WorldWideScience

Sample records for gene dsm1 mediates

  1. Electroporation-mediated gene delivery.

    Science.gov (United States)

    Young, Jennifer L; Dean, David A

    2015-01-01

    Electroporation has been used extensively to transfer DNA to bacteria, yeast, and mammalian cells in culture for the past 30 years. Over this time, numerous advances have been made, from using fields to facilitate cell fusion, delivery of chemotherapeutic drugs to cells and tissues, and most importantly, gene and drug delivery in living tissues from rodents to man. Electroporation uses electrical fields to transiently destabilize the membrane allowing the entry of normally impermeable macromolecules into the cytoplasm. Surprisingly, at the appropriate field strengths, the application of these fields to tissues results in little, if any, damage or trauma. Indeed, electroporation has even been used successfully in human trials for gene delivery for the treatment of tumors and for vaccine development. Electroporation can lead to between 100 and 1000-fold increases in gene delivery and expression and can also increase both the distribution of cells taking up and expressing the DNA as well as the absolute amount of gene product per cell (likely due to increased delivery of plasmids into each cell). Effective electroporation depends on electric field parameters, electrode design, the tissues and cells being targeted, and the plasmids that are being transferred themselves. Most importantly, there is no single combination of these variables that leads to greatest efficacy in every situation; optimization is required in every new setting. Electroporation-mediated in vivo gene delivery has proven highly effective in vaccine production, transgene expression, enzyme replacement, and control of a variety of cancers. Almost any tissue can be targeted with electroporation, including muscle, skin, heart, liver, lung, and vasculature. This chapter will provide an overview of the theory of electroporation for the delivery of DNA both in individual cells and in tissues and its application for in vivo gene delivery in a number of animal models.

  2. Vitamin D-mediated gene expression.

    Science.gov (United States)

    Lowe, K E; Maiyar, A C; Norman, A W

    1992-01-01

    The steroid hormone 1,25(OH)2D3 modulates the expression of a wide variety of genes in a tissue- and developmentally specific manner. It is well established that 1,25(OH)2D3 can up- or downregulate the expression of genes involved in cell proliferation, differentiation, and mineral homeostasis. The hormone exerts its genomic effects via interactions with the vitamin D receptor or VDR, a member of the superfamily of hormone-activated nuclear receptors which can regulate eukaryotic gene expression. The ligand-bound receptor acts as a transcription factor that binds to specific DNA sequences, HREs, in target gene promoters. The DNA-binding domains of the steroid hormone receptors are highly conserved and contain two zinc-finger motifs that recognize the HREs. The spacing and orientation of the HRE half-sites, as well as the HRE sequence, are critical for proper discrimination by the various receptors. Other nuclear factors such as fos and jun can influence vitamin D-mediated gene expression. A wide range of experimental techniques has been used to increase our understanding of how 1,25(OH)2D3 and its receptor play a central role in gene expression.

  3. Torsion-mediated interaction between adjacent genes.

    Directory of Open Access Journals (Sweden)

    Sam Meyer

    2014-09-01

    Full Text Available DNA torsional stress is generated by virtually all biomolecular processes involving the double helix, in particular transcription where a significant level of stress propagates over several kilobases. If another promoter is located in this range, this stress may strongly modify its opening properties, and hence facilitate or hinder its transcription. This mechanism implies that transcribed genes distant of a few kilobases are not independent, but coupled by torsional stress, an effect for which we propose the first quantitative and systematic model. In contrast to previously proposed mechanisms of transcriptional interference, the suggested coupling is not mediated by the transcription machineries, but results from the universal mechanical features of the double-helix. The model shows that the effect likely affects prokaryotes as well as eukaryotes, but with different consequences owing to their different basal levels of torsion. It also depends crucially on the relative orientation of the genes, enhancing the expression of eukaryotic divergent pairs while reducing that of prokaryotic convergent ones. To test the in vivo influence of the torsional coupling, we analyze the expression of isolated gene pairs in the Drosophila melanogaster genome. Their orientation and distance dependence is fully consistent with the model, suggesting that torsional gene coupling may constitute a widespread mechanism of (coregulation in eukaryotes.

  4. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    Directory of Open Access Journals (Sweden)

    Shyamal Kumar Kundu

    2014-01-01

    Full Text Available Advancement in the gene delivery system have resulted in clinical successes in gene therapy for patients with several genetic diseases, such as immunodeficiency diseases, X-linked adrenoleukodystrophy (X-ALD blindness, thalassemia, and many more. Among various delivery systems, liposomal mediated gene delivery route is offering great promises for gene therapy. This review is an attempt to depict a portrait about the polymer based liposomal gene delivery systems and their future applications. Herein, we have discussed in detail the characteristics of liposome, importance of polymer for liposome formulation, gene delivery, and future direction of liposome based gene delivery as a whole.

  5. CTCF-mediated chromatin loops enclose inducible gene regulatory domains

    NARCIS (Netherlands)

    Oti, M.O.; Falck, J.; Huynen, M.A.; Zhou, Huiqing

    2016-01-01

    BACKGROUND: The CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to

  6. Host genes involved in Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Soltani, Jalal

    2009-01-01

    Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the

  7. Host genes involved in Agrobacterium-mediated transformation

    NARCIS (Netherlands)

    Soltani, Jalal

    2009-01-01

    Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the w

  8. Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

    Science.gov (United States)

    2016-04-12

    34--- I lr_ Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Recent Development in Gene Therapy , 2007: 77-94...ISBN: 81-7895-262-9 Editor: Jim Xiang Adenovirus-mediated gene therapy against viral biothreat agents Josh Q.H. Wu Chemical Biological Defence... therapy , which introduces therapeutic genes into mammalian cells to achieve therapeutic effective, hds a great potential for use as a defensive

  9. Targeted gene knockout in chickens mediated by TALENs

    OpenAIRE

    Park, Tae Sub; Lee, Hong Jo; Kim, Ki Hyun; Kim, Jin-Soo; Han, Jae Yong

    2014-01-01

    Targeted gene knockout by editing specific loci in genome has revolutionized the field of functional genomics. Transcription activator-like effector nucleases (TALENs) are representative next-generation platforms for customized genomic editing in transgenic animals, as well as cultured cells in vitro. In this study, in combination with chicken primordial germ cell line with germ-line transmission capacity, we generated the ovalbumin gene knockout chickens by TALEN-mediated gene targeting. Our...

  10. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  11. 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.

  12. In vivo particle-mediated gene transfer for cancer therapy.

    Science.gov (United States)

    Rakhmilevich, A L; Yang, N S

    2000-01-01

    During the past several years, particle-mediated delivery techniques have been developed as a nonviral technology for gene transfer (1-7). For mammalian somatic tissues, this technology, popularly known as the gene gun method, has been shown effective for transfection of skin, liver, pancreas, muscle, spleen, and other organs in vivo (3,4), brain, mammary, and leukocyte primary cultures or tissue explants ex vivo (2,5-7), and a wide range of cell lines in vitro (3,6,7). In this chapter, we describe the general principles, mechanisms, protocols, and uses of the particle-mediated gene transfer technology for in vivo gene transfer, mainly into skin tissues. Specific applications of this technology to basic studies in molecular biology as well as to gene therapy and genetic immunization against cancer are addressed.

  13. Agrobacterium-mediated gene transfer to Chrysanthemum.

    NARCIS (Netherlands)

    Wordragen, van M.F.

    1991-01-01

    Genetic manipulation of plants is a technique that enables us to add to the plant genome, in a precise and well controlled manner, one or a few new genes, coding for desirable traits. In contrast to this, the conventional method for the introduction of new properties in plants, by cross breeding, is

  14. Alternative-splicing-mediated gene expression

    Science.gov (United States)

    Wang, Qianliang; Zhou, Tianshou

    2014-01-01

    Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

  15. Targeting of AID-mediated sequence diversification to immunoglobulin genes.

    Science.gov (United States)

    Kothapalli, Naga Rama; Fugmann, Sebastian D

    2011-04-01

    Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is probably a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes.

  16. Methods for particle-mediated gene transfer into skin.

    Science.gov (United States)

    Yang, N S; McCabe, D E; Swain, W F

    1997-01-01

    During the past 5 yr, particle-mediated delivery techniques have been developed as a physical means for gene transfer into various eukaryotic systems, including plants, insects, fish, and mammals (1-7). For mammalian somatic tissues, this technology, popularly known as the gene gun method, has been shown effective in transfection of skin, liver, pancreas, muscle, spleen, and other organs in vivo (3,4); brain, mammary, and leukocyte pnmary cultures or explants ex vivo (2,5-7); and a wide range of different mammalian cell lines in vitro (3,6,7).

  17. TEAD mediates YAP-dependent gene induction and growth control.

    Science.gov (United States)

    Zhao, Bin; Ye, Xin; Yu, Jindan; Li, Li; Li, Weiquan; Li, Siming; Yu, Jianjun; Lin, Jiandie D; Wang, Cun-Yu; Chinnaiyan, Arul M; Lai, Zhi-Chun; Guan, Kun-Liang

    2008-07-15

    The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial-mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

  18. DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...csml) Show Signalling pathways mediating type I interferon gene expression. PubmedID 17904888 Title Signalli...ng pathways mediating type I interferon gene expression. Authors Edwards MR, Slat

  19. RETROVIRAL-MEDIATED SUICIDE GENE THERAPY OF EXPERIMENTAL GLIOMA

    Institute of Scientific and Technical Information of China (English)

    Xu Lingfei; Ge Kai; Zheng Zhongcheng; Sun Lanying; Liu Xinyuan

    1998-01-01

    Objective: To establish a retroviral-mediated suicide gene therapy system for experimental glioma and test its efficacy. Methods: C6 rat glioma cells were infected with recombinant retrovirus containing HSV-tk gene. The C6/tk cell line which stably expressed tk was selected and cloned. The sensitivities of C6/tk cells to several nucleoside analogues, such as GCV, BVdU, ACV were compared by the growth inhibition studies. Antitumor effects were also observed after GCV treatment in nude mice bearing tumors derived from C6/tk cells. Results:The growth inhibition studies showed that GCV was the most efficient prodrug in this system. C6/tk cells were highly sensitive to GCV, with an IC50<0.2 μmol/L, being 500-fold less than that in tk-negative C6 cells. In vivo studies showed significant tumor inhibition in the treatment group. Conclusion: Glioma cells can be eradicated by using retroviral-mediated suicide gene system in vitro as well as in vivo.

  20. ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

    Science.gov (United States)

    de Pater, Sylvia; Pinas, Johan E; Hooykaas, Paul J J; van der Zaal, Bert J

    2013-05-01

    Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10⁻³ per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10⁻³ per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5' and the 3' end of the gene. One plant line contained a PPO gene repaired only at the 5' end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

  1. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

  2. Condensin-mediated chromosome organization and gene regulation

    Directory of Open Access Journals (Sweden)

    Alyssa Christine Lau

    2015-01-01

    Full Text Available In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In C. elegans, dosage compensation is achieved by the dosage compensation complex (DCC binding to both X chromosomes in hermaphrodites to downregulate gene expression by two fold. The DCC contains a subcomplex (condensin IDC similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 (H4K20 is increased, whereas acetylation of histone H4 lysine 16 (H4K16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.

  3. Adenovirus-mediated gene transfer to tumor cells.

    Science.gov (United States)

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  4. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    NARCIS (Netherlands)

    Michielse, C.B.; Arentshorst, M.; Ram, A.F.; Hondel, C.A. van den

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated tra

  5. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  6. Gene silencing: Double-stranded RNA mediated mRNA degradation and gene inactivation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that doublestranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.

  7. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers

    Directory of Open Access Journals (Sweden)

    Qiu-Lan Zhou

    2014-01-01

    Full Text Available With the development of nanotechnology, nanocarriers have been increasingly used for curative drug/gene delivery. Various nanocarriers are being introduced and assessed, such as polymer nanoparticles, liposomes, and micelles. As a novel theranostic system, nanocarriers hold great promise for ultrasound molecular imaging, targeted drug/gene delivery, and therapy. Nanocarriers, with the properties of smaller particle size, and long circulation time, would be advantageous in diagnostic and therapeutic applications. Nanocarriers can pass through blood capillary walls and cell membrane walls to deliver drugs. The mechanisms of interaction between ultrasound and nanocarriers are not clearly understood, which may be related to cavitation, mechanical effects, thermal effects, and so forth. These effects may induce transient membrane permeabilization (sonoporation on a single cell level, cell death, and disruption of tissue structure, ensuring noninvasive, targeted, and efficient drug/gene delivery and therapy. The system has been used in various tissues and organs (in vitro or in vivo, including tumor tissues, kidney, cardiac, skeletal muscle, and vascular smooth muscle. In this review, we explore the research progress and application of ultrasound-mediated local drug/gene delivery with nanocarriers.

  8. Stable oncogenic transformation induced by microcell-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    吕有勇; Donald G.Blair

    1995-01-01

    Oncogenes have been identified using DNA-mediated transfection, but the size of the transferable and unrearranged DNA, gene rearrangement and amplification which occur during the transfection process limit the use of the techniques. We have evaluated microcell-mediated gene transfer techniques for the transfer and analysis of dominant oncogenes. MNNG-HOS, a transformed human cell line which contained the met oncogene mapping to human chromosome 7 was infected with retroviruses carrying drug resistance markers and used to optimize microcell preparation and transfer. Stable and drug-resistant hybrids containing single human chromosomes as well as the foci of the transformed cells containing the activated met oncogene and intact hitman chromosomes were obtained. Hybridization analysis with probes (i.e. collA2, pJ3.11) mapping up to 1 Mb away from met shows that the cells from the individual focr contain different amounts of apparently unrearranged human DNA associated with the oncogene, and the microcell-g

  9. Isolated limb perfusion for local gene delivery: efficient and targeted adenovirus-mediated gene transfer into soft tissue sarcomas

    NARCIS (Netherlands)

    W.K. de Roos; J.H.W. de Wilt (Johannes); M.E. van der Kaaden; E.R. Manusama (Eric); M.W. de Vries; A. Bout; T.L.M. ten Hagen (Timo); D. Valerio (Dinko); A.M.M. Eggermont (Alexander)

    2000-01-01

    textabstractOBJECTIVE: To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA: A major concern in adenovirus-mediated gene therapy in cancer is the transfer of ge

  10. Kidney-specific transposon-mediated gene transfer in vivo

    Science.gov (United States)

    Woodard, Lauren E.; Cheng, Jizhong; Welch, Richard C.; Williams, Felisha M.; Luo, Wentian; Gewin, Leslie S.; Wilson, Matthew H.

    2017-01-01

    Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice. PMID:28317878

  11. Chromatin-mediated regulation of cytomegalovirus gene expression.

    Science.gov (United States)

    Reeves, Matthew B

    2011-05-01

    Following primary infection, whether Human cytomegalovirus (HCMV) enters either the latent or lytic lifecycle is dependent on the phenotype of the cell type infected. Multiple cell types are permissive for lytic infection with HCMV whereas, in contrast, well characterized sites of latency are restricted to a very specific population of CD34+ cells resident in the bone marrow and the immature myeloid cells they give rise to. It is becoming increasingly clear that one of the mechanisms that promote HCMV latency involves the recruitment of histone proteins to the major immediate early promoter (MIEP) which are subject to post-translational modifications that promote a transcriptionally inactive state. Integral to this, is the role of cellular transcriptional repressors that interact with histone modifying enzymes that promote and maintain this repressed state during latency. Crucially, the chromatin associated with the MIEP is dynamically regulated-myeloid cell differentiation triggers the acetylation of histones bound to the MIEP which is concomitant with the reactivation of IE gene expression and re-entry into lytic infection. Interestingly, this dynamic regulation of the MIEP by chromatin structure in latency extends not only into lytic infection but also for the regulation of multiple viral promoters in all phases of infection. HCMV lytic infection is characterised by a timely and co-ordinated pattern of gene expression that now has been shown to correlate with active post-translational modification of the histones associated with early and late promoters. These effects are mediated by the major IE products (IE72 and IE86) which physically and functionally interact with histone modifying enzymes resulting in the efficient activation of viral gene expression. Thus chromatin appears to play an important role in gene regulation in all phases of infection. Furthermore, these studies are highly suggestive that an intrinsic cellular anti-viral response to incoming viral

  12. Microhomology-mediated deletion and gene conversion in African trypanosomes.

    Science.gov (United States)

    Glover, Lucy; Jun, Junho; Horn, David

    2011-03-01

    Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.

  13. Induction of AhR-mediated gene transcription by coffee.

    Science.gov (United States)

    Ishikawa, Toshio; Takahashi, Satoshi; Morita, Koji; Okinaga, Hiroko; Teramoto, Tamio

    2014-01-01

    Aryl hydrocarbon receptor (AhR) is classically known to be activated by xenobiotics such as dioxins and polycyclic aromatic hydrocarbons (PAHs). Although it has been reported that PAHs are contained in roasted coffee beans, in general coffee beverages are not considered to be AhR activators. We tested whether exposure to coffee would activate AhR in cultured cells. HepG2 cells stably expressing an AhR-responsive reporter gene were treated with coffee samples. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was quantitated by RT-PCR and Western blotting in HepG2, Caco-2, and MCF-7 cells, after treatment with coffee. In order to obtain sensitive and reproducible results, all the experiments were performed with the cells placed in either phosphate-buffered saline (PBS) or pure serum, instead of routinely-used culture medium, whose intrinsic AhR-stimulating activity turned out to be so strong as to interfere with the analyses. All the coffee samples tested robustly stimulated AhR-mediated transcription in the reporter gene assays. Of note, to what extent coffee and other AhR agonists activated AhR was different, depending on whether the experiments were done in PBS or serum. CYP1A1 mRNA was induced by coffee, in HepG2, Caco-2, and MCF-7 cells placed in either PBS or serum. CYP1A1 protein expression, which was not detected in these cells incubated in PBS, was also increased by coffee in cells placed in serum. By using culture medium-free experimental settings, we have shown that coffee is a strong AhR activator. Our observation may help elucidate as-yet-unrecognized effects of coffee on human health.

  14. Efficient TALEN-mediated gene knockout in livestock.

    Science.gov (United States)

    Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G; Stverakova, Dana; Proudfoot, Chris; Christian, Michelle; Voytas, Daniel F; Long, Charles R; Whitelaw, C Bruce A; Fahrenkrug, Scott C

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

  15. Virally mediated gene manipulation in the adult CNS

    Directory of Open Access Journals (Sweden)

    Efrat eEdry

    2011-12-01

    Full Text Available Understanding how the CNS functions poses one of the greatest challenges in modern life science and medicine. Studying the brain is especially challenging because of its complexity, the heterogeneity of its cellular composition, and the substantial changes it undergoes throughout its life-span. The complexity of adult-brain neural networks results also from the diversity of properties and functions of neuronal cells, governed, inter alia, by temporally and spatially differential expression of proteins in mammalian brain cell populations. Hence, research into the biology of CNS activity and its implications to human and animal behavior must use novel scientific tools. One source of such tools is the field of molecular genetics – recently utilized more and more frequently in neuroscience research. Transgenic approaches in general, and gene targeting in rodents have become fundamental tools for elucidating gene function in the CNS. Although spectacular progress has been achieved over recent decades by using these approaches, it is important to note that they face a number of restrictions. One of the main challenges is presented by the temporal and spatial regulation of introduced genetic manipulations. Viral vectors provide an alternative approach to temporally regulated, localized delivery of genetic modifications into neurons. In this review we describe available technologies for gene transfer into the adult mammalian CNS that use both viral and non-viral tools. We discuss viral vectors frequently used in neuroscience, with emphasis on lentiviral vector (LV systems. We consider adverse effects of LVs, and the use of LVs for temporally and spatially controllable manipulations. Especially, we highlight the significance of viral vector-mediated genetic manipulations in studying learning and memory processes, and how they may be effectively used to separate out the various phases of learning: acquisition, consolidation, retrieval, and maintenance.

  16. Induction of AhR-mediated gene transcription by coffee.

    Directory of Open Access Journals (Sweden)

    Toshio Ishikawa

    Full Text Available Aryl hydrocarbon receptor (AhR is classically known to be activated by xenobiotics such as dioxins and polycyclic aromatic hydrocarbons (PAHs. Although it has been reported that PAHs are contained in roasted coffee beans, in general coffee beverages are not considered to be AhR activators. We tested whether exposure to coffee would activate AhR in cultured cells.HepG2 cells stably expressing an AhR-responsive reporter gene were treated with coffee samples. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was quantitated by RT-PCR and Western blotting in HepG2, Caco-2, and MCF-7 cells, after treatment with coffee. In order to obtain sensitive and reproducible results, all the experiments were performed with the cells placed in either phosphate-buffered saline (PBS or pure serum, instead of routinely-used culture medium, whose intrinsic AhR-stimulating activity turned out to be so strong as to interfere with the analyses.All the coffee samples tested robustly stimulated AhR-mediated transcription in the reporter gene assays. Of note, to what extent coffee and other AhR agonists activated AhR was different, depending on whether the experiments were done in PBS or serum. CYP1A1 mRNA was induced by coffee, in HepG2, Caco-2, and MCF-7 cells placed in either PBS or serum. CYP1A1 protein expression, which was not detected in these cells incubated in PBS, was also increased by coffee in cells placed in serum.By using culture medium-free experimental settings, we have shown that coffee is a strong AhR activator. Our observation may help elucidate as-yet-unrecognized effects of coffee on human health.

  17. Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of Immune Mediated Genes

    Directory of Open Access Journals (Sweden)

    Christopher A. Aoki

    2006-01-01

    Full Text Available Primary sclerosing cholangitis (PSC is a chronic disease of the bile ducts characterized by an inflammatory infiltrate and obliterative fibrosis. The precise role of the immune system in the pathogenesis of PSC remains unknown. We used RNA microarray analysis to identify immune-related genes and pathways that are differentially expressed in PSC. Messenger RNA (mRNA from peripheral blood mononuclear cells (PBMC was isolated from both patients with PSC and age and sex matched healthy controls. Samples from 5 PSC patients and 5 controls were analyzed by microarray and based upon rigorous statistical analysis of the data, relevant genes were chosen for confirmation by RT-PCR in 10 PSC patients and 10 controls. Using unsupervised hierarchical clustering, gene expression in PSC was statistically different from our control population. Interestingly, genes within the IL-2 receptor beta, IL-6 and MAP Kinase pathways were found to be differently expressed in patients with PSC compared to controls. Further, individual genes, TNF-α induced protein 6 (TNFaip6 and membrane-spanning 4-domains, subfamily A (ms4a were found to be upregulated in PSC while similar to Mothers against decapentaplegic homolog 5 (SMAD 5 was downregulated. In conclusion, several immune-related pathways and genes were differentially expressed in PSC compared to control patients, giving further evidence that this disease is systemic and immune-mediated.

  18. Mismatch-mediated error prone repair at the immunoglobulin genes.

    Science.gov (United States)

    Chahwan, Richard; Edelmann, Winfried; Scharff, Matthew D; Roa, Sergio

    2011-12-01

    The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood.

  19. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  20. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

    Directory of Open Access Journals (Sweden)

    Roopa Thapar

    2015-05-01

    Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

  1. PexRAP Inhibits PRDM16-Mediated Thermogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Irfan J. Lodhi

    2017-09-01

    Full Text Available How the nuclear receptor PPARγ regulates the development of two functionally distinct types of adipose tissue, brown and white fat, as well as the browning of white fat, remains unclear. Our previous studies suggest that PexRAP, a peroxisomal lipid synthetic enzyme, regulates PPARγ signaling and white adipogenesis. Here, we show that PexRAP is an inhibitor of brown adipocyte gene expression. PexRAP inactivation promoted adipocyte browning, increased energy expenditure, and decreased adiposity. Identification of PexRAP-interacting proteins suggests that PexRAP function extends beyond its role as a lipid synthetic enzyme. Notably, PexRAP interacts with importin-β1, a nuclear import factor, and knockdown of PexRAP in adipocytes reduced the levels of nuclear phospholipids. PexRAP also interacts with PPARγ, as well as PRDM16, a critical transcriptional regulator of thermogenesis, and disrupts the PRDM16-PPARγ complex, providing a potential mechanism for PexRAP-mediated inhibition of adipocyte browning. These results identify PexRAP as an important regulator of adipose tissue remodeling.

  2. Bacteriophage WO Can Mediate Horizontal Gene Transfer in Endosymbiotic Wolbachia Genomes

    Science.gov (United States)

    Wang, Guan H.; Sun, Bao F.; Xiong, Tuan L.; Wang, Yan K.; Murfin, Kristen E.; Xiao, Jin H.; Huang, Da W.

    2016-01-01

    Phage-mediated horizontal gene transfer (HGT) is common in free-living bacteria, and many transferred genes can play a significant role in their new bacterial hosts. However, there are few reports concerning phage-mediated HGT in endosymbionts (obligate intracellular bacteria within animal or plant hosts), such as Wolbachia. The Wolbachia-infecting temperate phage WO can actively shift among Wolbachia genomes and has the potential to mediate HGT between Wolbachia strains. In the present study, we extend previous findings by validating that the phage WO can mediate transfer of non-phage genes. To do so, we utilized bioinformatic, phylogenetic, and molecular analyses based on all sequenced Wolbachia and phage WO genomes. Our results show that the phage WO can mediate HGT between Wolbachia strains, regardless of whether the transferred genes originate from Wolbachia or other unrelated bacteria. PMID:27965627

  3. Many chromosomal genes modulate MarA-mediated multidrug resistance in Escherichia coli.

    Science.gov (United States)

    Ruiz, Cristian; Levy, Stuart B

    2010-05-01

    Multidrug resistance (MDR) in clinical isolates of Escherichia coli can be associated with overexpression of marA, a transcription factor that upregulates multidrug efflux and downregulates membrane permeability. Using random transposome mutagenesis, we found that many chromosomal genes and environmental stimuli affected MarA-mediated antibiotic resistance. Seven genes affected resistance mediated by MarA in an antibiotic-specific way; these were mostly genes encoding unrelated enzymes, transporters, and unknown proteins. Other genes affected MarA-mediated resistance to all antibiotics tested. These genes were acrA, acrB, and tolC (which encode the major MarA-regulated multidrug efflux pump AcrAB-TolC), crp, cyaA, hns, and pcnB (four genes involved in global regulation of gene expression), and the unknown gene damX. The last five genes affected MarA-mediated MDR by altering marA expression or MarA function specifically on acrA. These findings demonstrate that MarA-mediated MDR is regulated at multiple levels by different genes and stimuli, which makes it both complex and fine-tuned and interconnects it with global cell regulation and metabolism. Such a regulation could contribute to the adaptation and spread of MDR strains and may be targeted to treat antibiotic-resistant E. coli and related pathogens.

  4. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

    Science.gov (United States)

    Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

    2017-09-26

    Magnaportheoryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M.oryzae-derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M.oryzae was examined by targeting three predicted pathogenicity genes, MoABC1,MoMAC1 and MoPMK1. Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M.oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

  5. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.

    Science.gov (United States)

    Michielse, C B; Arentshorst, M; Ram, A F J; van den Hondel, C A M J J

    2005-01-01

    In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement.

  6. Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis

    Science.gov (United States)

    2015-10-01

    1 AWARD NUMBER: W81XWH-14-1-0314 TITLE: Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis ...19 Sep 2015 4. TITLE AND SUBTITLE Glucocorticoid Receptor-Mediated Repression of Pro- Inflammatory Genes in Rheumatoid Arthritis 5a. CONTRACT NUMBER... arthritis (RA) patients rely on glucocorticoids (GCs) at some point during the disease. GCs signal through the GC receptor (GR), a transcription factor that

  7. Nanoparticle-mediated p53 gene therapy for tumor inhibition

    OpenAIRE

    Sharma, Blanka; Ma, Wenxue; Adjei, Isaac Morris; Panyam, Jayanth; Dimitrijevic, Sanja; Labhasetwar, Vinod

    2011-01-01

    The p53 tumor suppressor gene is mutated in 50% of human cancers, resulting in more aggressive disease with greater resistance to chemotherapy and radiation therapy. Advances in gene therapy technologies offer a promising approach to restoring p53 function. We have developed polymeric nanoparticles (NPs), based on poly (lactic-co-glycolic acid), that provide sustained intracellular delivery of plasmid DNA, resulting in sustained gene expression without vector-associated toxicity. Our previous...

  8. DNA-mediated gene transfer in plant protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    U, Zang Kual; Riu, Key Zung; So, In Sup; Hong, Kyung Ae [Cheju National University, Cheju (Korea, Republic of)

    1994-12-31

    The neomycin phosphotransferase II gene(NPT-II) was introduced into geranium (Pelargonium zonale hybrids) protoplasts by using PEG or electroporation method. The presence of the introduced DNA in the protoplasts and the expressions of the gene in the transformed cells were examined. The presence of the NPT-II DNA in the protoplasts were detected by polymerase chain reaction. The expressions of NPT-II gene in the transformed cells were confirmed by the NPT-II assay. (author)

  9. The human desmin promoter drives robust gene expression for skeletal muscle stem cell-mediated gene therapy.

    Science.gov (United States)

    Jonuschies, Jacqueline; Antoniou, Michael; Waddington, Simon; Boldrin, Luisa; Muntoni, Francesco; Thrasher, Adrian; Morgan, Jennifer

    2014-01-01

    Lentiviral vectors (LVs) represent suitable candidates to mediate gene therapy for muscular dystrophies as they infect dividing and non-dividing cells and integrate their genetic material into the host genome, thereby theoretically mediating longterm expression. We evaluated the ability of LVs where a GFP reporter gene was under the control of five different promoters, to transduce and mediate expression in myogenic and non-myogenic cells in vitro and in skeletal muscle fibres and stem (satellite) cells in vivo. We further analysed lentivirally-transduced satellite cell-derived myoblasts following their transplantation into dystrophic, immunodeficient mouse muscles. The spleen focus-forming virus promoter mediated the highest gene expression in all cell types; the CBX3-HNRPA2B1 ubiquitously-acting chromatin opening element (UCOE) promoter was also active in all cells, whereas the human desmin promoter in isolation or fused with UCOE had lower activity in non-muscle cells. Surprisingly, the human skeletal muscle actin promoter was also active in immune cells. The human desmin promoter mediated robust, persistent reporter gene expression in myogenic cells in vitro, and satellite cells and muscle fibres in vivo. The human desmin promoter combined with UCOE did not significantly increase transgene expression. Therefore, our data indicate that the desmin promoter is suitable for the development of therapeutic purposes.

  10. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    Science.gov (United States)

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  11. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  12. In vivo electroporation mediated gene delivery to the beating heart.

    Directory of Open Access Journals (Sweden)

    Erick L Ayuni

    Full Text Available Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase and TNT (Troponin T were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.

  13. Vector-mediated cancer gene therapy: an overview.

    Science.gov (United States)

    Seth, Prem

    2005-05-01

    In recent years there has been a dramatic increase in developing gene therapy approaches for the treatment of cancer. The two events that have permitted the formulation of concept of cancer gene therapy are the new understanding of the molecular mechanisms underlying oncogenesis, and the development of the DNA-delivery vehicles or vectors. Many approaches to cancer gene therapy have been proposed, and several viral and non-viral vectors have been utilized. The purpose of this review article is to describe the various strategies of cancer gene therapy (transfer of tumor suppressor genes, suicide genes-enzyme/pro-drug approach, inhibition of dominant oncogenes, immunomodulation approaches, expression of molecules that affect angiogenesis, tumor invasion and metastasis, chemosensitization and radiosensitization approaches, and chemoprotection of stem cells). The chapter also reviews the commonly used vectors (retroviral vectors, adenoviral vectors, adeno-associated viral vectors, pox viruses, herpes simplex viruses, HIV- vectors, non-viral vectors and targetable vectors) for cancer gene therapy. Some of the important issues in cancer gene therapy, and the potential future directions are also being discussed.

  14. RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.

    Science.gov (United States)

    Drake, Lisa L; Price, David P; Aguirre, Sarah E; Hansen, Immo A

    2012-07-14

    This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

  15. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  16. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    Science.gov (United States)

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  17. Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

    Science.gov (United States)

    Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2015-03-01

    Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

  18. Magnetic Iron Oxide Nanoparticles Mediated Gene Therapy for Cancer An In Vitro Study

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The aim of this study was to evaluate the feasibility and efficacy of using TRAIL gene to treat breast cancer mediated with a novel carrier - magnetic iron oxide nanoparticles (polyMAG-1000) coated with PEI. The magnetic iron oxide nanoparticles were used as gene carrier to transfect TRAIL gene into MCF-7 cells. The polyMAG-1000 without TRAIL gene was transfected into the tumor cells as negative control. TRAIL gene transfection with liposome as carrier served as positive control. The apoptosis of cells was detected with TUNEL method. The apoptosis ratio of tumor cells was measured with flow cytometry (FCM). It was found that the apoptosis occurred in the tumor cells after transfection of TRAIL gene mediated by both polyMAG-1000 and liposome. The apoptosis ratio in the group with polyMAG-1000 as gene carrier was (25.11±2.85) %, whereas it was (5.06±1.05) % in the control group with polyMAG-1000 (P<0.01). The apoptosis ratio was as low as (18.31±2.44) % in the group with liposome as gene carrier (P<0.05, as compared with the group with polyMAG-1000 as gene carrier). It is suggested that TRAIL gene may induce apoptosis in MCF-7 breast cancer cells. The magnetic iron oxide nanoparticles coated with PEI may be a potential gene carrier with high transfection efficacy for cancer gene therapy.

  19. Baculovirus-mediated Gene Delivery and RNAi Applications

    Directory of Open Access Journals (Sweden)

    Kaisa-Emilia Makkonen

    2015-04-01

    Full Text Available Baculoviruses are widely encountered in nature and a great deal of data is available about their safety and biology. Recently, these versatile, insect-specific viruses have demonstrated their usefulness in various biotechnological applications including protein production and gene transfer. Multiple in vitro and in vivo studies exist and support their use as gene delivery vehicles in vertebrate cells. Recently, baculoviruses have also demonstrated high potential in RNAi applications in which several advantages of the virus make it a promising tool for RNA gene transfer with high safety and wide tropism.

  20. BRCA1-mediated repression of select X chromosome genes

    Directory of Open Access Journals (Sweden)

    Ropers H Hilger

    2004-09-01

    Full Text Available Abstract Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling. Significance was determined using parametric statistics with P

  1. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms.

  2. The Signature of Selection Mediated by Expression on Human Genes

    OpenAIRE

    Urrutia, Araxi O.; Hurst, Laurence D

    2003-01-01

    As the efficacy of natural selection is expected to be a function of population size, in humans it is usually presumed that selection is a weak force and hence that gene characteristics are mostly determined by stochastic forces. In contrast, in species with large population sizes, selection is expected to be a much more effective force. Evidence for this has come from examining how genic parameters vary with expression level, which appears to determine many of a gene's features, such as codo...

  3. Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper

    Directory of Open Access Journals (Sweden)

    Dong Ying

    2005-10-01

    Full Text Available Abstract Background Grasshopper serves as important model system in neuroscience, development and evolution. Representatives of this primitive insect group are also highly relevant targets of pest control efforts. Unfortunately, the lack of genetics or gene specific molecular manipulation imposes major limitations to the study of grasshopper biology. Results We investigated whether juvenile instars of the grasshopper species Schistocerca americana are conducive to gene silencing via the systemic RNAi pathway. Injection of dsRNA corresponding to the eye colour gene vermilion into first instar nymphs triggered suppression of ommochrome formation in the eye lasting through two instars equivalent to 10–14 days in absolute time. QRT-PCR analysis revealed a two fold decrease of target transcript levels in affected animals. Control injections of EGFP dsRNA did not result in detectable phenotypic changes. RT-PCR and in situ hybridization detected ubiquitous expression of the grasshopper homolog of the dsRNA channel protein gene sid-1 in embryos, nymphs and adults. Conclusion Our results demonstrate that systemic dsRNA application elicits specific and long-term gene silencing in juvenile grasshopper instars. The conservation of systemic RNAi in the grasshopper suggests that this pathway can be exploited for gene specific manipulation of juvenile and adult instars in a wide range of primitive insects.

