboxylase enzymes. The genes encoding these enzymes are

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.

Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

Science.gov (United States)

Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

2011-01-01

To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories. PMID:21508930

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics

DEFF Research Database (Denmark)

de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed

2015-01-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies...

Rescue of Learning and Memory Deficits in the Human Nonsyndromic Intellectual Disability Cereblon Knock-Out Mouse Model by Targeting the AMP-Activated Protein Kinase-mTORC1 Translational Pathway.

Science.gov (United States)

Bavley, Charlotte C; Rice, Richard C; Fischer, Delaney K; Fakira, Amanda K; Byrne, Maureen; Kosovsky, Maria; Rizzo, Bryant K; Del Prete, Dolores; Alaedini, Armin; Morón, Jose A; Higgins, Joseph J; D'Adamio, Luciano; Rajadhyaksha, Anjali M

2018-03-14

A homozygous nonsense mutation in the cereblon ( CRBN ) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out ( Crbn KO ) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and Crbn KO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that Crbn KO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult Crbn KO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, Crbn KO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory. SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon ( CRBN ) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an

Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

Science.gov (United States)

Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

2015-01-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster

DEFF Research Database (Denmark)

Eriksen, Kathrine Krageskov; Hauser, Frank; Schiøtt, Morten

2000-01-01

After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino aci...... knock-out mutants, where the DLGR-2 gene is interrupted by a P element insertion, die around the time of hatching. This finding, together with the expression data, strongly suggests that DLGR-2 is exclusively involved in development....

Optimal knockout strategies in genome-scale metabolic networks using particle swarm optimization.

Science.gov (United States)

Nair, Govind; Jungreuthmayer, Christian; Zanghellini, Jürgen

2017-02-01

Knockout strategies, particularly the concept of constrained minimal cut sets (cMCSs), are an important part of the arsenal of tools used in manipulating metabolic networks. Given a specific design, cMCSs can be calculated even in genome-scale networks. We would however like to find not only the optimal intervention strategy for a given design but the best possible design too. Our solution (PSOMCS) is to use particle swarm optimization (PSO) along with the direct calculation of cMCSs from the stoichiometric matrix to obtain optimal designs satisfying multiple objectives. To illustrate the working of PSOMCS, we apply it to a toy network. Next we show its superiority by comparing its performance against other comparable methods on a medium sized E. coli core metabolic network. PSOMCS not only finds solutions comparable to previously published results but also it is orders of magnitude faster. Finally, we use PSOMCS to predict knockouts satisfying multiple objectives in a genome-scale metabolic model of E. coli and compare it with OptKnock and RobustKnock. PSOMCS finds competitive knockout strategies and designs compared to other current methods and is in some cases significantly faster. It can be used in identifying knockouts which will force optimal desired behaviors in large and genome scale metabolic networks. It will be even more useful as larger metabolic models of industrially relevant organisms become available.

Next-generation text-mining mediated generation of chemical response-specific gene sets for interpretation of gene expression data

Science.gov (United States)

2013-01-01

Background Availability of chemical response-specific lists of genes (gene sets) for pharmacological and/or toxic effect prediction for compounds is limited. We hypothesize that more gene sets can be created by next-generation text mining (next-gen TM), and that these can be used with gene set analysis (GSA) methods for chemical treatment identification, for pharmacological mechanism elucidation, and for comparing compound toxicity profiles. Methods We created 30,211 chemical response-specific gene sets for human and mouse by next-gen TM, and derived 1,189 (human) and 588 (mouse) gene sets from the Comparative Toxicogenomics Database (CTD). We tested for significant differential expression (SDE) (false discovery rate -corrected p-values sets and the CTD-derived gene sets in gene expression (GE) data sets of five chemicals (from experimental models). We tested for SDE of gene sets for six fibrates in a peroxisome proliferator-activated receptor alpha (PPARA) knock-out GE dataset and compared to results from the Connectivity Map. We tested for SDE of 319 next-gen TM-derived gene sets for environmental toxicants in three GE data sets of triazoles, and tested for SDE of 442 gene sets associated with embryonic structures. We compared the gene sets to triazole effects seen in the Whole Embryo Culture (WEC), and used principal component analysis (PCA) to discriminate triazoles from other chemicals. Results Next-gen TM-derived gene sets matching the chemical treatment were significantly altered in three GE data sets, and the corresponding CTD-derived gene sets were significantly altered in five GE data sets. Six next-gen TM-derived and four CTD-derived fibrate gene sets were significantly altered in the PPARA knock-out GE dataset. None of the fibrate signatures in cMap scored significant against the PPARA GE signature. 33 environmental toxicant gene sets were significantly altered in the triazole GE data sets. 21 of these toxicants had a similar toxicity pattern as the

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)

Studies on functional roles of the histaminergic neuron system by using pharmacological agents, knockout mice and positron emission tomography

Energy Technology Data Exchange (ETDEWEB)

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)

Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

Science.gov (United States)

Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

2016-06-01

Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

OpenAIRE

Smithies, O; Maeda, N

1995-01-01

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These di...

Site-specific selfish genes as tools for the control and genetic engineering of natural populations.

Science.gov (United States)

Burt, Austin

2003-05-07

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Deficits in learning and memory in mice with a mutation of the candidate dyslexia susceptibility gene Dyx1c1.

Science.gov (United States)

Rendall, Amanda R; Tarkar, Aarti; Contreras-Mora, Hector M; LoTurco, Joseph J; Fitch, R Holly

2017-09-01

Dyslexia is a learning disability characterized by difficulty learning to read and write. The underlying biological and genetic etiology remains poorly understood. One candidate gene, dyslexia susceptibility 1 candidate 1 (DYX1C1), has been shown to be associated with deficits in short-term memory in dyslexic populations. The purpose of the current study was to examine the behavioral phenotype of a mouse model with a homozygous conditional (forebrain) knockout of the rodent homolog Dyx1c1. Twelve Dyx1c1 conditional homozygous knockouts, 7 Dyx1c1 conditional heterozygous knockouts and 6 wild-type controls were behaviorally assessed. Mice with the homozygous Dyx1c1 knockout showed deficits on memory and learning, but not on auditory or motor tasks. These findings affirm existing evidence that DYX1C1 may play an underlying role in the development of neural systems important to learning and memory, and disruption of this function could contribute to the learning deficits seen in individuals with dyslexia. Copyright © 2015 Elsevier Inc. All rights reserved.

Positive-negative-selection-mediated gene targeting in rice

Directory of Open Access Journals (Sweden)

Zenpei eShimatani

2015-01-01

Full Text Available Gene targeting (GT refers to the designed modification of genomic sequence(s through homologous recombination (HR. GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology (NPBT based on PNS are discussed.

Light-Dependent Expression of Four Cryptic Archaeal Circadian Gene Homologs

Directory of Open Access Journals (Sweden)

Michael eManiscalco

2014-03-01

Full Text Available Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. To investigate this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12 during balanced growth in H. volcanii. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes results in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

Epigenetic modifications by Trithorax group proteins during early embryogenesis: do members of Trx-G function as maternal effect genes?

Science.gov (United States)

Andreu-Vieyra, Claudia; Matzuk, Martin M

2007-02-01

Maternal effect genes encode transcripts that are expressed during oogenesis. These gene products are stored in the oocyte and become functional during resumption of meiosis and zygote genome activation, and in embryonic stem cells. To date, a few maternal effect genes have been identified in mammals. Epigenetic modifications have been shown to be important during early embryonic development and involve DNA methylation and post-translational modification of core histones. During development, two families of proteins have been shown to be involved in epigenetic changes: Trithorax group (Trx-G) and Polycomb group (Pc-G) proteins. Trx-G proteins function as transcriptional activators and have been shown to accumulate in the oocyte. Deletion of Trx-G members using conventional knockout technology results in embryonic lethality in the majority of the cases analysed to date. Recent studies using conditional knockout mice have revealed that at least one family member is necessary for zygote genome activation. We propose that other Trx-G members may also regulate embryonic genome activation and that the use of oocyte-specific deletor mouse lines will help clarify their roles in this process.

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Science.gov (United States)

de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed; White, Jacqui; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl Mj; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve Dm

2015-09-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates.

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Ida Elken Sønderby

Full Text Available BACKGROUND: Glucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools. METHODOLOGY/PRINCIPAL FINDINGS: MYB28 was identified as a candidate regulator of aliphatic glucosinolates based on its co-localization within a genomic region controlling variation both in aliphatic glucosinolate content (metabolite QTL and in transcript level for genes involved in the biosynthesis of aliphatic glucosinolates (expression QTL, as well as its co-expression with genes in aliphatic glucosinolate biosynthesis. A phylogenetic analysis with the R2R3 motif of MYB28 showed that it and two homologues, MYB29 and MYB76, were members of an Arabidopsis-specific clade that included three characterized regulators of indole glucosinolates. Over-expression of the individual MYB genes showed that they all had the capacity to increase the production of aliphatic glucosinolates in leaves and seeds and induce gene expression of aliphatic biosynthetic genes within leaves. Analysis of leaves and seeds of single knockout mutants showed that mutants of MYB29 and MYB76 have reductions in only short-chained aliphatic glucosinolates whereas a mutant in MYB28 has reductions in both short- and long-chained aliphatic glucosinolates. Furthermore, analysis of a double knockout in MYB28 and MYB29 identified an emergent property of the system since the absence of aliphatic glucosinolates in these plants could not be predicted by the chemotype of the single knockouts. CONCLUSIONS/SIGNIFICANCE: It seems that these cruciferous-specific MYB regulatory genes have evolved both overlapping and specific regulatory capacities. This provides a unique system within which to

Immunologic applications of conditional gene modification technology in the mouse.

Science.gov (United States)

Sharma, Suveena; Zhu, Jinfang

2014-04-02

Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources. Copyright © 2014 John Wiley & Sons, Inc.

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.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

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Ueda, Kohei; Nishimoto, Mitsuhiro; Hirohama, Daigoro; Ayuzawa, Nobuhiro; Kawarazaki, Wakako; Watanabe, Atsushi; Shimosawa, Tatsuo; Loffing, Johannes; Zhang, Ming-Zhi; Marumo, Takeshi; Fujita, Toshiro

2017-07-01

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl - cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl - cotransporter phosphorylation. Accordingly, a Na + -Cl - cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl - cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na + -Cl - cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the

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...... investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. Conclusions: There were no changes in the GPR39 knockout...

Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration

International Nuclear Information System (INIS)

Chowdhury, Uttam K.; Zakharyan, Robert A.; Hernandez, Alba; Avram, Mihaela D.; Kopplin, Michael J.; Aposhian, H. Vasken

2006-01-01

Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic + 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested. The livers of male knockout (KO) mice, in which 222 bp of Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate

Defects in ultrasonic vocalization of cadherin-6 knockout mice.

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Ryoko Nakagawa

Full Text Available BACKGROUND: Although some molecules have been identified as responsible for human language disorders, there is still little information about what molecular mechanisms establish the faculty of human language. Since mice, like songbirds, produce complex ultrasonic vocalizations for intraspecific communication in several social contexts, they can be good mammalian models for studying the molecular basis of human language. Having found that cadherins are involved in the vocal development of the Bengalese finch, a songbird, we expected cadherins to also be involved in mouse vocalizations. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether similar molecular mechanisms underlie the vocalizations of songbirds and mammals, we categorized behavioral deficits including vocalization in cadherin-6 knockout mice. Comparing the ultrasonic vocalizations of cadherin-6 knockout mice with those of wild-type controls, we found that the peak frequency and variations of syllables were differed between the mutant and wild-type mice in both pup-isolation and adult-courtship contexts. Vocalizations during male-male aggression behavior, in contrast, did not differ between mutant and wild-type mice. Open-field tests revealed differences in locomotors activity in both heterozygote and homozygote animals and no difference in anxiety behavior. CONCLUSIONS/SIGNIFICANCE: Our results suggest that cadherin-6 plays essential roles in locomotor activity and ultrasonic vocalization. These findings also support the idea that different species share some of the molecular mechanisms underlying vocal behavior.

β2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

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Jiang, Youde; Zhang, Qiuhua; Liu, Li; Tang, Jie; Kern, Timothy S.; Steinle, Jena J.

