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Sample records for gene transfer mediated

  1. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  2. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  3. Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays

    PubMed Central

    Raut, Shruti; Lei, Pedro; Padmashali, Roshan; Andreadis, Stelios T.

    2010-01-01

    We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine. PMID:20153386

  4. Viral mediated gene transfer to sprouting blood vessels during angiogenesis.

    PubMed

    Alian, Akram; Eldor, Amiram; Falk, Haya; Panet, Amos

    2002-08-01

    Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy. PMID:12176137

  5. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  6. AAV-mediated gene transfer to the mouse CNS

    PubMed Central

    Stoica, Lorelei; Ahmed, Seemin S.

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. PMID:23686825

  7. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  8. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  9. Electroporation-Mediated Gene Transfer Directly to the Swine Heart

    PubMed Central

    Hargrave, Barbara; Downey, Harre; Strange, Robert; Murray, Len; Cinnamond, Cade; Lundberg, Cathryn; Israel, Annelise; Chen, Yeong-Jer; Marshall, William; Heller, Richard

    2012-01-01

    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 3 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 ECG 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 euthanized 48 hours after injection and electroporation and gene expression was determined. Results were compared to 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 to 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. PMID:22456328

  10. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

    PubMed

    Gao, Caixia; Nielsen, Klaus K

    2013-01-01

    Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines. PMID:23104329

  11. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    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. PMID:14970588

  12. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  13. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  14. Kidney-specific Sonoporation-mediated Gene Transfer

    PubMed Central

    Ishida, Ryo; Kami, Daisuke; Kusaba, Tetsuro; Kirita, Yuhei; Kishida, Tsunao; Mazda, Osam; Adachi, Takaomi; Gojo, Satoshi

    2016-01-01

    Sonoporation can deliver agents to target local organs by systemic administration, while decreasing the associated risk of adverse effects. Sonoporation has been used for a variety of materials and in a variety of organs. Herein, we demonstrated that local sonoporation to the kidney can offer highly efficient transfer of oligonucleotides, which were systemically administrated to the tubular epithelium with high specificity. Ultrasonic wave irradiation to the kidney collapsed the microbubbles and transiently affected the glomerular filtration barrier and increased glomerular permeability. Oligonucleotides were passed through the barrier all at once and were absorbed throughout the tubular epithelium. Tumor necrosis factor alpha (TNFα), which plays a central role in renal ischemia–reperfusion injury, was targeted using small interfering RNA (siRNA) with renal sonoporation in a murine model. The reduction of TNFα expression after single gene transfer significantly inhibited the expression of kidney injury markers, suggesting that systemic administration of siRNA under temporary and local sonoporation could be applicable in the clinical setting of ischemic acute kidney injury. PMID:26419704

  15. Biomaterial-Mediated Retroviral Gene Transfer Using Self-Assembled Monolayers

    PubMed Central

    Gersbach, Charles A.; Coyer, Sean R.; Le Doux, Joseph M.; García, Andrés J.

    2007-01-01

    Biomaterial-mediated gene delivery has recently emerged as a promising alternative to conventional gene transfer technologies that focus on direct delivery of viral vectors or DNA-polymer/matrix complexes. However, biomaterial-based strategies have primarily targeted transient gene expression vehicles, including plasmid DNA and adenovirus particles. This study expands on this work by characterizing biomaterial properties conducive to the surface immobilization of retroviral particles and subsequent transduction of mammalian cells at the cell-material interface. Self-assembled monolayers (SAMs) of functionally-terminated alkanethiols on gold were used to establish biomaterial surfaces of defined chemical composition. Gene transfer was observed to be greater than 90% on NH2-terminated surfaces, approximately 50% on COOH-functionalized surfaces, and undetectable on CH3-terminated SAMs, similar to controls of tissue culture-treated polystyrene. Gene delivery via the NH2-SAM was further characterized as a function of coating time, virus concentration, and cell seeding density. Finally, SAM-mediated gene delivery was comparable to fibronectin- and poly-L-lysine-based methods for gene transfer. This work is significant to establishing safe and effective gene therapy strategies, developing efficient methods for gene delivery, and supporting recent progress in the field of biomaterial-mediated gene transfer. PMID:17698189

  16. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors.