  4. Advances in Ultrasound Mediated Gene Therapy Using Microbubble Contrast Agents

    Directory of Open Access Journals (Sweden)

    Shashank R. Sirsi, Mark A. Borden

    2012-01-01

    Full Text Available Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1 improving cell membrane permeability, 2 modulate vascular permeability, and 3 enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.

  5. Advances in ultrasound mediated gene therapy using microbubble contrast agents.

    Science.gov (United States)

    Sirsi, Shashank R; Borden, Mark A

    2012-01-01

    Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance in vivo gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve in vivo microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.

  6. Transcription mediated insulation and interference direct gene cluster expression switches.

    Science.gov (United States)

    Nguyen, Tania; Fischl, Harry; Howe, Françoise S; Woloszczuk, Ronja; Serra Barros, Ana; Xu, Zhenyu; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-11-19

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.

  7. Nanoparticle-mediated delivery of suicide genes in cancer therapy.

    Science.gov (United States)

    Vago, Riccardo; Collico, Veronica; Zuppone, Stefania; Prosperi, Davide; Colombo, Miriam

    2016-09-01

    Conventional chemotherapeutics have been employed in cancer treatment for decades due to their efficacy in killing the malignant cells, but the other side of the coin showed off-target effects, onset of drug resistance and recurrences. To overcome these limitations, different approaches have been investigated and suicide gene therapy has emerged as a promising alternative. This approach consists in the introduction of genetic materials into cancerous cells or the surrounding tissue to cause cell death or retard the growth of the tumor mass. Despite promising results obtained both in vitro and in vivo, this innovative approach has been limited, for long time, to the treatment of localized tumors, due to the suboptimal efficiency in introducing suicide genes into cancer cells. Nanoparticles represent a valuable non-viral delivery system to protect drugs in the bloodstream, to improve biodistribution, and to limit side effects by achieving target selectivity through surface ligands. In this scenario, the real potential of suicide genes can be translated into clinically viable treatments for patients. In the present review, we summarize the recent advances of inorganic nanoparticles as non-viral vectors in terms of therapeutic efficacy, targeting capacity and safety issues. We describe the main suicide genes currently used in therapy, with particular emphasis on toxin-encoding genes of bacterial and plant origin. In addition, we discuss the relevance of molecular targeting and tumor-restricted expression to improve treatment specificity to cancer tissue. Finally, we analyze the main clinical applications, limitations and future perspectives of suicide gene therapy.

  8. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Science.gov (United States)

    Srikhanta, Yogitha N; Dowideit, Stefanie J; Edwards, Jennifer L; Falsetta, Megan L; Wu, Hsing-Ju; Harrison, Odile B; Fox, Kate L; Seib, Kate L; Maguire, Tina L; Wang, Andrew H-J; Maiden, Martin C; Grimmond, Sean M; Apicella, Michael A; Jennings, Michael P

    2009-04-01

    Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes) that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression). In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion"), via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M) systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18) and modB (modB1, 2). These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11), differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates that

  9. Phasevarions mediate random switching of gene expression in pathogenic Neisseria.

    Directory of Open Access Journals (Sweden)

    Yogitha N Srikhanta

    2009-04-01

    Full Text Available Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression. In Haemophilus influenzae, the random switching of the modA gene controls expression of a phase-variable regulon of genes (a "phasevarion", via differential methylation of the genome in the modA ON and OFF states. Phase-variable mod genes are also present in Neisseria meningitidis and Neisseria gonorrhoeae, suggesting that phasevarions may occur in these important human pathogens. Phylogenetic studies on phase-variable mod genes associated with type III restriction modification (R-M systems revealed that these organisms have two distinct mod genes--modA and modB. There are also distinct alleles of modA (abundant: modA11, 12, 13; minor: modA4, 15, 18 and modB (modB1, 2. These alleles differ only in their DNA recognition domain. ModA11 was only found in N. meningitidis and modA13 only in N. gonorrhoeae. The recognition site for the modA13 methyltransferase in N. gonorrhoeae strain FA1090 was identified as 5'-AGAAA-3'. Mutant strains lacking the modA11, 12 or 13 genes were made in N. meningitidis and N. gonorrhoeae and their phenotype analyzed in comparison to a corresponding mod ON wild-type strain. Microarray analysis revealed that in all three modA alleles multiple genes were either upregulated or downregulated, some of which were virulence-associated. For example, in N. meningitidis MC58 (modA11, differentially expressed genes included those encoding the candidate vaccine antigens lactoferrin binding proteins A and B. Functional studies using N. gonorrhoeae FA1090 and the clinical isolate O1G1370 confirmed that modA13 ON and OFF strains have distinct phenotypes in antimicrobial resistance, in a primary human cervical epithelial cell model of infection, and in biofilm formation. This study, in conjunction with our previous work in H. influenzae, indicates

  10. Phasevarion mediated epigenetic gene regulation in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Yogitha N Srikhanta

    Full Text Available Many host-adapted bacterial pathogens contain DNA methyltransferases (mod genes that are subject to phase-variable expression (high-frequency reversible ON/OFF switching of gene expression. In Haemophilus influenzae and pathogenic Neisseria, the random switching of the modA gene, associated with a phase-variable type III restriction modification (R-M system, controls expression of a phase-variable regulon of genes (a "phasevarion", via differential methylation of the genome in the modA ON and OFF states. Phase-variable type III R-M systems are also found in Helicobacter pylori, suggesting that phasevarions may also exist in this key human pathogen. Phylogenetic studies on the phase-variable type III modH gene revealed that there are 17 distinct alleles in H. pylori, which differ only in their DNA recognition domain. One of the most commonly found alleles was modH5 (16% of isolates. Microarray analysis comparing the wild-type P12modH5 ON strain to a P12ΔmodH5 mutant revealed that six genes were either up- or down-regulated, and some were virulence-associated. These included flaA, which encodes a flagella protein important in motility and hopG, an outer membrane protein essential for colonization and associated with gastric cancer. This study provides the first evidence of this epigenetic mechanism of gene expression in H. pylori. Characterisation of H. pylori modH phasevarions to define stable immunological targets will be essential for vaccine development and may also contribute to understanding H. pylori pathogenesis.

  11. Electroporation-mediated gene transfer directly to the swine heart.

    Science.gov (United States)

    Hargrave, B; Downey, H; Strange, R; Murray, L; Cinnamond, C; Lundberg, C; Israel, A; Chen, Y-J; Marshall, W; Heller, R

    2013-02-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were killed 48 h after injection and electroporation and gene expression was determined. Results were compared with sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared with injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

  12. Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

    Directory of Open Access Journals (Sweden)

    Ji-Si Zhang

    Full Text Available The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'. Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS. Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution

  13. Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

    Science.gov (United States)

    Zhang, Ji-Si; Zhao, Jing; Zhang, Shaohua; He, Chaoying

    2014-01-01

    The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development

  14. miRNA-mediated functional changes through co-regulating function related genes.

    Directory of Open Access Journals (Sweden)

    Jie He

    Full Text Available BACKGROUND: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation. RESULTS: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1. CONCLUSIONS: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

  15. Adenovirus-mediated nitric oxide synthase gene transfer.

    Science.gov (United States)

    Raman, Kathleen G; Shapiro, Richard A; Tzeng, Edith; Kibbe, Melina R

    2004-01-01

    The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

  16. Transient gene expression mediated by integrase-defective retroviral vectors.

    Science.gov (United States)

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  17. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Energy Technology Data Exchange (ETDEWEB)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F [Department of Pharmaceutical Science, Zhujiang Hospital, Southern Medical University, Guangzhou 510282 (China); Yang, B, E-mail: andrewfxu1998@gmail.co [Department of Chemistry, Indiana University-Bloomington, Bloomington, IN 47405 (United States)

    2009-10-07

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  18. LONG DISTANCE POLLEN-MEDIATED GENE FLOW FROM CREEPING BENTGRASS

    Science.gov (United States)

    Researchers from USEPA WED have measured gene flow from experimental fields of Roundup? herbicide resistant genetically modified (GM) creeping bentgrass a grass used primarily on golf courses, to compatible non-crop relatives. Using a sampling design based on the estimated time ...

  19. A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants.

    Science.gov (United States)

    Li, Zhongsen; Xing, Aiqiu; Moon, Bryan P; Burgoyne, Susan A; Guida, Anthony D; Liang, Huiling; Lee, Catharina; Caster, Cheryl S; Barton, Joanne E; Klein, Theodore M; Falco, Saverio C

    2007-10-01

    Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

  20. Bistability in a stochastic RNA-mediated gene network

    Science.gov (United States)

    Lloyd-Price, Jason; Ribeiro, Andre S.

    2013-09-01

    Small regulatory RNAs (srRNAs) are important regulators of gene expression in eukaryotes and prokaryotes. A common motif containing srRNA is a bistable two-gene motif where one gene codes for a transcription factor (TF) which represses the transcription of the second gene, whose transcript is a srRNA which targets the first gene's transcript. Here, we investigate the properties of this motif in a stochastic model which takes the low copy numbers of the RNA components into account. First, we examine the conditions for stability of the two “noisy attractors.” We find that for realistic low copy numbers, extreme, but within realistic intervals, mutual repression strengths are required to compensate for the variability of the RNA numbers and thus, achieve long-term bistability. Second, the promoter initiation kinetics is found to strongly influence the bistability of the switch. Super-Poissonian RNA production disrupts the ability of the srRNA to silence its target, though sub-Poissonian RNA production does not rule out the need for strong mutual repression. Finally, we show that asymmetry between the two interactions forming the switch allows an external input to induce the transition from “high srRNA” to “‘high TF” more easily (i.e., with a shorter input) than in the opposite direction. We hypothesize that this asymmetric switching property allows these circuits to be more sensitive to one external input, without sacrificing the stability of one of the noisy attractors.

  1. Electroporation-mediated delivery of genes in rodent models of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David; Raghavendran, Krishnan

    2014-01-01

    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated gene delivery to the lung. Electroporation is a highly efficient and easily reproducible technique that allows circumvention of several of lung gene delivery challenges and safety issues present with other forms of lung gene therapy.

  2. Repressor-mediated tissue-specific gene expression in plants

    Science.gov (United States)

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  3. Cytokine gene-mediated immunotherapy: current status and future perspectives.

    Science.gov (United States)

    Jinushi, Masahisa; Tahara, Hideaki

    2009-08-01

    Recent understanding of the molecular events crucial in overcoming immunosuppressive tumor microenvironments and generating effective antitumor immunity provides us with the wreath opportunity to manipulate genes that have a key role in antitumor immune responses. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) are two indispensable cytokines for activating dendritic cells and boosting the strong immune responses against cancer. In this review, we describe the antitumor mechanisms and clinical application of gene-modified tumor cells and dendritic cells to secrete GM-CSF or IL-12, respectively, in various preclinical and clinical settings. The principles operative in these vaccination strategies may prove applicable to other immunotherapy strategies, especially in combination with other therapeutic modalities, such as chemotherapy and targeted therapy.

  4. Identifying genes that mediate anthracyline toxicity in immune cells

    Directory of Open Access Journals (Sweden)

    Amber eFrick

    2015-04-01

    Full Text Available The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS, we identified four genome-wide significant quantitative trait loci (QTL that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01x10-8. Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05.In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Thus, further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  5. Thermodynamic control of small RNA-mediated gene silencing

    Directory of Open Access Journals (Sweden)

    Kumiko eUi-Tei

    2012-06-01

    Full Text Available Small interfering RNAs (siRNAs and microRNAs (miRNAs are crucial regulators of posttranscriptional gene silencing, which is referred to as RNA interference (RNAi or RNA silencing. In RNAi, siRNA loaded onto the RNA-induced silencing complex (RISC downregulates target gene expression by cleaving mRNA whose sequence is perfectly complementary to the siRNA guide strand. We previously showed that highly functional siRNAs possessed the following characteristics: A or U residues at nucleotide position 1 measured from the 5’ terminal, four to seven A/Us in positions 1–7, and G or C residues at position 19. This finding indicated that an RNA strand with a thermodynamically unstable 5’ terminal is easily retained in the RISC and functions as a guide strand. In addition, it is clear that unintended genes with complementarities only in the seed region (positions 2–8 are also downregulated by off-target effects. siRNA efficiency is mainly determined by the Watson-Crick base-pairing stability formed between the siRNA seed region and target mRNA. siRNAs with a low seed-target duplex melting temperature (Tm have little or no seed-dependent off-target activity. Thus, important parts of the RNA silencing machinery may be regulated by nucleotide base-pairing thermodynamic stability. A mechanistic understanding of thermodynamic control may enable an efficient target gene-specific RNAi for functional genomics and safe therapeutic applications.

  6. Bacteriophage-mediated toxin gene regulation in Clostridium difficile.

    Science.gov (United States)

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D; Dupuy, Bruno; Fralick, Joe A

    2009-12-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by PhiCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in PhiCD119 lysogens. During this study we found that repR, a putative repressor gene of PhiCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in PhiCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.

  7. Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile▿

    Science.gov (United States)

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D.; Dupuy, Bruno; Fralick, Joe A.

    2009-01-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ΦCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ΦCD119 lysogens. During this study we found that repR, a putative repressor gene of ΦCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ΦCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen. PMID:19776116

  8. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  9. Sleeping Beauty-Mediated Drug Resistance Gene Transfer in Human Hematopoietic Progenitor Cells

    Science.gov (United States)

    Hyland, Kendra A.; Olson, Erik R.; McIvor, R. Scott

    2015-01-01

    The Sleeping Beauty (SB) transposon system can insert sequences into mammalian chromosomes, supporting long-term expression of both reporter and therapeutic genes. Hematopoietic progenitor cells (HPCs) are an ideal therapeutic gene transfer target as they are used in therapy for a variety of hematologic and metabolic conditions. As successful SB-mediated gene transfer into human CD34+ HPCs has been reported by several laboratories, we sought to extend these studies to the introduction of a therapeutic gene conferring resistance to methotrexate (MTX), potentially providing a chemoprotective effect after engraftment. SB-mediated transposition of hematopoietic progenitors, using a transposon encoding an L22Y variant dihydrofolate reductase fused to green fluorescent protein, conferred resistance to methotrexate and dipyridamole, a nucleoside transport inhibitor that tightens MTX selection conditions, as assessed by in vitro hematopoietic colony formation. Transposition of individual transgenes was confirmed by sequence analysis of transposon–chromosome junctions recovered by linear amplification-mediated PCR. These studies demonstrate the potential of SB-mediated transposition of HPCs for expression of drug resistance genes for selective and chemoprotective applications. PMID:26176276

  10. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Yamaguchi, Kazuki [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Feril, Loreto B., E-mail: ferilism@yahoo.com [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Tachibana, Katsuro [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Takahashi, Akira; Matsuo, Miki; Endo, Hitomi [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Harada, Yoshimi [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Nakayama, Juichiro [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  11. Genes That Mediate Arsenic and Heavy Metal Detoxification in Plants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, David A.; Gong, Ji-Ming; Schroeder, Julian I.

    2003-03-26

    To gain insight into the mechanisms of arsenic tolerance in plants, we developed a genetic screen to isolate Arabidopsis thaliana mutants with altered tolerance to arsenic. We report here on the isolation of ars1, a novel mutant with significantly increased tolerance to arsenate. ars1 accumulates similar levels of arsenic as wild type plants, but ars1 tolerance does not appear to be phytochelatin or glutathione dependent. ars1 plants do have a higher rate of phosphate uptake than wild type plants and plants grown with an excess of phosphate show increased tolerance to arsenate. Traditional models of arsenate tolerance in plants are based on the suppression of phosphate uptake pathways and, consequently, the reduced uptake of arsenate. Our data suggest that arsenate tolerance in ars1 is due to a new mechanism mediated by increased phosphate uptake in ars1. Results exploring increased metal tolerance through engineered phytochelatin expression will also be discussed.

  12. Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element.

    OpenAIRE

    1991-01-01

    Diabetes mellitus is characterized by insulin deficiency and high plasma glucagon levels, which can be normalized by insulin replacement. It has previously been reported that glucagon gene expression is negatively regulated by insulin at the transcriptional level. By transfection studies, I have now localized a DNA control element that mediates insulin effects on glucagon gene transcription. This element also confers insulin responsiveness to a heterologous promoter. DNA-binding proteins that...

  13. ISEcp1-Mediated Transposition of qnrB-Like Gene in Escherichia coli▿

    Science.gov (United States)

    Cattoir, Vincent; Nordmann, Patrice; Silva-Sanchez, Jesus; Espinal, Paula; Poirel, Laurent

    2008-01-01

    A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene. PMID:18519717

  14. Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge.

    Science.gov (United States)

    Shrestha, Sony; Park, Yoonseong; Stanley, David; Kim, Yonggyun

    2010-03-01

    We propose that expression of four genes encoding secretory phospholipases A(2) (sPLA(2)) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA(2)-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA(2) activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA(2) and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA(2)s. The recombinant sPLA(2)s were inhibited by sPLA(2) inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA(2) genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA(2)s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA(2)s showed the presence of the sPLA(2) enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA(2) genes that mediate nodulation reactions strongly supports our hypothesis that sPLA(2)s are central enzymes in insect cellular immune reactions. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  15. Chloroquine mediated modulation of Anopheles gambiae gene expression.

    Directory of Open Access Journals (Sweden)

    Patrícia Abrantes

    Full Text Available BACKGROUND: Plasmodium development in the mosquito is crucial for malaria transmission and depends on the parasite's interaction with a variety of cell types and specific mosquito factors that have both positive and negative effects on infection. Whereas the defensive response of the mosquito contributes to a decrease in parasite numbers during these stages, some components of the blood meal are known to favor infection, potentiating the risk of increased transmission. The presence of the antimalarial drug chloroquine in the mosquito's blood meal has been associated with an increase in Plasmodium infectivity for the mosquito, which is possibly caused by chloroquine interfering with the capacity of the mosquito to defend against the infection. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report a detailed survey of the Anopheles gambiae genes that are differentially regulated by the presence of chloroquine in the blood meal, using an A. gambiae cDNA microarray. The effect of chloroquine on transcript abundance was evaluated separately for non-infected and Plasmodium berghei-infected mosquitoes. Chloroquine was found to affect the abundance of transcripts that encode proteins involved in a variety of processes, including immunity, apoptosis, cytoskeleton and the response to oxidative stress. This pattern of differential gene expression may explain the weakened mosquito defense response which accounts for the increased infectivity observed in chloroquine-treated mosquitoes. CONCLUSIONS/SIGNIFICANCE: The results of the present study suggest that chloroquine can interfere with several putative mosquito mechanisms of defense against Plasmodium at the level of gene expression and highlight the need for a better understanding of the impacts of antimalarial agents on parasite transmission.

  16. Electric pulse-mediated gene delivery to various animal tissues

    DEFF Research Database (Denmark)

    Mir, Lluis M; Moller, Pernille H; André, Franck

    2005-01-01

    Electroporation designates the use of electric pulses to transiently permeabilize the cell membrane. It has been shown that DNA can be transferred to cells through a combined effect of electric pulses causing (1) permeabilization of the cell membrane and (2) an electrophoretic effect on DNA...... therapy, termed electrogenetherapy (EGT as well). By transfecting cells with a long lifetime, such as muscle fibers, a very long-term expression of genes can be obtained. A great variety of tissues have been transfected successfully, from muscle as the most extensively used, to both soft (e.g., spleen...

  17. The arginine vasopressin V1b receptor gene and prosociality: Mediation role of emotional empathy.

    Science.gov (United States)

    Wu, Nan; Shang, Siyuan; Su, Yanjie

    2015-09-01

    The vasopressin V1b receptor (AVPR1B) gene has been shown to be closely associated with bipolar disorder and depression. However, whether it relates to positive social outcomes, such as empathy and prosocial behavior, remains unknown. This study explored the possible role of the AVPR1B gene rs28373064 in empathy and prosociality. A total of 256 men, who were genetically unrelated, non-clinical ethnic Han Chinese college students, participated in the study. Prosociality was tested by measuring the prosocial tendencies of cognitive and emotional empathy using the Interpersonal Reactivity Index (IRI). The single nucleotide polymorphism (SNP), rs28373064, was genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results suggest that the AVPR1B gene rs28373064 is linked to emotional empathy and prosociality. The mediation analysis indicated that the effect of the AVPR1B gene on prosociality might be mediated by emotional empathy. This study demonstrated the link between the AVPR1B gene and prosociality and provided evidence that emotional empathy might mediate the relation between the AVPR1B gene and prosociality. © 2015 The Institute of Psychology, Chinese Academy of Sciences and Wiley Publishing Asia Pty Ltd.

  18. Long distance pollen-mediated flow of herbicide resistance genes in Lolium rigidum.

    Science.gov (United States)

    Busi, Roberto; Yu, Qin; Barrett-Lennard, Robert; Powles, Stephen

    2008-11-01

    Gene flow promotes genetic exchange among plant populations mediating evolutionary dynamics; yet, the importance of gene flow at distance via pollen movement is poorly understood. A field experiment at the landscape level was conducted with Lolium rigidum herbicide-susceptible individuals (population VLR1) placed into an otherwise Lolium-free bushland environment at increasing distances from adjacent large commercial crop fields infested with herbicide-resistant L. rigidum. Herbicide resistance was used as a marker to quantify the distance and the rate of pollen-mediated gene flow. About 21,245 seeds were produced on the isolated, susceptible mother plants of which 3,303 seedlings were tested for herbicide resistance and 664 seedlings were found to be resistant. Pollen-mediated gene flow occurred at 3,000 m (maximum tested distance). Both Mendelian and molecular analyses (sequencing and CAPS markers) confirmed the introgression of herbicide resistance genes. This is the first documented case of long-distance gene flow in L. rigidum. The results are important for future modeling simulations of herbicide resistance evolution and subsequent mobility. The adoption of integrated agronomic strategies, the control of potential receptor plants on fields' margins and conservative use of herbicides can be realistic options to minimize herbicide resistance spread.

  19. Toxin-mediated gene regulatory mechanism in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hwang-Soo Joo

    2016-12-01

    Full Text Available The dangerous human pathogen Staphylococcus aureus relies heavily on toxins to cause disease, but toxin production can put a strong burden on the bacteria’s energy balance. Thus, controlling the synthesis of proteins solely needed in times of toxin production represents a way for the bacteria to avoid wasting energy. One hypothetical manner to accomplish this sort of regulation is by gene regulatory functions of the toxins themselves. There have been several reports about gene regulation by toxins in S. aureus, but these were never verified on the molecular level. In our study published in MBio [Joo et al., 7(5. pii: e01579-16], we show that phenol-soluble modulins (PSMs, important peptide toxins of S. aureus, release a repressor from the promoter of the operon encoding the toxin export system, thereby enabling toxin secretion. This study describes the first molecular regulatory mechanism exerted by an S. aureus toxin, setting a paradigmatic example of how S. aureus toxins may influence cell functions to adjust them to times of toxin production.

  20. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  1. [Advances in research on radioiodine therapy of carcinoma mediated by gene transfer technology].

    Science.gov (United States)

    Mu, Da; Kuang, Anren

    2010-10-01

    Radioiodine therapy of carcinoma could be mediated by transferring the genes which participate in the process of iodine metabolism in thyroid. The correlative genes are sodium/iodine symporter gene, thyroid peroxidase gene and the specific thyroid transcription factors, and others. The objective gene can specifically express in carcinoma by inserting the tissue-specific promoter/enhancer upstream of them, so radioiodine could be used to treat varied carcinomas. The radioiodine uptake in carcinoma cells was obviously increased and the radioiodine therapy of carcinoma was effective after those genes had expressed in carcinoma cells. The main problem was that the effective half-time of radioiodine in cells was too short to produce the ideal effect of radioiodine therapy. Moreover, 211At and 188Re could be transferred by sodium/iodine symporter and they could be used to treat the carcinoma that is capable of radioiodine uptake.

  2. Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

    Directory of Open Access Journals (Sweden)

    Cuiyuan Huang

    2017-07-01

    Full Text Available Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.

  3. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Smialowska, Agata, E-mail: smialowskaa@gmail.com [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Djupedal, Ingela; Wang, Jingwen [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Kylsten, Per [School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Swoboda, Peter [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Ekwall, Karl, E-mail: Karl.Ekwall@ki.se [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden)

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  4. Adeno-associated virus mediated gene therapy for retinal degenerative diseases.

    Science.gov (United States)

    Stieger, Knut; Cronin, Therese; Bennett, Jean; Rolling, Fabienne

    2011-01-01

    Retinal gene therapy holds great promise for the treatment of inherited and noninherited blinding diseases such as retinitis pigmentosa and age-related macular degeneration. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because it mediates long-term transgene expression in a variety of retinal cell types and elicits minimal immune responses. Inherited retinal diseases are nonlethal and have a wide level of genetic heterogeneity. Many of the genes have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial.In this chapter, we focus on AAV-mediated specific gene therapy for inherited retinal degenerative diseases, describing the disease targets, the preclinical studies in animal models and the recent success of the LCA-RPE65 clinical trials.

  5. Computational modeling identifies key gene regulatory interactions underlying phenobarbital-mediated tumor promotion

    Science.gov (United States)

    Luisier, Raphaëlle; Unterberger, Elif B.; Goodman, Jay I.; Schwarz, Michael; Moggs, Jonathan; Terranova, Rémi; van Nimwegen, Erik

    2014-01-01

    Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and β-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesis. PMID:24464994

  6. Mechanisms of microRNA-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    microRNAs (miRNAs) are identified as a class of non-protein regulators and a new source for broad control of gene expression in eukaryotes. The past years have witnessed substantial progress in understanding miRNA functions and mechanisms, although a few controversies remain. Various hypotheses and models have been suggested for the mechanisms of miRNA repression, including translational inhibition at the level of initiation or elongation, rapid degradation of the nascent peptide, mRNA degradation, and mRNA sequestration into P bodies (processing bodies) and SGs (stress granules) for degradation or/and storage. Recently, some noncanonical miRNA regulation, such as miRNA activation and de-repression of miRNA inhibition, have been uncovered. This review discusses some recent advances about how miRNAs regulate their targets and various modes of miRNA function.

  7. Regulation of gene expression mediating indeterminate muscle growth in teleosts.

    Science.gov (United States)

    Ahammad, A K Shakur; Asaduzzaman, Md; Asakawa, Shuichi; Watabe, Shugo; Kinoshita, Shigeharu

    2015-08-01

    Teleosts are unique among vertebrates due to their indeterminate muscle growth, i.e., continued production of neonatal muscle fibers until death. However, the molecular mechanism(s) underlying this property is unknown. Here, we focused on the torafugu (Takifugu rubripes) myosin heavy chain gene, MYHM2528-1, which is specifically expressed in neonatal muscle fibers produced by indeterminate muscle growth. We examined the flanking region of MYHM2528-1 through an in vivo reporter assay using zebrafish (Danio rerio) and identified a 2100 bp 5'-flanking sequence that contained sufficient promoter activity to allow specific gene expression. The effects of enhanced promoter activity were observed at the outer region of the fast muscle and the dorsal edge of slow muscle in zebrafish larvae. At the juvenile stage, the promoter was specifically activated in small diameter muscle fibers scattered throughout fast muscle and in slow muscle near the septum separating slow and fast muscles. This spatio-temporal promoter activity overlapped with known myogenic zones involved in teleost indeterminate muscle growth. A deletion mutant analysis revealed that the -2100 to -600 bp 5'flanking sequence of MYHM2528-1 is essential for promoter activity. This region contains putative binding sites for several representative myogenesis-related transcription factors and nuclear factor of activated T-cell (NFAT), a transcription activator involved in regeneration of mammalian adult skeletal muscle. A significant reduction in the promoter activity of the MYHM2528-1 deletion constructs was observed in accordance with a reduction in the number of these binding sites, suggesting the involvement of specific transcription factors in indeterminate muscle growth.

  8. Adenovirus-mediated interteukin-13 gene therapy attenuates acute kidney allograft injury

    NARCIS (Netherlands)

    Sandovici, Maria; Deelmani, Leo E.; van Goor, Harry; Helfrich, Wijnand; de Zeeuw, Dick; Henning, Robert H.

    2007-01-01

    Background Kidney transplantation is possible by virtue of systemic immunosuppression, which is in turn accompanied by serious side effects. The search for novel therapeutic agents and strategies is ongoing. Here we investigate the effects of adenovirus-mediated gene therapy with interleukin (IL)-13

  9. Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA

    NARCIS (Netherlands)

    Huang, Yi; Deng, F.; Hu, Z.H.; Vlak, J.M.; Wang, H.

    2007-01-01

    Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA

  10. Role of Catechin Quinones in the Induction of EpRE-Mediated Gene Expression

    NARCIS (Netherlands)

    Muzolf-Panek, M.; Gliszczynska-Swiglo, A.; Haan, de L.H.J.; Aarts, J.M.M.J.G.; Szymusiak, H.; Vervoort, J.J.M.; Tyrakowska, B.; Rietjens, I.M.C.M.

    2008-01-01

    In the present study, the ability of green tea catechins to induce electrophile-responsive element (EpRE)-mediated gene expression and the role of their quinones in the mechanism of this induction were investigated. To this end, Hepa1c1c7 mouse hepatoma cells were used, stably transfected with a luc

  11. A Simple Laboratory Practical to Illustrate RNA Mediated Gene Interference Using Drosophila Cell Culture

    Science.gov (United States)

    Buluwela, Laki; Kamalati, Tahereh; Photiou, Andy; Heathcote, Dean A.; Jones, Michael D.; Ali, Simak

    2010-01-01

    RNA mediated gene interference (RNAi) is now a key tool in eukaryotic cell and molecular biology research. This article describes a five session laboratory practical, spread over a seven day period, to introduce and illustrate the technique. During the exercise, students working in small groups purify PCR products that encode "in vitro"…

  12. REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

    DEFF Research Database (Denmark)

    Svensson, Charlotte; Ceder, Jens; Iglesias Gato, Diego

    2014-01-01

    The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds...

  13. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    Science.gov (United States)

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar 'Morex'. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar 'Morex' or the full resistance reaction requires the presence of several PEI genes.

  14. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  15. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone...... or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  16. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone...... or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  17. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    Science.gov (United States)

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-30

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

  18. Recombinase-mediated Gene Stacking as a Transformation Operating System

    Institute of Scientific and Technical Information of China (English)

    David W. Ow

    2011-01-01

    The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of prewously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by sitespecific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.

  19. Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses.

    Directory of Open Access Journals (Sweden)

    Laila Gasmi

    2015-09-01

    Full Text Available Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.

  20. Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses.

    Science.gov (United States)

    Gasmi, Laila; Boulain, Helene; Gauthier, Jeremy; Hua-Van, Aurelie; Musset, Karine; Jakubowska, Agata K; Aury, Jean-Marc; Volkoff, Anne-Nathalie; Huguet, Elisabeth; Herrero, Salvador; Drezen, Jean-Michel

    2015-09-01

    Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.

  1. Agrobacterium tumefaciens-mediated GUS gene transformation of Robinia pseudoacacia 'Idaho'

    Institute of Scientific and Technical Information of China (English)

    Sun Hai-jun; Li Min; Chen Shou-yi; He Si-jie; Wang Hua-fang

    2006-01-01

    Based on the plant regeneration system, a GUS gene transformation system to Idaho locust (Robinia pseudoacacia 'Idaho')mediated by Agrobacterium tumefaciens was established. The successful transformation was confirmed by regenerating the shoots from the infected leaves in the presence of hygromysin; by histochemical X-gluc assays of β-glucuronidase (GUS) and by PCR and PCR-Southern blotting analysis. The ratio of positive transgenic plants is 5.8% (5 out of 86 plants). With this system, the target gene DREB was introduced into the leaves of Idaho locust. The transgenic plants regenerated, which was verified by PCR-Southem blotting. It is suggested that the transformation system could be a new, simple, reliable and practical route to gene transformation of R.pseudoacacia 'Idaho' mediated with A. tumefaciens.

  2. Inositol and Phosphatidylinositol Mediated Glucose Derepression, Gene Expression and Invertase Secretion in Yeasts

    Institute of Scientific and Technical Information of China (English)

    Zhen-Ming CHI; Jun-Feng LI; Xiang-Hong WANG; Shu-Min YAO

    2004-01-01

    Glucose repression occurs in many yeast species and some filamentous fungi, and it represses the expression and secretion of many intracellular and extracellular proteins. In recent years, it has been found that many biochemical reactions in yeast cells are mediated by phosphatidylinositol (PI)-type signaling pathway. However, little is known about the relationships between PI-type signaling and glucose repression,gene expression and invertase secretion in yeasts. Many evidences in our previous studies showed that glucose repression, invertase secretion, gene expression and cell growth were mediated by inositol and PI in Saccharomyces and Schizosaccharomyces. The elucidation of the new regulatory mechanisms of protein secretion, gene expression and glucose repression would be an entirely new aspect of inositol and PI-type signaling regulation in yeasts.

  3. Super-distant molecular hybridization of plant seeds by ion beam-mediated gene cluster

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The N beam-mediated distant molecular hybridization between Ginkgo biloba I and watermelon was studied. The results showed that the ester gene of Ginkgo biloba L was successfully expressed in two varieties of watermelon. 3-16 and SR2-14-2, in both of which the ester quantities were measured as 17.0756 μg/g and 45.9998 μg/g respectively. Meanwhile, superoxide dismutase (SOD) activity in leaves of the watennelon expressing ester gene was increased twofold as compared to that of the control, showing that ion beam could mediate distant and/or super-distant donor gene expression in the cells of a receptor. Furthermore, the molecular nechanism of distant hybridization was analyzed.

  4. Agrobacterium tumefaciens-mediated transformation of CryⅠA(b) gene to Trichoderma harzianum

    Institute of Scientific and Technical Information of China (English)

    GAO Xingxi; YANG Qian

    2004-01-01

    In this study, CryⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 106 spores by using Agrobacterium tumefaciens-mediated trans- formation. Putative transformants were analyzed to test the presence of CryⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the CryⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.

  5. Fsh controls gene expression in fish both independently of and through steroid mediation.

    Directory of Open Access Journals (Sweden)

    Elisabeth Sambroni

    Full Text Available The mechanisms and the mediators relaying Fsh action on testicular functions are poorly understood. Unlike in mammals, in fish both gonadotropins (Fsh and Lh are able to efficiently stimulate steroidogenesis, likely through a direct interaction with their cognate receptors present on the Leydig cells. In this context, it is crucial to understand if Fsh effects are mediated through the production of steroids. To address this issue we performed transcriptome studies after in vitro incubations of rainbow trout testis explants in the presence of Fsh alone or in combination with trilostane, an inhibitor of Δ4- steroidogenesis. Trilostane significantly reduced or suppressed the response of many genes to Fsh (like wisp1, testis gapdhs, cldn11, inha, vt1 or dmrt1 showing that, in fish, important aspects of Fsh action follow indirect pathways and require the production of Δ4-steroids. What is more, most of the genes regulated by Fsh through steroid mediation were similarly regulated by Lh (and/or androgens. In contrast, the response to Fsh of other genes was not suppressed in the presence of trilostane. These latter included genes encoding for anti-mullerian hormone, midkine a (pleiotrophin related, angiopoietine-related protein, cyclins E1 and G1, hepatocyte growth factor activator, insulin-like growth factor 1b/3. A majority of those genes were preferentially regulated by Fsh, when compared to Lh, suggesting that specific regulatory effects of Fsh did not depend on steroid production. Finally, antagonistic effects between Fsh and steroids were found, in particular for genes encoding key factors of steroidogenesis (star, hsd3b1, cyp11b2-2 or for genes of the Igf system (igf1b/3. Our study provides the first clear evidence that, in fish, Fsh exerts Δ4-steroid-independent regulatory functions on many genes which are highly relevant for the onset of spermatogenesis.

  6. C/EBPβ Mediates Growth Hormone-Regulated Expression of Multiple Target Genes

    Science.gov (United States)

    Cui, Tracy X.; Lin, Grace; LaPensee, Christopher R.; Calinescu, Anda-Alexandra; Rathore, Maanjot; Streeter, Cale; Piwien-Pilipuk, Graciela; Lanning, Nathan; Jin, Hui; Carter-Su, Christin; Qin, Zhaohui S.