2013-01-01

There is considerable evidence from our lab and others for a functional link between β-adrenergic receptor and insulin receptor signaling pathways in retina. Furthermore, we hypothesize that this link may contribute to lesions similar to diabetic retinopathy in that the loss of adrenergic input observed in diabetic retinopathy may disrupt normal anti-apoptotic insulin signaling, leading to retinal cell death. Our studies included assessment of neural retina function (ERG), vascular degeneration, and Müller glial cells (which express only β1 and β2-adrenergic receptor subtypes). In the current study, we produced β2-adrenergic receptor knockout mice to examine this deletion on retinal neurons and vasculature, and to identify specific pathways through which β2-adrenergic receptor modulates insulin signaling. As predicted from our hypothesis, β2-adrenergic receptor knockout mice display certain features similar to diabetic retinopathy. In addition, loss of β2-adrenergic input resulted in an increase in TNFα, a key inhibitor of insulin receptor signaling. Increased TNFα may be associated with insulin-dependent production of the anti-apoptotic factor, Akt. Since the effects occurred in vivo under normal glucose conditions, we postulate that aspects of the diabetic retinopathy phenotype might be triggered by loss of β2-adrenergic receptor signaling. PMID:23894672

Microfluidic measurement of effects of ACF7/MACF1 gene on the mechanics of primary cortical neurons

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Lee, Donghee; Ka, Minhan; Kim, Woo-Yang; Ryu, Sangjin

2014-03-01

Actin filaments and microtubules play important roles in determining the mechanics of cells, and ACF7/MACF1 (Actin Crosslinking Family 7/Microtubule And Actin Crosslinking Factor 1) gene seems to be closely related to connections between actin filaments and microtubules. To identify such roles of the ACF7/MACF1 gene of primary cortical neurons, we isolated neuronal cells from the cerebral cortex of the embryonic mouse brain, which is important in memory, language and perception. We exerted viscous shear flow to normal neuronal cells and ACF7/MACF1 gene knockout neuronal cells using rectangular microfluidic channels. While changing viscous shear stress on the cells, we recorded changes in the morphology of the two cell types using video microscopy. Having analyzed the deformation of the cells, we could quantitatively correlate differences in the morphological change between the both normal and ACF7/MACF1 gene knockout neuronal cells to the applied shear force, which will contribute toward identifying cell mechanical roles of the ACF7/MACF1 gene.

Knockout and fragmentation reactions using a broad range of tin isotopes

Science.gov (United States)

Rodríguez-Sánchez, J. L.; Benlliure, J.; Bertulani, C. A.; Vargas, J.; Ayyad, Y.; Alvarez-Pol, H.; Atkinson, J.; Aumann, T.; Beceiro-Novo, S.; Boretzky, K.; Caamaño, M.; Casarejos, E.; Cortina-Gil, D.; Díaz-Cortes, J.; Fernández, P. Díaz; Estrade, A.; Geissel, H.; Kelić-Heil, A.; Litvinov, Yu. A.; Mostazo, M.; Paradela, C.; Pérez-Loureiro, D.; Pietri, S.; Prochazka, A.; Takechi, M.; Weick, H.; Winfield, J. S.

2017-09-01

Production cross sections of residual nuclei obtained by knockout and fragmentation reactions of different tin isotopes accelerated at 1 A GeV have been measured with the fragment separator (FRS) at GSI, Darmstadt. The new measurements are used to investigate the neutron-excess dependence of the neutron- and proton-knockout cross sections. These cross sections are compared to Glauber model calculations coupled to a nuclear de-excitation code in order to investigate the role of the remnant excitations. This bench marking shows an overestimation of the cross sections for the removal of deeply bound nucleons. A phenomenological increase in the excitation energy induced in the remnants produced in these cases allows us to reproduce the measured cross sections.

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination.

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Astha Malik

Full Text Available Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG of the hippocampus and the subventricular zone (SVZ. Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte

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.

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

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

A modulatory role of the Rax homeobox gene in mature pineal gland function

DEFF Research Database (Denmark)

Rohde, Kristian; Bering, Tenna; Furukawa, Takahisa

2017-01-01

The retinal and anterior neural fold homeobox gene (Rax) controls development of the eye and the forebrain. Postnatal expression of Rax in the brain is restricted to the pineal gland, a forebrain structure devoted to melatonin synthesis. The role of Rax in pineal function is unknown. In order...... to investigate the role of Rax in pineal function while circumventing forebrain abnormalities of the global Rax knockout, we generated an eye and pineal-specific Rax conditional knockout mouse. Deletion of Rax in the pineal gland did not affect morphology of the gland, suggesting that Rax is not essential...... for the nucleus to develop. Telemetric analyses confirmed the lack of a functional circadian clock. Arylalkylamine N-acetyltransferase (Aanat) transcripts, encoding the melatonin rhythm-generating enzyme, were undetectable in the pineal gland of the Rax conditional knockout under normal conditions, whereas...

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

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Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

2013-01-01

SUMMARY 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-ERT 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. PMID:23471917

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.

Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans.

Science.gov (United States)

Sangaletti, Rachele; D'Amico, Massimo; Grant, Jeff; Della-Morte, David; Bianchi, Laura

2017-08-01

Sirtuins are NAD⁺-dependent deacetylases, lipoamidases, and ADP-ribosyltransferases that link cellular metabolism to multiple intracellular pathways that influence processes as diverse as cell survival, longevity, and cancer growth. Sirtuins influence the extent of neuronal death in stroke. However, different sirtuins appear to have opposite roles in neuronal protection. In Caenorhabditis elegans, we found that knock-out of mitochondrial sirtuin sir-2.3, homologous to mammalian SIRT4, is protective in both chemical ischemia and hyperactive channel induced necrosis. Furthermore, the protective effect of sir-2.3 knock-out is enhanced by block of glycolysis and eliminated by a null mutation in daf-16/FOXO transcription factor, supporting the involvement of the insulin/IGF pathway. However, data in Caenorhabditis elegans cell culture suggest that the effects of sir-2.3 knock-out act downstream of the DAF-2/IGF-1 receptor. Analysis of ROS in sir-2.3 knock-out reveals that ROS become elevated in this mutant under ischemic conditions in dietary deprivation (DD), but to a lesser extent than in wild type, suggesting more robust activation of a ROS scavenging system in this mutant in the absence of food. This work suggests a deleterious role of SIRT4 during ischemic processes in mammals that must be further investigated and reveals a novel pathway that can be targeted for the design of therapies aimed at protecting neurons from death in ischemic conditions.

Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

Science.gov (United States)

Szeberényi, József

2013-01-01

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

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.

A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.

Science.gov (United States)

Shibui, Tatsuro; Hara, Hiroyoshi

2017-09-01

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017. © 2017 American Institute of Chemical Engineers.

Altered gene expression in pulmonary tissue of tryptophan hydroxylase-1 knockout mice: implications for pulmonary arterial hypertension.

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Richard B Rothman

Full Text Available The use of fenfluramines can increase the risk of developing pulmonary arterial hypertension (PAH in humans, but the mechanisms responsible are unresolved. A recent study reported that female mice lacking the gene for tryptophan hydroxylase-1 (Tph1(-/- mice were protected from PAH caused by chronic dexfenfluramine, suggesting a pivotal role for peripheral serotonin (5-HT in the disease process. Here we tested two alternative hypotheses which might explain the lack of dexfenfluramine-induced PAH in Tph1(-/- mice. We postulated that: 1 Tph1(-/- mice express lower levels of pulmonary 5-HT transporter (SERT when compared to wild-type controls, and 2 Tph1(-/- mice display adaptive changes in the expression of non-serotonergic pulmonary genes which are implicated in PAH. SERT was measured using radioligand binding methods, whereas gene expression was measured using microarrays followed by quantitative real time PCR (qRT-PCR. Contrary to our first hypothesis, the number of pulmonary SERT sites was modestly up-regulated in female Tph1(-/- mice. The expression of 51 distinct genes was significantly altered in the lungs of female Tph1(-/- mice. Consistent with our second hypothesis, qRT-PCR confirmed that at least three genes implicated in the pathogenesis of PAH were markedly up-regulated: Has2, Hapln3 and Retlna. The finding that female Tph1(-/- mice are protected from dexfenfluramine-induced PAH could be related to compensatory changes in pulmonary gene expression, in addition to reductions in peripheral 5-HT. These observations emphasize the intrinsic limitation of interpreting data from studies conducted in transgenic mice that are not fully characterized.

Serotonin₂A/C receptors mediate the aggressive phenotype of TLX gene knockout mice.

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Juárez, Pablo; Valdovinos, Maria G; May, Michael E; Lloyd, Blair P; Couppis, Maria H; Kennedy, Craig H

2013-11-01

Deleting the tailless (TLX) gene in mice produces a highly aggressive phenotype yet to be characterized in terms of heterozygous animals or neurotransmitter mechanisms. We sought to establish pharmacological control over aggression and study the role of serotonin (5-HT)(2A/C) receptors in mediating changes in aggression. We analyzed aggression in mice heterozygous (+/-) or homozygous (-/-) for the TLX gene and wild-types (+/+) using a resident-intruder paradigm. No +/+ mice were aggressive, 36% of +/- TLX and 100% of -/- TLX mice showed aggression. Dose-effect functions were established for clozapine (0.1-1.5mg/kg, ip), ketanserin (0.3-1.25 mg/kg, ip), and (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI] (0.5-2.0 mg/kg, ip). Injecting clozapine decreased the frequency and duration of attacks for +/- TLX and -/- TLX mice. Clozapine did not decrease grooming in either +/- TLX or -/- TLX mice but may have increased locomotion for -/- TLX mice. Injecting ketanserin, a 5-HT(2A/C) receptor antagonist, produced differential decreases in frequency and latency to aggression between genotypes and corresponding increases in locomotor behavior. Injecting (±)DOI, a 5-HT(2A/C) receptor agonist, increased the frequency and duration of attacks, decreased the latency to attacks, and decreased locomotion in +/- and -/- TLX mice. Results of the current study suggest aggression displayed by TLX null and heterozygous mice involves 5-HT(2A/C) receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

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Benvenisti-Zarom Luna

2004-09-01

Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle

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Ruiz-Perera, Lucia M.; Kadhim, Hussamadin M.; Tertel, Tobias; Henkel, Elena; Hübner, Wolfgang; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian

2017-01-01

Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies. PMID:28767691

Trinucleon cluster knockout from 6Li

International Nuclear Information System (INIS)

Connelly, J.P.; Berman, B.L.; Briscoe, W.J.; Dhuga, K.S.; Mokhtari, A.; Zubanov, D.; Blok, H.P.; Ent, R.; Mitchell, J.H.; Lapikas, L.

1998-01-01

The momentum-transfer dependence of the 3 H and 3 He knockout reactions from 6 Li via exclusive electron scattering has been measured, and the two reactions are compared. In the absence of two-step processes, the ratio of the fivefold cross sections for these mirror reactions should simply scale by the ratio of the 3 H and 3 He electron-scattering cross sections. A significant deviation from this simple expectation is seen at low momentum transfer. Possible explanations for this dramatic difference in cross sections for these mirror reactions are discussed. copyright 1998 The American Physical Society

Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration

DEFF Research Database (Denmark)

Nunes, Ana Filipa; Montero, Maria; Franquinho, Filipa

2009-01-01

Transthyretin (TTR) has been regarded as a neuroprotective protein given that TTR knockout (KO) mice display increased susceptibility for amyloid beta deposition and memory deficits during aging. In parallel, TTR KO mice have increased levels of neuropeptide Y (NPY), which promotes neuroprotectio...

The Effect of Different Photoperiods in Circadian Rhythms of Per3 Knockout Mice

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D. S. Pereira

2014-01-01

Full Text Available The aim of this study was to analyse the circadian behavioural responses of mice carrying a functional knockout of the Per3 gene (Per3−/− to different light : dark (L : D cycles. Male adult wild-type (WT and Per3−/− mice were kept under 12-hour light : 12-hour dark conditions (12L : 12D and then transferred to either a short or long photoperiod and subsequently released into total darkness. All mice were exposed to both conditions, and behavioural activity data were acquired through running wheel activity and analysed for circadian characteristics during these conditions. We observed that, during the transition from 12L : 12D to 16L : 8D, Per3−/− mice take approximately one additional day to synchronise to the new L : D cycle compared to WT mice. Under these long photoperiod conditions, Per3−/− mice were more active in the light phase. Our results suggest that Per3−/− mice are less sensitive to light. The data presented here provides further evidence that Per3 is involved in the suppression of behavioural activity in direct response to light.

Transcriptional profiling of Foxo3a and Fancd2 regulated genes in mouse hematopoietic stem cells

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Xiaoli Li

2015-06-01

Full Text Available Functional maintenance of hematopoietic stem cells (HSCs is constantly challenged by stresses like DNA damage and oxidative stress. Foxo factors particularly Foxo3a function to regulate the self-renewal of HSCs and contribute to the maintenance of the HSC pool during aging by providing resistance to oxidative stress. Fancd2-deficient mice had multiple hematopoietic defects including HSC loss in early development and in response to cellular stresses including oxidative stress. The cellular mechanisms underlying HSC loss in Fancd2-deficient mice include abnormal cell cycle status loss of quiescence and compromised hematopoietic repopulating capacity of HSCs. To address on a genome wide level the genes and pathways that are impacted by deletion of the Fancd2 and Foxo3a we performed microarray analysis on phenotypic HSCs (Lin−ckit+Sca-1+CD150+CD48− from Fancd2 single knockout Foxo3a single knockout and Fancd2−/−Foxo3a−/− double-knockout (dKO mice. Here we provide detailed methods and analysis on these microarray data which has been deposited in Gene Expression Omnibus (GEO: GSE64215.

Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

International Nuclear Information System (INIS)

Holzer, Georg W.; Mayrhofer, Josef; Gritschenberger, Werner; Falkner, Falko G.

2005-01-01

The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

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Liang Zhu

Full Text Available Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB and a β-carotene hydroxylase gene (crtZ located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

Cloning and characterization of genes involved in nostoxanthin biosynthesis of Sphingomonas elodea ATCC 31461.

Science.gov (United States)

Zhu, Liang; Wu, Xuechang; Li, Ou; Qian, Chaodong; Gao, Haichun

2012-01-01

Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed.

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

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Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, Volker; Rülicke, Thomas; Rathkolb, Birgit; Hans, Wolfgang; Bohla, Alexander; Eickelberg, Oliver; Stoeger, Tobias; Wolf, Eckhard; Yildirim, Ali Önder; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Hozák, Pavel

2013-01-01

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.

Spectroscopy of 17C via one-neutron knockout reaction

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Kim Sunji

2016-01-01

Full Text Available A spectroscopic study of 17C was performed via the one-neutron knockout reaction of 18C on a carbon target at RIKEN-RIBF. Three unbound states at excitation energies of 2.66(2, 3.16(5, and 3.97(3 MeV (preliminary were observed. The energies are compared with shell-model calculations and existing measurements to deduce their spin-parities. From the comparison, the states at 2.66(2 and 3.97(3 MeV are suggested to be 1/2− and 3/2−, respectively. From its decay property, the state at 3.16(5 MeV is indicated to be 9/2+.

A novel radiation responsive cis-acting element regulates gene induction and mediates tissue injury

International Nuclear Information System (INIS)

Hallahan, Dennis E.; Virudachalam, Subbulakshmi; Kuchibahtla, Jaya

1997-01-01

Purpose: The intracellular adhesion molecule (ICAM-1) binds and activates inflammatory cells and thereby contributes to the pathogenesis of tissue injury. To characterize a model for radiation-induction of tissue injury, we studied radiation-mediated lung injury in mice deficient in the ICAM-1 gene. To study the mechanisms of x-ray mediated ICAM induction, we studied transcriptional activation of the ICAM promoter and nuclear protein binding to the 5' untranslated region of the ICAM gene. Methods: Immunohistochemistry and immunofluorescence were used to study the histologic pattern of ICAM expression in irradiated tissue. The ICAM-1 knockout mice were bred with wild type mice to create heterozygous mice with attenuated ICAM expression. ICAM -/-, ICAM+/- and ICAM +/+ mice were treated with thoracic irradiation and lung sections were stained for leukocyte common antigen (CD45) to study inflammation. To study the mechanism of x-ray induction of ICAM, we linked the 5' untranslated region of the ICAM gene to the luciferase reporter gene and delated DNA segments from the promoter to determine which elements are required for induction. We performed electrophoretic mobility shift analysis of nuclear proteins from irradiated endothelial cells to study transcription factor activation. Results: Immunohistochemistry showed dose and time dependent increases in ICAM protein expression in irradiated lungs which was prolonged as compared to endothelial cells in vitro. The histologic pattern of ICAM expression was in the capillary endothelium and was distinct from the pattern of expression of other radiation-inducible adhesion molecules. ICAM knockout mice had no ICAM expression and no inflammatory cell accumulation in the irradiated lung. ICAM+/+ mice developed leukocyte adhesion to irradiated endothelium within hours of irradiation and radiation pneumonitis 5 to 6 weeks later. The DNA sequence between -981 and -769 (relative to start codon) contains two 16-base pair repeats, each

The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones

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Satoshi Watanabe

2018-04-01

Full Text Available Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9 have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA, humanized Cas9 (hCas9 gene, or Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B4 lectin (IB4. Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were

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

Fundamental study of detection of muscle hypertrophy-oriented gene doping by myostatin knock down using RNA interference.

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Takemasa, Tohru; Yakushiji, Naohisa; Kikuchi, Dale Manjiro; Deocaris, Custer; Widodo; Machida, Masanao; Kiyosawa, Hidenori

2012-01-01

To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.

Impaired Eye-Blink Conditioning in waggler, a Mutant Mouse With Cerebellar BDNF Deficiency

OpenAIRE

Bao, Shaowen; Chen, Lu; Qiao, Xiaoxi; Knusel, Beat; Thompson, Richard F.

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expressi...

Choroid plexus transport: gene deletion studies

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Keep Richard F

2011-11-01

Full Text Available Abstract This review examines the use of transporter knockout (KO animals to evaluate transporter function at the choroid plexus (the blood-CSF barrier; BCSFB. Compared to the blood-brain barrier, there have been few such studies on choroid plexus (CP function. These have primarily focused on Pept2 (an oligopeptide transporter, ATP-binding cassette (ABC transporters, Oat3 (an organic anion transporter, Svct2 (an ascorbic acid transporter, transthyretin, ion transporters, and ion and water channels. This review focuses on the knowledge gained from such studies, both with respect to specific transporters and in general to the role of the CP and its impact on brain parenchyma. It also discusses the pros and cons of using KO animals in such studies and the technical approaches that can be used.

Ethanol self-administration in serotonin transporter knockout mice: unconstrained demand and elasticity.

Science.gov (United States)

Lamb, R J; Daws, L C

2013-10-01

Low serotonin function is associated with alcoholism, leading to speculation that increasing serotonin function could decrease ethanol consumption. Mice with one or two deletions of the serotonin transporter (SERT) gene have increased extracellular serotonin. To examine the relationship between SERT genotype and motivation for alcohol, we compared ethanol self-administration in mice with zero (knockout, KO), one (HET) or two copies (WT) of the SERT gene. All three genotypes learned to self-administer ethanol. The SSRI, fluvoxamine, decreased responding for ethanol in the HET and WT, but not the KO mice. When tested under a progressive ratio schedule, KO mice had lower breakpoints than HET or WT. As work requirements were increased across sessions, behavioral economic analysis of ethanol self-administration indicated that the decreased breakpoint in KO as compared to HET or WT mice was a result of lower levels of unconstrained demand, rather than differences in elasticity, i.e. the proportional decreases in ethanol earned with increasing work requirements were similar across genotypes. The difference in unconstrained demand was unlikely to result from motor or general motivational factors, as both WT and KO mice responded at high levels for a 50% condensed milk solution. As elasticity is hypothesized to measure essential value, these results indicate that KO value ethanol similarly to WT or HET mice despite having lower break points for ethanol. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice

Science.gov (United States)

Nakao, Akito; Miki, Takafumi; Shoji, Hirotaka; Nishi, Miyuki; Takeshima, Hiroshi; Miyakawa, Tsuyoshi; Mori, Yasuo

2015-01-01

Calcium (Ca2+) influx through voltage-gated Ca2+ channels (VGCCs) induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca2+ signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO) mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached “study-wide significance.” Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions. PMID:26136667

Deletion and Gene Expression Analyses Define the Paxilline Biosynthetic Gene Cluster in Penicillium paxilli

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Emily J. Parker

2013-08-01

Full Text Available The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse. This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

The alpha/beta carboxy-terminal domains of p63 are required for skin and limb development. New insights from the Brdm2 mouse which is not a complete p63 knockout but expresses p63 gamma-like proteins

DEFF Research Database (Denmark)

Wolff, S; Talos, F; Palacios, G

2009-01-01

p63, an ancestral transcription factor of the p53 family, has three C-terminal isoforms whose relative in vivo functions are elusive. The p63 gene is essential for skin and limb development, as vividly shown by two independent global knockout mouse models. Both strains, although constructed diffe...

NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program

DEFF Research Database (Denmark)

Hiraike, Yuta; Waki, Hironori; Yu, Jing

2017-01-01

. NFIA and the master transcriptional regulator of adipogenesis, PPARγ, co-localize at the brown-fat-specific enhancers. Moreover, the binding of NFIA precedes and facilitates the binding of PPARγ, leading to increased chromatin accessibility and active transcription. Introduction of NFIA into myoblasts...... results in brown adipocyte differentiation. Conversely, the brown fat of NFIA-knockout mice displays impaired expression of the brown-fat-specific genes and reciprocal elevation of muscle genes. Finally, expression of NFIA and the brown-fat-specific genes is positively correlated in human brown fat...

Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis.

Science.gov (United States)

Morrison, Christopher M; Dial, Sharon M; Day, William A; Joens, Lynn A

2012-04-01

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.

TAM receptor knockout mice are susceptible to retinal autoimmune induction.

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Ye, Fei; Li, Qiutang; Ke, Yan; Lu, Qingjun; Han, Lixia; Kaplan, Henry J; Shao, Hui; Lu, Qingxian

2011-06-16

TAM receptors are expressed mainly by dendritic cells and macrophages in the immune system, and mice lacking TAM receptors develop systemic autoimmune diseases because of inefficient negative control of the cytokine signaling in those cells. This study aims to test the susceptibility of the TAM triple knockout (tko) mice to the retina-specific autoantigen to develop experimental autoimmune uveoretinitis (EAU). TAM tko mice that were or were not immunized with interphotoreceptor retinoid-binding protein (IRBP) peptides were evaluated for retinal infiltration of the macrophages and CD3(+) T cells by immunohistochemistry, spontaneous activation of CD4(+) T cells, and memory T cells by flow cytometry and proliferation of IRBP-specific CD4(+) T cells by [(3)H]thymidine incorporation assay. Ocular inflammation induced by IRBP peptide immunization and specific T cell transfer were observed clinically by funduscopy and confirmed by histology. Tko mice were found to have less naive, but more activated, memory T cells, among which were exhibited high sensitivity to ocular IRBP autoantigens. Immunization with a low dose of IRBP and adoptive transfer of small numbers of IRBP-specific T cells from immunized tko mice caused the infiltration of lymphocytes, including CD3(+) T cells, into the tko retina. Mice without TAM receptor spontaneously develop IRBP-specific CD4(+) T cells and are more susceptible to retinal autoantigen immunization. This TAM knockout mouse line provides an animal model with which to study the role of antigen-presenting cells in the development of T cell-mediated uveitis.

A STAT-1 knockout mouse model for Machupo virus pathogenesis

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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.

Differential response of nNOS knockout mice to MDMA ("ecstasy")- and methamphetamine-induced psychomotor sensitization and neurotoxicity.

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Itzhak, Yossef; Anderson, Karen L; Ali, Syed F

2004-10-01

It has been shown that mice deficient in neuronal nitric oxide synthase (nNOS) gene are resistant to cocaine-induced psychomotor sensitization and methamphetamine (METH)-induced dopaminergic neurotoxicity. The present study was undertaken to investigate the hypothesis that nNOS has a major role in dopamine (DA)- but not serotonin (5-hydroxytryptamine; 5-HT)-mediated effects of psychostimulants. The response of nNOS knockout (KO) and wild-type (WT) mice to the psychomotor-stimulating and neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") and METH were investigated. Repeated administration of MDMA for 5 days resulted in psychomotor sensitization in both WT and nNOS KO mice, while repeated administration of METH caused psychomotor sensitization in WT but not in KO mice. Sensitization to both MDMA and METH was persistent for 40 days in WT mice, but not in nNOS KO mice. These findings suggest that the induction of psychomotor sensitization to MDMA and METH is NO independent and NO dependent, respectively, while the persistence of sensitization to both drugs is NO dependent. For the neurochemical studies, a high dose of MDMA caused marked depletion of 5-HT in several brain regions of both WT and KO mice, suggesting that the absence of the nNOS gene did not afford protection against MDMA-induced depletion of 5-HT. Striatal dopaminergic neurotoxicity caused by high doses of MDMA and METH in WT mice was partially prevented in KO mice administered with MDMA, but it was fully precluded in KO mice administered with METH. The differential response of nNOS KO mice to the behavioral and neurotoxic effects of MDMA and METH suggests that the nNOS gene is required for the expression and persistence of DA-mediated effects of METH and MDMA, while 5-HT-mediated effects of MDMA (induction of sensitization and 5-HT depletion) are not dependent on nNOS.

FUNDAMENTAL STUDY OF DETECTION OF MUSCLE HYPERTROPHY-ORIENTED GENE DOPING BY MYOSTATIN KNOCK DOWN USING RNA INTERFERENCE

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Tohru Takemasa

2012-06-01

Full Text Available To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control

Gene replacement in Penicillium roqueforti.