    PubMed

    Boviatsis, E J; Chase, M; Wei, M X; Tamiya, T; Hurford, R K; Kowall, N W; Tepper, R I; Breakefield, X O; Chiocca, E A

    1994-02-01

    Three vectors derived from retrovirus, herpes simplex virus type 1 (HSV), and adenovirus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a foreign gene (lacZ from Escherichia coli) into cells was more efficient with either the replication-defective retrovirus vector or the replication-conditional thymidine kinase (TK)-deficient HSV vector than with the replication-defective adenovirus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed three main histopathologic findings: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred into several types of endogenous neural cells, as well as into cells within the tumor; (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector, whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors. PMID:8186298

  17. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  18. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    PubMed

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  19. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera

    PubMed Central

    Qiu, Zhigang; Yu, Yunmei; Chen, Zhaoli; Jin, Min; Yang, Dong; Zhao, Zuguo; Wang, Jingfeng; Shen, Zhiqiang; Wang, Xinwei; Qian, Di; Huang, Aihua; Zhang, Buchang; Li, Jun-Wen

    2012-01-01

    Antibiotic resistance is a worldwide public health concern. Conjugative transfer between closely related strains or species of bacteria is an important method for the horizontal transfer of multidrug-resistance genes. The extent to which nanomaterials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic-resistance genes in bacteria, especially across genera, is still unknown. Here we show that nanomaterials in water can significantly promote the horizontal conjugative transfer of multidrug-resistance genes mediated by the RP4, RK2, and pCF10 plasmids. Nanoalumina can promote the conjugative transfer of the RP4 plasmid from Escherichia coli to Salmonella spp. by up to 200-fold compared with untreated cells. We also explored the mechanisms behind this phenomenon and demonstrate that nanoalumina is able to induce oxidative stress, damage bacterial cell membranes, enhance the expression of mating pair formation genes and DNA transfer and replication genes, and depress the expression of global regulatory genes that regulate the conjugative transfer of RP4. These findings are important in assessing the risk of nanomaterials to the environment, particularly from water and wastewater treatment systems, and in the estimation of the effect of manufacture and use of nanomaterials on the environment. PMID:22411796

  20. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  1. The agricultural antibiotic carbadox induces phage-mediated gene transfer in Salmonella

    PubMed Central

    Bearson, Bradley L.; Allen, Heather K.; Brunelle, Brian W.; Lee, In Soo; Casjens, Sherwood R.; Stanton, Thaddeus B.

    2013-01-01

    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 US 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 (STEC) 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 genes in the

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

    PubMed

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

    2015-10-01

    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

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

    SciTech Connect

    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.

  4. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    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. PMID:25520733

  5. Lentivirus-mediated gene transfer to the central nervous system: therapeutic and research applications.

    PubMed

    Wong, Liang-Fong; Goodhead, Lucy; Prat, Christine; Mitrophanous, Kyriacos A; Kingsman, Susan M; Mazarakis, Nicholas D

    2006-01-01

    The management of disorders of the nervous system remains a medical challenge. The key goals are to understand disease mechanisms, to validate therapeutic targets, and to develop new therapeutic strategies. Viral vector-mediated gene transfer can meet these goals and vectors based on lentiviruses have particularly useful features. Lentiviral vectors can deliver 8 kb of sequence, they mediate gene transfer into any neuronal cell type, expression and therapy are sustained, and normal cellular functions in vitro and in vivo are not compromised. After delivery into the nervous system they induce no significant immune responses, there are no unwanted side effects of the vectors per se to date, and manufacturing and safety testing for clinical applications are well advanced. There are now numerous examples of effective long-term treatment of animal models of neurological disorders, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, motor neuron diseases, lysosomal storage diseases, and spinal injury, using a range of therapeutic genes expressed in lentiviral vectors. Significant issues remain in some areas of neural gene therapy including defining the optimum therapeutic gene(s), increasing the specificity of delivery, regulating expression of potentially toxic genes, and designing clinically relevant strategies. We discuss the applications of lentiviral vectors in therapy and research and highlight the essential features that will ensure their translation to the clinic in the near future. PMID:16409120

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

    SciTech Connect

    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.

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

    PubMed Central

    Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

    2015-01-01

    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. PMID:26670247

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

    PubMed

    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. PMID:26683492

  9. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer.

    PubMed

    Walters, R; Welsh, M

    1999-11-01

    Delivery of a normal copy of CFTR cDNA to airway epithelia may provide a novel treatment for cystic fibrosis lung disease. Unfortunately, current vectors are inefficient because of limited binding to the apical surface of airway epithelia. We recently reported that incorporation of adenovirus in a calcium phosphate coprecipitate (Ad:CaPi) improves adenovirus-mediated gene transfer to airway epithelia in vitro and in vivo. To understand better how coprecipitation improves gene transfer, we tested the hypothesis that incorporation in a CaPi coprecipitate increases the binding of adenovirus to the apical surface of differentiated human airway epithelia. When a Cy3-labelled adenovirus was delivered in a coprecipitate, binding increased 54-fold as compared with adenovirus alone. Moreover, infection by Ad:CaPi was independent of fiber knob-CAR and penton base-integrin interactions. After binding to the cell surface, the virus must enter the cell in order to infect. We hypothesized that Ad:CaPi may stimulate fluid phase endocytosis, thereby facilitating entry. However, we found that neither adenovirus nor Ad:CaPi coprecipitates altered fluid phase endocytosis. Nevertheless, Ad:CaPi preferentially infected cells showing endocytosis. Thus, CaPi coprecipitation improves adenovirus-mediated gene transfer by coating the epithelial surface with a layer of virus which enters cells during the normal process of endocytosis. PMID:10602380