    2011-01-01

    Regulation of c-Fos transcription by GH is mediated by CCAAT/enhancer binding protein β (C/EBPβ). This study examines the role of C/EBPβ in mediating GH activation of other early response genes, including Cyr61, Btg2, Socs3, Zfp36, and Socs1. C/EBPβ depletion using short hairpin RNA impaired responsiveness of these genes to GH, as seen for c-Fos. Rescue with wild-type C/EBPβ led to GH-dependent recruitment of the coactivator p300 to the c-Fos promoter. In contrast, rescue with C/EBPβ mutated at the ERK phosphorylation site at T188 failed to induce GH-dependent recruitment of p300, indicating that ERK-mediated phosphorylation of C/EBPβ at T188 is required for GH-induced recruitment of p300 to c-Fos. GH also induced the occupancy of phosphorylated C/EBPβ and p300 on Cyr61, Btg2, and Socs3 at predicted C/EBP-cAMP response element-binding protein motifs in their promoters. Consistent with a role for ERKs in GH-induced expression of these genes, treatment with U0126 to block ERK phosphorylation inhibited their GH-induced expression. In contrast, GH-dependent expression of Zfp36 and Socs1 was not inhibited by U0126. Thus, induction of multiple early response genes by GH in 3T3-F442A cells is mediated by C/EBPβ. A subset of these genes is regulated similarly to c-Fos, through a mechanism involving GH-stimulated ERK 1/2 activation, phosphorylation of C/EBPβ, and recruitment of p300. Overall, these studies suggest that C/EBPβ, like the signal transducer and activator of transcription proteins, regulates multiple genes in response to GH. PMID:21292824

  7. Identification of certain cancer-mediating genes using Gaussian fuzzy cluster validity index

    Indian Academy of Sciences (India)

    Anupam Ghosh; Rajat K De

    2015-10-01

    In this article, we have used an index, called Gaussian fuzzy index (GFI), recently developed by the authors, based on the notion of fuzzy set theory, for validating the clusters obtained by a clustering algorithm applied on cancer gene expression data. GFI is then used for the identification of genes that have altered quite significantly from normal state to carcinogenic state with respect to their mRNA expression patterns. The effectiveness of the methodology has been demonstrated on three gene expression cancer datasets dealing with human lung, colon and leukemia. The performance of GFI is compared with 19 exiting cluster validity indices. The results are appropriately validated biologically and statistically. In this context, we have used biochemical pathways, -value statistics of GO attributes, -test and -score for the validation of the results. It has been reported that GFI is capable of identifying high-quality enriched clusters of genes, and thereby is able to select more cancer-mediating genes.

  8. Comparative Analysis of Cluster Validity Indices in Identifying Some Possible Genes Mediating Certain Cancers.

    Science.gov (United States)

    Ghosh, Anupam; Dhara, Bibhas Chandra; De, Rajat K

    2013-04-01

    In this article, we compare the performance of 19 cluster validity indices, in identifying some possible genes mediating certain cancers, based on gene expression data. For the purpose of this comparison, we have developed a method. The proposed method involves cluster generation, selection of the best k-value or c-values, cluster identification, identifying the altered gene cluster, scoring an altered gene cluster and determining the best k-value or c-value exploring through biological repositories. The effectiveness of the method has been demonstrated on three gene expression data sets dealing with human lung cancer, colon cancer, and leukemia. Here, we have used three clustering algorithms, i.e., k-means, PAM and fuzzy c-means. We have used biochemical pathways related to these cancers and p-value statistics for validating the study. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

    Directory of Open Access Journals (Sweden)

    Mansukhani Mahesh

    2006-05-01

    Full Text Available Abstract Background Cervical Cancer (CC exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo. These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. Results To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. Conclusion Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i play a role in CC development, (ii further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii form epigenetic signatures to identify precancerous lesions at risk to progression.

  10. Bacterial plasmid-mediated quinolone resistance genes in aquatic environments in China

    Science.gov (United States)

    Yan, Lei; Liu, Dan; Wang, Xin-Hua; Wang, Yunkun; Zhang, Bo; Wang, Mingyu; Xu, Hai

    2017-01-01

    Emerging antimicrobial resistance is a major threat to human’s health in the 21st century. Understanding and combating this issue requires a full and unbiased assessment of the current status on the prevalence of antimicrobial resistance genes and their correlation with each other and bacterial groups. In aquatic environments that are known reservoirs for antimicrobial resistance genes, we were able to reach this goal on plasmid-mediated quinolone resistance (PMQR) genes that lead to resistance to quinolones and possibly also to the co-emergence of resistance to β-lactams. Novel findings were made that qepA and aac-(6′)-Ib genes that were previously regarded as similarly abundant with qnr genes are now dominant among PMQR genes in aquatic environments. Further statistical analysis suggested that the correlation between PMQR and β-lactam resistance genes in the environment is still weak, that the correlations between antimicrobial resistance genes could be weakened by sufficient wastewater treatment, and that the prevalence of PMQR has been implicated in environmental, pathogenic, predatory, anaerobic, and more importantly, human symbiotic bacteria. This work provides a comprehensive analysis of PMQR genes in aquatic environments in Jinan, China, and provides information with which combat with the antimicrobial resistance problem may be fought. PMID:28094345

  11. Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting.

    Science.gov (United States)

    Lincecum, J M; Fannon, A; Song, K; Wang, Y; Sassoon, D A

    1998-07-01

    Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation.

  12. Nonsense-mediated mRNA decay among coagulation factor genes

    Directory of Open Access Journals (Sweden)

    Shirin Shahbazi

    2016-04-01

    Full Text Available Objective(s: Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulation factors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulation factor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulation factors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD. There are many pieces of evidence of decay among coagulation factor genes. However, the hemophilia gene (F8 does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade.

  13. Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays

    Science.gov (United States)

    Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

    2017-01-01

    Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods. PMID:28377783

  14. Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Perez, C.F.

    1984-08-01

    The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

  15. Application of GFAT as a novel selection marker to mediate gene expression.

    Directory of Open Access Journals (Sweden)

    Guogan Wu

    Full Text Available The enzyme glutamine: fructose-6-phosphate aminotransferase (GFAT, also known as glucosamine synthase (GlmS, catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. For the first time, the GFAT gene was proven to possess a function as an effective selection marker for genetically modified (GM microorganisms. This was shown by construction and analysis of two GFAT deficient strains, E. coli ΔglmS and S. pombe Δgfa1, and the ability of the GFAT encoding gene to mediate plasmid selection. The gfa1 gene of the fission yeast Schizosaccharomyces pombe was deleted by KanMX6-mediated gene disruption and the Cre-loxP marker removal system, and the glmS gene of Escherichia coli was deleted by using λ-Red mediated recombinase system. Both E. coli ΔglmS and S. pombe Δgfa1 could not grow normally in the media without addition of glucosamine. However, the deficiency was complemented by transforming the plasmids that expressed GFAT genes. The xylanase encoding gene, xynA2 from Thermomyces lanuginosus was successfully expressed and secreted by using GFAT as selection marker in S. pombe. Optimal glucosamine concentration for E. coli ΔglmS and S. pombe Δgfa1 growth was determined respectively. These findings provide an effective technique for the construction of GM bacteria without an antibiotic resistant marker, and the construction of GM yeasts to be applied to complex media.

  16. PPARalpha-mediated effects of dietary lipids on intestinal barrier gene expression

    Directory of Open Access Journals (Sweden)

    Bosch-Vermeulen Hanneke

    2008-05-01

    Full Text Available Abstract Background The selective absorption of nutrients and other food constituents in the small intestine is mediated by a group of transport proteins and metabolic enzymes, often collectively called 'intestinal barrier proteins'. An important receptor that mediates the effects of dietary lipids on gene expression is the peroxisome proliferator-activated receptor alpha (PPARα, which is abundantly expressed in enterocytes. In this study we examined the effects of acute nutritional activation of PPARα on expression of genes encoding intestinal barrier proteins. To this end we used triacylglycerols composed of identical fatty acids in combination with gene expression profiling in wild-type and PPARα-null mice. Treatment with the synthetic PPARα agonist WY14643 served as reference. Results We identified 74 barrier genes that were PPARα-dependently regulated 6 hours after activation with WY14643. For eicosapentaenoic acid (EPA, docosahexaenoic acid (DHA and oleic acid (OA these numbers were 46, 41, and 19, respectively. The overlap between EPA-, DHA-, and WY14643-regulated genes was considerable, whereas OA treatment showed limited overlap. Functional implications inferred form our data suggested that nutrient-activated PPARα regulated transporters and phase I/II metabolic enzymes were involved in a fatty acid oxidation, b cholesterol, glucose, and amino acid transport and metabolism, c intestinal motility, and d oxidative stress defense. Conclusion We identified intestinal barrier genes that were PPARα-dependently regulated after acute activation by fatty acids. This knowledge provides a better understanding of the impact dietary fat has on the barrier function of the gut, identifies PPARα as an important factor controlling this key function, and underscores the importance of PPARα for nutrient-mediated gene regulation in intestine.

  17. Nipbl and mediator cooperatively regulate gene expression to control limb development.

    Directory of Open Access Journals (Sweden)

    Akihiko Muto

    2014-09-01

    Full Text Available Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS, the most common "cohesinopathy". It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb, knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including fgfs, shha, hand2 and multiple hox genes. In limb buds of Nipbl-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of hox-gene expression depended on genomic location within the Hox clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish hoxda cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.

  18. Triptolide T10 enhances AAV-mediated gene transfer in mice striatum.

    Science.gov (United States)

    Ren, Xinmiao; Zhang, Ting; Hu, Jing; Ding, Wei; Wang, Xiaomin

    2010-08-02

    Adeno-associated virus (AAV) mediated gene transfer has been demonstrated to be an effective approach for treating Parkinson's disease (PD). Triptolide T10 is a monomeric compound isolated from tripterygium wilfordii Hook.f. (Thunder God vine), a traditional Chinese herb for anti-inflammatory medications. In the present study, we co-administered T10 with recombinant AAV2 in SH-SY5Y human neuroblastoma cells and in the striatum of C57BL/6 mice, and then evaluated the AAV-mediated gene expression levels. The results have shown that T10 significantly augmented the expression of AAV-mediated gene in a dose-dependent fashion without detectable cytotoxicity. As growing evidence indicated that inflammation contributed to the progression of PD, and the anti-inflammatory effect of T10 was shown in our previous studies, our data of T10 to enhance AAV transduction suggest that T10 might be potentially used as a facilitating reagent for the AAV gene therapy applications in neurodegenerative diseases.

  19. Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Olszewski, Pawel K., E-mail: olsze005@umn.edu [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Minnesota Obesity Center, Saint Paul, MN 55108 (United States); Fredriksson, Robert; Eriksson, Jenny D. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Mitra, Anaya [Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Radomska, Katarzyna J. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Gosnell, Blake A. [Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Solvang, Maria N. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden); Levine, Allen S. [Minnesota Obesity Center, Saint Paul, MN 55108 (United States); Department of Food Science and Nutrition, Saint Paul, MN 55108 (United States); Schioeth, Helgi B. [Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala (Sweden)

    2011-05-13

    Highlights: {yields} The majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto. {yields} The level of colocalization is similar in the male and female brain. {yields} Fto overexpression in hypothalamic neurons increases oxytocin mRNA levels by 50%. {yields} Oxytocin does not affect Fto expression through negative feedback mechanisms. -- Abstract: Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feeding-related peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin.

  20. The role of RNA structure at 5' untranslated region in microRNA-mediated gene regulation.

    Science.gov (United States)

    Gu, Wanjun; Xu, Yuming; Xie, Xueying; Wang, Ting; Ko, Jae-Hong; Zhou, Tong

    2014-09-01

    Recent studies have suggested that the secondary structure of the 5' untranslated region (5' UTR) of messenger RNA (mRNA) is important for microRNA (miRNA)-mediated gene regulation in humans. mRNAs that are targeted by miRNA tend to have a higher degree of local secondary structure in their 5' UTR; however, the general role of the 5' UTR in miRNA-mediated gene regulation remains unknown. We systematically surveyed the secondary structure of 5' UTRs in both plant and animal species and found a universal trend of increased mRNA stability near the 5' cap in mRNAs that are regulated by miRNA in animals, but not in plants. Intra-genome comparison showed that gene expression level, GC content of the 5' UTR, number of miRNA target sites, and 5' UTR length may influence mRNA structure near the 5' cap. Our results suggest that the 5' UTR secondary structure performs multiple functions in regulating post-transcriptional processes. Although the local structure immediately upstream of the start codon is involved in translation initiation, RNA structure near the 5' cap site, rather than the structure of the full-length 5' UTR sequences, plays an important role in miRNA-mediated gene regulation.

  1. Polyethyleneimine (PEI mediated siRNA gene silencing in the Schistosoma mansoni snail host, Biomphalaria glabrata.

    Directory of Open Access Journals (Sweden)

    Matty Knight

    2011-07-01

    Full Text Available An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI mediated delivery of long double-stranded (ds and small interfering (si RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.

  2. Plant-Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer ability

    Directory of Open Access Journals (Sweden)

    Satoko eNonaka

    2014-12-01

    Full Text Available Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium.

  3. The relationship of host-mediated induced resistance to polymorphism in gene-for-gene relationships.

    Science.gov (United States)

    Tellier, Aurélien; Brown, James K M

    2008-01-01

    Gene-for-gene relationships are a common feature of plant-parasite interactions. Polymorphism at host resistance and parasite avirulence loci is maintained if there is negative, direct frequency-dependent selection on alleles of either gene. More specifically, selection of this kind is generated when the disease is polycyclic with frequent auto-infection. When an incompatible interaction occurs between a resistant host and an avirulent parasite, systemic defenses are triggered, rendering the plant more resistant to a later attack by another parasite. However, induced resistance (IR) incurs a fitness cost to the plant. Here, the effect of IR on polymorphism in gene-for-gene interactions is investigated. First, in an infinite population model in which parasites have two generations per host generation, increasing the fitness cost of IR increases selection for susceptible plants at low disease severity, while increasing the effectiveness of IR against further parasite attacks enhances selection for resistant plants at high disease severity. This reduces the possibility of polymorphism being maintained in host and parasite populations. In finite population models, the number of plants varies over time as a function of the disease burden of the population. Polymorphism in gene-for-gene relationships is then more stable at high disease prevalence and severity if IR reactions are more costly when there is competition for resources between plants.

  4. Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

    Directory of Open Access Journals (Sweden)

    Alnawaz Rehemtulla

    2002-01-01

    Full Text Available Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD along with an optical reporter gene (luciferase. Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging

  5. Retinal functional change caused by adenoviral vector-mediated transfection of LacZ gene.

    Science.gov (United States)

    Sakamoto, T; Ueno, H; Goto, Y; Oshima, Y; Yamanaka, I; Ishibashi, T; Inomata, H

    1998-04-10

    We examined the effect of insertion of an exogenous gene on retinal function to assess the rationale of adenoviral vector-mediated gene transfer for future gene therapy. An adenoviral vector expressing bacterial LacZ (AdCALacZ) was injected into the eyes of adult rats either intravitreally (group A) or subretinally (group B), and the gene expression and retinal function were thus examined at different time points after gene transfer for 3 weeks. X-Gal histostaining showed that neural retinal cells were transfected in group A and that retinal pigment epithelial cells were transfected in group B. The gene transfer was more efficient in group B (54.4% of the fixed retinal area was stained) than in group A (10.4%). The electroretinogram (ERG) revealed retinal dysfunction in the AdCALacZ-transfected rats even at the stage in which the histological damage was not apparent by electron microscopy and immunohistochemical studies for cytokeratin, S-100 protein, and glial fibrillary acidic protein. The ERG change was correlated with the intensity of inflammation, and retinal function recovered to the original level by 3 weeks, along with a diminution of inflammation. Functional changes were more evident in eyes treated with AdCALacZ than in those infected with adenoviral vector with no exogenous gene; however, no histological difference was observed between these groups, indicating that the insertion of exogenous gene itself affects retinal function. The results showed that different kinds of retinal cells could be gene-transferred by an adenoviral vector, depending on the application method. The retinal dysfunction caused by each adenoviral transfection method was caused by inflammation and the insertion of exogenous gene, and this retinal dysfunction was recoverable. In future gene therapy, special attention should be given to the method of exogenous gene insertion in the retina.

  6. Conserved transcriptional responses to cyanobacterial stressors are mediated by alternate regulation of paralogous genes in Daphnia.

    Science.gov (United States)

    Asselman, Jana; Pfrender, Michael E; Lopez, Jacqueline A; De Coninck, Dieter I M; Janssen, Colin R; Shaw, Joseph R; De Schamphelaere, Karel A C

    2015-04-01

    Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles, whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of nonlineage-specific genes were conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment-specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families. © 2015 John Wiley & Sons Ltd.

  7. The requirement of multiple defense genes in soybean Rsv1-mediated extreme resistance to soybean mosaic virus.

    Science.gov (United States)

    Zhang, Chunquan; Grosic, Sehiza; Whitham, Steven A; Hill, John H

    2012-10-01

    Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the β-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

  8. Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria

    DEFF Research Database (Denmark)

    Zhang, Qingfeng; Siegel, T Nicolai; Martins, Rafael M

    2014-01-01

    malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin......-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non......-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of Pf...

  9. Agrobacterium-mediated Transformation of Rice (Oryza sativa L.) with Atrazine Chlorohydrolase Gene (atzA)

    Institute of Scientific and Technical Information of China (English)

    WANG Song-wen; SHI Li-li; SUN Zong-xiu; CAI Bao-li; FU Ya-ping; WANG Yang; SI Hua-min; LIU Xia; ZHANG Xin

    2005-01-01

    Atrazine chlorohydrolase gene (atzA) was cloned from Arthrobacter sp. AD1. A plant expression plasmid was constructed under the control of CaMV35s promoter and was used in rice transformation. The target gene was successfully introduced into mature embryos of a japonica rice cultivar Jindao 107 by Agrobacterium- mediated transformation and hundreds of transgenic plants were obtained. The exogenous atzA gene in the transgenic plants that expressed atrazine resistance was confirmed by Southern blot hybridization. The resistance experiments by spraying transgenic rice plants with 0.133% atrazine shown that most of the transgenic rice plants exhibited the resistance to herbicide atrazine. The segregation of exogenous atzA gene in T1 progeny corresponded to the Mendelian ratio.

  10. Polyethylenimine-mediated gene delivery to the lung and therapeutic applications

    Directory of Open Access Journals (Sweden)

    Sante Di Gioia

    2008-09-01

    Full Text Available Sante Di Gioia, Massimo ConeseDepartment of Biomedical Sciences, University of Foggia, Foggia, ItalyAbstract: Nonviral gene delivery is now considered a promising alternative to viral vectors. Among nonviral gene delivery agents, polyethylenimine (PEI has emerged as a potent candidate for gene delivery to the lung. PEI has some advantages over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. However, intracellular (mainly the nuclear membrane and extracellular obstacles still hamper its efficiency in vitro and in vivo, depending on the route of administration and the type of PEI. Nuclear delivery has been increased by adding nuclear localization signals. To overcome nonspecific interactions with biological fluids, extracellular matrix components and nontarget cells, strategies have been developed to protect polyplexes from these interactions and to increase target specificity and gene expression. When gene delivery into airway epithelial cells of the conducting airways is necessary, aerosolization of complexes seems to be better suited to guarantee higher transgene expression in the airway epithelial cells with lower toxicity than observed with either intratracheal or intravenous administration. Aerosolization, indeed, is useful to target the alveolar epithelium and pulmonary endothelium. Proof-of-principle that PEI-mediated gene delivery has therapeutic application to some genetic and acquired lung disease is presented, using as genetic material either plasmidic DNA or small-interfering RNA, although optimization of formulation and delivery protocols and limitation of toxicity need further studies.Keywords: gene transfer, gene therapy, polyethylenimine, airway epithelial cells, lung, RNA interference

  11. Euglossine bees mediate only limited long-distance gene flow in a tropical vine.

    Science.gov (United States)

    Opedal, Øystein H; Falahati-Anbaran, Mohsen; Albertsen, Elena; Armbruster, W Scott; Pérez-Barrales, Rocío; Stenøien, Hans K; Pélabon, Christophe

    2017-03-01

    Euglossine bees (Apidae: Euglossini) have long been hypothesized to act as long-distance pollinators of many low-density tropical plants. We tested this hypothesis by the analysis of gene flow and genetic structure within and among populations of the euglossine bee-pollinated vine Dalechampia scandens. Using microsatellite markers, we assessed historical gene flow by the quantification of regional-scale genetic structure and isolation by distance among 18 populations, and contemporary gene flow by the estimation of recent migration rates among populations. To assess bee-mediated pollen dispersal on a smaller scale, we conducted paternity analyses within a focal population, and quantified within-population spatial genetic structure in four populations. Gene flow was limited to certain nearby populations within continuous forest blocks, whereas drift appeared to dominate on larger scales. Limited long-distance gene flow was supported by within-population patterns; gene flow was biased towards nearby plants, and significant small-scale spatial genetic structure was detected within populations. These findings suggest that, although female euglossine bees might be effective at moving pollen within populations, and perhaps within forest blocks, their contribution to gene flow on the regional scale seems too limited to counteract genetic drift in patchily distributed tropical plants. Among-population gene flow might have been reduced following habitat fragmentation.

  12. Artificial microRNA mediated gene silencing in plants: progress and perspectives.

    Science.gov (United States)

    Tiwari, Manish; Sharma, Deepika; Trivedi, Prabodh Kumar

    2014-09-01

    Homology based gene silencing has emerged as a convenient approach for repressing expression of genes in order to study their functions. For this purpose, several antisense or small interfering RNA based gene silencing techniques have been frequently employed in plant research. Artificial microRNAs (amiRNAs) mediated gene silencing represents one of such techniques which can utilize as a potential tool in functional genomics. Similar to microRNAs, amiRNAs are single-stranded, approximately 21 nt long, and designed by replacing the mature miRNA sequences of duplex within pre-miRNAs. These amiRNAs are processed via small RNA biogenesis and silencing machinery and deregulate target expression. Holding to various refinements, amiRNA technology offers several advantages over other gene silencing methods. This is a powerful and robust tool, and could be applied to unravel new insight of metabolic pathways and gene functions across the various disciplines as well as in translating observations for improving favourable traits in plants. This review highlights general background of small RNAs, improvements made in RNAi based gene silencing, implications of amiRNA in gene silencing, and describes future themes for improving value of this technology in plant science.

  13. Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles.

    Directory of Open Access Journals (Sweden)

    Yi-Hong Wang

    Full Text Available BACKGROUND: Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in altered floral structure. METHODOLOGY/PRINCIPAL FINDINGS: Previously we identified two genes (LeTGA1 and SOLly GLB1 induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected. CONCLUSION: This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.

  14. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Directory of Open Access Journals (Sweden)

    Sha Lu

    Full Text Available In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  15. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Science.gov (United States)

    Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

    2015-01-01

    In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  16. Regulatory Factor X (RFX)-mediated transcriptional rewiring of ciliary genes in animals.

    Science.gov (United States)

    Piasecki, Brian P; Burghoorn, Jan; Swoboda, Peter

    2010-07-20

    Cilia were present in the last eukaryotic common ancestor (LECA) and were retained by most organisms spanning all extant eukaryotic lineages, including organisms in the Unikonta (Amoebozoa, fungi, choanoflagellates, and animals), Archaeplastida, Excavata, Chromalveolata, and Rhizaria. In certain animals, including humans, ciliary gene regulation is mediated by Regulatory Factor X (RFX) transcription factors (TFs). RFX TFs bind X-box promoter motifs and thereby positively regulate >50 ciliary genes. Though RFX-mediated ciliary gene regulation has been studied in several bilaterian animals, little is known about the evolutionary conservation of ciliary gene regulation. Here, we explore the evolutionary relationships between RFX TFs and cilia. By sampling the genome sequences of >120 eukaryotic organisms, we show that RFX TFs are exclusively found in unikont organisms (whether ciliated or not), but are completely absent from the genome sequences of all nonunikont organisms (again, whether ciliated or not). Sampling the promoter sequences of 12 highly conserved ciliary genes from 23 diverse unikont and nonunikont organisms further revealed that phylogenetic footprints of X-box promoter motif sequences are found exclusively in ciliary genes of certain animals. Thus, there is no correlation between cilia/ciliary genes and the presence or absence of RFX TFs and X-box promoter motifs in nonanimal unikont and in nonunikont organisms. These data suggest that RFX TFs originated early in the unikont lineage, distinctly after cilia evolved. The evolutionary model that best explains these observations indicates that the transcriptional rewiring of many ciliary genes by RFX TFs occurred early in the animal lineage.

  17. Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision.

    Science.gov (United States)

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Ohtsuki, Namie; Saika, Hiroaki; Toki, Seiichi

    2015-01-01

    Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive-negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.

  18. Expression of tumor necrosis factor-alpha-mediated genes predicts recurrence-free survival in lung cancer.

    Science.gov (United States)

    Wang, Baohua; Song, Ning; Yu, Tong; Zhou, Lianya; Zhang, Helin; Duan, Lin; He, Wenshu; Zhu, Yihua; Bai, Yunfei; Zhu, Miao

    2014-01-01

    In this study, we conducted a meta-analysis on high-throughput gene expression data to identify TNF-α-mediated genes implicated in lung cancer. We first investigated the gene expression profiles of two independent TNF-α/TNFR KO murine models. The EGF receptor signaling pathway was the top pathway associated with genes mediated by TNF-α. After matching the TNF-α-mediated mouse genes to their human orthologs, we compared the expression patterns of the TNF-α-mediated genes in normal and tumor lung tissues obtained from humans. Based on the TNF-α-mediated genes that were dysregulated in lung tumors, we developed a prognostic gene signature that effectively predicted recurrence-free survival in lung cancer in two validation cohorts. Resampling tests suggested that the prognostic power of the gene signature was not by chance, and multivariate analysis suggested that this gene signature was independent of the traditional clinical factors and enhanced the identification of lung cancer patients at greater risk for recurrence.

  19. Expression of tumor necrosis factor-alpha-mediated genes predicts recurrence-free survival in lung cancer.

    Directory of Open Access Journals (Sweden)

    Baohua Wang

    Full Text Available In this study, we conducted a meta-analysis on high-throughput gene expression data to identify TNF-α-mediated genes implicated in lung cancer. We first investigated the gene expression profiles of two independent TNF-α/TNFR KO murine models. The EGF receptor signaling pathway was the top pathway associated with genes mediated by TNF-α. After matching the TNF-α-mediated mouse genes to their human orthologs, we compared the expression patterns of the TNF-α-mediated genes in normal and tumor lung tissues obtained from humans. Based on the TNF-α-mediated genes that were dysregulated in lung tumors, we developed a prognostic gene signature that effectively predicted recurrence-free survival in lung cancer in two validation cohorts. Resampling tests suggested that the prognostic power of the gene signature was not by chance, and multivariate analysis suggested that this gene signature was independent of the traditional clinical factors and enhanced the identification of lung cancer patients at greater risk for recurrence.

  20. Method of Peptide Nucleic Acid (PNA)-Mediated Antisense Inhibition of Gene Expression in Campylobacter jejuni.

    Science.gov (United States)

    Oh, Euna; Jeon, Byeonghwa

    2017-01-01

    Peptide nucleic acid (PNA) is an oligonucleotide mimic that recognizes and binds to nucleic acids. The strong binding affinity of PNA to mRNA coupled with its high sequence specificity enable antisense PNA to selectively inhibit (i.e., knockdown) the protein synthesis of a target gene. This novel technology provides a powerful tool for Campylobacter studies because molecular techniques have been relatively less well-developed for this bacterium as compared to other pathogens, such as Escherichia coli and Salmonella. This chapter describes a protocol for PNA-mediated antisense inhibition of gene expression in Campylobacter jejuni.

  1. Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations o...

  2. Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.

  3. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  4. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  5. Electroporation-mediated Delivery of Genes in Rodent Models of Lung Contusion

    OpenAIRE

    2014-01-01

    Several of the biological processes involved in the pathogenesis of acute lung injury and acute respiratory distress syndrome after lung contusion are regulated at a genetic and epigenetic level. Thus, strategies to manipulate gene expression in this context are highly desirable not only to elucidate the mechanisms involved but also to look for potential therapies. In the present chapter, we describe mouse and rat models of inducing blunt thoracic injury followed by electroporation-mediated g...

  6. Genetic analysis of transgenome structure and size of chromosome—mediated gene transfer lines

    Institute of Scientific and Technical Information of China (English)

    XUWeIMING

    1992-01-01

    The TK-selected chromosome-mediate gene transfer lines were analysed using DNA dot blot method G-11 banding and in situ hybridization.The results showed that CMGT can provide a wide variety of intermediate size of the transgenome from greater than 80,000kb to less than 2,000kb,Some of transfectants are intergrated into mouse chromosome which can be detected by G-11 banding and in situ hybridization.

  7. Regulation of BDNF-mediated transcription of immediate early gene Arc by intracellular calcium and calmodulin

    OpenAIRE

    Zheng, Fei; Luo, Yongneng; Wang, Hongbing

    2009-01-01

    The induction of the immediate early gene Arc is strongly implicated in synaptic plasticity. Although the role of ERK was demonstrated, the regulation of Arc expression is largely unknown. In this study, we investigated the major signaling pathways underlying brain-derived neurotrophic factor (BDNF)-mediated Arc transcription in cultured cortical neurons. The BDNF-stimulated Arc transcription was solely regulated by the Ras-Raf-MAPK signaling through ERK, but not by phosphoinositide 3-kinase ...

  8. CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation.

    Directory of Open Access Journals (Sweden)

    Licheng Ren

    Full Text Available Chromatin loops play important roles in the dynamic spatial organization of genes in the nucleus. Growing evidence has revealed that the multivalent functional zinc finger protein CCCTC-binding factor (CTCF is a master regulator of genome spatial organization, and mediates the ubiquitous chromatin loops within the genome. Using circular chromosome conformation capture (4C methodology, we discovered that CTCF may be a master organizer in mediating the spatial organization of the kcnq5 gene locus. We characterized the cell-type specific spatial organization of the kcnq5 gene locus mediated by CTCF in detail using chromosome conformation capture (3C and 3C-derived techniques. Cohesion also participated in mediating the organization of this locus. RNAi-mediated knockdown of CTCF sharply diminished the interaction frequencies between the chromatin loops of the kcnq5 gene locus and down-regulated local gene expression. Functional analysis showed that the interacting chromatin loops of the kcnq5 gene locus can repress the gene expression in a luciferase reporter assay. These interacting chromatin fragments were a series of repressing elements whose contacts were mediated by CTCF. Therefore, these findings suggested that the dynamical spatial organization of the kcnq5 locus regulates local gene expression.

  9. Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

    Science.gov (United States)

    Peeters, Hilde; Voz, Marianne L; Verschueren, Kristin; De Cat, Bart; Pendeville, Hélène; Thienpont, Bernard; Schellens, Ann; Belmont, John W; David, Guido; Van De Ven, Wim J M; Fryns, Jean-Pierre; Gewillig, Marc; Huylebroeck, Danny; Peers, Bernard; Devriendt, Koen

    2006-11-15

    Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

  10. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  11. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  12. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    Science.gov (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  13. RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%~ 72.5%) than in supernatant system (33.1%~ 46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

  14. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

    Directory of Open Access Journals (Sweden)

    Marxa Figueiredo

    2012-01-01

    Full Text Available This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid (PLGA or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound composed either of polymers (PLGA, polystyrene or other contrast agent materials (Optison, SonoVue microbubbles. The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

  15. rAAV vector-mediated gene therapy for experimental ischemic stroke

    Directory of Open Access Journals (Sweden)

    Li Zhao-Jian

    2008-01-01

    Full Text Available The safest viral vector system for gene therapy is based on recombinant adeno-associated virus (rAAV up to date in Phase I clinical trials, which has been developed rapidly and applied for ischemic stroke gene therapy in animal experiments since the past seven years. rAAV vector has made great progress in improving gene delivery by modification of the capsid and increasing transgene expression by encapsidation of double-stranded rAAV genome. And in all, nine therapeutic genes in 12 animal studies were successfully delivered using rAAV vector to ischemic brain via different approaches in rat or mice stroke models for gene therapy and the results suggested that rAAV could mediate genes′ expression efficiently; most of them displayed evidently therapeutic efficacy with satisfactory biological safety. Gene therapy involving rAAV vector seems effective in attenuation of ischemic damage in stroke and has greatly promising potential use for patients in the future. In this review, we will focus on the basic biology and development of rAAV vector itself as well as the recent progress in the use of this vector for ischemic stroke gene therapy in animal experiments.

  16. Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes.

    Science.gov (United States)

    Wong, Piu; Hattangadi, Shilpa M; Cheng, Albert W; Frampton, Garrett M; Young, Richard A; Lodish, Harvey F

    2011-10-20

    It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA polymerase II (Pol II) occupancy, and multiple posttranslational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac, and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels, in particular, H3K79me2 and H4K16ac, were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, whereas gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data map the epigenetic landscape of terminal erythropoiesis and suggest that control of transcription elongation regulates gene expression during terminal erythroid differentiation.

  17. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  18. CRISPR-mediated direct mutation of cancer genes in the mouse liver

    Science.gov (United States)

    Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S.; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G.; Zhang, Feng; Anderson, Daniel G.; Sharp, Phillip A.; Jacks, Tyler

    2014-01-01

    The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells1. Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs)2–4 to the liver and directly target the tumor suppressor genes Pten5 and p536, alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology7, 8. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics. PMID:25119044

  19. RNA-mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus.

    Science.gov (United States)

    Leng, Yueqiang; Wu, Chengxiang; Liu, Zhaohui; Friesen, Timothy L; Rasmussen, Jack B; Zhong, Shaobin

    2011-04-01

    A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.

  20. CRISPR-mediated direct mutation of cancer genes in the mouse liver.

    Science.gov (United States)

    Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G; Zhang, Feng; Anderson, Daniel G; Sharp, Phillip A; Jacks, Tyler

    2014-10-16

    The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

  1. The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

    Directory of Open Access Journals (Sweden)

    Bradley L. Bearson

    2014-02-01

    Full Text Available Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the U.S. during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness

  2. 3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wang, Luwen; Jiang, Ning; Wang, Lin; Fang, Ou; Leach, Lindsey J; Hu, Xiaohua; Luo, Zewei

    2014-01-01

    Paired sense and antisense (S/AS) genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs) in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans) and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the transcriptional collision

  3. 3' Untranslated regions mediate transcriptional interference between convergent genes both locally and ectopically in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Luwen Wang

    2014-01-01

    Full Text Available Paired sense and antisense (S/AS genes located in cis represent a structural feature common to the genomes of both prokaryotes and eukaryotes, and produce partially complementary transcripts. We used published genome and transcriptome sequence data and found that over 20% of genes (645 pairs in the budding yeast Saccharomyces cerevisiae genome are arranged in convergent pairs with overlapping 3'-UTRs. Using published microarray transcriptome data from the standard laboratory strain of S. cerevisiae, our analysis revealed that expression levels of convergent pairs are significantly negatively correlated across a broad range of environments. This implies an important role for convergent genes in the regulation of gene expression, which may compensate for the absence of RNA-dependent mechanisms such as micro RNAs in budding yeast. We selected four representative convergent gene pairs and used expression assays in wild type yeast and its genetically modified strains to explore the underlying patterns of gene expression. Results showed that convergent genes are reciprocally regulated in yeast populations and in single cells, whereby an increase in expression of one gene produces a decrease in the expression of the other, and vice-versa. Time course analysis of the cell cycle illustrated the functional significance of this relationship for the three pairs with relevant functional roles. Furthermore, a series of genetic modifications revealed that the 3'-UTR sequence plays an essential causal role in mediating transcriptional interference, which requires neither the sequence of the open reading frame nor the translation of fully functional proteins. More importantly, transcriptional interference persisted even when one of the convergent genes was expressed ectopically (in trans and therefore does not depend on the cis arrangement of convergent genes; we conclude that the mechanism of transcriptional interference cannot be explained by the

  4. Gene expression profiling of aging reveals activation of a p53-mediated transcriptional program

    Directory of Open Access Journals (Sweden)

    Weindruch Richard

    2007-03-01

    Full Text Available Abstract Background Aging has been associated with widespread changes at the gene expression level in multiple mammalian tissues. We have used high density oligonucleotide arrays and novel statistical methods to identify specific transcriptional classes that may uncover biological processes that play a central role in mammalian aging. Results We identified 712 transcripts that are differentially expressed in young (5 month old and old (25-month old mouse skeletal muscle. Caloric restriction (CR completely or partially reversed 87% of the changes in expression. Examination of individual genes revealed a transcriptional profile indicative of increased p53 activity in the older muscle. To determine whether the increase in p53 activity is associated with transcriptional activation of apoptotic targets, we performed RT-PCR on four well known mediators of p53-induced apoptosis: puma, noxa, tnfrsf10b and bok. Expression levels for these proapoptotic genes increased significantly with age (P +/- and GPX4+/- mice, suggesting that oxidative stress does not induce the expression of these genes. Western blot analysis confirmed that protein levels for both p21 and GADD45a, two established transcriptional targets of p53, were higher in the older muscle tissue. Conclusion These observations support a role for p53-mediated transcriptional program in mammalian aging and suggest that mechanisms other than reactive oxygen species are involved in the age-related transcriptional activation of p53 targets.