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Goarin, Anne; Silar, Philippe; Malagnac, Fabienne

2015-05-01

Most cheese-making filamentous fungi lack suitable molecular tools to improve their biotechnology potential. Penicillium roqueforti, a species of high industrial importance, would benefit from functional data yielded by molecular genetic approaches. This work provides the first example of gene replacement by homologous recombination in P. roqueforti, demonstrating that knockout experiments can be performed in this fungus. To do so, we improved the existing transformation method to integrate transgenes into P. roqueforti genome. In the meantime, we cloned the PrNiaD gene, which encodes a NADPH-dependent nitrate reductase that reduces nitrate to nitrite. Then, we performed a deletion of the PrNiaD gene from P. roqueforti strain AGO. The ΔPrNiaD mutant strain is more resistant to chlorate-containing medium than the wild-type strain, but did not grow on nitrate-containing medium. Because genomic data are now available, we believe that generating selective deletions of candidate genes will be a key step to open the way for a comprehensive exploration of gene function in P. roqueforti.

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

CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability

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Martinez, Virginia; Lauritsen, Ida; Hobel, Tonja

2017-01-01

Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing pro...

MYT1L mutations cause intellectual disability and variable obesity by dysregulating gene expression and development of the neuroendocrine hypothalamus.

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Patricia Blanchet

2017-08-01

Full Text Available Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense. The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms "mental retardation". To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

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Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

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Dubey, J P; Sundar, N; Kwok, O C H; Saville, W J A

2013-09-01

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals. Published by Elsevier B.V.

Peptidomic analysis of the neurolysin-knockout mouse brain.

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Castro, Leandro M; Cavalcanti, Diogo M L P; Araujo, Christiane B; Rioli, Vanessa; Icimoto, Marcelo Y; Gozzo, Fábio C; Juliano, Maria; Juliano, Luiz; Oliveira, Vitor; Ferro, Emer S

2014-12-05

A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein-protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013

EFFECTS OF PHYSICAL TRAINING ON THE MYOCARDIUM OF FEMALE LDL KNOCKOUT OVARIECTOMIZED MICE

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Brianezi, Ledimar; Marques, Mara Rubia; Cardoso, Clever Gomes; Miranda, Maria Luiza de Jesus; Fonseca, Fernando Luiz Affonso; Maifrino, Laura Beatriz Mesiano

2017-01-01

ABSTRACT Introduction: The emergence of coronary heart disease increases with menopause, physical inactivity and with dyslipidemia. It is known that physical training promotes the improvement of cardiovascular functions. Objective: The purpose of this study was to investigate the effects of aerobic physical training on the left ventricle in female LDL knockout ovariectomized mice. Methods: Thirty animals were divided into 6 groups (n=5), namely, sedentary non-ovariectomized control; sedentary...

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder

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Claudia Fuchs

2018-01-01

Full Text Available CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/− mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/− mice. We found that Cdkl5 +/− mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/− mice show age-related alterations in protein kinase B (AKT and extracellular signal-regulated kinase (ERK signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/− mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.

Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex.

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Ventura, R; Pascucci, T; Catania, M V; Musumeci, S A; Puglisi-Allegra, S

2004-09-01

Fragile X syndrome is an X-linked form of mental retardation including, among others, symptoms such as stereotypic behaviour, hyperactivity, hyperarousal, and cognitive deficits. We hypothesized that hyperactivity and/or compromised attentional, cognitive functions may lead to impaired performance in cognitive tasks in Fmr1 knockout mice, the most widely used animal model of fragile X syndrome, and suggested that psychostimulant treatment may improve performance by acting on one or both components. Since hyperactivity and cognitive functions have been suggested to depend on striatal and prefrontal cortex dopaminergic dysfunction, we assessed whether amphetamine produced beneficial, positive effects by acting on dopaminergic corticostriatal systems. Our results show that Fmr1 knockout mice are not able to discriminate between a familiar object and a novel one in the object recognition test, thus showing a clear-cut cognitive impairment that, to date, has been difficult to demonstrate in other cognitive tasks. Amphetamine improved performance of Fmr1 knockout mice, leading to enhanced ability to discriminate novel versus familiar objects, without significantly affecting locomotor activity. In agreement with behavioural data, amphetamine produced a greater increase in dopamine release in the prefrontal cortex of Fmr1 knockout compared with the wild-type mice, while a weak striatal dopaminergic response was observed in Fmr1 knockout mice. Our data support the view that the psychostimulant ameliorates performance in Fmr1 knockout mice by improving merely cognitive functions through its action on prefrontal cortical dopamine, irrespective of its action on motor hyperactivity. These results indicate that prefrontal cortical dopamine plays a major role in cognitive impairments characterizing Fmr1 knockout mice, thus pointing to an important aetiological factor in the fragile X syndrome.

Knock-Outs, Stick-Outs, Cut-Outs: Clipping Paths Separate Objects from Background.

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Wilson, Bradley

1998-01-01

Outlines a six-step process that allows computer operators, using Photoshop software, to create "knock-outs" to precisely define the path that will serve to separate the object from the background. (SR)

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

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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

In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice.

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Río, Paula; Segovia, José Carlos; Hanenberg, Helmut; Casado, José Antonio; Martínez, Jesús; Göttsche, Kerstin; Cheng, Ngan Ching; Van de Vrugt, Henri J; Arwert, Fré; Joenje, Hans; Bueren, Juan A

2002-09-15

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect of Fanca(-/-) progenitors, either when total BM or when purified Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-) mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors of Fanca(-/-) mice.

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

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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.

Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause.

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Belo, Najara O; Sairam, M Ram; Dos Reis, Adelina M

2008-03-01

Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.

Variable Persister Gene Interactions with (pppGpp for Persister Formation in Escherichia coli

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Shuang Liu

2017-09-01

Full Text Available Persisters comprise a group of phenotypically heterogeneous metabolically quiescent bacteria with multidrug tolerance and contribute to the recalcitrance of chronic infections. Although recent work has shown that toxin-antitoxin (TA system HipAB depends on stringent response effector (pppGppin persister formation, whether other persister pathways are also dependent on stringent response has not been explored. Here we examined the relationship of (pppGpp with 15 common persister genes (dnaK, clpB, rpoS, pspF, tnaA, sucB, ssrA, smpB, recA, umuD, uvrA, hipA, mqsR, relE, dinJ using Escherichia coli as a model. By comparing the persister levels of wild type with their single gene knockout and double knockout mutants with relA, we divided their interactions into five types, namely A “dependent” (dnaK, recA, B “positive reinforcement” (rpoS, pspF, ssrA, recA, C “antagonistic” (clpB, sucB, umuD, uvrA, hipA, mqsR, relE, dinJ, D “epistasis” (clpB, rpoS, tnaA, ssrA, smpB, hipA, and E “irrelevant” (dnaK, clpB, rpoS, tnaA, sucB, smpB, umuD, uvrA, hipA, mqsR, relE, dinJ. We found that the persister gene interactions are intimately dependent on bacterial culture age, cell concentrations (diluted versus undiluted culture, and drug classifications, where the same gene may belong to different groups with varying antibiotics, culture age or cell concentrations. Together, this study represents the first attempt to systematically characterize the intricate relationships among the different mechanisms of persistence and as such provide new insights into the complexity of the persistence phenomenon at the level of persister gene network interactions.

Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

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Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

2013-09-01

Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

[Construction of Corynebacterium crenatum AS 1.542 δ argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway].

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Chen, Xuelan; Tang, Li; Jiao, Haitao; Xu, Feng; Xiong, Yonghua

2013-01-04

ArgR, coded by the argR gene from Corynebacterium crenatum AS 1.542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene: C. crenatum AS 1.542 Delta argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. We used marker-less knockout technology to construct C. crenatum AS 1.542 Delta argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. C. crenatum AS 1.542 Delta argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds. The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis

DEFF Research Database (Denmark)

Husted, L B; Harsløf, T; Stenkjær, L

2013-01-01

variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse......UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three...... of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted...

Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level

Directory of Open Access Journals (Sweden)

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.

Screen for genes involved in radiation survival of Escherichia coli and construction of a reference database

Energy Technology Data Exchange (ETDEWEB)

Sargentini, Neil J., E-mail: nsargentini@atsu.edu; Gularte, Nicholas P.; Hudman, Deborah A.

2016-11-15

Highlights: • 3907 Keio knockout mutants of E. coli screened for UV and X-radiation sensitivity. • 76 mutants showed significantly increased radiation sensitivity. • A database of 9 screening studies listed 352 genes only once; 103 genes, 2–7 times. • 33 genes from this study are uncommon and potentially novel. • Common and uncommon genes differ in gene function profile. - Abstract: A set of 3907 single-gene knockout (Keio collection) strains of Escherichia coli K-12 was examined for strains with increased susceptibility to killing by X- or UV-radiation. After screening with a high-throughput resazurin-based assay and determining radiation survival with triplicate clonogenic assays, we identified 76 strains (and associated deleted genes) showing statistically-significant increased radiation sensitivity compared to a control strain. To determine gene novelty, we constructed a reference database comprised of genes found in nine similar studies including ours. This database contains 455 genes comprised of 103 common genes (found 2–7 times), and 352 uncommon genes (found once). Our 76 genes includes 43 common genes and 33 uncommon (potentially novel) genes, i.e., appY, atoS, betB, bglJ, clpP, cpxA, cysB, cysE, ddlA, dgkA, dppF, dusB, elfG, eutK, fadD, glnA, groL, guaB, intF, prpR, queA, rplY, seqA, sufC,yadG, yagJ, yahD, yahO, ybaK, ybfA, yfaL, yhjV, and yiaL. Of our 33 uncommon gene mutants, 4 (12%) were sensitive only to UV-radiation, 10 (30%) only to X-radiation, and 19 (58%) to both radiations. Our uncommon mutants vs. our common mutants showed more radiation specificity, i.e., 12% vs. 9% (sensitive only to UV-); 30% vs. 16% (X-) and 58% vs. 74% (both radiations). Considering just our radiation-sensitive mutants, the median UV-radiation survival (75 J m{sup −2}) for 23 uncommon mutants was 6.84E-3 compared to 1.85E-3 for 36 common mutants (P = 0.025). Similarly, the average X-radiation survival for 29 uncommon mutants was 1.08E-2, compared to 6.19E

K Basins floor sludge retrieval system knockout pot basket fuel burn accident

International Nuclear Information System (INIS)

HUNT, J.W.

1998-01-01

The K Basins Sludge Retrieval System Preliminary Hazard Analysis Report (HNF-2676) identified and categorized a series of potential accidents associated with K Basins Sludge Retrieval System design and operation. The fuel burn accident was of concern with respect to the potential release of contamination resulting from a runaway chemical reaction of the uranium fuel in a knockout pot basket suspended in the air. The unmitigated radiological dose to an offsite receptor from this fuel burn accident is calculated to be much less than the offsite risk evaluation guidelines for anticipated events. However, because of potential radiation exposure to the facility worker, this accident is precluded with a safety significant lifting device that will prevent the monorail hoist from lifting the knockout pot basket out of the K Basin water pool

Beijing ambient particle exposure accelerates atherosclerosis in ApoE knockout mice.

Science.gov (United States)

Chen, Tian; Jia, Guang; Wei, Yongjie; Li, Jiucun

2013-11-25

Air pollution is associated with significant adverse health effects including increased cardiovascular morbidity and mortality. However research on the cardiovascular effect of "real-world" exposure to ambient particulate matter (PM) in susceptible animal model is very limited. In this study, we aimed to investigate the association between Beijing ambient particle exposure and the atherosclerosis development in the apolipoprotein E knockout mice (ApoE(-/-) mice). Two parallel exposure chambers were used for whole body exposure among ApoE knockout mice. One of the chambers was supplied with untreated ambient air (PM group) and the other chamber was treated with ambient air filtered by high-efficiency particulate air (HEPA) filter (FA group). Twenty mice were divided into two groups and exposed to ambient PM (n=10 for PM group) or filtered air (n=10 for FA group) for two months from January 18th to March 18th, 2010. During the exposure, the mass concentrations of PM2.5 and PM10 in the two chambers were continuously monitored. Additionally, a receptor source apportionment model of chemical mass balance using 19 organic tracers was applied to determine the contributions of sources on the PM2.5 in terms of natural gas, diesel vehicle, gasoline vehicle, coal burning, vegetable debris, biomass burning and cooking. At the end of the two-month exposure, biomarkers of oxidative stress, inflammation and lipid metabolism in bronchoalveolar lavage fluid (BAL) and blood samples were determined and the plaque area on the aortic endothelium was quantified. In the experiment, the concentrations of PM10 and PM2.5 in PM chamber were 99.45μg/m(3) and 61.0μg/m(3) respectively, while PM2.5 in FA chamber was 17.6μg/m(3). Source apportionment analysis by organic tracers showed that gasoline vehicle (39.9%) and coal burning (24.3%) emission were the two major sources contributing to the mass concentration of PM2.5 in Beijing. Among the ApoE knockout mice, the PM group were significantly

Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration.