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

    PubMed

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    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. PMID:23840834

  11. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    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. PMID:23840834

  12. Adenoviral-mediated Gene Transfer into the Canine Brain In Vivo

    PubMed Central

    Candolfi, Marianela; Kroeger, Kurt M.; Pluhar, G. Elizabeth; Bergeron, Josee; Puntel, Mariana; Curtin, James F.; McNiel, Elizabeth A.; Freese, Andrew B.; Ohlfest, John R.; Moore, Peter; Lowenstein, Pedro R.; Castro, Maria G.

    2007-01-01

    OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. PMID:17228266

  13. Inhibition of choroidal neovascularization by lentivirus-mediated PEDF gene transfer in rats

    PubMed Central

    Yu, Ya-Jie; Mo, Bin; Liu, Lu; Yue, Yan-Kun; Yue, Chang-Li; Liu, Wu

    2016-01-01

    AIM To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated PEDG gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1. PMID:27588264

  14. Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes.

    PubMed

    Grossman, M; Raper, S E; Wilson, J M

    1991-11-01

    Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans. PMID:1767337

  15. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice.

    PubMed

    Heckmann, M B; Bauer, R; Jungmann, A; Winter, L; Rapti, K; Strucksberg, K-H; Clemen, C S; Li, Z; Schröder, R; Katus, H A; Müller, O J

    2016-08-01

    Mutations of the human desmin (DES) gene cause autosomal dominant and recessive myopathies affecting skeletal and cardiac muscle tissue. Desmin knockout mice (DES-KO), which develop progressive myopathy and cardiomyopathy, mirror rare human recessive desminopathies in which mutations on both DES alleles lead to a complete ablation of desmin protein expression. Here, we investigated whether an adeno-associated virus-mediated gene transfer of wild-type desmin cDNA (AAV-DES) attenuates cardiomyopathy in these mice. Our approach leads to a partial reconstitution of desmin protein expression and the de novo formation of the extrasarcomeric desmin-syncoilin network in cardiomyocytes of treated animals. This finding was accompanied by reduced fibrosis and heart weights and improved systolic left-ventricular function when compared with control vector-treated DES-KO mice. Since the re-expression of desmin protein in cardiomyocytes of DES-KO mice restores the extrasarcomeric desmin-syncoilin cytoskeleton, attenuates the degree of cardiac hypertrophy and fibrosis, and improves contractile function, AAV-mediated desmin gene transfer may be a novel and promising therapeutic approach for patients with cardiomyopathy due to the complete lack of desmin protein expression. PMID:27101257

  16. Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer.

    PubMed Central

    Schlappi, M; Hohn, B

    1992-01-01

    Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation. PMID:12297627

  17. Phage-mediated transfer of a dextranase gene in Lactobacillus sanfranciscensis and characterization of the enzyme.

    PubMed

    Picozzi, Claudia; Meissner, Daniel; Chierici, Margherita; Ehrmann, Matthias A; Vigentini, Ileana; Foschino, Roberto; Vogel, Rudi F

    2015-06-01

    While phages of lactobacilli are extensively studied with respect to their structure and role in the dairy environment, knowledge about phages in bacteria residing in sourdough fermentation is limited. Based on the previous finding that the Lactobacillus sanfranciscensis phage EV3 carries a putative dextranase gene (dex), we have investigated the distribution of similar dex(+) phages in L. sanfranciscensis, the chance of gene transfer and the properties of the dextranase encoded by phage EV3. L. sanfranciscensis H2A (dex(-)), originally isolated from a wheat sourdough, expressed a Dex(+) phenotype upon infection with EV3. The dextranase gene was isolated from the transductant and heterologously expressed in Escherichia coli. The gene encoded a protein of 801 amino acids with a calculated molecular weight (Mw) of 89.09 kDa and a calculated pI of 5.62. Upon purification aided by a 6-His tag, enzyme kinetic parameters were determined. The Km value was 370 mM, and the Vmax was calculated in about 16 μmol of glucose released from dextran by 1 mg of enzyme in 1 min in a buffer solution at pH 5.0. The optimum conditions were 60 °C and pH 4.5. The enzyme retained its activity for >3h at 60 °C and exhibited only 40% activity at 30 °C; the highest homology of 72% was found to a dextranase gene from Lactobacillus fermentum phage φPYB5. Within 25 L. sanfransiscensis isolates tested, the strain 4B5 carried a similar prophage encoding a dextranase gene. Our data suggest a phage-mediated transfer of dextranase genes in the sourdough environment resulting in superinfection-resistant L. sanfranciscensis Dex(+) strains with a possible ecological advantage in dextran-containing sourdoughs. PMID:25771219

  18. Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer

    NASA Astrophysics Data System (ADS)

    Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.