  5. STAT4-mediated transcriptional repression of the IL5 gene in human memory Th2 cells.

    Science.gov (United States)

    Gonzales-van Horn, Sarah R; Estrada, Leonardo D; van Oers, Nicolai S C; Farrar, J David

    2016-06-01

    Type I interferon (IFN-α/β) plays a critical role in suppressing viral replication by driving the transcription of hundreds of interferon-sensitive genes (ISGs). While many ISGs are transcriptionally activated by the ISGF3 complex, the significance of other signaling intermediates in IFN-α/β-mediated gene regulation remains elusive, particularly in rare cases of gene silencing. In human Th2 cells, IFN-α/β signaling suppressed IL5 and IL13 mRNA expression during recall responses to T-cell receptor (TCR) activation. This suppression occurred through a rapid reduction in the rate of nascent transcription, independent of de novo expression of ISGs. Further, IFN-α/β-mediated STAT4 activation was required for repressing the human IL5 gene, and disrupting STAT4 dimerization reversed this effect. This is the first demonstration of STAT4 acting as a transcriptional repressor in response to IFN-α/β signaling and highlights the unique activity of this cytokine to acutely block the expression of an inflammatory cytokine in human T cells.

  6. PLGA-Chitosan nanoparticle-mediated gene delivery for oral cancer treatment: A brief review

    Science.gov (United States)

    Bakar, L. M.; Abdullah, M. Z.; Doolaanea, A. A.; Ichwan, S. J. A.

    2017-08-01

    Cancer becomes a serious issue on society with increasing of their growth and proliferation, either in well economic developed countries or not. Recent years, oral cancer is one of the most threatening diseases impairing the quality of life of the patient. Scientists have emphasised on application of gene therapy for oral cancer by using nanoparticle as transportation vectors as a new alternative platform in order to overcome the limitations of conventional approaches. In modern medicine, nanotechnologies’ application, such as nanoparticles-mediated gene delivery, is one of promising tool for therapeutic devices. The objective of this article is to present a brief review summarizes on the current progress of nanotechnology-based gene delivery treatment system targeted for oral cancer.

  7. Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.

    Science.gov (United States)

    Shen, Shensi; Rodrigo, Guillermo; Prakash, Satya; Majer, Eszter; Landrain, Thomas E; Kirov, Boris; Daròs, José-Antonio; Jaramillo, Alfonso

    2015-05-26

    Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.

  8. Growth Inhibition of Breast Cancer in Rat by AAV Mediated Angiostatin Gene

    Institute of Scientific and Technical Information of China (English)

    LI Ran; CHEN Hong; REN Chang-shan

    2007-01-01

    Objective: To observe growth inhibition effect of adeno-associated viral vectors (AAV) mediated angiostatin (ANG) gene on implanted breast cancer in rat and its mechanism. Methods: Gene transfer technique was used to transfer AAV-ANG to the tumor. Growth curves were drawn to observe the growth of breast cancer implanted in rat, and immunohistochemical method was used to detect the effects of angiostatin on microvesel density (MVD) of breast cancer implanted in rat. Results: Angiostatin inhibited the growth of breast cancer implanted in rat and decreased the microvessel density of tumor. Conclusion: Expression of an angiostatin transgene can suppress the growth of breast cancer implanted in rat through the inhibition of the growth of microvessels, surggesting that angiostatin gene transfer technique may be effective against breast cancer.

  9. Fluoroquinolone induction of phage-mediated gene transfer in multidrug-resistant Salmonella.

    Science.gov (United States)

    Bearson, Bradley L; Brunelle, Brian W

    2015-08-01

    Fluoroquinolones are broad-spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity, which can cause DNA damage and result in bacterial cell death. In response to DNA damage, bacteria induce an SOS response to stimulate DNA repair. However, the SOS response may also induce prophage with production of infectious virions. Salmonella strains typically contain multiple prophages, and certain strains including phage types DT120 and DT104 contain prophage that upon induction are capable of generalised transduction. In this study, strains of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium DT120 and DT104 were exposed to fluoroquinolones important for use in human and veterinary disease therapy to determine whether prophage(s) are induced that could facilitate phage-mediated gene transfer. Cultures of MDR S. Typhimurium DT120 and DT104 containing a kanamycin resistance plasmid were lysed after exposure to fluoroquinolones (ciprofloxacin, enrofloxacin and danofloxacin). Bacterial cell lysates were able to transfer the plasmid to a recipient kanamycin-susceptible Salmonella strain by generalised transduction. In addition, exposure of DT120 to ciprofloxacin induced the recA gene of the bacterial SOS response and genes encoded in a P22-like generalised transducing prophage. This research indicates that fluoroquinolone exposure of MDR Salmonella can facilitate horizontal gene transfer, suggesting that fluoroquinolone usage in human and veterinary medicine may have unintended consequences, including the induction of phage-mediated gene transfer from MDR Salmonella. Stimulation of gene transfer following bacterial exposure to fluoroquinolones should be considered an adverse effect, and clinical decisions regarding antibiotic selection for infectious disease therapy should include this potential risk. Published by Elsevier B.V.

  10. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer

    Institute of Scientific and Technical Information of China (English)

    TomohikoICHIKAWA; ShigeruHOSOKI; HiroyoshiSUZUKI; KoichiroAKAKURA; TatsuoIGARASHI; YuzoFURUYA; MitsuoOSHIMURA; CarrieW.RINKER-SCHAEFFER; NaokiNIHEI; JohnT.ISAACS; HaruoITO

    2000-01-01

    Aim: To identify the metastasis suppressor genes for prostate cancer. Methods: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene (s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. Results: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of ttmaorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal ann deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. Conclusion: This assay system is useful to identify metastasis suppressor gene (s) for prostate cancer.

  11. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

  12. Avidin-biotin interaction mediated peptide assemblies as efficient gene delivery vectors for cancer therapy.

    Science.gov (United States)

    Qu, Wei; Chen, Wei-Hai; Kuang, Ying; Zeng, Xuan; Cheng, Si-Xue; Zhou, Xiang; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-01-01

    Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin-biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel self-assembled vector TAC developed is a promising gene vector for cancer therapy.

  13. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    B. Radhika

    2016-01-01

    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  14. Pollen-Mediated Movement of Herbicide Resistance Genes in Lolium rigidum.

    Science.gov (United States)

    Loureiro, Iñigo; Escorial, María-Concepción; Chueca, María-Cristina

    2016-01-01

    The transfer of herbicide resistance genes by pollen is a major concern in cross-pollinated species such as annual ryegrass (Lolium rigidum). A two-year study was conducted in the greenhouse, under favorable conditions for pollination, to generate information on potential maximum cross-pollination. This maximum cross-pollination rate was 56.1%. A three-year field trial was also conducted to study the cross-pollination rates in terms of distance and orientation to an herbicide-resistant pollen source. Under field conditions, cross-pollination rates varied from 5.5% to 11.6% in plants adjacent to the pollen source and decreased with increasing distances (1.5 to 8.9% at 15 m distance and up to 4.1% at 25 m in the downwind direction). Environmental conditions influenced the cross-pollination both under greenhouse and field conditions. Data were fit to an exponential decay model to predict gene flow at increasing distances. This model predicted an average gene flow of 7.1% when the pollen donor and recipient plants were at 0 m distance from each other. Pollen-mediated gene flow declined by 50% at 16.7 m from the pollen source, yet under downwind conditions gene flow of 5.2% was predicted at 25 m, the farthest distance studied. Knowledge of cross-pollination rates will be useful for assessing the spread of herbicide resistance genes in L. rigidum and in developing appropriate strategies for its mitigation.

  15. Mutant acetolactate synthase (ALS) gene as a selectable marker for Agrobacterium-mediated transformation of soybean

    Institute of Scientific and Technical Information of China (English)

    Chen Shiyun; Zhang Yong

    2006-01-01

    Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean transformation systems with different selectable marker genes have been reported, e.g. antibiotic (kanamycin or hygromycin) resistant genes and herbicide ( glufosinate, glyphosate) resistant selectable marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase (ALS) gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide selection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant.PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.

  16. Genome-Wide Overexpression Screen Identifies Genes Able to Bypass p16-Mediated Senescence in Melanoma.

    Science.gov (United States)

    Lee, Won Jae; Škalamera, Dubravka; Dahmer-Heath, Mareike; Shakhbazov, Konstanin; Ranall, Max V; Fox, Carly; Lambie, Duncan; Stevenson, Alexander J; Yaswen, Paul; Gonda, Thomas J; Gabrielli, Brian

    2017-03-01

    Malignant melanomas often arise from nevi, which result from initial oncogene-induced hyperproliferation of melanocytes that are maintained in a CDKN2A/p16-mediated senescent state. Thus, genes that can bypass this senescence barrier are likely to contribute to melanoma development. We have performed a gain-of-function screen of 17,030 lentivirally expressed human open reading frames (ORFs) in a melanoma cell line containing an inducible p16 construct to identify such genes. Genes known to bypass p16-induced senescence arrest, including the human papilloma virus 18 E7 gene ( HPV18E7), and genes such as the p16-binding CDK6 with expected functions, as well as panel of novel genes, were identified, including high-mobility group box (HMGB) proteins. A number of these were further validated in two other models of p16-induced senescence. Tissue immunohistochemistry demonstrated higher levels of CDK6 in primary melanomas compared with normal skin and nevi. Reduction of CDK6 levels drove melanoma cells expressing functional p16 into senescence, demonstrating its contribution to bypass senescence.

  17. ADA1 and NET1 Genes of Yeast Mediate Both Chromosome Maintenance and Mitochondrial $\\rho^{-}$ Mutagenesis

    CERN Document Server

    Koltovaya, N A; Tchekhouta, I A; Devin, A B

    2002-01-01

    An increase in the mitochondrial (mt) rho^- mutagenesis is a well-known respose of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho^- mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho^- mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well as on ce...

  18. Ocular gene therapy: an evaluation of recombinant adeno-associated virus-mediated gene therapy interventions for the treatment of ocular disease.

    Science.gov (United States)

    Roy, Kamolika; Stein, Linda; Kaushal, Shalesh

    2010-08-01

    Both gene replacement therapy and alteration of host gene expression are playing increasingly important roles in the treatment of ocular diseases. Ocular gene therapy may provide alternatives to current treatments for eye diseases that are either greatly invasive and thus run the risk of complications, that offer only short-term relief from disease symptoms, or that are unable to directly treat vision loss. The success of three separate phase I clinical trials investigating a gene therapy intervention for the treatment of the retinal degenerative disorder Leber's congenital amaurosis (LCA) has unveiled the therapeutic potential of gene therapy. Preliminary results have demonstrated ocular gene transfer, using nonpathogenic recombinant adeno-associated viral (rAAV) vectors specifically, to be a safe, effective, and long-term treatment for LCA, a previously untreatable disorder. Nonpathogenic rAAV vectors offer the potential for long-term treatment. Many of the genes implicated in human ocular diseases have been identified, and animal models for such diseases have been developed, which have greatly facilitated the application of experimental rAAV-mediated gene therapy. This review highlights the key features of rAAV-mediated gene therapy that make it the most suitable gene therapy treatment approach for ocular diseases. Furthermore, it summarizes the current progress of rAAV-mediated gene therapy interventions/applications for a wide variety of ophthalmologic disorders.

  19. AAV2-mediated in vivo immune gene therapy of solid tumours

    LENUS (Irish Health Repository)

    Collins, Sara A

    2010-12-20

    Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb\\/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour

  20. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  1. The mechanism underlying Ler-mediated alleviation of gene repression by H-NS.

    Science.gov (United States)

    Shin, Minsang

    2017-01-29

    Secretion of effector proteins in Enteropathogeneic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5 promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on LEE5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of H-NS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on LEE5 promoter resulting in a derepression. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement.

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes' encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired "safe" harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant

  3. Probing the Limits to MicroRNA-Mediated Control of Gene Expression.

    Directory of Open Access Journals (Sweden)

    Araks Martirosyan

    2016-01-01

    Full Text Available According to the 'ceRNA hypothesis', microRNAs (miRNAs may act as mediators of an effective positive interaction between long coding or non-coding RNA molecules, carrying significant potential implications for a variety of biological processes. Here, inspired by recent work providing a quantitative description of small regulatory elements as information-conveying channels, we characterize the effectiveness of miRNA-mediated regulation in terms of the optimal information flow achievable between modulator (transcription factors and target nodes (long RNAs. Our findings show that, while a sufficiently large degree of target derepression is needed to activate miRNA-mediated transmission, (a in case of differential mechanisms of complex processing and/or transcriptional capabilities, regulation by a post-transcriptional miRNA-channel can outperform that achieved through direct transcriptional control; moreover, (b in the presence of large populations of weakly interacting miRNA molecules the extra noise coming from titration disappears, allowing the miRNA-channel to process information as effectively as the direct channel. These observations establish the limits of miRNA-mediated post-transcriptional cross-talk and suggest that, besides providing a degree of noise buffering, this type of control may be effectively employed in cells both as a failsafe mechanism and as a preferential fine tuner of gene expression, pointing to the specific situations in which each of these functionalities is maximized.

  4. Methylation mediated silencing of TMS1/ASC gene in prostate cancer

    Directory of Open Access Journals (Sweden)

    Gopisetty Gopal

    2006-07-01

    Full Text Available Abstract Background Transcriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing, also known as ASC (Apoptosis Speck like protein containing a CARD is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation. Results Loss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP assay showed enrichment of MBD3 (methyl binding domain protein 3 to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002 while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91. Conclusion Our study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.

  5. Silver nanoparticles mediated altered gene expression of melanin biosynthesis genes in Bipolaris sorokiniana.

    Science.gov (United States)

    Mishra, Sandhya; Singh, H B

    2015-03-01

    Melanin production in many fungal phytopathogens has been investigated to play direct or indirect role in pathogenesis. However, in Bipolaris sorokiniana, the spot blotch pathogen of wheat, much less is known about the role melanin play in pathogenesis. As an extension of our previous report, the present study aims to investigate the plausible association between melanin production and virulence factor in B. sorokiniana. In the previous study, we carried out analysis on the antifungal efficacy of biosynthesized silver nanoparticles (AgNPs) against B. sorokiniana. The present investigation revealed the gene expression analysis of melanin biosynthesis genes viz. polyketide synthase (PKS1) and scytalone dehydratase (SCD1) under the influence of AgNPs. The 0.05mg/ml concentration of AgNPs yielded noticeable inhibition of B. sorokiniana growth, while 0.1mg/ml concentration of AgNPs accounted for complete inhibition of pathogen growth. In addition, the semiquantitative RT-PCR analysis exhibited reduced expression of PKS1 and SCD1 under the influence of AgNPs treatment. Furthermore, the qRT-PCR demonstrated 6.47 and 1.808 fold significant decrease in the expression pattern of PKS1 and SCD1, respectively, in B. sorokiniana treated with AgNPs. The present study provides probable understanding of molecular events underlying the antifungal role of AgNPs against B. sorokiniana.

  6. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons.

    Directory of Open Access Journals (Sweden)

    Christoph Straub

    Full Text Available The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.

  7. Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

    Science.gov (United States)

    Sawamura, N; Ando, T; Maruyama, Y; Fujimuro, M; Mochizuki, H; Honjo, K; Shimoda, M; Toda, H; Sawamura-Yamamoto, T; Makuch, L A; Hayashi, A; Ishizuka, K; Cascella, N G; Kamiya, A; Ishida, N; Tomoda, T; Hai, T; Furukubo-Tokunaga, K; Sawa, A

    2008-12-01

    Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

  8. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    Science.gov (United States)

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  9. Epigenetic silencing of the XAF1 gene is mediated by the loss of CTCF binding

    Science.gov (United States)

    Victoria-Acosta, Georgina; Vazquez-Santillan, Karla; Jimenez-Hernandez, Luis; Muñoz-Galindo, Laura; Maldonado, Vilma; Martinez-Ruiz, Gustavo Ulises; Melendez-Zajgla, Jorge

    2015-01-01

    XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding. PMID:26443201

  10. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    Science.gov (United States)

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  11. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available BACKGROUND: Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. METHODOLOGY/PRINCIPAL FINDINGS: RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. CONCLUSIONS: SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  12. Molecular Network Analysis Suggests Aberrant CREB-Mediated Gene Regulation in the Alzheimer Disease Hippocampus

    Directory of Open Access Journals (Sweden)

    Jun-ichi Satoh

    2009-01-01

    Full Text Available The pathogenesis of Alzheimer disease (AD involves the complex interaction between genetic and environmental factors affecting multiple cellular pathways. Recent advances in systems biology provide a system-level understanding of AD by elucidating the genome-wide molecular interactions. By using KeyMolnet, a bioinformatics tool for analyzing molecular interactions on the curated knowledgebase, we characterized molecular network of 2,883 all stages of AD-related genes (ADGs and 559 incipient AD-related genes (IADGs identified by global gene expression profiling of the hippocampal CA1 region of AD brains in terms of significant clinical and pathological correlations (Blalock et al., Proc Natl Acad Sci USA 101: 2173-2178, 2004. By the common upstream search, KeyMolnet identified cAMP-response element-binding protein (CREB as the principal transcription factor exhibiting the most significant relevance to molecular networks of both ADGs and IADGs. The CREB-regulated transcriptional network included upregulated and downregulated sets of ADGs and IADGs, suggesting an involvement of generalized deregulation of the CREB signaling pathway in the pathophysiology of AD, beginning at the early stage of the disease. To verify the in silico observations in vivo, we conducted immunohistochemical studies of 11 AD and 13 age-matched control brains by using anti-phoshorylated CREB (pCREB antibody. An abnormal accumulation of pCREB imunoreactivity was identified in granules of granulovacuolar degeneration (GVD in the hippocampal neurons of AD brains. These observations suggest that aberrant CREB-mediated gene regulation serves as a molecular biomarker of AD-related pathological processes, and support the hypothesis that sequestration of pCREB in GVD granules is in part responsible for deregulation of CREB-mediated gene expression in AD hippocampus.

  13. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  14. Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection.

    Science.gov (United States)

    Ogawa, Ryohei; Kondo, Takashi; Honda, Hidemi; Zhao, Qing Li; Fukuda, Shigekazu; Riesz, Peter

    2002-09-01

    The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated. HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene. Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium. When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively. When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases. These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases.

  15. Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

    Directory of Open Access Journals (Sweden)

    Michelle R. Campbell

    2013-01-01

    Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

  16. Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

    Science.gov (United States)

    Bertoni, Carmen; Rustagi, Arjun; Rando, Thomas A.

    2009-01-01

    Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested. PMID:19854937

  17. Understanding the role of ETS-mediated gene regulation in complex biological processes.

    Science.gov (United States)

    Findlay, Victoria J; LaRue, Amanda C; Turner, David P; Watson, Patricia M; Watson, Dennis K

    2013-01-01

    Ets factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, which when perturbed contribute to tumor progression. The well-documented alterations in ETS factor expression and function during cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. The anti- and prometastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such an understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection, as well as better diagnosis and staging of disease; (2) detection of minimal residual disease recurrences and evaluation of response to therapy; (3) prevention; and (4) novel treatment strategies. Increased understanding of ETS-regulated biological pathways will directly impact these areas. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Conservation of the Nrf2-Mediated Gene Regulation of Proteasome Subunits and Glucose Metabolism in Zebrafish

    Directory of Open Access Journals (Sweden)

    Vu Thanh Nguyen

    2016-01-01

    Full Text Available The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Besides the exogenous stress response, Nrf2 has been found to regulate numerous cellular functions, including protein turnover and glucose metabolism; however, the evolutionary origins of these functions remain unknown. In the present study, we searched for novel target genes associated with the zebrafish Nrf2 to answer this question. A microarray analysis of zebrafish embryos that overexpressed Nrf2 revealed that 115 candidate genes were targets of Nrf2, including genes encoding proteasome subunits and enzymes involved in glucose metabolism. A real-time quantitative PCR suggested that the expression of 3 proteasome subunits (psma3, psma5, and psmb7 and 2 enzymes involved in glucose metabolism (pgd and fbp1a were regulated by zebrafish Nrf2. We thus next examined the upregulation of these genes by an Nrf2 activator, diethyl maleate, using Nrf2 mutant zebrafish larvae. The results of real-time quantitative PCR and whole-mount in situ hybridization showed that all of these 5 genes were upregulated by diethyl maleate treatment in an Nrf2-dependent manner, especially in the liver. These findings implied that the Nrf2-mediated regulation of the proteasome subunits and glucose metabolism is evolutionarily conserved among vertebrates.

  19. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  20. Mechanisms of HO-1 mediated attenuation of renal immune injury: a gene profiling study.

    Science.gov (United States)

    Duann, Pu; Lianos, Elias A

    2011-10-01

    Using a mouse model of immune injury directed against the renal glomerular vasculature and resembling human forms of glomerulonephritis (GN), we assessed the effect of targeted expression of the cytoprotective enzyme heme oxygenase (HO)-1. A human (h) HO-1 complementary DNAN (cDNA) sequence was targeted to glomerular epithelial cells (GECs) using a GEC-specific murine nephrin promoter. Injury by administration of antibody against the glomerular basement membrane (anti-GBM) to transgenic (TG) mice with GEC-targeted hHO-1 was attenuated compared with wild-type (WT) controls. To explore changes in the expression of genes that could mediate this salutary effect, we performed gene expression profiling using a microarray analysis of RNA isolated from the renal cortex of WT or TG mice with or without anti-GBM antibody-induced injury. Significant increases in expression were detected in 9 major histocompatibility complex (MHC)-class II genes, 2 interferon-γ (IFN-γ)-inducible guanosine triphosphate (GTP)ases, and 3 genes of the ubiquitin-proteasome system. The increase in MHC-class II and proteasome gene expression in TG mice with injury was validated by real-time polymerase chain reaction (PCR) or Western blot analysis. The observations point to novel mechanisms underlying the cytoprotective effect of HO-1 in renal immune injury. Copyright © 2011. Published by Mosby, Inc.

  1. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    Directory of Open Access Journals (Sweden)

    Amanda Donnelly

    2015-12-01

    Full Text Available The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i provides enhanced phage-mediated gene transfer; (ii is applicable for laboratory transfection processes and (iii shows promise within industry for large-scale gene transfer applications.

  2. siRNA mediated gene silencing in Fusarium sp. HKF15 for overproduction of bikaverin.

    Science.gov (United States)

    Deshmukh, Radhika; Purohit, Hemant J

    2014-04-01

    Fusarium sp. HKF15 is an isolate from effluent treatment plant which produces bikaverin. Bikaverin is a polyketide having antitumor and antibiotic potential. Acetyl coenzyme A is a common precursor for bikaverin as well as carotenoids and gibberellins. A polyketide synthase gene bik1 is responsible for bikaverin production whereas, hydroxymethyl glutaryl coenzyme A reductase (hmgR) and farnesyl pyrophosphate synthase (fpps) are carotenoid and gibberellin pathway genes. Aim of this study was assessing siRNA mediated gene silencing for bikaverin overproduction with down-regulation of carotenoid and gibberellin pathway. HKF15 protoplasts derived from glucose grown culture were treated with 200pmolml(-1)hmgR and fpps siRNAs separately. Along with down-regulation of target genes, there was 2.4-fold increase in bik1 gene expression. The silencing was effective till 48h with a 41% increase in bikaverin production. The study proposes a strategy for manipulation of physiology towards desired secondary metabolite overproduction.

  3. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in

  4. Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement

    Directory of Open Access Journals (Sweden)

    Yongwei Sun

    2016-12-01

    Full Text Available Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks (DSBs by sequence-specific nucleases (SSNs to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ or homology-directed repair (HDR. While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired ‘safe’ harbor in a predefined manner. The emergence of three programmable SSNs such as zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated protein 9 (Cas9 systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential

  5. Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereus ATCC 14579

    Directory of Open Access Journals (Sweden)

    Buist Girbe

    2008-04-01

    Full Text Available Abstract Background The catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain. Results Comparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe and hemolytic enterotoxin (Hbl was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent. Conclusion The catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control

  6. Parentage versus two-generation analyses for estimating pollen-mediated gene flow in plant populations.

    Science.gov (United States)

    Burczyk, Jaroslaw; Koralewski, Tomasz E

    2005-07-01

    Assessment of contemporary pollen-mediated gene flow in plants is important for various aspects of plant population biology, genetic conservation and breeding. Here, through simulations we compare the two alternative approaches for measuring pollen-mediated gene flow: (i) the NEIGHBORHOOD model--a representative of parentage analyses, and (ii) the recently developed TWOGENER analysis of pollen pool structure. We investigate their properties in estimating the effective number of pollen parents (N(ep)) and the mean pollen dispersal distance (delta). We demonstrate that both methods provide very congruent estimates of N(ep) and delta, when the methods' assumptions considering the shape of pollen dispersal curve and the mating system follow those used in data simulations, although the NEIGHBORHOOD model exhibits generally lower variances of the estimates. The violations of the assumptions, especially increased selfing or long-distance pollen dispersal, affect the two methods to a different degree; however, they are still capable to provide comparable estimates of N(ep). The NEIGHBORHOOD model inherently allows to estimate both self-fertilization and outcrossing due to the long-distance pollen dispersal; however, the TWOGENER method is particularly sensitive to inflated selfing levels, which in turn may confound and suppress the effects of distant pollen movement. As a solution we demonstrate that in case of TWOGENER it is possible to extract the fraction of intraclass correlation that results from outcrossing only, which seems to be very relevant for measuring pollen-mediated gene flow. The two approaches differ in estimation precision and experimental efforts but they seem to be complementary depending on the main research focus and type of a population studied.

  7. Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yuan; ZHAI Suo-qiang; SONG Wei; GUO Wei; ZHENG Gui-liang; HU Yin-yan

    2008-01-01

    Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possibl utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT-PCR analysis. The Hathl gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differenti-ate into hair cell-like cells when forced to express Hathl. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

  8. Adenovirus-mediated Gene Transfer of MMP-2 into Cultured Porcine Trabecular Meshwork Cells

    OpenAIRE

    2012-01-01

    This study aimed to use adenoviral gene transfer to express matrix metalloproteinase (MMP)-2 in cultured porcine trabecular meshwork cells and to evaluate the duration of adenovirus-mediated MMP-2 expression and its enzymatic activity. MMP-2 cDNA was synthesized by ligating three segments of MMP-2 cDNA obtained by reverse transcription-polymerase chain reaction (RT-PCR) with mRNA extracted from mouse lungs. MMP-2 cDNA was inserted into replication-deficient adenoviral vectors. Western blottin...

  9. Gene Targets in Prostate Tumor Cells that Mediate Aberrant Growth and Invasiveness

    Science.gov (United States)

    2005-02-01

    named EIAF ( Higashino et al. 1993). Finally, PEA3/EI AF was found to be the fourth Ets factor involved in chromosomal fusions with EWS (Kaneko et al. 1996...in transcriptional repression mediated by the t(8;21), t(16;21), t(12;21), and inv(16) fusion proteins. Curr Opin Hematol 8:197-200 Higashino F...Yoshida K, Handa M, Toyoda Y, Nishihira H, Tanaka Y, Sasaki Y, Ishida S, Higashino F, Fuj inaga K (1996) Fusion of an ETS-family gene, EIAF, to EWS by t

  10. Agrobacterium tumefaciens-mediated GUS gene transfer to Sophora japonica L.

    Institute of Scientific and Technical Information of China (English)

    Zhang Xiao-ying; Wang Hua-fang; Yin Wei-lun; Zhu Zhen

    2006-01-01

    Agrobacterium-mediated genetic transformation of Sophorajaponica was standardized using the Agrobacterium tumefaciens strain LBA4404 that harbored the binary vector pBI121 containing genes for β-glucuronidase (GUS) and neomycin phosphotransterase (npt Ⅱ). S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets from the explants. The transformed plants resembled their parents in morphology.

  11. Homeotic Gene teashirt (tsh has a neuroprotective function in amyloid-beta 42 mediated neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Michael T Moran

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a debilitating age related progressive neurodegenerative disorder characterized by the loss of cognition, and eventual death of the affected individual. One of the major causes of AD is the accumulation of Amyloid-beta 42 (Aβ42 polypeptides formed by the improper cleavage of amyloid precursor protein (APP in the brain. These plaques disrupt normal cellular processes through oxidative stress and aberrant signaling resulting in the loss of synaptic activity and death of the neurons. However, the detailed genetic mechanism(s responsible for this neurodegeneration still remain elusive. METHODOLOGY/ PRINCIPLE FINDINGS: We have generated a transgenic Drosophila eye model where high levels of human Aβ42 is misexpressed in the differentiating photoreceptor neurons of the developing eye, which phenocopy Alzheimer's like neuropathology in the neural retina. We have utilized this model for a gain of function screen using members of various signaling pathways involved in the development of the fly eye to identify downstream targets or modifiers of Aβ42 mediated neurodegeneration. We have identified the homeotic gene teashirt (tsh as a suppressor of the Aβ42 mediated neurodegenerative phenotype. Targeted misexpression of tsh with Aβ42 in the differentiating retina can significantly rescue neurodegeneration by blocking cell death. We found that Tsh protein is absent/ downregulated in the neural retina at this stage. The structure function analysis revealed that the PLDLS domain of Tsh acts as an inhibitor of the neuroprotective function of tsh in the Drosophila eye model. Lastly, we found that the tsh paralog, tiptop (tio can also rescue Aβ42 mediated neurodegeneration. CONCLUSIONS/SIGNIFICANCE: We have identified tsh and tio as new genetic modifiers of Aβ42 mediated neurodegeneration. Our studies demonstrate a novel neuroprotective function of tsh and its paralog tio in Aβ42 mediated neurodegeneration. The

  12. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    Science.gov (United States)

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.

  13. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

    Directory of Open Access Journals (Sweden)

    Pravas Ranjan Sahoo

    2016-05-01

    Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  14. Agrobacterium-mediated transformation of chickpea with -amylase inhibitor gene for insect resistance

    Indian Academy of Sciences (India)

    S Ignacimuthu; S Prakash

    2006-09-01

    Chickpea is the world’s third most important pulse crop and India produces 75% of the world’s supply. Chickpea seeds are attacked by Callosobruchus maculatus and C. chinensis which cause extensive damage. The -amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into chickpea cultivar K850 through Agrobacterium-mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb -amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of -amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevil C. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1.

  15. RNAi-mediated knocking- down of rlpk2 gene retarded soybean leaf senescence

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoping; MA Yuanyuan; LI Pengli; ZHANG Liwen; WANG Yong; ZHANG Ren; WANG Ningning

    2005-01-01

    Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence and the way the senescence signal is transduced. In a previous study on artificially induced soybean leaf senescence, we cloned a novel gene designated rlpk2 (Genbank Accession No. AY687391) that encodes a leucine-rich repeat (LRR) receptor like protein kinase. The expression level of rlpk2 gene was shown to be strongly up-regulated during both the natural leaf senescence process in this report and the artificially induced primary-leaf-senescence process in our previous work. The RNA interference (RNAi)-mediated knocking-down of rlpk2 dramatically retarded both the natural and nutrient deficiency-induced leaf senescence in transgenic soybean. The transgenic leaves showed more cell-aggregated surface structure and higher content of chlorophyll.

  16. Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

    Science.gov (United States)

    Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

    2014-10-01

    Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient.

  17. Regeneration and gene transformation systems of Robinia pseudoacacia 'Idaho' mediated by Agrobacterium tumefaciens

    Institute of Scientific and Technical Information of China (English)

    Li Min; Cai Zao; Sun De-you; Yin Wei-lun; Chen Shou-yi; Wang Hua-fang

    2006-01-01

    Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.

  18. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    Science.gov (United States)

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  19. Thyroid hormone-responsive genes mediate otolith growth and development during flatfish metamorphosis.

    Science.gov (United States)

    Wang, X; Tan, Y; Sievers, Q; Sievers, B; Lee, M; Burrall, K; Schreiber, A M

    2011-01-01

    Flatfish begin life as up-right swimming, bilaterally symmetrical larvae that metamorphose into asymmetrically shaped juveniles that swim with a highly lateralized posture. We have previously shown that TH induces abrupt growth and mineralization of one component of the vestibular system, the otoliths, during early larval development and metamorphosis. Here we report that four of five vestibular-specific genes that we tested (alpha-tectorin, otogelin, otolith matrix protein, and otopetrins 1 and 2 that are known to be associated with otolith development in other vertebrates are up-regulated 1.5- to 7-fold in larval flatfish during spontaneous metamorphosis and/or following 72 h of TH treatment. These findings suggest that otolith growth and development are mediated by diverse TH-responsive genes during flatfish metamorphosis.

  20. A non-surgical approach for male germ cell mediated gene transmission through transgenesis.

    Science.gov (United States)

    Usmani, Abul; Ganguli, Nirmalya; Sarkar, Hironmoy; Dhup, Suveera; Batta, Suryaprakash R; Vimal, Manoj; Ganguli, Nilanjana; Basu, Sayon; Nagarajan, P; Majumdar, Subeer S

    2013-01-01

    Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

  1. RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression

    Science.gov (United States)

    Lebreton, Alice; Cossart, Pascale

    2017-01-01

    ABSTRACT The model opportunistic pathogen Listeria monocytogenes has been the object of extensive research, aiming at understanding its ability to colonize diverse environmental niches and animal hosts. Bacterial transcriptomes in various conditions reflect this efficient adaptability. We review here our current knowledge of the mechanisms allowing L. monocytogenes to respond to environmental changes and trigger pathogenicity, with a special focus on RNA-mediated control of gene expression. We highlight how these studies have brought novel concepts in prokaryotic gene regulation, such as the ‘excludon’ where the 5′-UTR of a messenger also acts as an antisense regulator of an operon transcribed in opposite orientation, or the notion that riboswitches can regulate non-coding RNAs to integrate complex metabolic stimuli into regulatory networks. Overall, the Listeria model exemplifies that fine RNA tuners act together with master regulatory proteins to orchestrate appropriate transcriptional programmes. PMID:27217337

  2. Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

    Directory of Open Access Journals (Sweden)

    Bin eRen MD, Phd, FAHA

    2016-05-01

    Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  3. Ultrasound-Mediated Drug/Gene Delivery in Solid Tumor Treatment

    Directory of Open Access Journals (Sweden)

    Yufeng Zhou

    2013-01-01

    Full Text Available Ultrasound is an emerging modality for drug delivery in chemotherapy. This paper reviews this novel technology by first introducing the designs and characteristics of three classes of drug/gene vehicles, microbubble (including nanoemulsion, liposomes, and micelles. In comparison to conventional free drug, the targeted drug-release and delivery through vessel wall and interstitial space to cancerous cells can be activated and enhanced under certain sonication conditions. In the acoustic field, there are several reactions of these drug vehicles, including hyperthermia, bubble cavitation, sonoporation, and sonodynamics, whose physical properties are illustrated for better understanding of this approach. In vitro and in vivo results are summarized, and future directions are discussed. Altogether, ultrasound-mediated drug/gene delivery under imaging guidance provides a promising option in cancer treatment with enhanced agent release and site specificity and reduced toxicity.