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Yuan Wang

Full Text Available With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635.

Effects of strain and age on hepatic gene expression profiles in murine models of HFE-associated hereditary hemochromatosis.

Science.gov (United States)

Lee, Seung-Min; Loguinov, Alexandre; Fleming, Robert E; Vulpe, Christopher D

2015-01-01

Hereditary hemochromatosis is an iron overload disorder most commonly caused by a defect in the HFE gene. While the genetic defect is highly prevalent, the majority of individuals do not develop clinically significant iron overload, suggesting the importance of genetic modifiers. Murine hfe knockout models have demonstrated that strain background has a strong effect on the severity of iron loading. We noted that hepatic iron loading in hfe-/- mice occurs primarily over the first postnatal weeks (loading phase) followed by a timeframe of relatively static iron concentrations (plateau phase). We thus evaluated the effects of background strain and of age on hepatic gene expression in Hfe knockout mice (hfe-/-). Hepatic gene expression profiles were examined using cDNA microarrays in 4- and 8-week-old hfe-/- and wild-type mice on two different genetic backgrounds, C57BL/6J (C57) and AKR/J (AKR). Genes differentially regulated in all hfe-/- mice groups, compared with wild-type mice, including those involved in cell survival, stress and damage responses and lipid metabolism. AKR strain-specific changes in lipid metabolism genes and C57 strain-specific changes in cell adhesion and extracellular matrix protein genes were detected in hfe-/- mice. Mouse strain and age are each significantly associated with hepatic gene expression profiles in hfe-/- mice. These affects may underlie or reflect differences in iron loading in these mice.

Low cadmium (LCD), a novel gene related to cadmium tolerance and accumulation in rice

OpenAIRE

Shimo, Hugo; Ishimaru, Yasuhiro; An, Gynheung; Yamakawa, Takashi; Nakanishi, Hiromi; Nishizawa, Naoko K.

2011-01-01

The contamination of food crops by cadmium (Cd) is a major concern in food production because it can reduce crop yields and threaten human health. In this study, knockout rice plants (Oryza sativa) tagged with the gene trap vector pGA2707 were screened for Cd tolerance, and the tolerant line lcd was obtained. The lcd mutant showed tolerance to Cd on agar plates and in hydroponic culture during early plant development. Metal concentration measurements in hydroponically grown plants revealed si...

Impact of Campylobacter jejuni cj0268c knockout mutation on intestinal colonization, translocation, and induction of immunopathology in gnotobiotic IL-10 deficient mice.

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Markus M Heimesaat

Full Text Available BACKGROUND: Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c, or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i. revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis, mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls. CONCLUSION/SIGNIFICANCE: We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further

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

Knockout mouse model for Fxr2: a model for mental retardation

NARCIS (Netherlands)

C.J.M. Bontekoe (Carola); L. Kirkpatrick; C.E. Bakker (Cathy); A.T. Hoogeveen (Andre); R. McAninch; M. Merriweather; B.A. Oostra (Ben); N.C. Cheng (Ngan Ching); K.L. McIlwain; I.M. Nieuwenhuizen (Ingeborg); L.A. Yuva-Paylor; R. Paylor; A. Nellis; R. Willemsen (Rob); Z. Fang; D. Nelson

2002-01-01

textabstractFragile X syndrome is a common form of mental retardation caused by the absence of the FMR1 protein, FMRP. Fmr1 knockout mice exhibit a phenotype with some similarities to humans, such as macro-orchidism and behavioral abnormalities. Two homologs of FMRP have been

Medium effects on spin observables of proton knockout reactions

International Nuclear Information System (INIS)

Krein, G.; Maris, T.A.J.; Rodrigues, B.B.; Veit, E.A.

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

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.

RF-knockout Extraction System for the CNAO Synchrotron

CERN Document Server

Carmignani, Nicola; Serio, Mario; Balbinot, Giovanni; Bressi, Erminia; Caldara, Michele; Pullia, Marco; Bosser, Jacques; Venchi, Giuseppe

2010-01-01

The National Centre for Oncological Hadrontherapy (CNAO) is a centre in Italy for the treatment of patients affected by tumours with proton and carbon ions beams accelerated in a synchrotron. The synchrotron extraction method is based on the use of a betatron core. This work aims to verify, through a theoretical study and a simulation, the possibility of using the RF-knockout extraction method exploiting the existing hardware. A simulation program has been written to simulate the extraction system of the synchrotron with the purpose to define the parameters of the radio frequency. Two types of radio frequencies have been compared in order to obtain a constant spill with the minimum ripple: a carrier wave with a frequency and amplitude modulation, and a gaussian narrow band noise modulated in amplitude. Results of the simulation and considerations on the kicker characteristics are presented

Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

Science.gov (United States)

Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

2016-03-01

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

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

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

Conditional RARα Knockout Mice Reveal Acute Requirement for Retinoic Acid and RARα in Homeostatic Plasticity

Directory of Open Access Journals (Sweden)

Federica eSarti

2012-02-01

Full Text Available All-trans retinoic acid (RA plays important roles in brain development through regulating gene transcription. Recently, a novel postdevelopmental role of RA in mature brain was proposed. Specifically, RA rapidly enhanced excitatory synaptic transmission independent of transcriptional regulation. RA synthesis was induced when excitatory synaptic transmission was chronically blocked, and RA then activated dendritic protein synthesis and synaptic insertion of homomeric GluA1 AMPA receptors, thereby compensating for the loss of neuronal activity in a homeostatic fashion. This action of RA was suggested to be mediated by its canonical receptor RARα but no genetic evidence was available. Thus, we here tested the fundamental requirement of RARα in homeostatic plasticity using conditional RARα knockout mice, and additionally performed a structure-function analysis of RARα. We show that acutely deleting RARα in neurons eliminated RA’s effect on excitatory synaptic transmission, and inhibited activity blockade-induced homeostatic synaptic plasticity. By expressing various RARα rescue constructs in RARα knockout neurons, we found that the DNA-binding domain of RARα was dispensable for its role in regulating synaptic strength, further supporting the notion that RA and RARα act in a non-transcriptional manner in this context. By contrast, the ligand-binding domain (LBD and the mRNA-binding domain (F-domain are both necessary and sufficient for the function of RARα in homeostatic plasticity. Furthermore, we found that homeostatic regulation performed by the LBD/F domains leads to insertion of calcium-permeable AMPA receptors. Our results confirm with unequivocal genetic approaches that RA and RARα perform essential non-transcriptional functions in regulating synaptic strength, and establish a functional link between the various domains of RARα and their involvement in regulating protein synthesis and excitatory synaptic transmission during

[Effect of Huanglian Jiedu Decoction on Monocyte Development in apoE Gene Knockout Mice].

Science.gov (United States)

Chen, Bing; Kong, Ya-xian; Ll, Yu-mei; Xue, Xin; Zhang, Jian-ping; Zeng, Hui; Hu, Jing- qing; Ma, Ya-luan

2016-01-01

To observe monocyte (Mo) development in wild type C57BL/6 mice and apoE gene knockout (apoE(-/-)) mice, and to evaluate the immuno-regulatory effect of Huanglian Jiedu Decoction (HJD) on peripheral Mo development in apoE(-/-) mice. Four, 8, 12, and 16 weeks old female C57BL/6 mice were set up as control groups of different ages, while 4, 8, 12, and 16 weeks old female apoE(-/-) mice were set up as hyperlipidemia groups of different ages. Four-week old female C57BL/6 mice were recruited as a blank group. Four-week old female apoE(-/-) mice were randomly divided into the control group, the Western medicine group, and the Chinese medicine group by paired comparison, 5 in each group. Equivalent clinical dose was administered to mice according to body weight. Mice in the Western medicine group were administered with Atrovastatin at the daily dose of 10 mg/kg by gastrogavage, while those in the Chinese medicine group were administered with HJD at the daily dose of 5 g/kg by gastrogavage. Body weight was detected each week. After 4 weeks blood lipids levels (such as TG, TC, LDL-C, and HDL-C), and the proportions of Mo and Ly6c(hi) were detected. Compared with 4-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05). Levels of TC and TG, and the proportion of Ly6c(hi) subtype increased, but the proportion of Mo de- creased in 8-week-old apoE(-/-) mice (P <0. 05). Levels of TC, TG, and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05). Levels of TC, TG, LDL-C, and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with 8-week-old homogenic mice, the proportion of Mo decreased in 16-week-old C57BL/6 mice (P < 0.05); levels of TC and LDL-C increased in 12-week-old apoE(-/-) mice (P < 0.05); levels of TC and HDL-C increased in 16-week-old apoE(-/-) mice (P < 0.05, P < 0.01). Compared with C57BL/6 mice of the same age, TC and TG increased, HDL-C decreased (P < 0.01) in 4-and 8-week-old apoE(-/-) mice (P

Haplotype-based case-control study on human apurinic/apyrimidinic endonuclease 1/redox effector factor-1 gene and essential hypertension.

Science.gov (United States)

Naganuma, Takahiro; Nakayama, Tomohiro; Sato, Naoyuki; Fu, Zhenyan; Soma, Masayoshi; Yamaguchi, Mai; Shimodaira, Masanori; Aoi, Noriko; Usami, Ron

2010-02-01

Oxidative DNA damage is involved in the pathophysiology of essential hypertension (EH), which is a multifactorial disorder. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/REF-1) is an essential endonuclease in the base excision repair pathway of oxidatively damaged DNA, in addition to having reducing properties that promote the binding of redox-sensitive transcription factors. Blood pressure in APE1/REF-1-knockout mice is reported to be significantly higher than in wild-type mice. The aim of this study was to investigate the relationship between EH and the human APE1/REF-1 gene through a haplotype-based case-control study using single-nucleotide polymorphisms (SNPs). We selected five SNPs in the human APE1/REF-1 gene (rs1760944, rs3136814, rs17111967, rs3136817, and rs1130409), and performed case-control studies in 265 EH patients and 266 age-matched normotensive (NT) subjects. rs17111967 was found to show nonheterogeneity among Japanese subjects. There were no significant differences in the overall distribution of genotypes or alleles for each SNP between EH and NT groups. In the overall distribution of the haplotype-based case-control study constructed based on rs1760944, rs3136817, and rs1130409, the frequency of the G-T-T haplotype was significantly higher in the EH group than in the NT group (2.1% vs. 0.0%, P = 0.001). Multiple logistic regression analysis also revealed significant differences for the G-T-T haplotype, even after adjustment for confounding factors (OR = 8.600, 95% CI: 1.073-68.951, P = 0.043). Based on the present results, the G-T-T haplotype appears to be a genetic marker of EH, and the APE1/REF-1 gene appears to be a susceptibility gene for EH.

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

Disruption of the GH Receptor Gene in Adult Mice Increases Maximal Lifespan in Females

DEFF Research Database (Denmark)

Junnila, Riia K.; Duran-Ortiz, Silvana; Suer, Ozan

2016-01-01

GH and IGF-1 are important for a variety of physiological processes including growth, development, and aging. Mice with reduced levels of GH and IGF-1 have been shown to live longer than wild-type controls. Our laboratory has previously found that mice with a GH receptor gene knockout (GHRKO) fro...

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

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Science.gov (United States)

Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

2015-05-01

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

DEFF Research Database (Denmark)

Sørensen, B.; Lamm, Trine Tandrup; Koltzenburg, M.

2003-01-01

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...

RNA-Seq analysis of Gtf2ird1 knockout epidermal tissue provides potential insights into molecular mechanisms underpinning Williams-Beuren syndrome.

Science.gov (United States)

Corley, Susan M; Canales, Cesar P; Carmona-Mora, Paulina; Mendoza-Reinosa, Veronica; Beverdam, Annemiek; Hardeman, Edna C; Wilkins, Marc R; Palmer, Stephen J

2016-06-13

Williams-Beuren Syndrome (WBS) is a genetic disorder associated with multisystemic abnormalities, including craniofacial dysmorphology and cognitive defects. It is caused by a hemizygous microdeletion involving up to 28 genes in chromosome 7q11.23. Genotype/phenotype analysis of atypical microdeletions implicates two evolutionary-related transcription factors, GTF2I and GTF2IRD1, as prime candidates for the cause of the facial dysmorphology. Using a targeted Gtf2ird1 knockout mouse, we employed massively-parallel sequencing of mRNA (RNA-Seq) to understand changes in the transcriptional landscape associated with inactivation of Gtf2ird1 in lip tissue. We found widespread dysregulation of genes including differential expression of 78 transcription factors or coactivators, several involved in organ development including Hey1, Myf6, Myog, Dlx2, Gli1, Gli2, Lhx2, Pou3f3, Sox2, Foxp3. We also found that the absence of GTF2IRD1 is associated with increased expression of genes involved in cellular proliferation, including growth factors consistent with the observed phenotype of extreme thickening of the epidermis. At the same time, there was a decrease in the expression of genes involved in other signalling mechanisms, including the Wnt pathway, indicating dysregulation in the complex networks necessary for epidermal differentiation and facial skin patterning. Several of the differentially expressed genes have known roles in both tissue development and neurological function, such as the transcription factor Lhx2 which regulates several genes involved in both skin and brain development. Gtf2ird1 inactivation results in widespread gene dysregulation, some of which may be due to the secondary consequences of gene regulatory network disruptions involving several transcription factors and signalling molecules. Genes involved in growth factor signalling and cell cycle progression were identified as particularly important for explaining the skin dysmorphology observed in this

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.

Inferring transcriptional gene regulation network of starch metabolism in Arabidopsis thaliana leaves using graphical Gaussian model

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Ingkasuwan Papapit

2012-08-01

Full Text Available Abstract Background Starch serves as a temporal storage of carbohydrates in plant leaves during day/night cycles. To study transcriptional regulatory modules of this dynamic metabolic process, we conducted gene regulation network analysis based on small-sample inference of graphical Gaussian model (GGM. Results Time-series significant analysis was applied for Arabidopsis leaf transcriptome data to obtain a set of genes that are highly regulated under a diurnal cycle. A total of 1,480 diurnally regulated genes included 21 starch metabolic enzymes, 6 clock-associated genes, and 106 transcription factors (TF. A starch-clock-TF gene regulation network comprising 117 nodes and 266 edges was constructed by GGM from these 133 significant genes that are potentially related to the diurnal control of starch metabolism. From this network, we found that β-amylase 3 (b-amy3: At4g17090, which participates in starch degradation in chloroplast, is the most frequently connected gene (a hub gene. The robustness of gene-to-gene regulatory network was further analyzed by TF binding site prediction and by evaluating global co-expression of TFs and target starch metabolic enzymes. As a result, two TFs, indeterminate domain 5 (AtIDD5: At2g02070 and constans-like (COL: At2g21320, were identified as positive regulators of starch synthase 4 (SS4: At4g18240. The inference model of AtIDD5-dependent positive regulation of SS4 gene expression was experimentally supported by decreased SS4 mRNA accumulation in Atidd5 mutant plants during the light period of both short and long day conditions. COL was also shown to positively control SS4 mRNA accumulation. Furthermore, the knockout of AtIDD5 and COL led to deformation of chloroplast and its contained starch granules. This deformity also affected the number of starch granules per chloroplast, which increased significantly in both knockout mutant lines. Conclusions In this study, we utilized a systematic approach of microarray

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

Science.gov (United States)

Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

2017-01-01

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

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

A neuroanatomical and physiological study of the non-image forming visual system of the cone-rod homeobox gene (Crx) knock out mouse

DEFF Research Database (Denmark)

Rovsing, Louise; Rath, Martin F; Lund-Andersen, Casper

2010-01-01

The anatomy and physiology of the non-image forming visual system was investigated in a visually blind cone-rod homeobox gene (Crx) knock-out mouse (Crx(-)(/)(-)), which lacks the outer segments of the photoreceptors. We show that the suprachiasmatic nuclei (SCN) in the Crx(-/-) mouse exhibit...... melanopsin neurons or the SCN may be necessary for a normal function of the non-image forming system of the mouse. However, a change in the SCN of the Crx(-/-) mouse might also explain the observed circadian differences between the knock out mouse and wild type mouse....

High-resolution gene expression profiling using RNA sequencing in patients with inflammatory bowel disease and in mouse models of colitis

DEFF Research Database (Denmark)

Holgersen, Kristine; Kutlu, Burak; Fox, Brian

2015-01-01

pathways and assess the similarity between the experimental models and human disease. RNA sequencing was performed on colon biopsies from CD patients, UC patients and non-IBD controls. Genes shown to be significantly dysregulated in human IBD were used to study gene expression in colons from a piroxicam......Proper interpretation of data from preclinical animal studies requires a thorough knowledge about the pathophysiology of both the human disease and animal models. In this study, the expression of IBD-associated genes was characterised in mouse models of colitis to examine the underlying molecular......-accelerated colitis interleukin-10 knockout (PAC IL-10 k.o.), an adoptive transfer (AdTr) and a dextran sulfate sodium (DSS) colitis mouse model. 92 out of 115 literature-defined genes linked to IBD were significantly differentially expressed in inflamed mucosa of CD and/or UC patients compared with non-IBD controls...

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.

Isoflurane Damages the Developing Brain of Mice and Induces Subsequent Learning and Memory Deficits through FASL-FAS Signaling

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Xiuwen Yi

2015-01-01

Full Text Available Background. Isoflurane disrupts brain development of neonatal mice, but its mechanism is unclear. We explored whether isoflurane damaged developing hippocampi through FASL-FAS signaling pathway, which is a well-known pathway of apoptosis. Method. Wild type and FAS- or FASL-gene-knockout mice aged 7 days were exposed to either isoflurane or pure oxygen. We used western blotting to study expressions of caspase-3, FAS (CD95, and FAS ligand (FASL or CD95L proteins, TUNEL staining to count apoptotic cells in hippocampus, and Morris water maze (MWM to evaluate learning and memory. Result. Isoflurane increased expression of FAS and FASL proteins in wild type mice. Compared to isoflurane-treated FAS- and FASL-knockout mice, isoflurane-treated wild type mice had higher expression of caspase-3 and more TUNEL-positive hippocampal cells. Expression of caspase-3 in wild isoflurane group, wild control group, FAS/FASL-gene-knockout control group, and FAS/FASL-gene-knockout isoflurane group showed FAS or FASL gene knockout might attenuate increase of caspase-3 caused by isoflurane. MWM showed isoflurane treatment of wild type mice significantly prolonged escape latency and reduced platform crossing times compared with gene-knockout isoflurane-treated groups. Conclusion. Isoflurane induces apoptosis in developing hippocampi of wild type mice but not in FAS- and FASL-knockout mice and damages brain development through FASL-FAS signaling.

Iron homeostasis in Arabidopsis thaliana: transcriptomic analyses reveal novel FIT-regulated genes, iron deficiency marker genes and functional gene networks.

Science.gov (United States)

Mai, Hans-Jörg; Pateyron, Stéphanie; Bauer, Petra

2016-10-03

FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is the central regulator of iron uptake in Arabidopsis thaliana roots. We performed transcriptome analyses of six day-old seedlings and roots of six week-old plants using wild type, a fit knock-out mutant and a FIT over-expression line grown under iron-sufficient or iron-deficient conditions. We compared genes regulated in a FIT-dependent manner depending on the developmental stage of the plants. We assembled a high likelihood dataset which we used to perform co-expression and functional analysis of the most stably iron deficiency-induced genes. 448 genes were found FIT-regulated. Out of these, 34 genes were robustly FIT-regulated in root and seedling samples and included 13 novel FIT-dependent genes. Three hundred thirty-one genes showed differential regulation in response to the presence and absence of FIT only in the root samples, while this was the case for 83 genes in the seedling samples. We assembled a virtual dataset of iron-regulated genes based on a total of 14 transcriptomic analyses of iron-deficient and iron-sufficient wild-type plants to pinpoint the best marker genes for iron deficiency and analyzed this dataset in depth. Co-expression analysis of this dataset revealed 13 distinct regulons part of which predominantly contained functionally related genes. We could enlarge the list of FIT-dependent genes and discriminate between genes that are robustly FIT-regulated in roots and seedlings or only in one of those. FIT-regulated genes were mostly induced, few of them were repressed by FIT. With the analysis of a virtual dataset we could filter out and pinpoint new candidates among the most reliable marker genes for iron deficiency. Moreover, co-expression and functional analysis of this virtual dataset revealed iron deficiency-induced and functionally distinct regulons.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

Energy Technology Data Exchange (ETDEWEB)

Harrill, Joshua A. [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States); Hukkanen, Renee R.; Lawson, Marie; Martin, Greg [The Dow Chemical Company, Midland, MI 48640 (United States); Gilger, Brian [North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27606 (United States); Soldatow, Valerie [University of North Carolina, Department of Environmental Sciences and Engineering, Chapel Hill, NC 27599 (United States); LeCluyse, Edward L. [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States); Budinsky, Robert A.; Rowlands, J. Craig [The Dow Chemical Company, Midland, MI 48640 (United States); Thomas, Russell S., E-mail: RThomas@thehamner.org [The Hamner Institute for Health Sciences, Institute for Chemical Safety Sciences, RTP, NC 27709 (United States)

2013-10-15

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague–Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ∼ 30–45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. - Highlights: • An AHR knockout rat was generated on a Sprague–Dawley outbred background. • AHR-KO rats lack expression of AHR protein. • AHR-KO rats are insensitive to TCDD-mediated effects. • Data suggests difference in the role of AHR in tissue development of rats and mice. • Abnormalities in vascular

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice

International Nuclear Information System (INIS)

Harrill, Joshua A.; Hukkanen, Renee R.; Lawson, Marie; Martin, Greg; Gilger, Brian; Soldatow, Valerie; LeCluyse, Edward L.; Budinsky, Robert A.; Rowlands, J. Craig; Thomas, Russell S.

2013-01-01

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague–Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 μg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ∼ 30–45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species. - Highlights: • An AHR knockout rat was generated on a Sprague–Dawley outbred background. • AHR-KO rats lack expression of AHR protein. • AHR-KO rats are insensitive to TCDD-mediated effects. • Data suggests difference in the role of AHR in tissue development of rats and mice. • Abnormalities in vascular

Tissue-Specific Contributions of Paternally Expressed Gene 3 in Lactation and Maternal Care of Mus musculus.

Directory of Open Access Journals (Sweden)

Wesley D Frey

Full Text Available Paternally Expressed Gene 3 (Peg3 is an imprinted gene that controls milk letdown and maternal-caring behaviors. In this study, a conditional knockout allele has been developed in Mus musculus to further characterize these known functions of Peg3 in a tissue-specific manner. The mutant line was first crossed with a germline Cre. The progeny of this cross displayed growth retardation phenotypes. This is consistent with those seen in the previous mutant lines of Peg3, confirming the usefulness of the new mutant allele. The mutant line was subsequently crossed individually with MMTV- and Nkx2.1-Cre lines to test Peg3's roles in the mammary gland and hypothalamus, respectively. According to the results, the milk letdown process was impaired in the nursing females with the Peg3 mutation in the mammary gland, but not in the hypothalamus. This suggests that Peg3's roles in the milk letdown process are more critical in the mammary gland than in the hypothalamus. In contrast, one of the maternal-caring behaviors, nest-building, was interrupted in the females with the mutation in both MMTV- and Nkx2.1-driven lines. Overall, this is the first study to introduce a conditional knockout allele of Peg3 and to further dissect its contribution to mammalian reproduction in a tissue-specific manner.

Functional Profiling of Transcription Factor Genes in Neurospora crassa

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Alexander J. Carrillo

2017-09-01

Full Text Available Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa. We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6 binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed, followed by asexual sporulation (38%, and the various stages of sexual development (19%. Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.

Development of an intracellular glycolytic flux sensor for high throughput applications in E.coli

DEFF Research Database (Denmark)

Lehning, Christina Eva

The aim of this PhD project was to construct, test and apply an intracellular, growth-­‐ independent and direct measureable glycolytic flux biosensor in E. coli. Studying the metabolic flux of bacterial cells is of growing interest as it is of fundamental importance to bacterial physiology as well...... to study the flux-­‐altering effects of gene knockouts in E. coli at the single cell level in a vastly parallelized and high-­‐throughput manner. After growth for several generations in rich and minimal media, 2126 gene knockouts, mainly outside of the core metabolism, could be screened. 3 gene knockouts...

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

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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

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.

TIEG1-null tenocytes display age-dependent differences in their gene expression, adhesion, spreading and proliferation properties

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Haddad, Oualid; Gumez, Laurie [Laboratoire de Biomecanique et Bioingenierie UMR CNRS 6600, Universite de Technologie de Compiegne, Compiegne (France); Hawse, John R.; Subramaniam, Malayannan; Spelsberg, Thomas C. [Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN (United States); Bensamoun, Sabine F., E-mail: sabine.bensamoun@utc.fr [Laboratoire de Biomecanique et Bioingenierie UMR CNRS 6600, Universite de Technologie de Compiegne, Compiegne (France)

2011-07-15

The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.