  4. Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants?

    Science.gov (United States)

    Laxa, Miriam

    2017-01-01

    Many plant promoters were characterized and used for transgene expression in plants. Even though these promoters drive high levels of transgene expression in plants, the expression patterns are rarely constitutive but restricted to some tissues and developmental stages. In terms of crop improvement not only the enhancement of expression per se but, in particular, tissue-specific and spatial expression of genes plays an important role. Introns were used to boost expression in transgenic plants in the field of crop improvement for a long time. However, the mechanism behind this so called intron-mediated enhancement (IME) is still largely unknown. This review highlights the complexity of IME on the levels of its regulation and modes of action and gives an overview on IME methodology, examples in fundamental research and models of proposed mechanisms. In addition, the application of IME in heterologous gene expression is discussed. PMID:28111580

  5. RNAi-mediated silencing of fungal acuD gene attenuates the virulence of Penicillium marneffei.

    Science.gov (United States)

    Sun, Jiufeng; Li, Xiqing; Feng, Peiying; Zhang, Junmin; Xie, Zhi; Song, Erwei; Xi, Liyan

    2014-02-01

    A number of pathogens, most of them intracellular, employ the glyoxylate cycle in order to ingest fatty acids as carbon sources as a way of coping with nutrient deprivation during the infection process. Isocitrate lyase, which is encoded by the pathogen's acuD gene, plays a pivotal role in the glyoxylate cycle, which has been implicated in fungal pathogenesis. In this study, the acuD gene of Penicillium marneffei was knocked down using siRNA expressed by a filamentous fungi expression system. The acuD siRNA reduced the acuD gene's mRNA and protein expression by 21.5 fold and 3.5 fold, respectively. When macrophages were infected with different transformants of P. marneffei, the knockdown of acuD expression with RNA interference was lethal to the pathogens. In addition, the secretion of tumor necrosis factor-alpha and interferon-gamma from the infected macrophages was reduced. Moreover, the RNAi-mediated silencing of acuD expression reduced the fungal burden in the nude mice infected with P. marneffei; inhibited the inflammatory response in the lungs, livers, and spleens during the chronic phase instead of the acute phase of infection; and thus prolonged survival of the infected animals. Collectively, our data indicate that the RNAi-mediated silencing of acuD expression could attenuate virulence of P. marneffei. The endogenous expression of the delivered siRNA vector could be used to evaluate the role of functional genes by continuous and stable expression of siRNA.

  6. Gene Transfection Mediated by Ultrasound and Pluronic P85 in HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Fen; LI Kaiyan; CHEN Yunchao; DENG Yuan; HONG Kai

    2007-01-01

    In order to assess whether gene transfection could be mediated by ultrasound in associa- tion with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irra- diated by ultrasound at 1 MHz, 0.4-2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal trans- fection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.051. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.

  7. Immunity and AAV-mediated gene therapy for muscular dystrophies in large animal models and human trials

    OpenAIRE

    2011-01-01

    Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery ...

  8. Establishing a Gene Trap System Mediated by T-DNA(GUS) in Rice

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Chen; Ai-Min Wang; Wei Li; Zong-Yang Wang; Xiu-Ling Cai

    2008-01-01

    Two plasmids, p13GUS and p13GUS2, were constructed to create a gene trap system containing the promoterless β-glucuronidase (GUS) reporter gene in the T-DNA region. Transformation of these two plasmids into the rice variety Zhonghua 11 (Oryza sativa ssp. japonica cv.), mediated by Agrobacterium tumefaciens, resulted in 942 independent transgenic lines. Histochemical GUS assays revealed that 31 To plants had various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. Hygromycin-resistant (hygr) homozygotes were screened and the copy number of the T-DNA inserts was determined in the GUS-positivs transgenic plants. The flanking sequences of the T-DNA were isolated by inverse-polymerase chain reaction and the insert positions on the rice genome of T-DNA were determined by a basic local alignment search tool in the GUS-positive transgenic plants transformed with plasmid p13GUS. Moreover, calii induced from the seeds of the T1 generation of 911 GUS-negative transgenic lines were subjected to stress and hormone treatments. Histochemical GUS assays were carried out on the calli before and after treatment. The results revealed that calli from 21 lines displayed differential GUS expression after treatment. All of these data demonstrated that this trap system is suitable for identifying rice genes, including those that are sensitive to induction.

  9. Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

    Institute of Scientific and Technical Information of China (English)

    PENG Li-ping; JIN Ning-yi; LI Xiao; SUN Li-li; WEN Zhong-mei; LIU Yan; GAO Peng; HUANG Hai-yan; PIAO Bing-guo; JIN Jing

    2011-01-01

    Hemagglutinin-neuramidinase(HN) is one of the most important surface structure proteins of the Newcastle disease virus(NDV). HN not only mediates receptor recognition but also possesses neuraminidase(NA) activity,which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma(LLC) solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene(Ad-HN), and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Thl/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4+ and CD8+ T cell populations and cytotoxic T lymphocyte(CTL) and killer cell response, but also skews the immune response toward Thl. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.

  10. Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation.

    Science.gov (United States)

    Lo, Chia-Wen; Desjouy, Cyril; Chen, Shing-Ru; Lee, Jyun-Lin; Inserra, Claude; Béra, Jean-Christophe; Chen, Wen-Shiang

    2014-03-01

    It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.

  11. Epigenome mapping highlights chromatin-mediated gene regulation in the protozoan parasite Trichomonas vaginalis

    Science.gov (United States)

    Song, Min-Ji; Kim, Mikyoung; Choi, Yeeun; Yi, Myung-hee; Kim, Juri; Park, Soon-Jung; Yong, Tai-Soon; Kim, Hyoung-Pyo

    2017-01-01

    Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes trichomoniasis, one of the most common non-viral sexually transmitted diseases. To survive and to maintain infection, T. vaginalis adapts to a hostile host environment by regulating gene expression. However, the mechanisms of transcriptional regulation are poorly understood for this parasite. Histone modification has a marked effect on chromatin structure and directs the recruitment of transcriptional machinery, thereby regulating essential cellular processes. In this study, we aimed to outline modes of chromatin-mediated gene regulation in T. vaginalis. Inhibition of histone deacetylase (HDAC) alters global transcriptional responses and induces hyperacetylation of histones and hypermethylation of H3K4. Analysis of the genome of T. vaginalis revealed that a number of enzymes regulate histone modification, suggesting that epigenetic mechanisms are important to controlling gene expression in this organism. Additionally, we describe the genome-wide localization of two histone H3 modifications (H3K4me3 and H3K27Ac), which we found to be positively associated with active gene expression in both steady and dynamic transcriptional states. These results provide the first direct evidence that histone modifications play an essential role in transcriptional regulation of T. vaginalis, and may help guide future epigenetic research into therapeutic intervention strategies against this parasite. PMID:28345651

  12. Ferripyochelin uptake genes are involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.

    Science.gov (United States)

    Michel, Laurent; Bachelard, Aude; Reimmann, Cornelia

    2007-05-01

    In response to iron starvation, Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted to the extracellular environment, pyochelin chelates iron and transports it to the bacterial cytoplasm via its specific outer-membrane receptor FptA and the inner-membrane permease FptX. Exogenously added pyochelin also acts as a signal which induces the expression of the pyochelin biosynthesis and uptake genes by activating PchR, a cytoplasmic regulatory protein of the AraC/XylS family. The importance of ferripyochelin uptake genes in this regulation was evaluated. The fptA and fptX genes were shown to be part of the fptABCX ferripyochelin transport operon, which is conserved in Burkholderia sp. and Rhodospirillum rubrum. The fptB and fptC genes were found to be dispensable for utilization of pyochelin as an iron source, for signalling and for pyochelin production. By contrast, mutations in fptA and fptX not only interfered with pyochelin utilization, but also affected signalling and diminished siderophore production. It is concluded from this that pyochelin-mediated signalling operates to a large extent via the ferripyochelin transport system.

  13. Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

    Science.gov (United States)

    Keravala, Annahita; Liu, Dexi; Lechman, Eric R; Wolfe, Darren; Nash, Joan A; Lampe, David J; Robbins, Paul D

    2006-10-01

    The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

  14. Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

    Science.gov (United States)

    Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

    2016-06-01

    Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

  15. Pollen-Mediated Movement of Herbicide Resistance Genes in Lolium rigidum.

    Directory of Open Access Journals (Sweden)

    Iñigo Loureiro

    Full Text Available The transfer of herbicide resistance genes by pollen is a major concern in cross-pollinated species such as annual ryegrass (Lolium rigidum. A two-year study was conducted in the greenhouse, under favorable conditions for pollination, to generate information on potential maximum cross-pollination. This maximum cross-pollination rate was 56.1%. A three-year field trial was also conducted to study the cross-pollination rates in terms of distance and orientation to an herbicide-resistant pollen source. Under field conditions, cross-pollination rates varied from 5.5% to 11.6% in plants adjacent to the pollen source and decreased with increasing distances (1.5 to 8.9% at 15 m distance and up to 4.1% at 25 m in the downwind direction. Environmental conditions influenced the cross-pollination both under greenhouse and field conditions. Data were fit to an exponential decay model to predict gene flow at increasing distances. This model predicted an average gene flow of 7.1% when the pollen donor and recipient plants were at 0 m distance from each other. Pollen-mediated gene flow declined by 50% at 16.7 m from the pollen source, yet under downwind conditions gene flow of 5.2% was predicted at 25 m, the farthest distance studied. Knowledge of cross-pollination rates will be useful for assessing the spread of herbicide resistance genes in L. rigidum and in developing appropriate strategies for its mitigation.

  16. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  17. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

    Directory of Open Access Journals (Sweden)

    Tatiana I Novobrantseva

    2012-01-01

    Full Text Available Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP for durable and potent in vivo RNA interference (RNAi-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA. In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.

  18. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

    Science.gov (United States)

    Novobrantseva, Tatiana I; Borodovsky, Anna; Wong, Jamie; Klebanov, Boris; Zafari, Mohammad; Yucius, Kristina; Querbes, William; Ge, Pei; Ruda, Vera M; Milstein, Stuart; Speciner, Lauren; Duncan, Rick; Barros, Scott; Basha, Genc; Cullis, Pieter; Akinc, Akin; Donahoe, Jessica S; Narayanannair Jayaprakash, K; Jayaraman, Muthusamy; Bogorad, Roman L; Love, Kevin; Whitehead, Katie; Levins, Chris; Manoharan, Muthiah; Swirski, Filip K; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G; de Fougerolles, Antonin; Nahrendorf, Matthias; Koteliansky, Victor

    2012-01-01

    Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells. PMID:23344621

  19. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    Directory of Open Access Journals (Sweden)

    Srivathsa C Venugopal

    2009-07-01

    Full Text Available Resistance (R protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1, non-race-specific disease resistance 1 (NDR1, phytoalexin deficient 4 (PAD4, senescence associated gene 101 (SAG101, and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  20. Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling.

    Science.gov (United States)

    Venugopal, Srivathsa C; Jeong, Rae-Dong; Mandal, Mihir K; Zhu, Shifeng; Chandra-Shekara, A C; Xia, Ye; Hersh, Matthew; Stromberg, Arnold J; Navarre, DuRoy; Kachroo, Aardra; Kachroo, Pradeep

    2009-07-01

    Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.

  1. Glucose availability is a decisive factor for Nrf2-mediated gene expression

    Directory of Open Access Journals (Sweden)

    Elke H. Heiss

    2013-01-01

    Full Text Available Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2 is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells.

  2. EDS1 in tomato is required for resistance mediated by TIR-class R genes and the receptor-like R gene Ve.

    Science.gov (United States)

    Hu, Gongshe; deHart, Amy K A; Li, Yansu; Ustach, Carolyn; Handley, Vanessa; Navarre, Roy; Hwang, Chin-Feng; Aegerter, Brenna J; Williamson, Valerie M; Baker, Barbara

    2005-05-01

    In tobacco and other Solanaceae species, the tobacco N gene confers resistance to tobacco mosaic virus (TMV), and leads to induction of standard defense and resistance responses. Here, we report the use of N-transgenic tomato to identify a fast-neutron mutant, sun1-1 (suppressor of N), that is defective in N-mediated resistance. Induction of salicylic acid (SA) and expression of pathogenesis-related (PR) genes, each signatures of systemic acquired resistance, are both dramatically suppressed in sun1-1 plants after TMV treatment compared to wild-type plants. Application of exogenous SA restores PR gene expression, indicating that SUN1 acts upstream of SA. Upon challenge with additional pathogens, we found that the sun1-1 mutation impairs resistance mediated by certain resistance (R) genes, (Bs4, I, and Ve), but not others (Mi-1). In addition, sun1-1 plants exhibit enhanced susceptibility to TMV, as well as to virulent pathogens. sun1-1 has been identified as an EDS1 homolog present on chromosome 6 of tomato. The discovery of enhanced susceptibility in the sun1-1 (Le_eds1-1) mutant plant, which contrasts to reports in Nicotiana benthamiana using virus-induced gene silencing, provides evidence that the intersection of R gene-mediated pathways with general resistance pathways is conserved in a Solanaceous species. In tomato, EDS1 is important for mediating resistance to a broad range of pathogens (viral, bacterial, and fungal pathogens), yet shows specificity in the class of R genes that it affects (TIR-NBS-LRR as opposed to CC-NBS-LRR). In addition, a requirement for EDS1 for Ve-mediated resistance in tomato exposes that the receptor-like R gene class may also require EDS1.

  3. cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.

    Science.gov (United States)

    Patrizio, Mario; Vago, Valerio; Musumeci, Marco; Fecchi, Katia; Sposi, Nadia Maria; Mattei, Elisabetta; Catalano, Liviana; Stati, Tonino; Marano, Giuseppe

    2008-12-01

    The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling

  4. Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.

    Science.gov (United States)

    Gilles, Anna F; Schinko, Johannes B; Averof, Michalis

    2015-08-15

    Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.

  5. Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhong-Min GUO; Kang XU; Ying YUE; Bing HUANG; Xin-Yan DENG; Nü-Qi ZHONG; Xun HONG; Xi-Gu CHEN; Dong XIAO

    2005-01-01

    Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTAtetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur.Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

  6. Efficient Gene Transfer Mediated by HIV-1-based Defective Lentivector and Inhibition of HIV-1 Replication

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.

  7. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

    Directory of Open Access Journals (Sweden)

    Eroglu E

    2013-04-01

    Full Text Available Erdal Eroglu,1 Pooja M Tiwari,1 Alain B Waffo,1 Michael E Miller,2 Komal Vig,1 Vida A Dennis,1 Shree R Singh1 1Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL, USA; 2Research Instrumentation Facility, Auburn University, AL, USA Abstract: The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs, which consisted of poly (2-hydroxyethyl methacrylate nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV-F gene construct (a model for a DNA vaccine. The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM, fluorescence activated cell sorting (FACS, and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR, we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo. Keywords: pHEMA+chitosan nanoparticles, nonviral vector

  8. CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ronda, Carlotta; Maury, Jérôme; Jakočiunas, Tadas; Jacobsen, Simo Abdessamad Baallal; Germann, Susanne Manuela; Harrison, Scott James; Borodina, Irina; Keasling, Jay D; Jensen, Michael Krogh; Nielsen, Alex Toftgaard

    2015-07-07

    One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers. Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

  9. Efficiency of adenoviral vector mediated CTLA4Ig gene delivery into mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    邓宇斌; 郭小荑; 原清涛; 李树浓

    2003-01-01

    Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. Methods We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. Results Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P<0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation. Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.

  10. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    Directory of Open Access Journals (Sweden)

    Himangi G Marathe

    Full Text Available SOX10 is a Sry-related high mobility (HMG-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ and the gene that encodes myelin basic protein (MBP. Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate

  11. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    Science.gov (United States)

    Marathe, Himangi G; Mehta, Gaurav; Zhang, Xiaolu; Datar, Ila; Mehrotra, Aanchal; Yeung, Kam C; de la Serna, Ivana L

    2013-01-01

    SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to

  12. Area-Specific Cell Stimulation via Surface-Mediated Gene Transfer Using Apatite-Based Composite Layers

    Directory of Open Access Journals (Sweden)

    Yushin Yazaki

    2015-04-01

    Full Text Available Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA–fibronectin–apatite composite (DF-Ap layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in theunderlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.

  13. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, Navdeep [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Vijayan, Mathilakath M., E-mail: mvijayan@uwaterloo.ca [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada)

    2011-05-15

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  14. Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression.

    Science.gov (United States)

    Fernandes, Fabiana; Vidigal, João; Dias, Mafalda M; Prather, Kristala L J; Coroadinha, Ana S; Teixeira, Ana P; Alves, Paula M

    2012-11-01

    Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

  15. Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy

    Directory of Open Access Journals (Sweden)

    Siemionow Maria

    2006-08-01

    Full Text Available Abstract Background The aim of the present study was to develop and characterize a novel in vivo cancer gene therapy model in which intra-arterial adenoviral gene delivery can be characterized. In this model, the rat cremaster muscle serves as the site for tumor growth and provides convenient and isolated access to the tumor parenchyma with discrete control of arterial and venous access for delivery of agents. Results Utilizing adenovirus encoding the green fluorescent protein we demonstrated broad tumor transfection. We also observed a dose dependant increment in luciferase activity at the tumor site using an adenovirus encoding the luciferase reporter gene. Finally, we tested the intra-arterial adenovirus dwelling time required to achieve optimal tumor transfection and observed a minimum time of 30 minutes. Conclusion We conclude that adenovirus mediated tumor transfection grown in the cremaster muscle of athymic nude rats via an intra-arterial route could be achieved. This model allows definition of the variables that affect intra-arterial tumor transfection. This particular study suggests that allowing a defined intra-tumor dwelling time by controlling the blood flow of the affected organ during vector infusion can optimize intra-arterial adenoviral delivery.

  16. RNA structures facilitate recombination-mediated gene swapping in HIV-1.

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P; Weeks, Kevin M; Negroni, Matteo

    2010-12-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.

  17. RNA Structures Facilitate Recombination-Mediated Gene Swapping in HIV-1 ▿

    Science.gov (United States)

    Simon-Loriere, Etienne; Martin, Darren P.; Weeks, Kevin M.; Negroni, Matteo

    2010-01-01

    Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny. PMID:20881047

  18. Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.

    Science.gov (United States)

    Münch, Steffen; Ludwig, Nancy; Floss, Daniela S; Sugui, Janyce A; Koszucka, Anna M; Voll, Lars M; Sonnewald, Uwe; Deising, Holger B

    2011-01-01

    A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function. © 2010 The Authors. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.

  19. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  20. Insulin mediated hemodynamic responses in spontaneous hypertensive rats (SHRs): effect of chromosome 4 gene transfer.

    Science.gov (United States)

    Rao, Sumangala P; McRae, Crystal; Lapanowski, Karen; Churchill, Monique; Kurtz, Theodore W; Dunbar, Joseph C

    2003-02-01

    The spontaneous hypertensive rat (SHR) is a widely studied model of essential hypertension and has been reported to exhibit alterations in carbohydrate and lipid metabolism. Genetic linkage studies implicated that SHR carries deletion variant of Cd36 gene of chromosome 4, the gene that encodes fatty acid transporter. Thus it could be possible that primary genetic defect in SHR is compromised tissue utilization of fatty acid that would form the basis for the pathogenesis of hyperinsulinemia, insulin resistance and insulin-mediated responses. We measured both the hemodynamic and metabolic responses to insulin in SHR in comparison with the chromosome congenic spontaneous hypertensive rats (cSHRs) (rats in which piece of chromosome 4 containing wild type Cd36 was integrated into the SHR genome). A bolus infusion of insulin increased iliac conductance and decreased blood pressure in Wistar Kyoto (WKY) rats. However, in SHR insulin did not reduce blood pressure as in WKY but after about 15 min it significantly enhanced blood pressure and reduced iliac conductance. Whereas in cSHR insulin did not reduce blood pressure as in WKY rats. However, pressor responses to insulin were eliminated by chromosome 4 gene transfer. Glucose clearance was significantly slower in both SHR and cSHR. Glucose tolerance test revealed that SHR are hyperinsulinemic and insulin resistant. These findings indicate that transfer of segment of chromosome 4 from Brown Norway rats onto spontaneous hypertensive background eliminates hyperinsulinemia and pressor effects of insulin.

  1. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J; Park, Sung-Min; Cai, Dongsheng

    2011-07-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  2. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Directory of Open Access Journals (Sweden)

    Hai Zhang

    2011-07-01

    Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  3. Oligonucleotide-mediated gene modification and its promise for animal agriculture.

    Science.gov (United States)

    Laible, Götz; Wagner, Stefan; Alderson, Jon

    2006-01-17

    One of the great aspirations in modern biology is the ability to utilise the expanding knowledge of the genetic basis of phenotypic diversity through the purposeful tailoring of the mammalian genome. A number of technologies are emerging which have the capacity to modify genes in their chromosomal context. Not surprisingly, the major thrust in this area has come from the evaluation of gene therapy applications to correct mutations implicated in human genetic diseases. The recent development of somatic cell nuclear transfer (SCNT) provides access to these technologies for the purposeful modification of livestock animals. The enormous phenotypic variety existent in contemporary livestock animals has in many cases been linked to quantitative trait loci (QTL) and their underlying point mutations, often referred to as single-nucleotide polymorphisms (SNPs). Thus, the ability for the targeted genetic modification of livestock animals constitutes an attractive opportunity for future agricultural applications. In this review, we will summarize attempts and approaches for oligonucleotide-mediated gene modification (OGM) strategies for the site-specific modification of the genome, with an emphasis on chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligonucletides (ssODNs). The potential of this approach for the directed genetic improvement of livestock animals is illustrated through examples, outlining the effects of point mutations on important traits, including meat and milk production, reproductive performance, disease resistance and superior models of human diseases. Current technological hurdles and potential strategies that might remove these barriers in the future are discussed.

  4. Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Frandsen, Mette; Giese, Nanna Henriette

    2012-01-01

    Genome sequence data on fungal pathogens provide the opportunity to carry out a reverse genetics approach to uncover gene function. Efficient methods for targeted genome modifications such as knockout and in locus over-expression are in high demand. Here we describe two efficient single-step clon......Genome sequence data on fungal pathogens provide the opportunity to carry out a reverse genetics approach to uncover gene function. Efficient methods for targeted genome modifications such as knockout and in locus over-expression are in high demand. Here we describe two efficient single......-step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT). Targeted genome modifications require integration by a homologous double crossover event, which is achieved by placing target sequences on either side of a selection marker gene in the vector....... Protocols are given for two single-step vector construction techniques. The In-Fusion cloning technique is independent of compatible restriction enzyme sites in the vector and the fragment to be cloned. The method can be directly applied to any vector of choice and it is possible to carry out four fragment...

  5. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  6. Construction of a genetic map of human chromosome 17 by use of chromosome-mediated gene transfer

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Weiming; Gorman, P.A.; Rider, S.H.; Hedge, P.J.; Moore, G.; Prichard, C.; Sheer, D.; Solomon, E. (Imperial Cancer Research Fund, London (England))

    1988-11-01

    The authors used somatic-cell hybrids, containing as their only human genetic contribution part or all of chromosome 17, as donors for chromosome-mediated gene transfer. A total of 54 independent transfectant clones were isolated and analyzed by use of probes or isoenzymes for >20 loci located on chromosome 17. By combining the data from this chromosome-mediated gene transfer transfectant panel, conventional somatic-cell hybrids containing well-defined breaks on chromosome 17, and in situ hybridization they propose the following order for these loci; pter-(TP53-RNP2-D17S1)-(MYH2-MYH1)-D17Z1-CRYB1-(ERBA1-GCSF-NGL)-acute promyelocytic leukemia breakpoint-RNU2-HOX2-(NGFR-COLIAI-MPO)-GAA-UMPH-GHC-TK1-GALK-qter. Using chromosome-mediated gene transfer, they have also regionally localized the random probes D17S6 to D17S19 on chromosome 17.

  7. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

    Science.gov (United States)

    Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

    2017-04-01

    In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements.

  8. Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection.

    Science.gov (United States)

    Loupy, Alexandre; Duong Van Huyen, Jean Paul; Hidalgo, Luis; Reeve, Jeff; Racapé, Maud; Aubert, Olivier; Venner, Jeffery M; Falmuski, Konrad; Bories, Marie Cécile; Beuscart, Thibaut; Guillemain, Romain; François, Arnaud; Pattier, Sabine; Toquet, Claire; Gay, Arnaud; Rouvier, Philippe; Varnous, Shaida; Leprince, Pascal; Empana, Jean Philippe; Lefaucheur, Carmen; Bruneval, Patrick; Jouven, Xavier; Halloran, Philip F

    2017-03-07

    Antibody-mediated rejection (AMR) contributes to heart allograft loss. However, an important knowledge gap remains in terms of the pathophysiology of AMR and how detection of immune activity, injury degree, and stage could be improved by intragraft gene expression profiling. We prospectively monitored 617 heart transplant recipients referred from 4 French transplant centers (January 1, 2006-January 1, 2011) for AMR. We compared patients with AMR (n=55) with a matched control group of 55 patients without AMR. We characterized all patients using histopathology (ISHLT [International Society for Heart and Lung Transplantation] 2013 grades), immunostaining, and circulating anti-HLA donor-specific antibodies at the time of biopsy, together with systematic gene expression assessments of the allograft tissue, using microarrays. Effector cells were evaluated with in vitro human cell cultures. We studied a validation cohort of 98 heart recipients transplanted in Edmonton, AB, Canada, including 27 cases of AMR and 71 controls. A total of 240 heart transplant endomyocardial biopsies were assessed. AMR showed a distinct pattern of injury characterized by endothelial activation with microcirculatory inflammation by monocytes/macrophages and natural killer (NK) cells. We also observed selective changes in endothelial/angiogenesis and NK cell transcripts, including CD16A signaling and interferon-γ-inducible genes. The AMR-selective gene sets accurately discriminated patients with AMR from those without and included NK transcripts (area under the curve=0.87), endothelial activation transcripts (area under the curve=0.80), macrophage transcripts (area under the curve=0.86), and interferon-γ transcripts (area under the curve=0.84; P<0.0001 for all comparisons). These 4 gene sets showed increased expression with increasing pathological AMR (pAMR) International Society for Heart and Lung Transplantation grade (P<0.001) and association with donor-specific antibody levels. The

  9. Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    Science.gov (United States)

    Fukuda, Aya; Shimada, Miho; Nakadai, Tomoyoshi; Nishimura, Ken; Hisatake, Koji

    2013-01-01

    The c-fos gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R) facilitates transcription reinitiation of the c-fos promoter in vitro in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH) as well as TFIIB, which recruits RNA polymerase II (Pol II) and TFIIF to Scaffold. The cooperative action of hnRNP R and Mediator is diminished by the cyclin-dependent kinase 8 (CDK8) module, which is comprised of CDK8, Cyclin C, MED12 and MED13 of the Mediator subunits. Interestingly, we find that the length of the G-free cassettes, and thereby their transcripts, influences the hnRNP R-mediated facilitation of reinitiation. Indeed, indicative of a possible role of the transcript in facilitating transcription reinitiation, the RNA transcript produced from the G-free cassette interacts with hnRNP R through its RNA recognition motifs (RRMs) and arginine-glycine-glycine (RGG) domain. Mutational analyses of hnRNP R indicate that facilitation of initiation and reinitiation requires distinct domains of hnRNP R. Knockdown of hnRNP R in mouse cells compromised rapid induction of the c-fos gene but did not affect transcription of constitutive genes. Together, these results suggest an important role for hnRNP R in regulating robust response of the c-fos gene. PMID:23967313

  10. Heterogeneous nuclear ribonucleoprotein R cooperates with mediator to facilitate transcription reinitiation on the c-Fos gene.

    Directory of Open Access Journals (Sweden)

    Aya Fukuda

    Full Text Available The c-fos gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R facilitates transcription reinitiation of the c-fos promoter in vitro in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH as well as TFIIB, which recruits RNA polymerase II (Pol II and TFIIF to Scaffold. The cooperative action of hnRNP R and Mediator is diminished by the cyclin-dependent kinase 8 (CDK8 module, which is comprised of CDK8, Cyclin C, MED12 and MED13 of the Mediator subunits. Interestingly, we find that the length of the G-free cassettes, and thereby their transcripts, influences the hnRNP R-mediated facilitation of reinitiation. Indeed, indicative of a possible role of the transcript in facilitating transcription reinitiation, the RNA transcript produced from the G-free cassette interacts with hnRNP R through its RNA recognition motifs (RRMs and arginine-glycine-glycine (RGG domain. Mutational analyses of hnRNP R indicate that facilitation of initiation and reinitiation requires distinct domains of hnRNP R. Knockdown of hnRNP R in mouse cells compromised rapid induction of the c-fos gene but did not affect transcription of constitutive genes. Together, these results suggest an important role for hnRNP R in regulating robust response of the c-fos gene.

  11. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    Science.gov (United States)

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  12. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    -mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo...

  13. Antitumor effect of TRAIL on oral squamous cell carcinoma using magnetic nanoparticle-mediated gene expression.

    Science.gov (United States)

    Miao, Leiying; Liu, Chao; Ge, Jiuyu; Yang, Weidong; Liu, Jinzhong; Sun, Weibin; Yang, Bai; Zheng, Changyu; Sun, Hongchen; Hu, Qingang

    2014-07-01

    We developed a new magnetic nanovector to improve the efficiency and targeting of transgene therapy for oral squamous cell carcinoma (OSCC). Positively charged polymer PEI-modified Fe(3)O(4) magnetic nanoparticles were tested as gene transfer vectors in the presence of a magnetic field. The Fe(3)O(4) nanoparticles were prepared by a co-precipitation method and had good dispersibility in water. These nanoparticles modified by PEI were combined with negatively charged pACTERT-EGFP via electrostatic interaction. The transfection efficiency of the magnetic nano-gene vector with the magnetic field was determined by a fluorescence-inverted microscope and flow cytometry. The results showed significant improvement compared with the control group (p < 0.05). The magnetic complexes also exhibited up to 6-times higher transfection efficiency compared with commonly used PEI or lipofectin. On the basis of these results, the antitumor effect with suicide gene therapy using pACTERT-TRAIL in vitro and vivo was evaluated. In vitro apoptosis was determined with the Annexin V-FITC Apoptosis Detection Kit. The results suggested that PEI-modified Fe(3)O(4) nanoparticles could mediate the killing of Tca83 cells. Furthermore, treatment with pACTERT-TRAIL delivered by magnetic nanoparticles showed a significant cytostatic effect through the induction of apoptosis in a xenograft model. This indicates that magnetic nano-gene vectors could improve the transgene efficiency for Tca83 cells and could exhibit antitumor functions with the plasmid pACTERT-TRAIL. This may be a new way to treat OSCC.

  14. Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer.

    Science.gov (United States)

    Li, Lan; Shen, Wei; Min, Lingjiang; Dong, Huansheng; Sun, Yujiang; Pan, Qingjie

    2006-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat beta-casein gene 5' flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 microg mL(-1) DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO-sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 +/- 31 microg mL(-1), and the mean expression level in all of the transgenic rabbits was 103 +/- 20 microg mL(-1) in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO-SMGT were able to express human LF protein in the correct tissue.

  15. GROWTH INHIBITION OF HUMAN LARYNGEAL CANCER CELL WITH THE ADENOVIRUS-MEDIATED p53 GENE

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; HAN De-min; WANG Wen-ge; WU Zu-ze; ZHANG Wei

    1999-01-01

    Objective: In most laryngeal cancers, the function of p53 gene is down regulated. To explore the potential use of p53 in gene therapy of laryngeal cancer, by introducing wild-type p53 into laryngeal cancer cell line via a recombinant adenoviral vector, Ad5CMV-p53 and analyzing its effects on cell and tumor growth. Methods: A human laryngeal cancer cell line Hep-2 was used.Recombinant cytomegalovirus-promoted adenoviruses containing human wild-type p53 cDNA was transiently introduced into Hep-2 line. The growth suppression of the Hep-2 cells and established s.c. squamous carcinoma model was examined. The p53 protein expression was detected using immunohistochemical analysis. Results: The transduction efficiencies of Hep-2 cell line were 100% at a multiplicity of 100 or greater. The p53 protein expression peaked on day 2 after infection and lasted far 5 days. In vitro growth assays revealed cell death following Ad5CMV-p53 infected. In vivo studies, Ad5CMV-p53 inhibited the tumorigenicity of Hep-2 cell, and in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. Conclusion: Adenovirus-mediated antitumor therapy carrying the p53 gene is an efficient method to inhibit laryngeal cancer growth. Transfection of laryngeal cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential novel approach to the therapy of laryngeal cancer.

  16. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.

    Science.gov (United States)

    Tang, Lichun; Zeng, Yanting; Du, Hongzi; Gong, Mengmeng; Peng, Jin; Zhang, Buxi; Lei, Ming; Zhao, Fang; Wang, Weihua; Li, Xiaowei; Liu, Jianqiao

    2017-03-01

    Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

  17. Caregiver communication to the child as moderator and mediator of genes for language.

    Science.gov (United States)

    Onnis, Luca

    2017-05-15

    Human language appears to be unique among natural communication systems, and such uniqueness impinges on both nature and nurture. Human babies are endowed with cognitive abilities that predispose them to learn language, and this process cannot operate in an impoverished environment. To be effectively complete the acquisition of human language in human children requires highly socialised forms of learning, scaffolded over years of prolonged and intense caretaker-child interactions. How genes and environment operate in shaping language is unknown. These two components have traditionally been considered as independent, and often pitted against each other in terms of the nature versus nurture debate. This perspective article considers how innate abilities and experience might instead work together. In particular, it envisages potential scenarios for research, in which early caregiver verbal and non-verbal attachment practices may mediate or moderate the expression of human genetic systems for language. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Loss of the tumour suppressor gene AIP mediates the browning of human brown fat tumours.

    Science.gov (United States)

    Magnusson, Linda; Hansen, Nils; Saba, Karim H; Nilsson, Jenny; Fioretos, Thoas; Rissler, Pehr; Nord, Karolin H

    2017-10-01

    Human brown fat tumours (hibernomas) show concomitant loss of the tumour suppressor genes MEN1 and AIP. We hypothesized that the brown fat phenotype is attributable to these mutations. Accordingly, in this study, we demonstrate that silencing of AIP in human brown preadipocytic and white fat cell lines results in the induction of the brown fat marker UCP1. In human adipocytic tumours, loss of MEN1 was found both in white (one of 51 lipomas) and in brown fat tumours. In contrast, concurrent loss of AIP was always accompanied by a brown fat morphology. We conclude that this white-to-brown phenotype switch in brown fat tumours is mediated by the loss of AIP. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Functional analysis of the white gene of Drosophila by P-factor-mediated transformation.

    Science.gov (United States)

    Gehring, W J; Klemenz, R; Weber, U; Kloter, U

    1984-09-01

    A 12-kb DNA segment spanning the white (w) locus of Drosophila has been inserted into a P-transposon vector and used for P-factor-mediated germ-line transformation. Several red-eyed transformants were recovered which complement the white mutant phenotype. Analysis of the eye pigments and the interaction with the zeste mutation indicates that the w gene inserted at several new chromosomal sites is expressed normally. The tissue-specific accumulation of w transcripts, as studied by in situ hybridization to tissue sections, is the same in transformant and wild-type larvae. This indicates that all the genetic information specified by the w locus is contained within this 12-kb segment of DNA. By secondary mobilization it was shown that the w sequences have been inserted as a functional P(w) transposon which is capable of further transposition.

  20. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system.

    Science.gov (United States)

    Molina-Cruz, Alvaro; Garver, Lindsey S; Alabaster, Amy; Bangiolo, Lois; Haile, Ashley; Winikor, Jared; Ortega, Corrie; van Schaijk, Ben C L; Sauerwein, Robert W; Taylor-Salmon, Emma; Barillas-Mury, Carolina

    2013-05-24

    Plasmodium falciparum transmission by Anopheles gambiae mosquitoes is remarkably efficient, resulting in a very high prevalence of human malaria infection in sub-Saharan Africa. A combination of genetic mapping, linkage group selection, and functional genomics was used to identify Pfs47 as a P. falciparum gene that allows the parasite to infect A. gambiae without activating the mosquito immune system. Disruption of Pfs47 greatly reduced parasite survival in the mosquito, and this phenotype could be reverted by genetic complementation of the parasite or by disruption of the mosquito complement-like system. Pfs47 suppresses midgut nitration responses that are critical to activate the complement-like system. We provide direct experimental evidence that immune evasion mediated by Pfs47 is critical for efficient human malaria transmission by A. gambiae.

  1. Apelin mediates the induction of profibrogenic genes in human hepatic stellate cells.

    Science.gov (United States)

    Melgar-Lesmes, Pedro; Casals, Gregori; Pauta, Montserrat; Ros, Josefa; Reichenbach, Vedrana; Bataller, Ramon; Morales-Ruiz, Manuel; Jimenez, Wladimiro

    2010-11-01

    Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor β (PDGFRβ) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRβ expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRβ in fibrotic rats. apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

  2. AAV5-mediated gene transfer to the parotid glands of non-human primates

    Science.gov (United States)

    Voutetakis, A; Zheng, C; Cotrim, AP; Mineshiba, F; Afione, S; Roescher, N; Swaim, WD; Metzger, M; Eckhaus, MA; Donahue, RE; Dunbar, CE; Chiorini, JA; Baum, BJ

    2010-01-01

    Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 1010 or 3 × 1011 particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by ~80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (~1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands. PMID:19759566

  3. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    Science.gov (United States)

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  4. Cancer progression mediated by horizontal gene transfer in an in vivo model.

    Directory of Open Access Journals (Sweden)

    Catalina Trejo-Becerril

    Full Text Available It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.

  5. AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

    Science.gov (United States)

    Bradbury, Allison M; Peterson, Tiffany A; Gross, Amanda L; Wells, Stephen Z; McCurdy, Victoria J; Wolfe, Karen G; Dennis, John C; Brunson, Brandon L; Gray-Edwards, Heather; Randle, Ashley N; Johnson, Aime K; Morrison, Edward E; Cox, Nancy R; Baker, Henry J; Sena-Esteves, Miguel; Martin, Douglas R

    2017-01-06

    Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

    Science.gov (United States)

    Katter, Katharina; Geurts, Aron M; Hoffmann, Orsolya; Mátés, Lajos; Landa, Vladimir; Hiripi, László; Moreno, Carol; Lazar, Jozef; Bashir, Sanum; Zidek, Vaclav; Popova, Elena; Jerchow, Boris; Becker, Katja; Devaraj, Anantharam; Walter, Ingrid; Grzybowksi, Michael; Corbett, Molly; Filho, Artur Rangel; Hodges, Matthew R; Bader, Michael; Ivics, Zoltán; Jacob, Howard J; Pravenec, Michal; Bosze, Zsuzsanna; Rülicke, Thomas; Izsvák, Zsuzsanna

    2013-03-01

    Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

  7. Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

    Science.gov (United States)

    Trejo-Becerril, Catalina; Pérez-Cárdenas, Enrique; Taja-Chayeb, Lucía; Anker, Philippe; Herrera-Goepfert, Roberto; Medina-Velázquez, Luis A.; Hidalgo-Miranda, Alfredo; Pérez-Montiel, Delia; Chávez-Blanco, Alma; Cruz-Velázquez, Judith; Díaz-Chávez, José; Gaxiola, Miguel; Dueñas-González, Alfonso

    2012-01-01

    It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy. PMID:23285175

  8. Combining cytotoxic and immune-mediated gene therapy to treat brain tumors.

    Science.gov (United States)

    Curtin, James F; King, Gwendalyn D; Candolfi, Marianela; Greeno, Remy B; Kroeger, Kurt M; Lowenstein, Pedro R; Castro, Maria G

    2005-01-01

    Glioblastoma (GBM) is a type of intracranial brain tumor, for which there is no cure. In spite of advances in surgery, chemotherapy and radiotherapy, patients die within a year of diagnosis. Therefore, there is a critical need to develop novel therapeutic approaches for this disease. Gene therapy, which is the use of genes or other nucleic acids as drugs, is a powerful new treatment strategy which can be developed to treat GBM. Several treatment modalities are amenable for gene therapy implementation, e.g. conditional cytotoxic approaches, targeted delivery of toxins into the tumor mass, immune stimulatory strategies, and these will all be the focus of this review. Both conditional cytotoxicity and targeted toxin mediated tumor death, are aimed at eliminating an established tumor mass and preventing further growth. Tumors employ several defensive strategies that suppress and inhibit anti-tumor immune responses. A better understanding of the mechanisms involved in eliciting anti-tumor immune responses has identified promising targets for immunotherapy. Immunotherapy is designed to aid the immune system to recognize and destroy tumor cells in order to eliminate the tumor burden. Also, immune-therapeutic strategies have the added advantage that an activated immune system has the capability of recognizing tumor cells at distant sites from the primary tumor, therefore targeting metastasis distant from the primary tumor locale. Pre-clinical models and clinical trials have demonstrated that in spite of their location within the central nervous system (CNS), a tissue described as 'immune privileged', brain tumors can be effectively targeted by the activated immune system following various immunotherapeutic strategies. This review will highlight recent advances in brain tumor immunotherapy, with particular emphasis on advances made using gene therapy strategies, as well as reviewing other novel therapies that can be used in combination with immunotherapy. Another important

  9. A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2012-08-01

    Full Text Available Abstract Background Transcription activator-like effectors (TALEs are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP applications. Conclusion We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.

  10. Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

    Science.gov (United States)

    Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

    2003-07-11

    Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

  11. EFFECT OF TITANIUM-ALLOY PARTICLES ON EXPRESSION OF MEDIATING-RELATED GENES IN HUMAN OSTEOBLASTS

    Institute of Scientific and Technical Information of China (English)

    杨旭; 杨庆铭; 邓廉夫; 许福平; 张玥

    2004-01-01

    Objective To study mRNA expression of the mediating-related genes in osteoblasts after exposure to Titanium-alloy particles and cytochalasin-D. Methods Human osteoblasts were isolated from trabecular fragments and exposed to 0. 01% (v/v) titanium-alloy particles for 8h. Pretreatment of osteoblasts with cytochalasinD( 5μmol/L ) to prevent phagocytosis prior to the addition of Titanium-alloy particles, mRNA expression of RANKL,OPG, and IL-6 was semi-quantified by RT-PCR. Results Expressions of RANKL, OPG, and lL-6 mRNA at respective levels were found in osteoblasts under normal condition, which did not show significant changes in the samples treated with cytochalasin-D but without particles. Exposure of osteoblasts to particles after being treated with or without cytochalasin-D altered mRNA expression of these genes, however, an increase in the ratio of RANKL/ OPG mRNA was only found in samples without cytochalassin-D treatment. Conclusion Changes of expression of RANKL, OPG, and IL-6 mRNA in osteoblasts after exposure to titanium-alloy particles were independent of phagocytosis, whereas phagocytosis has a direct effect on their ability to stimulate osteoclast differentiation and function.

  12. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene

    Directory of Open Access Journals (Sweden)

    Yu Li

    2005-08-01

    Full Text Available Abstract Background White spot syndrome virus (WSSV is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection.

  13. Tuning riboswitch-mediated gene regulation by rational control of aptamer ligand binding properties.

    Science.gov (United States)

    Rode, Ambadas B; Endoh, Tamaki; Sugimoto, Naoki

    2015-01-12

    Riboswitch-mediated control of gene expression depends on ligand binding properties (kinetics and affinity) of its aptamer domain. A detailed analysis of interior regions of the aptamer, which affect the ligand binding properties, is important for both understanding natural riboswitch functions and for enabling rational design of tuneable artificial riboswitches. Kinetic analyses of binding reaction between flavin mononucleotide (FMN) and several natural and mutant aptamer domains of FMN-specific riboswitches were performed. The strong dependence of the dissociation rate (52.6-fold) and affinity (100-fold) on the identities of base pairs in the aptamer stem suggested that the stem region, which is conserved in length but variable in base-pair composition and context, is the tuning region of the FMN-specific aptamer. Synthetic riboswitches were constructed based on the same aptamer domain by rationally modifying the tuning regions. The observed 9.31-fold difference in the half-maximal effective concentration (EC50) corresponded to a 11.6-fold difference in the dissociation constant (K(D)) of the aptamer domains and suggested that the gene expression can be controlled by rationally adjusting the tuning regions.

  14. MicroRNA-mediated gene regulation: potential applications for plant genetic engineering.

    Science.gov (United States)

    Zhou, Man; Luo, Hong

    2013-09-01

    Food security is one of the most important issues challenging the world today. Any strategies to solve this problem must include increasing crop yields and quality. MicroRNA-based genetic modification technology (miRNA-based GM tech) can be one of the most promising solutions that contribute to agricultural productivity directly by developing superior crop cultivars with enhanced biotic and abiotic stress tolerance and increased biomass yields. Indirectly, the technology may increase usage of marginal soils and decrease pesticide use, among other benefits. This review highlights the most recent progress of transgenic studies utilizing various miRNAs and their targets for plant trait modifications, and analyzes the potential of miRNA-mediated gene regulation for use in crop improvement. Strategies for manipulating miRNAs and their targets in transgenic plants including constitutive, stress-induced, or tissue-specific expression of miRNAs or their targets, RNA interference, expressing miRNA-resistant target genes, artificial target mimic and artificial miRNAs were discussed. We also discussed potential risks of utilizing miRNA-based GM tech. In general, miRNAs and their targets not only provide an invaluable source of novel transgenes, but also inspire the development of several new GM strategies, allowing advances in breeding novel crop cultivars with agronomically useful characteristics.

  15. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    Directory of Open Access Journals (Sweden)

    Shih Ping Yao

    2002-04-01

    Full Text Available Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C, is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57 of transgenic pigs (F0 generation. Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

  16. [Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

    Science.gov (United States)

    Zhou, Yue; Zhao, Xupeng; Wu, Xiuhua; Zhang, Yanling; Zhang, Lin; Luo, Keming; Tang, Shaohu

    2014-06-01

    Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

  17. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    Science.gov (United States)

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  18. Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage

    Directory of Open Access Journals (Sweden)

    Guan-gui Chen

    2015-01-01

    Full Text Available Polyethyleneimine-polyethylene glycol (PEI-PEG, a novel nanocarrier, has been used for transfection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Auditory brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

  19. A translatable, closed recirculation system for AAV6 vector-mediated myocardial gene delivery in the large animal.

    Science.gov (United States)

    Swain, JaBaris D; Katz, Michael G; White, Jennifer D; Thesier, Danielle M; Henderson, Armen; Stedman, Hansell H; Bridges, Charles R

    2011-01-01

    Current strategies for managing congestive heart failure are limited, validating the search for an alternative treatment modality. Gene therapy holds tremendous promise as both a practical and translatable technology platform. Its effectiveness is evidenced by the improvements in cardiac function observed in vector-mediated therapeutic transgene delivery to the murine myocardium. A large animal model validating these results is the likely segue into clinical application. However, controversy still exists regarding a suitable method of vector-mediated cardiac gene delivery that provides for efficient, global gene transfer to the large animal myocardium that is also clinically translatable and practical. Intramyocardial injection and catheter-based coronary delivery techniques are attractive alternatives with respect to their clinical applicability; yet, they are fraught with numerous challenges, including concerns regarding collateral gene expression in other organs, low efficiency of vector delivery to the myocardium, inhomogeneous expression, and untoward immune response secondary to gene delivery. Cardiopulmonary bypass (CPB) delivery with dual systemic and isolated cardiac circuitry precludes these drawbacks and has the added advantage of allowing for control of the pharmacological milieu, multiple pass recirculation through the coronary circulation, the selective addition of endothelial permeabilizing agents, and an increase in vector residence time. Collectively, these mechanics significantly improve the efficiency of global, vector-mediated cardiac gene delivery to the large animal myocardium, highlighting a potential therapeutic strategy to be extended to some heart failure patients.

  20. Gene transfection mediated by polyethyleneimine-polyethylene glycol nanocarrier prevents cisplatin-induced spiral ganglion cell damage

    Institute of Scientific and Technical Information of China (English)

    Guan-gui Chen; Min Mao; Li-zi Qiu; Qi-ming Liu

    2015-01-01

    Polyethyleneimine-polyethylene glycol (PEI-PEG), a novel nanocarrier, has been used for trans-fection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein (XIAP) in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats,via scala tympani fenestration, before daily cisplatin injections. Audito-ry brainstem relfex tests demonstrated the protective effects ofXIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels ofXIAP mRNA expression in the cochlea. The present ifndings suggest that PEI-PEG nanocarrier-mediatedXIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

  1. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  2. Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing.

    Science.gov (United States)

    Chen, Yurong; Zhou, Xue-Rong; Zhang, Zhi-Jun; Dribnenki, Paul; Singh, Surinder; Green, Allan

    2015-04-01

    Simultaneous gene silencing of both FAD2 genes in high linoleic acid flax leads to high level of oleic acid, which is stable across multiple generations. High oleic oil is one of the preferred traits in oil crop engineering due to its stability and multiple applications as an industrial feedstock. Flax possesses two isoforms of FAD2 enzymes that desaturate monounsaturated oleic acid to polyunsaturated linoleic acid. These two enzymes are encoded by two FAD2 genes. By simultaneous gene silencing both FAD2 genes in high linoleic acid flax, Linola, high level of oleic acid up to 80% was achieved in 69 silencing lines. The high oleic trait was stable across multiple generations with oleic acid reaching up to 77% in homozygote T3 progeny. The RNAi-mediated gene-silencing approach generated high oleic linseed oil, as well as a high oleic platform that can be exploited for further fatty acid engineering.

  3. Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

    Science.gov (United States)

    Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-02-08

    Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

  4. Viral small interfering RNAs target host genes to mediate disease symptoms in plants.

    Directory of Open Access Journals (Sweden)

    Neil A Smith

    2011-05-01

    Full Text Available The Cucumber mosaic virus (CMV Y-satellite RNA (Y-Sat has a small non-protein-coding RNA genome that induces yellowing symptoms in infected Nicotiana tabacum (tobacco. How this RNA pathogen induces such symptoms has been a longstanding question. We show that the yellowing symptoms are a result of small interfering RNA (siRNA-directed RNA silencing of the chlorophyll biosynthetic gene, CHLI. The CHLI mRNA contains a 22-nucleotide (nt complementary sequence to the Y-Sat genome, and in Y-Sat-infected plants, CHLI expression is dramatically down-regulated. Small RNA sequencing and 5' RACE analyses confirmed that this 22-nt sequence was targeted for mRNA cleavage by Y-Sat-derived siRNAs. Transformation of tobacco with a RNA interference (RNAi vector targeting CHLI induced Y-Sat-like symptoms. In addition, the symptoms of Y-Sat infection can be completely prevented by transforming tobacco with a silencing-resistant variant of the CHLI gene. These results suggest that siRNA-directed silencing of CHLI is solely responsible for the Y-Sat-induced symptoms. Furthermore, we demonstrate that two Nicotiana species, which do not develop yellowing symptoms upon Y-Sat infection, contain a single nucleotide polymorphism within the siRNA-targeted CHLI sequence. This suggests that the previously observed species specificity of Y-Sat-induced symptoms is due to natural sequence variation in the CHLI gene, preventing CHLI silencing in species with a mismatch to the Y-Sat siRNA. Taken together, these findings provide the first demonstration of small RNA-mediated viral disease symptom production and offer an explanation of the species specificity of the viral disease.

  5. The PLA2 gene mediates the humoral immune responses in Bactrocera dorsalis (Hendel).

    Science.gov (United States)

    Li, Qiujia; Dong, Xiaolong; Zheng, Weiwei; Zhang, Hongyu

    2017-02-01

    The phospholipase A2 (PLA2) gene encodes the enzyme that catalyzes the hydrolysis of phospholipids (PLs) from the sn-2 position. However, little is known about its role in humoral immune responses. In this study, we investigated the expression profile of PLA2 in different tissues and developmental stages in Bactrocera dorsalis (Hendel), and the results showed that the transcriptional level of PLA2 was high in the egg and mature stage and in the testis tissue. Bacterial infection increased the expression of PLA2, and the highest degree of up-regulation appeared in the fat body. Silencing PLA2 influenced the expression of immune-related genes, including MyD88 and defensin in the Toll pathway and relish and diptericin in the Imd pathway. Moreover, the expression of MyD88 and defensin was down-regulated significantly in the ds-PLA2 group compared with those in the ds-egfp group when B. dorsalis was infected with L. monocytogenes and S. aureus, indicating that PLA2 was involved in the activation of the Toll pathway. Meanwhile, infection with L. monocytogenes and E. coli, which activate the Imd pathway, does not increase the mRNA levels of relish and diptericin in the ds-PLA2 group as severely as it increases those in the ds-egfp group, indicating that the Imd pathway was also repressed after silencing PLA2. Notably, the development of lipid droplets in fat body cells was influenced by silencing PLA2, implying that PLA2 affects the function of fat body tissue. These results suggest that the PLA2 gene may mediate humoral immune responses by reducing lipid storage in fat body cells in B. dorsalis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Nutrient-mediated modulation of incretin gene expression: a systematic review

    Directory of Open Access Journals (Sweden)

    R. Martínez-Rodríguez

    Full Text Available Incretins are a cluster of hormones which are secreted and released into the bloodstream after food intake by gut enteroendocrine cells, reaching to pancreas where produce a potentiating effect on insulin release. The aim of this study was to perform a systematic review of incretins gene expression mediated by nutrients using specific search equations in the PubMed database. The two most relevant incretins are GLP-1 and GIP, which come from proglucagon and proGIP precursor respectively. GLP-1 is mainly synthesized and released by ileum and colon L cells, in contrast to GIP which does it by K cells in duodenum and proximal jejunum. It has been shown that canonical Wnt signalling pathway is closely related to the production of these hormones, since transcription factor TCF7L2 affects proglucagon and proGIP gene expression in L and K enteroendocrine cells. On the other hand, it has been shown that the hexosamine biosynthetic pathway can produce N-linked glycosylation of -catenin, an essential component of canonical Wnt signalling. This process hinders β-catenin phosphorylation and, thereby prevents proteasome degradation. Increasing glucose concentration enhances the hexosamine pathway and thus β-catenin glycosylation. This causes a β-catenin cytoplasmic accumulation allowing entry into nucleus, where it exerts its action by binding to a clump of molecules and transcription factors, allowing to express the target genes, including the incretin hormones. There is also evidence that glucose, through the hexosamine pathway, can induces autocrine activation of Wnt signalling pathway by stimulating secretion of Wnt proteins.

  7. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    Science.gov (United States)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  8. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    Science.gov (United States)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  9. Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens.

    Directory of Open Access Journals (Sweden)

    Wenjun Zha

    Full Text Available BACKGROUND: RNA interference (RNAi is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. METHODOLOGY/PRINCIPAL FINDINGS: The Hemipteran insect brown planthopper (Nilaparvata lugens Stål is a typical phloem sap feeder specific to rice (Oryza sativa L.. To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. CONCLUSIONS: Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

  10. Development of a Loop-Mediated Isothermal Amplification Assay Targeting the mpb64 Gene for Diagnosis of Intraocular Tuberculosis

    Science.gov (United States)

    Balne, Praveen Kumar; Barik, Manas Ranjan; Sharma, Savitri

    2013-01-01

    A loop-mediated isothermal amplification (LAMP) assay targeting the mpb64 gene for the diagnosis of intraocular tuberculosis was highly specific (100%), sensitive (85.7%), rapid, and easy to perform. The LAMP assay can be an alternative to conventional PCR for the diagnosis of ocular tuberculosis in resource-limited settings. PMID:23966513

  11. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Marton, L.

    1996-02-01

    Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

  12. Preliminary screening of differentially expressed genes involved in methyl-CpG-binding protein 2 gene-mediated proliferation in human osteosarcoma cells.

    Science.gov (United States)

    Meng, Gang; Li, Yi; Lv, YangFan; Dai, Huanzi; Zhang, Xi; Guo, Qiao-Nan

    2015-04-01

    Methyl-CpG-binding protein 2 (MeCP2) is essential in human brain development and has been linked to several cancer types and neuro-developmental disorders. This study aims to screen the MeCP2 related differentially expressed genes and discover the therapeutic targets for osteosarcoma. CCK8 assay was used to detect the proliferation and SaOS2 and U2OS cells. Apoptosis of cells was detected by flow cytometry analysis that monitored Annexin V-APC/7-DD binding and 7-ADD uptake simultaneously. Denaturing formaldehyde agarose gel electrophoresis was employed to examine the quality of total RNA 18S and 28S units. Gene chip technique was utilized to discover the differentially expressed genes correlated with MeCP2 gene. Differential gene screening criteria were used to screen the changed genes. The gene up-regulation or down-regulation more than 1.5 times was regarded as significant differential expression genes. The CCK8 results indicated that the cell proliferation of MeCP2 silencing cells (LV-MeCP2-RNAi) was significantly decreased compared to non-silenced cells (LV-MeCP2-RNAi-CN) (P genes were screened from a total of 49,395 transcripts. Among the total 107 transcripts, 34 transcripts were up-regulated and 73 transcripts were down-regulated. There were five significant differentially expressed genes, including IGFBP4, HOXC8, LMO4, MDK, and CTGF, which correlated with the MeCP2 gene. The methylation frequency of CpG in IGFBP4 gene could achieve 55%. In conclusion, the differentially expressed IGFBP4, HOXC8, LMO4, MDK, and CTGF genes may be involved in MeCP2 gene-mediated proliferation and apoptosis in osteosarcoma cells.

  13. Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons.

    OpenAIRE

    1991-01-01

    The gene encoding a 67-kDa cytoplasmic guanylate-binding protein (GBP) is transcriptionally induced in cells exposed to interferon of either type I (alpha interferon [IFN-alpha] or type II (IFN-gamma). The promoter of the GBP gene was cloned and found to contain an IFN-alpha-stimulated response element, which mediated the response of the GBP gene to IFN-alpha. On the basis of transfection experiments with recombinant plasmids, two different elements were delineated. Both were required to obta...

  14. Pharmacological and genetic modulation of Wnt-targeted Cre-Lox-mediated gene expression in colorectal cancer cells

    OpenAIRE

    Bordonaro, Michael; Lazarova, Darina L.; Sartorelli, Alan C.

    2004-01-01

    Wnt-targeted gene therapy has been proposed as a treatment for human colorectal cancer (CRC). The Cre-Lox system consists of methodology for enhancing targeted expression from tissue-specific or cancer-specific promoters. We analyzed the efficiency of Wnt-specific promoters as drivers of the Cre-mediated activity of a luciferase reporter gene or cell death effector gene in CRC cell lines in the presence and absence of two modulators of Wnt activity, sodium butyrate and lithium chloride. Butyr...

  15. Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

    Science.gov (United States)

    Nomikou, Nikolitsa; Feichtinger, Georg A; Redl, Heinz; McHale, Anthony P

    2016-01-01

    It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration.

  16. Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy.

    Science.gov (United States)

    Derrick-Roberts, Ainslie L K; Panir, Kavita; Pyragius, Carmen E; Zarrinkalam, Krystyna H; Atkins, Gerald J; Byers, Sharon

    2016-11-01

    Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine β-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

    Science.gov (United States)

    Bire, Solenne; Gosset, David; Jégot, Gwenhael; Midoux, Patrick; Pichon, Chantal; Rouleux-Bonnin, Florence

    2013-09-26

    Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration

  18. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  19. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

    Science.gov (United States)

    Lavitrano, Marialuisa; Bacci, Maria Laura; Forni, Monica; Lazzereschi, Davide; Di Stefano, Carla; Fioretti, Daniela; Giancotti, Paola; Marfé, Gabriella; Pucci, Loredana; Renzi, Luigina; Wang, Hongjun; Stoppacciaro, Antonella; Stassi, Giorgio; Sargiacomo, Massimo; Sinibaldi, Paola; Turchi, Valeria; Giovannoni, Roberto; Della Casa, Giacinto; Seren, Eraldo; Rossi, Giancarlo

    2002-01-01

    A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models. PMID:12393815

  20. Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials.

    Science.gov (United States)

    Wang, Zejing; Tapscott, Stephen J; Chamberlain, Jeffrey S; Storb, Rainer

    2011-01-01

    Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery specific to muscles for treating muscular dystrophies and non-muscle diseases in large animal models and human trials, factors that influence the intensity of the immune responses, and immune modulatory strategies to prevent unwanted immune responses and induce tolerance to the vector and therapeutic gene for a successful gene therapy.

  1. Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer

    Institute of Scientific and Technical Information of China (English)

    WANG Hai-jun; YU Chang-long; Kishi Hiroyuki; Motoki Kazumi; MAO Ze-bin; Muraguchi Atsushi

    2006-01-01

    Background Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor- α (TNF- α ) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-α receptor type Ⅰ (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. Methods Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligment plus medial meniscectomy. Five days after surgery, approximately 1×108 plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1×108 pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1×108 pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue

  2. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  3. Necdin, a p53-target gene, is an inhibitor of p53-mediated growth arrest.

    Directory of Open Access Journals (Sweden)

    Julie Lafontaine

    Full Text Available In vitro, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT, a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability.

  4. Relaxin treatment of solid tumors: effects on electric field-mediated gene delivery.

    Science.gov (United States)

    Henshaw, Joshua; Mossop, Brian; Yuan, Fan

    2008-08-01

    Pulsed electric fields have been shown to enhance interstitial transport of plasmid DNA (pDNA) in solid tumors in vivo. However, the extent of enhancement is still limited partly due to the collagen component in extracellular matrix. To this end, effects of collagen remodeling on interstitial electrophoresis were investigated by pretreatment of tumor-bearing mice with a recombinant human relaxin (rh-Rlx). In the study, two tumor lines (4T1 and B16.F10) were examined and implanted s.c. to establish two murine models: dorsal skin-fold chamber (DSC) and hind leg. Effects of rh-Rlx on pDNA electrophoresis were measured either directly in the DSC model or indirectly in the hind leg model via reporter gene expression. It was observed that rh-Rlx treatment reduced collagen levels in the hind leg tumors but not in the DSC tumors. The observation correlated with the results from electromobility experiments, where rh-Rlx treatment enhanced transgene expression in 4T1 hind leg tumors but did not increase the electromobility of pDNA in the DSC tumors. In addition, it was observed that pDNA binding to collagen could block its diffusion in collagen gel in vitro. These observations showed that effects of rh-Rlx on the collagen content depended on microenvironment in solid tumors and that rh-Rlx treatment would enhance electric field-mediated gene delivery only if it could effectively reduce the collagen content in collagen-rich tumors.

  5. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression.

    Science.gov (United States)

    Kagale, Sateesh; Rozwadowski, Kevin

    2011-02-01

    Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-mediated transcriptional repression is emerging as one of the principal mechanisms of plant gene regulation. The EAR motif, defined by the consensus sequence patterns of either LxLxL or DLNxxP, is the most predominant form of transcriptional repression motif so far identified in plants. Additionally, this active repression motif is highly conserved in transcriptional regulators known to function as negative regulators in a broad range of developmental and physiological processes across evolutionarily diverse plant species. Recent discoveries of co-repressors interacting with EAR motifs, such as TOPLESS (TPL) and AtSAP18, have begun to unravel the mechanisms of EAR motif-mediated repression. The demonstration of genetic interaction between mutants of TPL and AtHDA19, co-complex formation between TPL-related 1 (TPR1) and AtHDA19, as well as direct physical interaction between AtSAP18 and AtHDA19 support a model where EAR repressors, via recruitment of chromatin remodeling factors, facilitate epigenetic regulation of gene expression. Here, we discuss the biological significance of EAR-mediated gene regulation in the broader context of plant biology and present literature evidence in support of a model for EAR motif-mediated repression via the recruitment and action of chromatin modifiers. Additionally, we discuss the possible influences of phosphorylation and ubiquitination on the function and turnover of EAR repressors.

  6. Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium.

    Science.gov (United States)

    Yuan, Lei; Janes, Lauren; Beeler, David; Spokes, Katherine C; Smith, Joshua; Li, Dan; Jaminet, Shou-Ching; Oettgen, Peter; Aird, William C

    2013-05-23

    We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we used transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human β-globin, mouse Down syndrome critical region 1, or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a posttranscriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

  7. An Sp185/333 gene cluster from the purple sea urchin and putative microsatellite-mediated gene diversification

    Directory of Open Access Journals (Sweden)

    Buckley Katherine M

    2010-10-01

    Full Text Available Abstract Background The immune system of the purple sea urchin, Strongylocentrotus purpuratus, is complex and sophisticated. An important component of sea urchin immunity is the Sp185/333 gene family, which is significantly upregulated in immunologically challenged animals. The Sp185/333 genes are less than 2 kb with two exons and are members of a large diverse family composed of greater than 40 genes. The S. purpuratus genome assembly, however, contains only six Sp185/333 genes. This underrepresentation could be due to the difficulties that large gene families present in shotgun assembly, where multiple similar genes can be collapsed into a single consensus gene. Results To understand the genomic organization of the Sp185/333 gene family, a BAC insert containing Sp185/333 genes was assembled, with careful attention to avoiding artifacts resulting from collapse or artificial duplication/expansion of very similar genes. Twelve candidate BAC assemblies were generated with varying parameters and the optimal assembly was identified by PCR, restriction digests, and subclone sequencing. The validated assembly contained six Sp185/333 genes that were clustered in a 34 kb region at one end of the BAC with five of the six genes tightly clustered within 20 kb. The Sp185/333 genes in this cluster were no more similar to each other than to previously sequenced Sp185/333 genes isolated from three different animals. This was unexpected given their proximity and putative effects of gene homogenization in closely linked, similar genes. All six genes displayed significant similarity including both 5' and 3' flanking regions, which were bounded by microsatellites. Three of the Sp185/333 genes and their flanking regions were tandemly duplicated such that each repeated segment consisted of a gene plus 0.7 kb 5' and 2.4 kb 3' of the gene (4.5 kb total. Both edges of the segmental duplications were bounded by different microsatellites. Conclusions The high sequence

  8. Suppression of gastric cancer growth by adenovirus-mediated transfer of the PTEN gene

    Institute of Scientific and Technical Information of China (English)

    Ying Hang; Yong-Chen Zheng; Yan Cao; Qing-Shan Li; Yu-Jie Sui

    2005-01-01

    AIM: To investigate the tumor-suppressive effect of the phosphatase and tensin homologue deleted from chromosome (PTEN) in human gastric cancer cells th atwere wild type for PTEN.METHODS: Adenoviruses expressing PTEN or luciferase as a control were introduced into gastric cancer cells.The effect of exogenous PTEN gene on the growth and apoptosis of gastric cancer cells that are wtPTEN were examined in vitro and in vivo.RESULTS: Adenovirus-mediated transfer of PTEN (AdPTEN) suppressed cell growth and induced apoptosis significantly in gastric cancer cells (MGC-803, SGC-7901)carrying wtPTEN in comparison with that in normal gastric epithelial cells (GES-1) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase and mitogen-activated protein kinase and cell-cycle arrest at the G2/M phase but not at the G1 phase. Furthermore,treatment of human gastric tumor xenografts (MGC-803,SGC-7901) with Ad-PTEN resulted in a significant (P<0.01)suppression of tumor growth.CONCLUSION: These results indicate a significant tumorsuppressive effect of Ad-PTEN against human gastric cancer cells. Thus, Ad-PTEN may be used as a potential therapeutic strategy for treatment of gastric cancers.

  9. Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

    Science.gov (United States)

    Le, Huy Quang; Ghatak, Sushmita; Yeung, Ching-Yan Chloé; Tellkamp, Frederik; Günschmann, Christian; Dieterich, Christoph; Yeroslaviz, Assa; Habermann, Bianca; Pombo, Ana; Niessen, Carien M; Wickström, Sara A

    2016-08-01

    Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

  10. Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma

    Directory of Open Access Journals (Sweden)

    Margarita Gutova

    2017-03-01

    Full Text Available Despite improved survival for children with newly diagnosed neuroblastoma (NB, recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs expressing a modified rabbit carboxylesterase (rCE can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11 to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093 and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

  11. Angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium

    Institute of Scientific and Technical Information of China (English)

    DONG Shu-qiang; ZHANG Bao-ren; MEI Ju; XU Zhi-yun; ZOU Liang-jian; HUANG Sheng-dong

    2002-01-01

    Objective: To study the angiogenesis effects of adenovirus-mediated gene transfer of VEGF-B on chronic ischemic myocardium. Methods: Domestic pigs underwent thoracotomy and placement of an ameroid constrictor on the circumflex coronary artery. Four weeks later, Ad. VEGF-B, Ad. LacZ or PBS were administrated directly into the myocardium at 10 sites in the circumflex distribution (109 PFU or 100 μl) according to groups. Echocardiography and ex vivo coronary angiography were performed. The injection sites around myocardium were harvested and subjected to histological analysis and immunochemical staining. Results: Echocardiography assessment 4 weeks after vector administration demonstrated significant improvement of regional wall systolic function. Collateral vesseldevelopment assessed by angiography was also significantly greater in Ad. VEGF-B animals than that in control animals. Vascular density analysis revealed a mean of 43±5 neovessels per high-power field in Ad.VEGF-B group versus 19±4 and 17±6 in Ad.LacZ and PBS group. Conclusion:Direct intramyocardial administration of Ad.VEGF-B can induce focal angiogenesis and result in improvement in regional myocardial function, which may be useful in patients with ischemic heart disease who are not eligible for conventional therapies.

  12. Genetic environments of the transferable plasmid-mediated blaCTX-M-3 gene in Serratia marcescens isolates.

    Science.gov (United States)

    Chu, Pei-Yu; Peng, Chien-Fang

    2014-01-01

    In this study, genetic environments of the transferable plasmid-mediated blaCTX-M-3 gene were characterized among 14 isolates of cefotaxime-resistant Serratia marcescens using PCR and BLAST DNA sequence analysis. A total of 3 types of genetic architectures in the regions surrounding this blaCTX-M-3 gene were identified. Type I architecture was characterized by the presence of a complete insertion sequence of tnpA-ISEcp1, identified as interrupting a reverse IS26 sequence in the upstream region of the blaCTX-M-3 gene. A reverse-directional orf477 fragment was located downstream of the blaCTX-M-3 gene, which was in the same direction of the mucA gene. A common region containing the orf513 element was located upstream of the mucA gene. Moreover, a copy of the 3'-CS2 element was located immediately upstream of the orf513 element. A novel complex class 1 integron was characterized by the presence of the dfrA19 gene, which was flanked by two copies of class 1 integrons. This is the first report to describe the dfrA19 gene within a novel complex class 1 integron in S. marcescens isolates from Taiwan. This novel complex class 1 integron structure was located distantly upstream of the blaCTX-M-3 gene.

  13. Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua.

    Science.gov (United States)

    Li, Hang; Jiang, Weihua; Zhang, Zan; Xing, Yanru; Li, Fei

    2013-01-01

    The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1(st) to 5(th) instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4(th) instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20-94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (Ppest control.

  14. Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus.

    Science.gov (United States)

    Suzuki, Ayako; Fujii, Hiroshi; Hoshida, Hisashi; Akada, Rinji

    2015-09-01

    Gene expression analysis provides valuable information to evaluate cellular state. Unlike quantitative mRNA analysis techniques like reverse-transcription PCR and microarray, expression analysis using a reporter gene has not been commonly used for multiple-gene analysis, probably due to the difficulty in preparing multiple reporter-gene constructs. To circumvent this problem, we developed a novel one-step reporter-gene construction system mediated by non-homologous end joining (NHEJ) in the yeast Kluyveromyces marxianus. As a selectable reporter gene, the ScURA3 selection marker was fused in frame with a red fluorescent gene yEmRFP (ScURA3:yEmRFP). The N-terminally truncated ScURA3:yEmRFP fragment was prepared by PCR. Promoter sequences were also prepared by PCR using primers containing the sequence of the deleted ScURA3 N-terminus to attach at their 3(') ends. The two DNA fragments were used for the transformation of a ura3(-) strain of K. marxianus, in which two DNA fragments are randomly joined and integrated into the chromosome through NHEJ. Only the correctly aligned fragments produced transformants on uracil-deficient medium and expressed red fluorescence under the control of the introduced promoters. A total of 36 gene promoters involved in glycolysis and other pathways were analyzed. Fluorescence measurements of these strains allowed real-time gene expression analysis in different culture conditions.