Impact of SO(2) on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing.

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Hamisch, Domenica; Randewig, Dörte; Schliesky, Simon; Bräutigam, Andrea; Weber, Andreas P M; Geffers, Robert; Herschbach, Cornelia; Rennenberg, Heinz; Mendel, Ralf R; Hänsch, Robert

2012-12-01

High concentrations of sulfur dioxide (SO(2) ) as an air pollutant, and its derivative sulfite, cause abiotic stress that can lead to cell death. It is currently unknown to what extent plant fumigation triggers specific transcriptional responses. To address this question, and to test the hypothesis that sulfite oxidase (SO) is acting in SO(2) detoxification, we compared Arabidopsis wildtype (WT) and SO knockout lines (SO-KO) facing the impact of 600 nl l(-1) SO(2) , using RNAseq to quantify absolute transcript abundances. These transcriptome data were correlated to sulfur metabolism-related enzyme activities and metabolites obtained from identical samples in a previous study. SO-KO plants exhibited remarkable and broad regulative responses at the mRNA level, especially in transcripts related to sulfur metabolism enzymes, but also in those related to stress response and senescence. Focusing on SO regulation, no alterations were detectable in the WT, whereas in SO-KO plants we found up-regulation of two splice variants of the SO gene, although this gene is not functional in this line. Our data provide evidence for the highly specific coregulation between SO and sulfur-related enzymes like APS reductase, and suggest two novel candidates for involvement in SO(2) detoxification: an apoplastic peroxidase, and defensins as putative cysteine mass storages. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

More than genes: the advanced fetal programming hypothesis.

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Hocher, Berthold

2014-10-01

Many lines of data, initial epidemiologic studies as well as subsequent extensive experimental studies, indicate that early-life events play a powerful role in influencing later suceptibility to certain chronic diseases. Such events might be over- or undernutrition, exposure to environmental toxins, but also changes in hormones, in particular stress hormones. Typically, those events are triggered by the environmental challenges of the mother. However, recent studies have shown that paternal environmental or nutritional factors affect the phenotype of the offspring as well. The maternal and paternal environmental factors act on the phenotype of the offspring via epigenetic modification of its genome. The advanced fetal programming hypothesis proposes an additional non-environmentally driven mechanism: maternal and also paternal genes may influence the maturating sperm, the oocyte, and later the embryo/fetus, leading to their epigenetic alteration. Thus, the observed phenotype of the offspring may be altered by maternal/paternal genes independent of the fetal genome. Meanwhile, several independent association studies in humans dealing with metabolic and neurological traits also suggest that maternal genes might affect the offspring phenotype independent of the transmission of that particular gene to the offspring. Considering the implications of this hypothesis, some conclusions drawn from transgenic or knockout animal models and based on the causality between a genetic alteration and a phenotype, need to be challenged. Possible implications for the development, diagnostic and therapy of human genetic diseases have to be investigated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Identification of Hematopoietic Stem Cell Engraftment Genes in Gene Therapy Studies.

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Powers, John M; Trobridge, Grant D

2013-09-01

Hematopoietic stem cell (HSC) therapy using replication-incompetent retroviral vectors is a promising approach to provide life-long correction for genetic defects. HSC gene therapy clinical studies have resulted in functional cures for several diseases, but in some studies clonal expansion or leukemia has occurred. This is due to the dyregulation of endogenous host gene expression from vector provirus insertional mutagenesis. Insertional mutagenesis screens using replicating retroviruses have been used extensively to identify genes that influence oncogenesis. However, retroviral mutagenesis screens can also be used to determine the role of genes in biological processes such as stem cell engraftment. The aim of this review is to describe the potential for vector insertion site data from gene therapy studies to provide novel insights into mechanisms of HSC engraftment. In HSC gene therapy studies dysregulation of host genes by replication-incompetent vector proviruses may lead to enrichment of repopulating clones with vector integrants near genes that influence engraftment. Thus, data from HSC gene therapy studies can be used to identify novel candidate engraftment genes. As HSC gene therapy use continues to expand, the vector insertion site data collected will be of great interest to help identify novel engraftment genes and may ultimately lead to new therapies to improve engraftment.

Leukocytosis and enhanced susceptibility to endotoxemia but not atherosclerosis in adrenalectomized APOE knockout mice.

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Menno Hoekstra

Full Text Available Hyperlipidemic apolipoprotein E (APOE knockout mice show an enhanced level of adrenal-derived anti-inflammatory glucocorticoids. Here we determined in APOE knockout mice the impact of total removal of adrenal function through adrenalectomy (ADX on two inflammation-associated pathologies, endotoxemia and atherosclerosis. ADX mice exhibited 91% decreased corticosterone levels (P<0.001, leukocytosis (WBC count: 10.0 ± 0.4 x 10E9/L vs 6.5 ± 0.5 x 10E9/L; P<0.001 and an increased spleen weight (P<0.01. FACS analysis on blood leukocytes revealed increased B-lymphocyte numbers (55 ± 2% vs 46 ± 1%; P<0.01. T-cell populations in blood appeared to be more immature (CD62L+: 26 ± 2% vs 19 ± 1% for CD4+ T-cells, P<0.001 and 58 ± 7% vs 47 ± 4% for CD8+ T-cells, P<0.05, which coincided with immature CD4/CD8 double positive thymocyte enrichment. Exposure to lipopolysaccharide failed to increase corticosterone levels in ADX mice and was associated with a 3-fold higher (P<0.05 TNF-alpha response. In contrast, the development of initial fatty streak lesions and progression to advanced collagen-containing atherosclerotic lesions was unaffected. Plasma cholesterol levels were decreased by 35% (P<0.001 in ADX mice. This could be attributed to a decrease in pro-atherogenic very-low-density lipoproteins (VLDL as a result of a diminished hepatic VLDL secretion rate (-24%; P<0.05. In conclusion, our studies show that adrenalectomy induces leukocytosis and enhances the susceptibility for endotoxemia in APOE knockout mice. The adrenalectomy-associated rise in white blood cells, however, does not alter atherosclerotic lesion development probably due to the parallel decrease in plasma levels of pro-atherogenic lipoproteins.

Dopamine D2 receptor function is compromised in the brain of the methionine sulfoxide reductase A knockout mouse

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Oien, Derek B.; Ortiz, Andrea N.; Rittel, Alexander G.; Dobrowsky, Rick T.; Johnson, Michael A.; Levant, Beth; Fowler, Stephen C.; Moskovitz, Jackob

2010-01-01

Previous research suggests that brain oxidative stress and altered rodent locomotor behavior are linked. We observed bio-behavioral changes in methionine sulfoxide reductase A knockout mice associated with abnormal dopamine signaling. Compromised ability of these knockout mice to reduce methionine sulfoxide enhances accumulation of sulfoxides in proteins. We examined the dopamine D2-receptor function and expression, which has an atypical arrangement and quantity of methionine residues. Indeed...

The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

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Xu, Bowen; Cai, Ling; Butler, Jason M; Chen, Dongliang; Lu, Xiongdong; Allison, David F; Lu, Rui; Rafii, Shahin; Parker, Joel S; Zheng, Deyou; Wang, Gang Greg

2018-03-13

Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC "stemness" genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of "stemness" gene-expression programs and proper function of adult HSCs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Learning gene networks under SNP perturbations using eQTL datasets.

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Lingxue Zhang

2014-02-01

Full Text Available The standard approach for identifying gene networks is based on experimental perturbations of gene regulatory systems such as gene knock-out experiments, followed by a genome-wide profiling of differential gene expressions. However, this approach is significantly limited in that it is not possible to perturb more than one or two genes simultaneously to discover complex gene interactions or to distinguish between direct and indirect downstream regulations of the differentially-expressed genes. As an alternative, genetical genomics study has been proposed to treat naturally-occurring genetic variants as potential perturbants of gene regulatory system and to recover gene networks via analysis of population gene-expression and genotype data. Despite many advantages of genetical genomics data analysis, the computational challenge that the effects of multifactorial genetic perturbations should be decoded simultaneously from data has prevented a widespread application of genetical genomics analysis. In this article, we propose a statistical framework for learning gene networks that overcomes the limitations of experimental perturbation methods and addresses the challenges of genetical genomics analysis. We introduce a new statistical model, called a sparse conditional Gaussian graphical model, and describe an efficient learning algorithm that simultaneously decodes the perturbations of gene regulatory system by a large number of SNPs to identify a gene network along with expression quantitative trait loci (eQTLs that perturb this network. While our statistical model captures direct genetic perturbations of gene network, by performing inference on the probabilistic graphical model, we obtain detailed characterizations of how the direct SNP perturbation effects propagate through the gene network to perturb other genes indirectly. We demonstrate our statistical method using HapMap-simulated and yeast eQTL datasets. In particular, the yeast gene network

The Small RNA ErsA of Pseudomonas aeruginosa Contributes to Biofilm Development and Motility through Post-transcriptional Modulation of AmrZ

DEFF Research Database (Denmark)

Falcone, Marilena; Ferrara, Silvia; Rossi, Elio

2018-01-01

. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P....... aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the Amr...

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.

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Seong-Bin Kim

Full Text Available Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega. To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Macrophage migration inhibitory factor (MIF) knockout preserves cardiac homeostasis through alleviating Akt-mediated myocardial autophagy suppression in high-fat diet-induced obesity.

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Xu, X; Ren, J

2015-03-01

Macrophage migration inhibitory factor (MIF) has a role in the development of obesity and diabetes. However, whether MIF has a role in fat diet-induced obesity and associated cardiac anomalies still remains unknown. The aim of this study was to examine the impact of MIF knockout on high-fat diet-induced obesity, obesity-associated cardiac anomalies and the underlying mechanisms involved with a focus on Akt-mediated autophagy. Adult male wild-type (WT) and MIF knockout (MIF(-/-)) mice were placed on 45% high-fat diet for 5 months. Oxygen consumption, CO2 production, respiratory exchange ratio, locomotor activity and heat generation were measured using energy calorimeter. Echocardiographic, cardiomyocyte mechanical and intracellular Ca2+ properties were assessed. Apoptosis was examined using terminal dUTP nick end labeling staining and western blot analysis. Akt signaling pathway and autophagy markers were evaluated. Cardiomyocytes isolated from WT and MIF(-/-) mice were treated with recombinant mouse MIF (rmMIF). High-fat diet feeding elicited increased body weight gain, insulin resistance and caloric disturbance in WT and MIF(-/-) mice. High-fat diet induced unfavorable geometric, contractile and histological changes in the heart, the effects of which were alleviated by MIF knockout. In addition, fat diet-induced cardiac anomalies were associated with Akt activation and autophagy suppression, which were nullified by MIF deficiency. In cardiomyocytes from WT mice, autophagy was inhibited by exogenous rmMIF through Akt activation. In addition, MIF knockout rescued palmitic acid-induced suppression of cardiomyocyte autophagy, the effect of which was nullified by rmMIF. These results indicate that MIF knockout preserved obesity-associated cardiac anomalies without affecting fat diet-induced obesity, probably through restoring myocardial autophagy in an Akt-dependent manner. Our findings provide new insights for the role of MIF in obesity and associated cardiac

Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145

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Olga Tsypik

2017-01-01

Full Text Available Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.

Relevant feature set estimation with a knock-out strategy and random forests

DEFF Research Database (Denmark)

Ganz, Melanie; Greve, Douglas N; Fischl, Bruce

2015-01-01

unintuitive and difficult to determine. In this article, we propose a novel MVPA method for group analysis of high-dimensional data that overcomes the drawbacks of the current techniques. Our approach explicitly aims to identify all relevant variations using a "knock-out" strategy and the Random Forest...

The FKBP5 Gene Affects Alcohol Drinking in Knockout Mice and Is Implicated in Alcohol Drinking in Humans

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Bin Qiu

2016-08-01

Full Text Available FKBP5 encodes FK506-binding protein 5, a glucocorticoid receptor (GR-binding protein implicated in various psychiatric disorders and alcohol withdrawal severity. The purpose of this study is to characterize alcohol preference and related phenotypes in Fkbp5 knockout (KO mice and to examine the role of FKBP5 in human alcohol consumption. The following experiments were performed to characterize Fkpb5 KO mice. (1 Fkbp5 KO and wild-type (WT EtOH consumption was tested using a two-bottle choice paradigm; (2 The EtOH elimination rate was measured after intraperitoneal (IP injection of 2.0 g/kg EtOH; (3 Blood alcohol concentration (BAC was measured after 3 h limited access of alcohol; (4 Brain region expression of Fkbp5 was identified using LacZ staining; (5 Baseline corticosterone (CORT was assessed. Additionally, two SNPs, rs1360780 (C/T and rs3800373 (T/G, were selected to study the association of FKBP5 with alcohol consumption in humans. Participants were college students (n = 1162 from 21–26 years of age with Chinese, Korean or Caucasian ethnicity. The results, compared to WT mice, for KO mice exhibited an increase in alcohol consumption that was not due to differences in taste sensitivity or alcohol metabolism. Higher BAC was found in KO mice after 3 h of EtOH access. Fkbp5 was highly expressed in brain regions involved in the regulation of the stress response, such as the hippocampus, amygdala, dorsal raphe and locus coeruleus. Both genotypes exhibited similar basal levels of plasma corticosterone (CORT. Finally, single nucleotide polymorphisms (SNPs in FKBP5 were found to be associated with alcohol drinking in humans. These results suggest that the association between FKBP5 and alcohol consumption is conserved in both mice and humans.