  15. Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu 8988

    Institute of Scientific and Technical Information of China (English)

    PAN Xue; LI Zhao-shen; XU Guo-ming; CUI Long; ZHANG Su-zhen; GONG Yan-fang; TU Zhen-xing

    2001-01-01

    Objective: To evaluate the in vitro killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic carcinoma. Methods: Cytosine Deaminase (CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria, and the products containing green fluorescent protein (GFP)were propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line 8988 were infected with this virus, then 5-FC was added; XTT assay was used to estimate the relative numbers of viable cells. Results: The positive clones were confirmed by using endonuclease digestion, and the titer of the virus containing CD gene was 2 × 1011 pfu/ml. It was found that 5-FC possessed significant cytotoxic activities for CD gene transfected 8988cell line, but had little effects on non-transfected pancreatic carcinoma cells. Conclusion: CD gene mediated by adenovirus has a high infectivity and is efficient for killing cultured pancreatic carcinoma cells, indicating suicide gene may be effective for pancreatic cancer in furure.

  16. Suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific-1 gene

    Institute of Scientific and Technical Information of China (English)

    Wei Huang; Xiang-Long Tian; Yun-Lin Wu; Jie Zhong; Li-Fen Yu; Sheng-Ping Hu; Biao Li

    2008-01-01

    AIM: To study the inhibitory effect of baculovirus-mediated normal epithelial celt specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo.METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry.RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.

  17. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Directory of Open Access Journals (Sweden)

    Ashok Sharma

    Full Text Available BACKGROUND: Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. METHODOLOGY/PRINCIPAL FINDINGS: WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV. We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes, endoplasmic reticulum (22 genes and vacuole (8 genes were significantly over represented. CONCLUSIONS/SIGNIFICANCE: In this study we have identified key molecular pathways and downstream

  18. Hepatic gene expression profiling reveals key pathways involved in leptin-mediated weight loss in ob/ob mice.

    Science.gov (United States)

    Sharma, Ashok; Bartell, Shoshana M; Baile, Clifton A; Chen, Bo; Podolsky, Robert H; McIndoe, Richard A; She, Jin-Xiong

    2010-08-16

    Leptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss. WE UTILIZED MICROARRAY TECHNOLOGY TO COMPARE HEPATIC GENE EXPRESSION CHANGES AFTER TWO TYPES OF LEPTIN ADMINISTRATION: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ) and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV). We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes), endoplasmic reticulum (22 genes) and vacuole (8 genes) were significantly over represented. In this study we have identified key molecular pathways and downstream genes which respond to leptin treatment and are involved in leptin-mediated weight

  19. [Experimental study of MAT1 gene silencing mediated by siRNA in pancreatic cancer].

    Science.gov (United States)

    Liu, Jian-ping; Yuan, Shi-zhen; Zhang, Shi-neng

    2007-10-16

    To investigate the inhibitory effect of gene silencing mediated by MAT1-siRNA constructed in vitro transcription for pancreatic cancer in vivo and in vitro. 21-nt double strand siRNA targeting MAT1 gene was constructed and labeled with Cy3 fluorescent labeling reagent. Human pancreatic cancer cells of the line BxPC3 were cultured and divided into 4 groups: MAT1-siRNA transfected group, negative siRNA control group, lipid control group, and blank control group. The rate of cell duplication was determined by counting the cells for three consecutive days. Cell cycle profiles were determined by flow cytometry. Semi-quantitative analysis of the level of MAT1-mRNA expression was performed using the RT-PCR technique. The level of MAT1 protein expression was analyzed by Western-blotting. 18 nude mice were injected subcutaneously with BxPC3 cells to establish mouse tumor models, and then divided randomly into 3 equal groups: MAT1-siRNA group undergoing injection of MAT1-siRNA directly into the tumors 2 times a week for 4 weeks, blank control group, and negative MAT1-siRNA group. 4 weeks later the mice were killed to observe the weight and size of tumor and to calculate the tumor inhibition rate. Two of the 4 designed MAT1-siRNAs significantly suppressed the growth of the BxPC3 cells. 72 h after transfection the cell duplication was inhibited by 34.9% in the MAT1-siRNA transfection group. The cell cycle profile showed 83.9% of the MAT1-siRNA transfected cells were in the G0/G1 phase, a rate significantly higher than that in the blank control group (59.86%, P < 0.01). 48 h later the content of MAT1-mRNA of the MAT1-siRNA transfected cells was significantly reduced by 80.12%, and 72 h after the transfection the content of MAT1 protein was reduced by 50.12%, a rate significantly higher than those of the 2 control groups (both P < 0.01). The weight and volume of the transplant tumors in the MAT1-siRNA injected nude mice were significantly reduced compare with the negative si

  20. Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune-mediated diseases

    Directory of Open Access Journals (Sweden)

    Walker Neil M

    2006-03-01

    Full Text Available Abstract Background The identification of the HLA class II, insulin (INS, CTLA-4 and PTPN22 genes as determinants of type 1 diabetes (T1D susceptibility indicates that fine tuning of the immune system is centrally involved in disease development. Some genes have been shown to affect several immune-mediated diseases. Therefore, we tested the hypothesis that alleles of susceptibility genes previously associated with other immune-mediated diseases might perturb immune homeostasis, and hence also associate with predisposition to T1D. Methods We resequenced and genotyped tag single nucleotide polymorphisms (SNPs from two genes, CRP and FCER1B, and genotyped 27 disease-associated polymorphisms from thirteen gene regions, namely FCRL3, CFH, SLC9A3R1, PADI4, RUNX1, SPINK5, IL1RN, IL1RA, CARD15, IBD5-locus (including SLC22A4, LAG3, ADAM33 and NFKB1. These genes have been associated previously with susceptibility to a range of immune-mediated diseases including rheumatoid arthritis (RA, systemic lupus erythematosus (SLE, Graves' disease (GD, psoriasis, psoriatic arthritis (PA, atopy, asthma, Crohn disease and multiple sclerosis (MS. Our T1D collections are divided into three sample subsets, consisting of set 1 families (up to 754 families, set 2 families (up to 743 families, and a case-control collection (ranging from 1,500 to 4,400 cases and 1,500 to 4,600 controls. Each SNP was genotyped in one or more of these subsets. Our study typically had approximately 80% statistical power for a minor allele frequency (MAF >5% and odds ratios (OR of 1.5 with the type 1 error rate, α = 0.05. Results We found no evidence of association with T1D at most of the loci studied 0.02 P ADAM33, rs2787094, was any evidence of association obtained, P = 0.0004 in set 1 families (relative risk (RR = 0.78, but further support was not observed in the 4,326 cases and 4,610 controls, P = 0.57 (OR = 1.02. Conclusion Polymorphisms in a variety of genes previously associated with

  1. Radiochemotherapy of hepatocarcinoma via lentivirus-mediated transfer of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen Libo, E-mail: libochen888@hotmail.com [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China); Guo Guoying [Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Liu Tianjing; Guo Lihe [Division of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Zhu Ruisen [Department of Nuclear Medicine, Shanghai Sixth People' s Hospital, Shanghai Jiao Tong University, Shanghai 200233 (China)

    2011-07-15

    Herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system has been widely used as a traditional gene therapy modality, and the sodium/iodide symporter gene (NIS) has been found to be a novel therapeutic gene. Since the therapeutic effects of radioiodine therapy or prodrug chemotherapy on cancers following NIS or HSV-TK gene transfer need to be enhanced, this study was designed to investigate the feasibility of radiochemotherapy for hepatocarcinoma via coexpression of NIS gene and HSV-TK gene. Methods: HepG2 cells were stably transfected with NIS, TK and GFP gene via recombinant lentiviral vector and named HepG2/NTG. Gene expression was examined by reverse transcriptase polymerase chain reaction, fluorescence imaging and iodide uptake. The therapeutic effects were assessed by MTT assay and clonogenic assay. Results: HepG2/NTG cells concentrated {sup 125}I{sup -} up to 76-fold higher than the wild-type cells within 20 min, and the efflux happened with a T{sub 1/2eff} of less than 10 min. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dose-dependent toxicity to HepG2/NTG cells by either GCV or {sup 131}I was revealed by clonogenic assay and MTT assay, respectively. The survival rate of HepG2/NTG cells decreased to 49.7%{+-}2.5%, 43.4%{+-}2.8% and 8.6%{+-}1.2% after exposure to {sup 131}I, GCV and combined therapy, respectively. Conclusion: We demonstrate that radiochemotherapy of hepatocarcinoma via lentiviral-mediated coexpression of NIS gene and HSV-TK gene leads to stronger killing effect than single treatment, and in vivo studies are needed to verify these findings.

  2. Combating oncogene activation associated with retrovirus-mediated gene therapy of X-linked severe combined immunodeficiency

    Directory of Open Access Journals (Sweden)

    B.E. Strauss

    2007-05-01

    Full Text Available A successful gene therapy clinical trial that also encountered serious adverse effects has sparked extensive study and debate about the future directions for retrovirus-mediated interventions. Treatment of X-linked severe combined immunodeficiency with an oncoretrovirus harboring a normal copy of the gc gene was applied in two clinical trials, essentially curing 13 of 16 infants, restoring a normal immune system without the need for additional immune-related therapies. Approximately 3 years after their gene therapy, tragically, 3 of these children, all from the same trial, developed leukemia as a result of this experimental treatment. The current understanding of the mechanism behind this leukemogenesis involves three critical and cooperating factors, i.e., viral integration, oncogene activation, and the function of the therapeutic gene. In this review, we will explore the causes of this unwanted event and some of the possibilities for reducing the risk of its reoccurrence.

  3. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium -mediated system

    Institute of Scientific and Technical Information of China (English)

    翟文学; 李晓兵; 田文忠; 周永力; 潘学彪; 曹守云; 赵显峰; 赵彬; 章琦; 朱立煌

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  4. Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.

  5. Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.) using the expansin 10 (CsEXP10) gene.

    Science.gov (United States)

    Sun, Y D; Luo, W R; Sun, S Y; Ni, L

    2015-12-08

    The cucumber expansin 10 (CsEXP10) gene was previously cloned from young cucumber fruits but its role has not been defined. To determine the role of this gene in plant growth and development, a CsEXP10 gene transformation system was established. The open reading frame of the gene was inserted behind the CaMV35S promoter of vector pCAMBIA1301, and the construct was introduced into tomato plants by Agrobacterium-mediated transformation. In total, 19 kanamycin-positive lines were produced and nine independent transgenic lines were identified by β-glucuronidase and polymerase chain reaction (PCR) analysis. Quantitative real-time PCR analysis showed that levels of the CsEXP10 transcript were higher in transgenic lines than in a non-transgenic line.

  6. Delivery of surface-mediated non-viral gene nanoparticles from ultrathin layer-by-layer multilayers

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    An efficient and safe gene delivery system remains a challenge in the development of gene therapy.Polycation-based gene nanoparticles are a typical non-viral gene delivery system,which are able to transfect cells in vitro and in vivo.This paper reported a facile method for constructing biodegradable multilayers via layer-by-layer self-assembly,in which the polycation-based gene nanoparticles were loaded.Through this surface-mediated delivery system,adherent cells on the multilayer could be transfected in situ.Gene nanoparticles-loaded multilayers transfect cells with higher efficiency than naked DNA-loaded multilayers because of the complex configuration of the DNA.DNA nanoparticles/PGA multilayers constructed on the scaffold surface could also realize in situ transfection on the adherent cells.The well-structured,easy-processed multilayers may provide a novel approach to precisely controlled delivery of gene nanoparticles,which may have potential applications for gene therapy in tissue engineering and medical implants.

  7. Core clock, SUB1, and ABAR genes mediate flooding and drought responses via alternative splicing in soybean.

    Science.gov (United States)

    Syed, Naeem H; Prince, Silvas J; Mutava, Raymond N; Patil, Gunvant; Li, Song; Chen, Wei; Babu, Valliyodan; Joshi, Trupti; Khan, Saad; Nguyen, Henry T

    2015-12-01

    Circadian clocks are a great evolutionary innovation and provide competitive advantage during the day/night cycle and under changing environmental conditions. The circadian clock mediates expression of a large proportion of genes in plants, achieving a harmonious relationship between energy metabolism, photosynthesis, and biotic and abiotic stress responses. Here it is shown that multiple paralogues of clock genes are present in soybean (Glycine max) and mediate flooding and drought responses. Differential expression of many clock and SUB1 genes was found under flooding and drought conditions. Furthermore, natural variation in the amplitude and phase shifts in PRR7 and TOC1 genes was also discovered under drought and flooding conditions, respectively. PRR3 exhibited flooding- and drought-specific splicing patterns and may work in concert with PRR7 and TOC1 to achieve energy homeostasis under flooding and drought conditions. Higher expression of TOC1 also coincides with elevated levels of abscisic acid (ABA) and variation in glucose levels in the morning and afternoon, indicating that this response to abiotic stress is mediated by ABA, endogenous sugar levels, and the circadian clock to fine-tune photosynthesis and energy utilization under stress conditions. It is proposed that the presence of multiple clock gene paralogues with variation in DNA sequence, phase, and period could be used to screen exotic germplasm to find sources for drought and flooding tolerance. Furthermore, fine tuning of multiple clock gene paralogues (via a genetic engineering approach) should also facilitate the development of flooding- and drought-tolerant soybean varieties. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Changes in nucleosome position at transcriptional start sites of specific genes in Zea mays mediator of paramutation1 mutants.

    Science.gov (United States)

    Labonne, Jonathan D J; Dorweiler, Jane E; McGinnis, Karen M

    2013-04-01

    Nucleosomes facilitate compaction of DNA within the confines of the eukaryotic nucleus. This packaging of DNA and histone proteins must accommodate cellular processes, such as transcription and DNA replication. The repositioning of nucleosomes to facilitate cellular processes is likely regulated by several factors. In Zea mays, Mediator of paramutation1 (MOP1) has been demonstrated to be an epigenetic regulator of gene expression. Based on sequence orthology and mutant phenotypes, MOP1 is likely to function in an RNA-dependent pathway to mediate changes to chromatin. High-resolution microarrays were used to assay the distribution of nucleosomes across the transcription start sites (TSSs) of ~400 maize genes in wild type and mutant mop1-1 tissues. Analysis of nucleosome distribution in leaf, immature tassel and ear shoot tissues resulted in the identification of three genes showing consistent differences in nucleosome positioning and occupancy between wild type and mutant mop1-1. These specific changes in nucleosome distribution were located upstream as well as downstream of the TSS. No direct relationship between the specific changes in nucleosome distribution and transcription were observed through quantitative expression analysis in these tissues. In silico prediction suggests that nucleosome positioning is not dictated by intrinsic DNA sequence signals in the TSSs of two of the identified genes, suggesting a role for chromatin remodeling proteins in MOP1-mediated pathways. These results also indicate that MOP1 contributions to nucleosome position may be either separate from changes in gene expression, or cooperative with development and other levels of regulation in coordinating gene expression.

  9. Spatial and temporal assessment of pollen- and seed-mediated gene flow from genetically engineered plum Prunus domestica.

    Science.gov (United States)

    Scorza, Ralph; Kriss, Alissa B; Callahan, Ann M; Webb, Kevin; Demuth, Mark; Gottwald, Tim

    2013-01-01

    Pollen flow from a 0.46 ha plot of genetically engineered (GE) Prunus domestica located in West Virginia, USA was evaluated from 2000-2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera) were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree) to 0.033-0.017% at longer distances (384-998 m). Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards.

  10. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma

    Institute of Scientific and Technical Information of China (English)

    Cheng YI; Ying HUANG; Zhi-ying GUO; Shu-ren WANG

    2005-01-01

    Aim: To construct a Bifidobacterium infantis/CD targeting gene therapy system and observe the antitumor effect of cytosine deaminase/5-fluorocytosine (CD/5-FC) suicide gene therapy system mediated by Bifidobacterium infantis on melanoma in vitro and in vivo. Methods: A recombinant CD/pGEX- 1LamdaT plasmid was transfected into Bifidobacterium infantis by electroporation. Bifidobac terium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid was in cubated with 5-FC anaerobically. Then the supernatant fluid was collected and added to melanoma B16-F10 cells to observe the killing effect for B16-F10 cells.Mice were inoculated with melanoma B 16-F10 cells to establish animal models.The mice were then injected with 5-FC and Bifidobacterium infantis transfected by recombinant CD/pGEX-1LamdaT plasmid. Results:Two segments of approxi mate 4.9 kb and 1.3 kb were extracted from the 6.2 kb recombinant plasmid, which were equal to the size of the pGEX-1LamdaT plasmid and CD gene, respectively.Sequencing results showed that the full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. In vitro, B 16-F10 cells treated by supernatant fluid were remarkably damaged morphologically, and the cell growth was significantly inhibited. Experi ments on the mice melanoma model showed that after treatment with a combination of transfected Bifidobacterium infantis and 5-FC, the tumor volume was significantly inhibited compared with controls. Conclusion: The foreign gene,CD gene, was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium infantis. CD/5-FC suicide gene therapy system mediated by Bifidobacterium infantis demonstrated a good antitumor effect on melanoma in vitro and in vivo.

  11. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  12. Spatial and temporal assessment of pollen- and seed-mediated gene flow from genetically engineered plum Prunus domestica.

    Directory of Open Access Journals (Sweden)

    Ralph Scorza

    Full Text Available Pollen flow from a 0.46 ha plot of genetically engineered (GE Prunus domestica located in West Virginia, USA was evaluated from 2000-2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree to 0.033-0.017% at longer distances (384-998 m. Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards.

  13. Clinical and Microbiological Aspects of Linezolid Resistance Mediated by the cfr Gene Encoding a 23S rRNA Methyltransferase▿

    Science.gov (United States)

    Arias, Cesar A.; Vallejo, Martha; Reyes, Jinnethe; Panesso, Diana; Moreno, Jaime; Castañeda, Elizabeth; Villegas, Maria V.; Murray, Barbara E.; Quinn, John P.

    2008-01-01

    The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method. PMID:18174304

  14. A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport of genes.

    Science.gov (United States)

    Henshaw, Joshua W; Zaharoff, David A; Mossop, Brian J; Yuan, Fan

    2006-10-01

    The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.

  15. Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

    Institute of Scientific and Technical Information of China (English)

    ZHOU Xi-yuan; LIAO Qing; PU Yi-min; TANG Yong-qiang; GONG Xiao; LI Jia; XU Yan; WANG Zhi-gang

    2009-01-01

    Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with

  16. SYNERGISTIC EFFICACY OF ADENOVIRUS-MEDIATED BCL-XS GENE TRANSFER AND TOPOTECAN IN OVARIAN CANCER CELL

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To observe the synergistic efficacy between Adenovirus-mediated bcl-Xs(Adv-bcl-Xs) gene transfer and chemotherapy on ovarian cancer cell growth. Methods: NuTu-19 cells were infected by different titers of Adv-bcl-Xs and treated with topotecan in the meantime. Cell proliferation was measured 3 days later by MTT. Graphical representations and statistical analyses for their interaction in tumor cells were done. Results: The statistical result and Graphical representations of the statistical modeling showed synergy effect on cell growth inhibition (P<0.01). Conclusion: There were synergistic efficacies between Adv-bcl-Xs gene therapy and Topotecan in ovarian cancer cell growth.

  17. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish

    2010-07-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  18. Lentiviral Mediating Genetic Engineered Mesenchymal Stem Cells for Releasing IL-27 as a Gene Therapy Approach for Autoimmune Diseases

    OpenAIRE

    Shohreh Hajizadeh-Sikaroodi; Ahmad Hosseini; Ali Falla; Hajar Estiri; Zahra Noormohammadi; Mohammad Salehi; Sayyed Mohammad Hossein Ghaderian; Haleh Akhavan Niaki; Masoud Soleimani; Bahram Kazemi

    2014-01-01

    Objective: Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in...

  19. Mulberroside A suppresses PXR-mediated transactivation and gene expression of P-gp in LS174T cells.

    Science.gov (United States)

    Li, Yuhua; Huang, Ling; Sun, Jiahong; Wei, Xiaohua; Wen, Jinhua; Zhong, Guoping; Huang, Min; Bi, Huichang

    2016-12-05

    Mulberroside A (Mul A) is the main bioactive constituents of Sangbaipi, which is officially listed in the Chinese Pharmacopoeia. The pregnane X receptor (PXR) has been recognized as the critical mediator of human P-glycoprotein (P-gp) gene transactivation. In this study, the effect of Mul A on PXR-mediated transactivation and gene expression of P-gp was investigated. It was found that Mul A significantly suppressed PXR-mediated P-gp luciferase activity induced by rifampicin (Rif). Furthermore, Rif induced an elevation of P-gp expression and transport activity, which was apparently suppressed by Mul A. However, Mul A did not suppress the P-gp luciferase activity, P-gp expression, and function in the absence of Rif. These findings suggest that Mul A suppresses PXR-mediated transactivation and P-gp expression induced by Rif. This should be taken into consideration to predict any potential herb-drug interactions when Mul A or Sangbaipi are co-administered with Rif or other PXR agonist drugs.

  20. Model-based calculating tool for pollen-mediated gene flow frequencies in plants.

    Science.gov (United States)

    Lei, Wang; Bao-Rong, Lu

    2016-12-30

    The potential social-economic and environmental impacts caused by transgene flow from genetically engineered (GE) crops have stimulated worldwide biosafety concerns. To determine transgene flow frequencies resulted from pollination is the first critical step for assessing such impacts, in addition to the determination of transgene expression and fitness in crop-wild hybrid descendants. Two methods are commonly used to estimate pollen-mediated gene flow (PMGF) frequencies: field experimenting and mathematical modeling. Field experiments can provide relatively accurate results but are time/resource consuming. Modeling offers an effective complement for PMGF experimental assessment. However, many published models describe PMGF by mathematical equations and are practically not easy to use. To increase the application of PMGF modeling for the estimation of transgene flow, we established a tool to calculate PMGF frequencies based on a quasi-mechanistic PMGF model for wind-pollination species. This tool includes a calculating program displayed by an easy-operating interface. PMGF frequencies of different plant species can be quickly calculated under different environmental conditions by including a number of biological and wind speed parameters that can be measured in the fields/laboratories or obtained from published data. The tool is freely available in the public domain (http://ecology.fudan.edu.cn/userfiles/cn/files/Tool_Manual.zip). Case studies including rice, wheat, and maize demonstrated similar results between the calculated frequencies based on this tool and those from published PMGF data. This PMGF calculating tool will provide useful information for assessing and monitoring social-economic and environmental impacts caused by transgene flow from GE crops. This tool can also be applied to determine the isolation distances between GE and non-GE crops in a coexistence agro-ecosystem, and to ensure the purity of certified seeds by setting proper isolation distances

  1. Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation.

    Science.gov (United States)

    Wang, Jianbo; Hamblet, Natasha S; Mark, Sharayne; Dickinson, Mary E; Brinkman, Brendan C; Segil, Neil; Fraser, Scott E; Chen, Ping; Wallingford, John B; Wynshaw-Boris, Anthony

    2006-05-01

    The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.

  2. Citral and eugenol modulate DNA damage and pro-inflammatory mediator genes in murine peritoneal macrophages.

    Science.gov (United States)

    Porto, Marilia de Paula; da Silva, Glenda Nicioli; Luperini, Bruno Cesar Ottoboni; Bachiega, Tatiana Fernanda; de Castro Marcondes, João Paulo; Sforcin, José Maurício; Salvadori, Daisy Maria Fávero

    2014-11-01

    Citral and eugenol have been broadly studied because of their anti-inflammatory, antioxidant and antiparasitic potentials. In this study, the effects of citral (25, 50 and 100 µg/mL) and eugenol (0.31, 0.62, 1.24 and 2.48 µg/mL) on the expression (RT-PCR) of the pro-inflammatory mediator genes NF-κB1, COX-2 and TNF-α were evaluated in mouse peritoneal macrophages with or without activation by a bacterial lipopolysaccharide (LPS). Additionally, the genotoxic potentials of two compounds and their capacities to modulate the DNA damage induced by doxorubicin (DXR) were investigated using the comet assay. The data revealed that neither citral nor eugenol changed COX-2, NF-κB1 or TNF-α expression in resting macrophages. However, in LPS-activated cells, citral induced the hypoexpression of COX-2 (100 µg/mL) and TNF-α (50 and 100 µg/mL). Hypoexpression of TNF-α was also detected after cellular exposure to eugenol at the highest concentration (2.48 µg/mL). Both compounds exhibited genotoxic potential (citral at 50 and 100 µg/mL and eugenol at all concentrations) but also showed chemopreventive effects, in various treatment protocols. Both citral and eugenol might modulate inflammatory processes and DXR-induced DNA damage, but the use of these compounds must be viewed with caution because they are also able to induce primary DNA lesions.

  3. Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer

    Indian Academy of Sciences (India)

    Tiago Collares; Vinicius Farias Campos; Fabiana Kömmling Seixas; Paulo V Cavalcanti; Odir A Dellagostin; Heden Luiz M Moreira; João Carlos Deschamps

    2010-03-01

    The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E, INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups ( > 0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology

  4. Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

    Science.gov (United States)

    Rey-Rico, Ana; Cucchiarini, Magali

    2016-04-01

    Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

  5. Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

    Science.gov (United States)

    Herzog, Katja; Flachowsky, Henryk; Deising, Holger B; Hanke, Magda-Viola

    2012-04-25

    Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene.

  6. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

    Science.gov (United States)

    Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

    2002-02-05

    Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

  7. [Study on the effect of Klotho gene interferred by plasmid-mediated short hairpin RNA (shRNA) on sinoatrial node pacing channel gene].

    Science.gov (United States)

    Cai, Yingying; Wang, Han; Hou, Yanbin; Fang, Chenli; Tian, Peng; Wang, Guihua; Li, Lu; Deng, Juelin

    2013-06-01

    The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  8. Pollen-mediated gene flow from glyphosate-resistant common waterhemp (Amaranthus rudis Sauer): consequences for the dispersal of resistance genes

    Science.gov (United States)

    Sarangi, Debalin; Tyre, Andrew J.; Patterson, Eric L.; Gaines, Todd A.; Irmak, Suat; Knezevic, Stevan Z.; Lindquist, John L.; Jhala, Amit J.

    2017-01-01

    Gene flow is an important component in evolutionary biology; however, the role of gene flow in dispersal of herbicide-resistant alleles among weed populations is poorly understood. Field experiments were conducted at the University of Nebraska-Lincoln to quantify pollen-mediated gene flow (PMGF) from glyphosate-resistant (GR) to -susceptible (GS) common waterhemp using a concentric donor-receptor design. More than 130,000 common waterhemp plants were screened and 26,199 plants were confirmed resistant to glyphosate. Frequency of gene flow from all distances, directions, and years was estimated with a double exponential decay model using Generalized Nonlinear Model (package gnm) in R. PMGF declined by 50% at pollen source, whereas 90% reduction was found at 88 m (maximum) depending on the direction of the pollen-receptor blocks. Amplification of the target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), was identified as the mechanism of glyphosate resistance in parent biotype. The EPSPS gene amplification was heritable in common waterhemp and can be transferred via PMGF, and also correlated with glyphosate resistance in pseudo-F2 progeny. This is the first report of PMGF in GR common waterhemp and the results are critical in explaining the rapid dispersal of GR common waterhemp in Midwestern United States. PMID:28327669

  9. Effect of neutralizing sera on factor X-mediated adenovirus serotype 5 gene transfer

    NARCIS (Netherlands)

    Parker, A.L.; Waddington, S.N.; Buckley, S.M.K.; Custers, J.; Havenga, M.J.E.; Rooijen, N. van; Goudsmit, J.; McVey, J.H.; Nicklin, S.A.; Baker, A.H.

    2009-01-01

    The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated

  10. Induction of electrophile-responsive element (EpRE)-mediated gene expression by tomato extracts in vitro.

    Science.gov (United States)

    Gijsbers, Linda; van Eekelen, Henriëtte D L M; Nguyen, Thuy H; de Haan, Laura H J; van der Burg, Bart; Aarts, Jac M M J G; Rietjens, Ivonne M C M; Bovy, Arnaud G

    2012-12-01

    The market for food products with additional health benefits is increasing rapidly and tools for identification of bio-functional characteristics of food items are essential. To facilitate the detection of beneficial effects of tomato on gene expression, methods to prepare tomato extracts suitable to test in the EpRE LUX assay and other cell-based reporter gene assays for health-related bioactivity mechanisms, were developed. An isoprenoid-containing chloroform extract of tomato fruit and most individual isoprenoids did not induce electrophile-responsive element (EpRE)-mediated gene expression. A semi-polar extract of tomato fruits, enzymatically hydrolysed to remove the glycosyl residues from the phenolic ingredients was able to induce EpRE-mediated luciferase expression at both mRNA and protein level, which might be partly due to the presence of quercetin, kaempferol, naringenin and naringenin chalcone. It was concluded that induction of EpRE-regulated genes, such as detoxifying phase II and antioxidant enzymes, may contribute to the beneficial health effects of tomato.

  11. HDAC2 selectively regulates FOXO3a-mediated gene transcription during oxidative stress-induced neuronal cell death.

    Science.gov (United States)

    Peng, Shengyi; Zhao, Siqi; Yan, Feng; Cheng, Jinbo; Huang, Li; Chen, Hong; Liu, Qingsong; Ji, Xunming; Yuan, Zengqiang

    2015-01-21

    All neurodegenerative diseases are associated with oxidative stress-induced neuronal death. Forkhead box O3a (FOXO3a) is a key transcription factor involved in neuronal apoptosis. However, how FOXO3a forms complexes and functions in oxidative stress processing remains largely unknown. In the present study, we show that histone deacetylase 2 (HDAC2) forms a physical complex with FOXO3a, which plays an important role in FOXO3a-dependent gene transcription and oxidative stress-induced mouse cerebellar granule neuron (CGN) apoptosis. Interestingly, we also found that HDAC2 became selectively enriched in the promoter region of the p21 gene, but not those of other target genes, and inhibited FOXO3a-mediated p21 transcription. Furthermore, we found that oxidative stress reduced the interaction between FOXO3a and HDAC2, leading to an increased histone H4K16 acetylation level in the p21 promoter region and upregulated p21 expression in a manner independent of p53 or E2F1. Phosphorylation of HDAC2 at Ser 394 is important for the HDAC2-FOXO3a interaction, and we found that cerebral ischemia/reperfusion reduced phosphorylation of HDAC2 at Ser 394 and mitigated the HDAC2-FOXO3a interaction in mouse brain tissue. Our study reveals the novel regulation of FOXO3a-mediated selective gene transcription via epigenetic modification in the process of oxidative stress-induced cell death, which could be exploited therapeutically.

  12. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    Science.gov (United States)

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  13. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.

  14. Ex-Vivo Gene Therapy Using Lentiviral Mediated Gene Transfer Into Umbilical Cord Blood Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Hanieh Jalali

    2016-02-01

    Full Text Available Background Introduction of therapeutic genes into the injured site of nervous system can be achieved using transplantation of cellular vehicles containing desired gene. To transfer exogenous genes into the cellular vehicles, lentiviral vectors are one of interested vectors because of advantages such high transduction efficiency of dividing and non-dividing cells. Unrestricted somatic stem cells are subclasses of umbilical cord blood derived stem cells which are appreciate candidates to use as cellular vehicles for ex vivo gene therapy of nervous system. Objectives In current study we investigated the effect of lentiviral vector transduction on the neuronal related features of unrestricted somatic stem cells to indicate the probable and unwanted changes related to transduction procedure. Materials and Methods In this experimental study, lentiviral vector containing green fluorescent protein (GFP were transduced into unrestricted somatic stem cells and its effect was investigated with using MTT assay, qPCR and immunohistochemistry techniques. For statistical comparison of real time PCR results, REST software (2009, Qiagen was used. Results Obtained results showed lentiviral vector transduction did not have cytotoxic effects on unrestricted somatic stem cells and did not change neuronal differentiation capacity of them as well the expression of some neuronal related genes and preserved them in multilineage situation. Conclusions In conclusion, we suggested that lentiviral vectors could be proper vectors to transfer therapeutic gene into unrestricted somatic stem cells to provide a cellular vehicle for ex vivo gene therapy of nervous system disorders.

  15. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  16. A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

    Institute of Scientific and Technical Information of China (English)

    Xiao-Fu Zhou; Peng-Da Ma; Ren-Hou Wang; Bo Liu; Xing-Zhi Wang

    2006-01-01

    A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunlt (rbcS) gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena,transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

  17. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

  18. Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control

    Science.gov (United States)

    Diop, Soda Balla; Bertaux, Karine; Vasanthi, Dasari; Sarkeshik, Ali; Goirand, Benjamin; Aragnol, Denise; Tolwinski, Nicholas S; Cole, Michael D; Pradel, Jacques; Yates, John R; Mishra, Rakesh K; Graba, Yacine; Saurin, Andrew J

    2008-01-01

    Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output. PMID:18259215

  19. Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

    Institute of Scientific and Technical Information of China (English)

    HUANG Jia-quan; SUN Zhong-hai

    2005-01-01

    The Arabidopsis ICE1 (inducer of CBF expression 1) gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon (Citrus Limon (L.) Burm. F. cv. Eureka) genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICE1 gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

  20. Post operative infection and sepsis in humans is associated with deficient gene expression of gammac cytokines and their apoptosis mediators.

    LENUS (Irish Health Repository)

    White, Mary

    2011-06-28

    Abstract Introduction Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators. Methods The study population consisted of a total of 60 patients with severe sepsis, 15 with gram negative bacteraemia, 10 healthy controls and 60 patients undergoing elective lung resection surgery. Pneumonia was diagnosed by CDC NNIC criteria. Gene expression in peripheral blood leukocytes (PBLs) of interleukin (IL)-2, 7, 15 and interferon (IFN)-γ, Bax, Bim, Bcl-2 was determined by qRT-PCR and IL-2 and IL-7 serum protein levels by ELISA. Gene expression of IL-2, 7 and IFN-γ was measured in peripheral blood leukocytes (PBL), cultured in the presence of lipopolysacharide (LPS) and CD3 binding antibody (CD3ab) Results IL-2 gene expression was lower in the bacteraemia group compared with controls, and lower still in the sepsis group (P < 0.0001). IL-7 gene expression was similar in controls and bacteraemia, but lower in sepsis (P < 0.0001). IL-15 gene expression was similar in the three groups. Bcl-2 gene expression was less (P < 0.0001) and Bim gene expression was greater (P = 0.0003) in severe sepsis compared to bacteraemic and healthy controls. Bax gene expression was similar in the three groups. In lung resection surgery patients, post-operative pneumonia was associated with a perioperative decrease in IL-2 mRNA (P < 0.0001) and IL-7 mRNA (P = 0.003). IL-2 protein levels were reduced in sepsis and bacteraemia compared to controls (P = 0.02) but similar in pneumonia and non-pneumonia groups. IL-7 protein levels were similar in all groups. In cultured PBLs, IFN-γ gene expression was decreased in response to LPS and increased in response to CD3ab with sepsis: IL-7 gene expression increased in response to LPS in controls and to CD3ab with sepsis; Bcl-2 gene expression decreased in response to combined CD3ab and IL-2 with sepsis

  1. Post-operative infection and sepsis in humans is associated with deficient gene expression of γc cytokines and their apoptosis mediators.

    LENUS (Irish Health Repository)

    White, Mary

    2011-06-01

    Lymphocyte homeostasis is dependent on the γc cytokines. We hypothesised that sepsis in humans is associated with differential gene expression of the γc cytokines and their associated apoptosis mediators.