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

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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.

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana.

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Lee, Se Jin; Lee, Mi Rong; Kim, Sihyeon; Kim, Jong Cheol; Park, So Eun; Shin, Tae Young; Kim, Jae Su

2018-03-01

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhancing Understanding of the Visual Cycle by Applying CRISPR/Cas9 Gene Editing in Zebrafish

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Rebecca Ward

2018-04-01

Full Text Available During the vertebrate visual cycle, all-trans-retinal is exported from photoreceptors to the adjacent RPE or Müller glia wherein 11-cis-retinal is regenerated. The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient

Deficient Mechanical Activation of Anabolic Transcripts and Post-Traumatic Cartilage Degeneration in Matrilin-1 Knockout Mice.

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Yupeng Chen

Full Text Available Matrilin-1 (Matn1, a cartilage-specific peri-cellular and extracellular matrix (ECM protein, has been hypothesized to regulate ECM interactions and transmit mechanical signals in cartilage. Since Matn1 knock-out (Matn1-/- mice exhibit a normal skeleton, its function in vivo is unclear. In this study, we found that the anabolic Acan and Col2a transcript levels were significantly higher in wildtype (Matn1+/+ mouse cartilage than that of MATN1-/- mice in vivo. However, such difference was not observed between Matn1+/+ and MATN1-/- chondrocytes cultured under stationary conditions in vitro. Cyclic loading significantly stimulated Acan and Col2a transcript levels in Matn1+/+ but not in MATN1-/- chondrocytes. This suggests that, while Matn1+/+ chondrocytes increase their anabolic gene expression in response to mechanical loading, the MATN1-/- chondrocytes fail to do so because of the deficiency in mechanotransduction. We also found that altered elastic modulus of cartilage matrix in Matn1-/- mice, suggesting the mechanotransduction has changed due to the deficiency of Matn1. To understand the impact of such deficiency on joint disease, mechanical loading was altered in vivo by destabilization of medial meniscus. While Matn1+/+ mice exhibited superficial fissures and clefts consistent with mechanical damage to the articular joint, Matn1-/- mice presented more severe cartilage lesions characterized by proteoglycan loss and disorganization of cells and ECM. This suggests that Matn1 deficiency affects pathogenesis of post-traumatic osteoarthritis by failing to up-regulate anabolic gene expression. This is the first demonstration of Matn1 function in vivo, which suggests its protective role in cartilage degeneration under altered mechanical environment.

Deleting the Arntl clock gene in the granular layer of the mouse cerebellum

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Bering, Tenna; Carstensen, Mikkel Bloss; Rath, Martin Fredensborg

2017-01-01

nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable. To investigate the hypothesis that the circadian oscillator of the cerebellum...... is involved in circadian physiology and food anticipation, we therefore by use of Cre-LoxP technology generated a conditional knockout mouse with the core clock gene Arntl deleted specifically in granule cells of the cerebellum, since expression of clock genes in the cerebellar cortex is mainly located...

Empirical study of supervised gene screening

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Ma Shuangge

2006-12-01

Full Text Available Abstract Background Microarray studies provide a way of linking variations of phenotypes with their genetic causations. Constructing predictive models using high dimensional microarray measurements usually consists of three steps: (1 unsupervised gene screening; (2 supervised gene screening; and (3 statistical model building. Supervised gene screening based on marginal gene ranking is commonly used to reduce the number of genes in the model building. Various simple statistics, such as t-statistic or signal to noise ratio, have been used to rank genes in the supervised screening. Despite of its extensive usage, statistical study of supervised gene screening remains scarce. Our study is partly motivated by the differences in gene discovery results caused by using different supervised gene screening methods. Results We investigate concordance and reproducibility of supervised gene screening based on eight commonly used marginal statistics. Concordance is assessed by the relative fractions of overlaps between top ranked genes screened using different marginal statistics. We propose a Bootstrap Reproducibility Index, which measures reproducibility of individual genes under the supervised screening. Empirical studies are based on four public microarray data. We consider the cases where the top 20%, 40% and 60% genes are screened. Conclusion From a gene discovery point of view, the effect of supervised gene screening based on different marginal statistics cannot be ignored. Empirical studies show that (1 genes passed different supervised screenings may be considerably different; (2 concordance may vary, depending on the underlying data structure and percentage of selected genes; (3 evaluated with the Bootstrap Reproducibility Index, genes passed supervised screenings are only moderately reproducible; and (4 concordance cannot be improved by supervised screening based on reproducibility.

An extremely high dietary iodide supply forestalls severe hypothyroidism in Na+/I- symporter (NIS) knockout mice.

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Ferrandino, Giuseppe; Kaspari, Rachel R; Reyna-Neyra, Andrea; Boutagy, Nabil E; Sinusas, Albert J; Carrasco, Nancy

2017-07-13

The sodium/iodide symporter (NIS) mediates active iodide (I - ) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I - transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I - . Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate ( 99m TcO 4 - ). NIS KO mice showed undetectable serum T 4 and very low serum T 3 levels when fed a diet supplying the minimum I - requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I - diet partially rescued TH biosynthesis, demonstrating that, at high I - concentrations, I - enters the thyroid through routes other than NIS.

Genes Involved in the Evolution of Herbivory by a Leaf-Mining, Drosophilid Fly

DEFF Research Database (Denmark)

Whiteman, Noah K.; Gloss, Andrew D.; Sackton, Timothy B.

2012-01-01

a total of 341 transcripts that were differentially regulated by glucosinolate uptake in larval S. flava. Of these, approximately a third corresponded to homologs of Drosophila melanogaster genes associated with starvation, dietary toxin-, heat-, oxidation-, and aging-related stress. The upregulated...... the widely studied reference plant, Arabidopsis thaliana (Arabidopsis). Scaptomyza flava is phylogenetically nested within the paraphyletic genus Drosophila, and the whole genome sequences available for 12 species of Drosophila facilitated phylogenetic analysis and assembly of a transcriptome for S. flava....... The presence of glucosinolates in wild-type (WT) Arabidopsis plants reduced S. flava larval weight gain and increased egg-adult development time relative to flies reared in glucosinolate knockout (GKO) plants. An analysis of gene expression differences in 5-day-old larvae reared on WT versus GKO plants showed...

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

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Cheng, Yaofeng; Chen, Shenjue; Freeden, Chris; Chen, Weiqi; Zhang, Yueping; Abraham, Pamela; Nelson, David M; Humphreys, W Griffith; Gan, Jinping; Lai, Yurong

2017-09-01

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice

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Manzini, S.; Pinna, C.; Busnelli, M.; Cinquanta, P.; Rigamonti, E.; Ganzetti, G.S.; Dellera, F.; Sala, A.; Calabresi, L.; Franceschini, G.; Parolini, C.; Chiesa, G.

2015-01-01

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcatwt) and LCAT knockout (LcatKO) mice exposed to noradrenaline showed reduced contractility in LcatKO mice (P < 0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in LcatKO mice (P < 0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in LcatKO mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcatwt and LcatKO mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

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Manzini, S; Pinna, C; Busnelli, M; Cinquanta, P; Rigamonti, E; Ganzetti, G S; Dellera, F; Sala, A; Calabresi, L; Franceschini, G; Parolini, C; Chiesa, G

2015-11-01

Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. Copyright © 2015. Published by Elsevier Inc.

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

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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.

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...

Disruption of Slc52a3 gene causes neonatal lethality with riboflavin deficiency in mice

OpenAIRE

Yoshimatsu, Hiroki; Yonezawa, Atsushi; Yamanishi, Kaori; Yao, Yoshiaki; Sugano, Kumiko; Nakagawa, Shunsaku; Imai, Satoshi; Omura, Tomohiro; Nakagawa, Takayuki; Yano, Ikuko; Masuda, Satohiro; Inui, Ken-ichi; Matsubara, Kazuo

2016-01-01

Homeostasis of riboflavin should be maintained by transporters. Previous in vitro studies have elucidated basic information about riboflavin transporter RFVT3 encoded by SLC52A3 gene. However, the contribution of RFVT3 to the maintenance of riboflavin homeostasis and the significance in vivo remain unclear. Here, we investigated the physiological role of RFVT3 using Slc52a3 knockout (Slc52a3−/−) mice. Most Slc52a3−/− mice died with hyperlipidemia and hypoglycemia within 48 hr after birth. The...

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

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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…

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

Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

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Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

2013-09-01

Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

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Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

2018-06-01

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

Diabetes-causing gene, kruppel-like factor 11, modulates the antinociceptive response of chronic ethanol intake.

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Ou, Xiao-Ming; Udemgba, Chinelo; Wang, Niping; Dai, Xiaoli; Lomberk, Gwen; Seo, Seungmae; Urrutia, Raul; Wang, Junming; Duncan, Jeremy; Harris, Sharonda; Fairbanks, Carolyn A; Zhang, Xiao

2014-02-01

Alcohol (EtOH [ethanol]) is an antinociceptive agent, working in part, by reducing sensitivity to painful stimuli. The transcription factor Kruppel-like factor 11 (KLF11), a human diabetes-causing gene that also regulates the neurotransmitter metabolic enzymes monoamine oxidase (MAO), has recently been identified as an EtOH-inducible gene. However, its role in antinociception remains unknown. Consequently, we investigated the function of KLF11 in chronic EtOH-induced antinociception using a genetically engineered knockout mouse model. Wild-type (Klf11(+/+) ) and KLF11 knockout (Klf11(-/-) ) mice were fed a liquid diet containing EtOH for 28 days with increasing amounts of EtOH from 0% up to a final concentration of 6.4%, representing a final diet containing 36% of calories primarily from EtOH. Control mice from both genotypes were fed liquid diet without EtOH for 28 days. The EtOH-induced antinociceptive effect was determined using the tail-flick test before and after EtOH exposure (on day 29). In addition, the enzyme activity and mRNA levels of MAO A and MAO B were measured by real-time RT-PCR and enzyme assays, respectively. EtOH produced an antinociceptive response to thermal pain in Klf11(+/+) mice, as expected. In contrast, deletion of KLF11 in the Klf11(-/-) mice abolished the EtOH-induced antinociceptive effect. The mRNA and protein levels of KLF11 were significantly increased in the brain prefrontal cortex of Klf11(+/+) mice exposed to EtOH compared with control Klf11(+/+) mice. Furthermore, MAO enzyme activities were affected differently in Klf11 wild-type versus Klf11 knockout mice exposed to chronic EtOH. Chronic EtOH intake significantly increased MAO B activity in Klf11(+/+) mice. The data show KLF11 modulation of EtOH-induced antinociception. The KLF11-targeted MAO B enzyme may contribute more significantly to EtOH-induced antinociception. Thus, this study revealed a new role for the KLF11 gene in the mechanisms underlying the antinociceptive

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.

Loss of CO2 sensing by the olfactory system of CNGA3 knockout mice

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Jinlong HAN, Minmin LUO

2010-12-01

Full Text Available Atmospheric CO2 can signal the presence of food, predators or environmental stress and trigger stereotypical behaviors in both vertebrates and invertebrates. Recent studies have shown that the necklace olfactory system in mice sensitively detects CO2 in the air. Olfactory CO2 neurons are believed to rely on cyclic guanosine monophosphate (cGMP as the key second messenger; however, the specific ion channel underlying CO­2 responses remains unclear. Here we show that CO2-evoked neuronal and behavioral responses require cyclic nucleotide-gated (CNG channels consisting of the CNGA3 subunit. Through Ca2+-imaging, we found that CO2-triggered Ca2+ influx was abolished in necklace olfactory sensory neurons (OSNs of CNGA3-knockout mice. Olfactory detection tests using a Go/No-go paradigm showed that these knockout mice failed to detect 0.5% CO2. Thus, sensitive detection of atmospheric CO2 depends on the function of CNG channels consisting of the CNGA3 subunit in necklace OSNs. These data support the important role of the necklace olfactory system in CO2 sensing and extend our understanding of the signal transduction pathway mediating CO2 detection in mammals [Current Zoology 56 (6: 793–799, 2010].

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

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Tatiana Flisikowska

Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.