  2. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

    Science.gov (United States)

    Gantz, Valentino M; Jasinskiene, Nijole; Tatarenkova, Olga; Fazekas, Aniko; Macias, Vanessa M; Bier, Ethan; James, Anthony A

    2015-12-08

    Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

  3. Do candidate genes mediating conspecific sperm precedence affect sperm competitive ability within species? A test case in Drosophila.

    Science.gov (United States)

    Civetta, Alberto; Finn, Scott

    2014-07-16

    When females mate to multiple males, the last male to mate fathers the majority of progeny. When males of different species inseminate a female, the sperm of the male conspecific to the female is favored in fertilization in a process known as conspecific sperm precedence (CSP). A large number of studies in Drosophila have assayed the genetic basis of sperm competition, with a main focus on D. melanogaster and accessory gland protein genes. Only a few studies have attempted to disentangle the genetic basis of CSP between related species of Drosophila. Although there is no a priori reason to believe that genes influencing intraspecific sperm competitive ability might also mediate conspecific sperm precedence, no study has addressed the question. Here, we test a group of candidate CSP genes between D. simulans and D. mauritiana for their effect on sperm competition in D. melanogaster. The use of P-element insertion lines identified CG14891 gene disruption as the only one causing a significant decrease in second male paternity success relative to wild-type and ebony tester males. The gene disruption affected both sperm displacement and the sperm fertilizing ability. Out of five genes tested using RNA interference, only gene knockdown of CG6864(Mst89B) [corrected] significantly reduced the male's ability to father progeny when second to mate. Our results suggest that CG14891 and CG6468 might have been co-opted from an intraspecies gene function (i.e., sperm competition) into an interspecies avoidance phenotype (i.e., CSP). Alternatively, the dual role of these genes could be a consequence of their pleiotropic roles. Copyright © 2014 Civetta and Finn.

  4. Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

    Science.gov (United States)

    Kwon, Young-Ju; Park, Mi-Jeong; Kim, Sang-Gyu; Baldwin, Ian T; Park, Chung-Mo

    2014-05-19

    The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5' splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress

  5. IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

    Science.gov (United States)

    Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar

    2014-01-01

    Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system.

  6. ShRNA-mediated gene silencing of β-catenin inhibits growth of human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205.METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry.RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level.The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing.CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.

  7. Enhancing effect of ultrasound-mediated microbubble destruction on gene delivery into rat kidney via different administration routes

    Institute of Scientific and Technical Information of China (English)

    Jun-Xiang Chen; Qiang Ma; Hong Wu; An Zhou; Xing Chen; You-Ming Peng; Fu-You Liu; Mei-Chu Cheng

    2012-01-01

    ABSTRACT Objective:To investigate the efficiency of -galactosidase gene transfer into rat kidney with ultrasound-mediated microbubble destruction via different injection routes.Methods:A total of25 Wistar rats were randomly divided into5 groups. Four groups received a mixture of optison microbubbles (0.2 mL) and lacz plasmids (25 g) injection via renal artery, tail vein, anterior tibial muscle and renal parenchyma, respectively. The control group received a mixture ofPBS (xx mL) and lacz plasmids (25 g) via renal artery. Three days after the gene transfer, ultrasound with fixed frequency and power (1 MHz, xxW) was delivered to the kidneys for3 min. The efficiency of the gene transfer and expression was evaluated on the basis of β-galactosidase expression. The side effects of this method were evaluated by immunohistological method. Results:β-galactosidase expression could be observed only in tubules but not in glomeruli and interstitial area. The efficiency of renal artery group was higher than that of tail vein, anterior tibial muscle and renal parenchyma group (P<0.05). Immunohistochemical analysis revealed co-expression of -galactosidase with a roximal tubule marker, megalin, which suggested that ultrasound enhanced gene transfer into the proximal tubular epithelial cells. No -galactosidase expression was observed in the extrarenal organs. There were no evident pathological and biochemical changes after gene transfer.Conclusions:Ultrasound-mediated microbubble destruction can transfer gene into kidney via renal artery, tail vein, anterior tibial muscle and renal parenchyma. Compared with renal artery, administrating microbubbles via tail vein and anterior tibial muscle are more convenient and less vulnerarious.

  8. Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

    Institute of Scientific and Technical Information of China (English)

    LI Xiu-ying; LANG Zhi-hong; ZHANG Jie; HE Kang-lai; ZHU Li; HUANG Da-fang

    2014-01-01

    A novel insecticidal gene cry1Ah was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active Cry1Ah toxin has a toxicity level similar to that of the full-length Cry1Ah toxin. In this study, plant expression vector pMhGM harboring truncated cry1Ah gene was transformed into maize (Zea mays L.) immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions (madMARs) were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efifciency of 5%around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer (Ostrinia furnacalis). These two events were further conifrmed by molecular analysis. Southern blot suggested that a single copy of the cry1Ah gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene cry1Ah was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T1-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

  9. A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination

    Directory of Open Access Journals (Sweden)

    Vassetzky Yegor S

    2008-12-01

    Full Text Available Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418 and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kγ replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. Conclusion Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.

  10. AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

    Directory of Open Access Journals (Sweden)

    Arjen J Boender

    Full Text Available Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5 in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.

  11. AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

    Science.gov (United States)

    Boender, Arjen J; Koning, Nivard A; van den Heuvel, José K; Luijendijk, Mieneke C M; van Rozen, Andrea J; la Fleur, Susanne E; Adan, Roger A H

    2014-01-01

    Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.

  12. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata: an important tool for functional study of genes and crop improvement

    Directory of Open Access Journals (Sweden)

    Evans eNyaboga

    2014-09-01

    Full Text Available Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp. with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by PCR, Southern blot analysis and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by RT-PCR analysis. Transformation efficiency varied from 9.4% to 18.2% depending on the cultivars, selectable marker genes and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop.

  13. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement.

    Science.gov (United States)

    Nyaboga, Evans; Tripathi, Jaindra N; Manoharan, Rajesh; Tripathi, Leena

    2014-01-01

    Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3-4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important

  14. Viral vector-mediated gene transfer in fundamental and applied cardiovascular research

    NARCIS (Netherlands)

    Swildens, Jim

    2012-01-01

    To treat various cardiac diseases, modification of gene expression for the purpose of increased or decreased expression of a particular gene, is regarded as a potential therapy. As a vehicle to introduce the gene of choice into the heart cell, virus vectors have given the most promising results. Thi

  15. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes

    NARCIS (Netherlands)

    Romano, A.; Raemakers, C.J.J.M.; Bernardi, J.; Visser, R.G.F.; Mooibroek, A.

    2003-01-01

    Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transf

  16. Genome-wide expression analysis of rice aquaporin genes and development of a functional gene network mediated by aquaporin expression in roots.

    Science.gov (United States)

    Nguyen, Minh Xuan; Moon, Sunok; Jung, Ki-Hong

    2013-10-01

    The world population continually faces challenges of water scarcity for agriculture. A common strategy called water-balance control has evolved to adapt plant growth to these challenges. Aquaporins are a family of integral membrane proteins that play a central role in water-balance control. In this study, we identified 34 members of the rice aquaporin gene family, adding a novel member to the previous list. A combination of phylogenetic tree and anatomical meta-expression profiling data consisting of 983 Affymetrix arrays and 209 Agilent 44 K arrays was used to identify tissue-preferred aquaporin genes and evaluate functional redundancy among aquaporin family members. Eight aquaporins showed root-preferred expression in the vegetative growth stage, while 4 showed leaf/shoot-preferred expression. Integrating stress-induced expression patterns into phylogenetic tree and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that 3 rice aquaporin genes were markedly downregulated and 4 were upregulated by water deficiency in the root, suggesting that these candidate genes are key regulators of water uptake from the soil. Finally, we constructed a functional network of genes mediated by water stress and refined the network by confirming the differential expression using RT-PCR and real-time PCR. Our data will be useful to elucidate the molecular mechanism of water-balance control in rice root.

  17. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    Science.gov (United States)

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  18. Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: functional characterization of the CYP82E10 gene.

    Science.gov (United States)

    Lewis, Ramsey S; Bowen, Steven W; Keogh, Matthew R; Dewey, Ralph E

    2010-12-01

    In most tobacco (Nicotiana tabacum L.) plants, nornicotine is a relatively minor alkaloid, comprising about 2-5% of the total pyridine alkaloid pool in the mature leaf. Changes in gene expression at an unstable locus, however, can give rise to plants that produce high levels of nornicotine, specifically during leaf senescence and curing. Minimizing the nornicotine content in tobacco is highly desirable, because this compound serves as the direct precursor in the synthesis of N'-nitrosonornicotine, a potent carcinogen in laboratory animals. Nornicotine is likely produced almost entirely via the N-demethylation of nicotine, in a process called nicotine conversion that is catalyzed by the enzyme nicotine N-demethylase (NND). Previous studies have identified CYP82E4 as the specific NND gene responsible for the unstable conversion phenomenon, and CYP82E5v2 as a putative minor NND gene. Here, by discovery and characterization of CYP82E10, a tobacco NND gene, is reported. PCR amplification studies showed that CYP82E10 originated from the N. sylvestris ancestral parent of modern tobacco. Using a chemical mutagenesis strategy, knockout mutations were induced and identified in all three tobacco NND genes. By generating a series of mutant NND genotypes, the relative contribution of each NND gene toward the nornicotine content of the plant was assessed. Plants possessing knockout mutations in all three genes displayed nornicotine phenotypes that were much lower (∼0.5% of total alkaloid content) than that found in conventional tobacco cultivars. The introduction of these mutations into commercial breeding lines promises to be a viable strategy for reducing the levels of one of the best characterized animal carcinogens found in tobacco products.

  19. Literature-Based Discovery of IFN-γ and Vaccine-Mediated Gene Interaction Networks

    Directory of Open Access Journals (Sweden)

    Arzucan Özgür

    2010-01-01

    Full Text Available Interferon-gamma (IFN-γ regulates various immune responses that are often critical for vaccine-induced protection. In order to annotate the IFN-γ-related gene interaction network from a large amount of IFN-γ research reported in the literature, a literature-based discovery approach was applied with a combination of natural language processing (NLP and network centrality analysis. The interaction network of human IFN-γ (Gene symbol: IFNG and its vaccine-specific subnetwork were automatically extracted using abstracts from all articles in PubMed. Four network centrality metrics were further calculated to rank the genes in the constructed networks. The resulting generic IFNG network contains 1060 genes and 26313 interactions among these genes. The vaccine-specific subnetwork contains 102 genes and 154 interactions. Fifty six genes such as TNF, NFKB1, IL2, IL6, and MAPK8 were ranked among the top 25 by at least one of the centrality methods in one or both networks. Gene enrichment analysis indicated that these genes were classified in various immune mechanisms such as response to extracellular stimulus, lymphocyte activation, and regulation of apoptosis. Literature evidence was manually curated for the IFN-γ relatedness of 56 genes and vaccine development relatedness for 52 genes. This study also generated many new hypotheses worth further experimental studies.

  20. The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes

    Directory of Open Access Journals (Sweden)

    Johnson Julie K

    2012-06-01

    Full Text Available Abstract Background Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product 1. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI, has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs from multiple-case, non- BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. Methods We applied GINI to a total of 24 LCLs, established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition. Results The expression profiling identified a total of eight candidate genes from these three families. One gene, PPARGC1A, was a candidate in two separate families. We performed semi-quantitative real-time reverse transcriptase PCR of all candidate genes but only PPARGC1A showed successful validation by being stabilised in individuals with breast cancer but not in many unaffected members of the same family. Sanger sequencing of all coding and splice site regions of PPARGC1A did not reveal any protein

  1. Achieving high gene delivery performance with caveolae-mediated endocytosis pathway by (l)-arginine/(l)-histidine co-modified cationic gene carriers.

    Science.gov (United States)

    Li, Hui; Luo, Ting; Sheng, Ruilong; Sun, Jingjing; Wang, Zhao; Cao, Amin

    2016-12-01

    Developing new amphiphilic polymers with natural product moieties has been regarded as a promising way to achieve biocompatibility and certain biological functions. In prior work, we developed some natural (l)-arginine modified cationic polymers (PAHMAA-Rs) as cationic gene carriers. For the sake of continuing optimize the gene delivery performance, herein, a new series of (l)-arginine and (l)-histidine co-modified cationic poly (ω-aminohexyl methacrylamide)s (PAHMAA-R-H) were synthesized and characterized with (1)H NMR, GPC-SLS and FT-IR. Their proton buffering capacities were studied by acid-base titration assay. pDNA binding affinity and self-assembly properties of the polyplexes were analyzed by agarose gel retardation assay, DLS and AFM, respectively. In vitro cytotoxicity of the PAHMAA-R-H was determined by MTT and LDH assays in H1299 cells, the gene transfection efficacy and intracellular uptake capability were evaluated by luciferase assay and FACS, respectively. Moreover, the endocytosis pathways and intracellular distribution of the polyplexes were investigated by using specific endocytic inhibitors and fluorescent co-localization techniques. The results demonstrated that co-modification of (l)-arginine and (l)-histidine onto the PAHMAA polymer could enhance proton buffering capacity, shield surface charge, decrease cytotoxicity, and improve gene transfection efficiency and serum-compatibility. Moreover, the gene transfection and intracellular uptake behaviors were disclosed strongly rely on the (l)-arginine/(l)-histidine modification ratios. The polyplexes tend to be internalized through caveolae-mediated endocytosis gateway and localized with endosomes/lysosomes in H1299 cells. Notably, among the polymers, the PAHMAA-R18-H6 exhibited remarkable gene delivery efficiency and serum compatibility, which made it promising gene transfection agent for practical application.

  2. Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

    Institute of Scientific and Technical Information of China (English)

    刘启功; 陆再英; 岳远坤; 林立; 张卫东; 颜进

    2004-01-01

    This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer.The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

  3. Retroviral-mediated transfer and functional expression of multidrug resistance gene in human placenta mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    HAN Li-ying; YE Ming-zhu; LI Ya-ping; WANG Bo-wei; WANG Qiang; ZHAO Shu-hua; LI He-lian

    2008-01-01

    Background Most of gynecologic malignancies are sensitive to chemotherapy. Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdrl) gene is well known for its ability to confer drug resistance. This study aimed to explore the feasibility of expression and resistance of mdrl gene transduction into human placenta mesenchymal stem cells (P-MSCs) by retrovirus vector.Methods Human P-MSCs were isolated from trypsin-digested term placentas, and their immunophenotypes and differentiation potential were evaluated. Human P-MSCs were transduced by reconstructed retroviral vector containing the mdrl gene and green fluorescent protein (GFP) reporter gene. The integration and expression of the mdrl gene were observed indirectly by the expression of GFP, and fluorescence-activated cell sorter was used to evaluate the functional activity of permeability glycoprotein (P-gp) encoded by the mdrl gene. The stimulating test was made in vitro to show pleiotropic drug resistance of transfected cells.Results The isolated, cultured and expanded P-MSCs expressed stem cell markers such as CD29, CD44 and CD73,and showed osteogenic and adipogenic differentiation potentials under appropriate conditions. The expression of P-gp in the non-transfected P-MSCs cells was (0.4±0.1)%, but increased to (28.1±4.7)% after gene transfection (P<0.01). And positive staining of P-gp located mainly at cell membrane and cytoplasm. Accumulation and extrusion assays showed that P-gp expressed by the transfected cells had pump-functional activity and could efflux daunomycin out of cells. The analysis of cell survival confirmed that transfected P-MSCs had a characteristic of multidrug resistance with a significant increase in the resistance to anticancer agents.Conclusions Transfer and expression of human mdrl gene mediated by retrovirus vector conferred P-MSCs drug resistance. It might provide a new alternative to chemoprotection strategies.

  4. I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

    Science.gov (United States)

    Ouedraogo, Jean Paul; Arentshorst, Mark; Nikolaev, Igor; Barends, Sharief; Ram, Arthur F J

    2015-12-01

    Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.

  5. A search theory model of patch-to-patch forager movement with application to pollinator-mediated gene flow.

    Science.gov (United States)

    Hoyle, Martin; Cresswell, James E

    2007-09-07

    We present a spatially implicit analytical model of forager movement, designed to address a simple scenario common in nature. We assume minimal depression of patch resources, and discrete foraging bouts, during which foragers fill to capacity. The model is particularly suitable for foragers that search systematically, foragers that deplete resources in a patch only incrementally, and for sit-and-wait foragers, where harvesting does not affect the rate of arrival of forage. Drawing on the theory of job search from microeconomics, we estimate the expected number of patches visited as a function of just two variables: the coefficient of variation of the rate of energy gain among patches, and the ratio of the expected time exploiting a randomly chosen patch and the expected time travelling between patches. We then consider the forager as a pollinator and apply our model to estimate gene flow. Under model assumptions, an upper bound for animal-mediated gene flow between natural plant populations is approximately proportional to the probability that the animal rejects a plant population. In addition, an upper bound for animal-mediated gene flow in any animal-pollinated agricultural crop from a genetically modified (GM) to a non-GM field is approximately proportional to the proportion of fields that are GM and the probability that the animal rejects a field.

  6. An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype.

    Science.gov (United States)

    Koda, Y; Soejima, M; Johnson, P H; Smart, E; Kimura, H

    2000-01-01

    Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.

  7. Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

    Science.gov (United States)

    Lee, K H; Song, S U; Hwang, T S; Yi, Y; Oh, I S; Lee, J Y; Choi, K B; Choi, M S; Kim, S J

    2001-09-20

    Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

  8. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles.

    Science.gov (United States)

    Lopes, Cátia Df; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies.

  9. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    Science.gov (United States)

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

  10. Pollen-mediated gene flow and seed exchange in small-scale Zambian maize farming, implications for biosafety assessment

    Science.gov (United States)

    Bøhn, Thomas; Aheto, Denis W.; Mwangala, Felix S.; Fischer, Klara; Bones, Inger Louise; Simoloka, Christopher; Mbeule, Ireen; Schmidt, Gunther; Breckling, Broder

    2016-10-01

    Gene flow in agricultural crops is important for risk assessment of genetically modified (GM) crops, particularly in countries with a large informal agricultural sector of subsistence cultivation. We present a pollen flow model for maize (Zea mays), a major staple crop in Africa. We use spatial properties of fields (size, position) in three small-scale maize farming communities in Zambia and estimate rates of cross-fertilisation between fields sown with different maize varieties (e.g. conventional and transgene). As an additional factor contributing to gene flow, we present data on seed saving and sharing among farmers that live in the same communities. Our results show that: i) maize fields were small and located in immediate vicinity of neighboring fields; ii) a majority of farmers saved and shared seed; iii) modeled rates of pollen-mediated gene flow showed extensive mixing of germplasm between fields and farms and iv) as a result, segregation of GM and non-GM varieties is not likely to be an option in these systems. We conclude that the overall genetic composition of maize, in this and similar agricultural contexts, will be strongly influenced both by self-organised ecological factors (pollen flow), and by socially mediated intervention (seed recycling and sharing).

  11. T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis.

    Science.gov (United States)

    Sequeira, Ana Filipa; Guerreiro, Catarina I P D; Vincentelli, Renaud; Fontes, Carlos M G A

    2016-09-01

    Efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. Here, we analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, data presented here reveal that incorporation of a mutation-removal step including T7 endonuclease I can effectively improve the fidelity of artificial gene synthesis.

  12. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer.

    Science.gov (United States)

    Rivella, Stefano; May, Chad; Chadburn, Amy; Rivière, Isabelle; Sadelain, Michel

    2003-04-15

    Patients affected by beta-thalassemia major require lifelong transfusions because of insufficient or absent production of the beta chain of hemoglobin (Hb). A minority of patients are cured by allogeneic bone marrow transplantation. In the most severe of the hitherto available mouse models of beta-thalassemia, a model for human beta-thalassemia intermedia, we previously demonstrated that globin gene transfer in bone marrow cells is curative, stably raising Hb levels from 8.0-8.5 to 11.0-12.0 g/dL in long-term chimeras. To fully assess the therapeutic potential of gene therapy in the context of a lethal anemia, we now have created an adult model of beta(0)-thalassemia major. In this novel model, mice engrafted with beta-globin-null (Hbb(th3/th3)) fetal liver cells succumb to ineffective erythropoiesis within 60 days. These mice rapidly develop severe anemia (2-4 g/dL), massive splenomegaly, extramedullary hematopoiesis (EMH), and hepatic iron overload. Remarkably, most mice (11 of 13) treated by lentivirus-mediated globin gene transfer were rescued. Long-term chimeras with an average 1.0-2.4 copies of the TNS9 vector in their hematopoietic and blood cells stably produced up to 12 g/dL chimeric Hb consisting of mu alpha(2):hu beta(2) tetramers. Pathologic analyses indicated that erythroid maturation was restored, while EMH and iron overload dramatically decreased. Thus, we have established an adult animal model for the most severe of the hemoglobinopathies, Cooley anemia, which should prove useful to investigate both genetic and pharmacologic treatments. Our findings demonstrate the remarkable efficacy of lentivirus-mediated globin gene transfer in treating a fulminant blood disorder and strongly support the efficacy of gene therapy in the severe hemoglobinopathies.

  13. Overexpression of two α-esterase genes mediates metabolic resistance to malathion in the oriental fruit fly, Bactrocera dorsalis (Hendel).

    Science.gov (United States)

    Wang, L-L; Huang, Y; Lu, X-P; Jiang, X-Z; Smagghe, G; Feng, Z-J; Yuan, G-R; Wei, D; Wang, J-J

    2015-08-01

    Esterase has been reported to be involved in malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel). However, the underlying molecular mechanism of the esterase-mediated resistance remains largely unknown in this species. Here, with the use of a strain selected for malathion resistance in the laboratory (MR), we found that two overexpressed α-esterase genes, namely BdCarE4 and BdCarE6, predominant in the adult midgut and fat body, function in conferring malathion resistance in B. dorsalis. Notably, these two genes were found to be mostly close to the esterase E3, which are usually implicated in detoxifying organophosphate insecticides. The transcript levels of BdCarE4 and BdCarE6 were investigated and compared between the MR and a susceptible (MS) strain of B. dorsalis. Both genes were significantly up-regulated in the MR strain, which was consistent with the enhanced esterase activity in the MR strain. However, no changes in either the coding sequence or gene copy number were observed between the two strains. Subsequently, heterologous expression combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE4 and BdCarE6 can probably detoxify malathion. Furthermore, RNA interference-mediated knockdown of each of these two genes significantly increased malathion susceptibility in the MR strain adults. In conclusion, these results expand our molecular understanding of the important role of α-esterases during the development of resistance to organophosphorous insecticides in B. dorsalis. © 2015 The Royal Entomological Society.

  14. Effect of bile on nisin-mediated antibacterial activity and the expression of nisin genes of Lactococcus lactis W8.

    Science.gov (United States)

    Mitra, Suranjita; Mukhopadhyay, Bidhan Chandra; Chakrabartty, Pran Krishna; Biswas, Swadesh Ranjan

    2013-12-01

    The capability of Lactococcus lactis to produce nisin in the presence of bile in the intestinal environment remains an intriguing question. The aim of this study was to determine the effects of bile on production of nisin and the mRNA expression of nisin genes of L. lactis W8. The strain L. lactis W8 was grown on glucose in the absence and presence of bile (0.005-0.08 %) and the antibacterial activities of culture supernatants were determined. In culture with 0.035 % bile, the nisin activity was significantly reduced (400 AU/mL) within 5 h compared to that in the control without bile (2000 AU/mL), while growth of the cells was only slightly affected. In the presence of 0.07 % bile no nisin activity of the strain was manifested. Consistent with these results, mRNA expression of nisin-biosynthetic genes nisZ, nisRK, nisI, and nisF was down-regulated by 7.5-, 2.5-, 1.7-, and 6.0-fold, respectively in cells grown in the presence of bile (0.07 %) as compared to control culture without bile. The present study suggested that bile inhibited transcription of nisin genes. Nisin-production in intestine by orally administered L. lactis, thus, does not occur since complete inhibition of nisin-production by bile is observed at a concentration much lower than the physiological concentration (0.3 %) of bile present in the human intestine. The molecular mechanism underlying the bile-mediated inhibition of nisin genes remains to be elucidated. This is the first report on bile-mediated inhibition of nisin genes.

  15. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  16. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus.

    Science.gov (United States)

    Liu, Yule; Schiff, Michael; Marathe, Rajendra; Dinesh-Kumar, S P

    2002-05-01

    The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.

  17. Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes

    DEFF Research Database (Denmark)

    Lykke-Andersen, Søren; Chen, Yun; Ardal, Britt;

    2014-01-01

    Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogen......Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities...... yield alternative transcript isoforms that allow for differential expression of individual coencoded snoRNAs. Based on our findings, we hypothesize that snoRNA host genes need to be highly transcribed to accommodate high levels of snoRNA production and that the expression of individual sno...

  18. A single qrr gene is necessary and sufficient for LuxO-mediated regulation in Vibrio fischeri.

    Science.gov (United States)

    Miyashiro, Tim; Wollenberg, Michael S; Cao, Xiaodan; Oehlert, Dane; Ruby, Edward G

    2010-09-01

    All members of the Vibrionaceae harbour LuxO, a response regulator that integrates outputs from various signalling systems, ultimately controlling specific traits that are crucial to the distinct biology of each species. LuxO is phosphorylated in response to low cell density, activating the transcription of a family of small RNAs called Qrrs, which in turn, control the levels of a global regulatory protein conserved within the Vibrionaceae. Although the function of each Qrr is similar, the number of qrr genes varies among the different species. Using a bioinformatics approach, we have determined the number of qrr genes in fully sequenced Vibrionaceae members. Phylogenetic analysis suggests the most recent common ancestor of all Vibrionaceae shared a single, ancestral qrr gene, which duplicated and diverged into multiple qrr genes in some present-day vibrio lineages. To demonstrate that a single qrr gene is sufficient to mediate repression of LitR, the global regulator in Vibrio fischeri, we have performed a series of genetic and phenotypic analyses of the LuxO pathway and its output. Our studies contribute to a better understanding of the ancestral state of these pathways in vibrios, as well as to the evolution and divergence of other sRNAs within different bacterial lineages. © 2010 Blackwell Publishing Ltd.

  19. Midkine promoter-driven suicide gene expression and -mediated adenovirus replication produced cytotoxic effects to immortalised and tumour cells.

    Science.gov (United States)

    Yu, L; Hamada, K; Namba, M; Kadomatsu, K; Muramatsu, T; Matsubara, S; Tagawa, M

    2004-07-01

    We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

  20. A RGD-Containing Oligopeptide (K)16GRGDSPC: A Novel Vector for Integrin-Mediated Targeted Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    PAN Haitao; ZHENG Qixin; GUO Xiaodong; LIU Yong; LI Changwen; SONG Yulin

    2006-01-01

    A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). Synthesis of the peptide (K)16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by (K)16GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of (K)16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs' attachment to the 96-well plates pretreated with fibronectin or vitronectin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide (K)16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

  1. Responses of plasmid-mediated quinolone resistance genes and bacterial taxa to (fluoro)quinolones-containing manure in arable soil.

    Science.gov (United States)

    Xiong, Wenguang; Sun, Yongxue; Ding, Xueyao; Zhang, Yiming; Zhong, Xiaoxia; Liang, Wenfei; Zeng, Zhenling

    2015-01-01

    The aim of the present study was to investigate the fate of plasmid-mediated quinolone resistance (PMQR) genes and the disturbance of soil bacterial communities posed by (fluoro)quinolones (FQNs)-containing manure in arable soil. Representative FQNs (enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)), PMQR genes (qepA, oqxA, oqxB, aac(6')-Ib-cr and qnrS) and bacterial communities in untreated soil, +manure and +manure+FQNs groups were analyzed using culture independent methods. The significantly higher abundance of oqxA, oqxB and aac(6')-Ib-cr, and significantly higher abundance of qnrS in +manure group than those in untreated soil disappeared at day 30 and day 60, respectively. All PMQR genes (oqxA, oqxB, aac(6')-Ib-cr and qnrS) dissipated 1.5-1.7 times faster in +manure group than those in +manure+FQNs group. The disturbance of soil bacterial communities posed by FQNs-containing manure was also found. The results indicated that significant effects of PMQR genes (oqxA, oqxB, aac(6')-Ib and qnrS) on arable soils introduced by manure disappeared 2 month after manure application. FQNs introduced by manure slowed down the dissipation of PMQR genes. The presence of high FQNs provided a selective advantage for species affiliated to the phylum including Acidobacteria, Verrucomicrobia and Planctomycetes while suppressing Proteobacteria and Actinobacteria.

  2. Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

    Institute of Scientific and Technical Information of China (English)

    Lei Zhou; Jun Zhao; Wangzhen Guo; Tianzhen Zhang

    2013-01-01

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi.However,the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood.Here,we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes,VdATG8 and VdATG12,by means of targeted gene replacement and complementadon.Transformation of a cotton-infecting Verticilliun dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 106 conidia.V.dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production.Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants,compared with the wildtype and gene complemented strains.Surprisingly,in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants.These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V.dahliae.

  3. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    Directory of Open Access Journals (Sweden)

    Ziga Zebec

    2016-10-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  4. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  5. Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum.

    Science.gov (United States)

    Wolabu, Tezera W; Zhang, Fei; Niu, Lifang; Kalve, Shweta; Bhatnagar-Mathur, Pooja; Muszynski, Michael G; Tadege, Million

    2016-05-01

    Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.

  6. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

    Science.gov (United States)

    Wan, Fengyi; Anderson, D Eric; Barnitz, Robert A; Snow, Andrew; Bidere, Nicolas; Zheng, Lixin; Hegde, Vijay; Lam, Lloyd T; Staudt, Louis M; Levens, David; Deutsch, Walter A; Lenardo, Michael J

    2007-11-30

    NF-kappaB is a DNA-binding protein complex that transduces a variety of activating signals from the cytoplasm to specific sets of target genes. To understand the preferential recruitment of NF-kappaB to specific gene regulatory sites, we used NF-kappaB p65 in a tandem affinity purification and mass spectrometry proteomic screen. We identified ribosomal protein S3 (RPS3), a KH domain protein, as a non-Rel subunit of p65 homodimer and p65-p50 heterodimer DNA-binding complexes that synergistically enhances DNA binding. RPS3 knockdown impaired NF-kappaB-mediated transcription of selected p65 target genes but not nuclear shuttling or global protein translation. Rather, lymphocyte-activating stimuli caused nuclear translocation of RPS3, parallel to p65, to form part of NF-kappaB bound to specific regulatory sites in chromatin. Thus, RPS3 is an essential but previously unknown subunit of NF-kappaB involved in the regulation of key genes in rapid cellular activation responses. Our observations provide insight into how NF-kappaB selectively controls gene expression.

  7. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    Science.gov (United States)

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  8. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    Science.gov (United States)

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-04-26

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.

  9. Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer

    Directory of Open Access Journals (Sweden)

    Choi Jin-Ok

    2010-04-01

    Full Text Available Abstract Background Enzyme replacement therapy (ERT with α-galactosidase A (α-Gal A is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding α-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The α-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of α-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the α-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. α-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher α-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more α-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The α-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated α-Gal A gene

  10. Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate

    DEFF Research Database (Denmark)

    Hasman, Henrik; Kempf, I.; Chidaine, B.;

    2006-01-01

    The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets...... in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes...

  11. Combining Cytotoxic and Immune-Mediated Gene Therapy to Treat Brain Tumors

    OpenAIRE

    Curtin, James; King, Gwendalyn; Candolfi, Marianela; Greeno, Remy; Kroeger, Kurt; Lowenstein, Pedro; Castro,Maria

    2005-01-01

    Glioblastoma (GBM) is a type of intracranial brain tumor, for which there is no cure. In spite of advances in surgery, chemotherapy and radiotherapy, patients die within a year of diagnosis. Therefore, there is a critical need to develop novel therapeutic approaches for this disease. Gene therapy, which is the use of genes or other nucleic acids as drugs, is a powerful new treatment strategy which can be developed to treat GBM. Several treatment modalities are amenable for gene therapy implem...

  12. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    Science.gov (United States)

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

  13. Transcriptome analysis reveals response regulator SO2426-mediated gene expression in Shewanella oneidensis MR-1 under chromate challenge

    Directory of Open Access Journals (Sweden)

    Chourey Karuna

    2008-08-01

    Full Text Available Abstract Background Shewanella oneidensis MR-1 exhibits diverse metal ion-reducing capabilities and thus is of potential utility as a bioremediation agent. Knowledge of the molecular components and regulatory mechanisms dictating cellular responses to heavy metal stress, however, remains incomplete. In a previous work, the S. oneidensis so2426 gene, annotated as a DNA-binding response regulator, was demonstrated to be specifically responsive at both the transcript and protein levels to acute chromate [Cr(VI] challenge. To delineate the cellular function of SO2426 and its contribution to metal stress response, we integrated genetic and physiological approaches with a genome-wide screen for target gene candidates comprising the SO2426 regulon. Results Inactivation of so2426 by an in-frame deletion resulted in enhanced chromate sensitivity and a reduced capacity to remove extracellular Cr(VI relative to the parental strain. Time-resolved microarray analysis was used to compare transcriptomic profiles of wild-type and SO2426-deficient mutant S. oneidensis under conditions of chromate exposure. In total, 841 genes (18% of the arrayed genome were up- or downregulated at least twofold in the Δso2426 mutant for at least one of six time-point conditions. Hierarchical cluster analysis of temporal transcriptional profiles identified a distinct cluster (n = 46 comprised of co-ordinately regulated genes exhibiting significant downregulated expression (p e.g., siderophore biosynthetic enzymes, TonB-dependent receptors, and the iron-storage protein ferritin. A conserved hypothetical operon (so1188-so1189-so1190, previously identified as a potential target of Fur-mediated repression, as well as a putative bicyclomycin resistance gene (so2280 and cation efflux family protein gene (so2045 also were repressed in the so2426 deletion mutant. Furthermore, the temporal expression profiles of four regulatory genes including a cpxR homolog were perturbed in the

  14. Estrogen receptor-mediated neuroprotection: The role of the Alzheimer’s disease-related gene seladin-1

    Directory of Open Access Journals (Sweden)

    Alessandro Peri

    2008-09-01

    Full Text Available Alessandro Peri, Mario SerioDepartment of Clinical Physiopathology, Endocrine Unit, Center for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative and Neoplastic Disorders for the Development of Novel Therapies (DENOThe, University of Florence, Florence, ItalyAbstract: Experimental evidence supports a protective role of estrogen in the brain. According to the fact that Alzheimer’s disease (AD is more common in postmenopausal women, estrogen treatment has been proposed. However, there is no general consensus on the beneficial effect of estrogen or selective estrogen receptor modulators in preventing or treating AD. It has to be said that several factors may markedly affect the efficacy of the treatment. A few years ago, the seladin-1 gene (for selective Alzheimer’s disease indicator-1 has been isolated and found to be down-regulated in brain regions affected by AD. Seladin-1 has been found to be identical to the gene encoding the enzyme 3-beta-hydroxysterol delta-24-reductase, involved in the cholesterol biosynthetic pathway, which confers protection against β-amyloid-mediated toxicity and from oxidative stress, and is an effective inhibitor of caspase-3 activity, a key mediator of apoptosis. Interestingly, we found earlier that the expression of this gene is up-regulated by estrogen. Furthermore, our very recent data support the hypothesis that seladin-1 is a mediator of the neuroprotective effects of estrogen. This review will summarize the current knowledge regarding the neuroprotective effects of seladin-1 and the relationship between this protein and estrogen.Keywords: seladin-1, DHCR24, estrogen, brain, Alzheimer’s disease

  15. Precise gene modification mediated by TALEN and single-stranded oligodeoxynucleotides in human cells.

    Directory of Open Access Journals (Sweden)

    Xiaoling Wang

    Full Text Available The development of human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs facilitates in vitro studies of human disease mechanisms, speeds up the process of drug screening, and raises the feasibility of using cell replacement therapy in clinics. However, the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs spurred interest due to the ease of assembly, high efficiency and faithful gene targeting. In this study, we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21 gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall, our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application.

  16. Complementary Information Derived