    1997-10-01

    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.

  19. Integrase-Deficient Lentiviral Vectors Mediate Efficient Gene Transfer to Human Vascular Smooth Muscle Cells with Minimal Genotoxic Risk

    PubMed Central

    Chick, Helen E.; Nowrouzi, Ali; Fronza, Raffaele; McDonald, Robert A.; Kane, Nicole M.; Alba, Raul; Delles, Christian; Sessa, William C.; Schmidt, Manfred; Thrasher, Adrian J.

    2012-01-01

    Abstract We have previously shown that injury-induced neointima formation was rescued by adenoviral-Nogo-B gene delivery. Integrase-competent lentiviral vectors (ICLV) are efficient at gene delivery to vascular cells but present a risk of insertional mutagenesis. Conversely, integrase-deficient lentiviral vectors (IDLV) offer additional benefits through reduced mutagenesis risk, but this has not been evaluated in the context of vascular gene transfer. Here, we have investigated the performance and genetic safety of both counterparts in primary human vascular smooth muscle cells (VSMC) and compared gene transfer efficiency and assessed the genotoxic potential of ICLVs and IDLVs based on their integration frequency and insertional profile in the human genome. Expression of enhanced green fluorescent protein (eGFP) mediated by IDLVs (IDLV-eGFP) demonstrated efficient transgene expression in VSMCs. IDLV gene transfer of Nogo-B mediated efficient overexpression of Nogo-B in VSMCs, leading to phenotypic effects on VSMC migration and proliferation, similar to its ICLV version and unlike its eGFP control and uninfected VSMCs. Large-scale integration site analyses in VSMCs indicated that IDLV-mediated gene transfer gave rise to a very low frequency of genomic integration compared to ICLVs, revealing a close-to-random genomic distribution in VSMCs. This study demonstrates for the first time the potential of IDLVs for safe and efficient vascular gene transfer. PMID:22931362

  20. Plasmid-mediated VEGF gene transfer induces cardiomyogenesis and reduces myocardial infarct size in sheep.

    PubMed

    Vera Janavel, G; Crottogini, A; Cabeza Meckert, P; Cuniberti, L; Mele, A; Papouchado, M; Fernández, N; Bercovich, A; Criscuolo, M; Melo, C; Laguens, R

    2006-08-01

    We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8 mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1 h after coronary artery ligation. After 15 days infarct area was 11.3+/-1.3% of the left ventricle in the VEGF group and 18.2+/-2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration. PMID:16572192

  1. Cellular Immune Response Against Firefly Luciferase After Sleeping Beauty–Mediated Gene Transfer In Vivo

    PubMed Central

    Podetz-Pedersen, Kelly M.; Vezys, Vaiva; Somia, Nikunj V.; Russell, Stephen J.

    2014-01-01

    Abstract The Sleeping Beauty (SB) transposon system has been shown to mediate new gene sequence integration resulting in long-term expression. Here the effectiveness of hyperactive SB100X transposase was tested, and we found that hydrodynamic co-delivery of a firefly luciferase transposon (pT2/CaL) along with SB100X transposase (pCMV-SB100X) resulted in remarkably sustained, high levels of luciferase expression. However, after 4 weeks there was a rapid, animal-by-animal loss of luciferase expression that was not observed in immunodeficient mice. We hypothesized that this sustained, high-level luciferase expression achieved using the SB100X transposase elicits an immune response in pT2/CaL co-administered mice, which was supported by the rapid loss of luciferase expression upon challenge of previously treated animals and in naive animals adoptively transferred with splenocytes from previously treated animals. Specificity of the immune response to luciferase was demonstrated by increased cytokine expression in splenocytes after exposure to luciferase peptide in parallel with MHC I–luciferase peptide tetramer binding. This anti-luciferase immune response observed following continuous, high-level luciferase expression in vivo clearly impacts its use as an in vivo reporter. As both an immunogen and an extremely sensitive reporter, luciferase is also a useful model system for the study of immune responses following in vivo gene transfer and expression. PMID:25093708

  2. Regulation of competence-mediated horizontal gene transfer in the natural habitat of Vibrio cholerae.

    PubMed

    Metzger, Lisa C; Blokesch, Melanie

    2016-04-01

    The human pathogen Vibrio cholerae is an autochthonous inhabitant of aquatic environments where it often interacts with zooplankton and their chitinous molts. Chitin induces natural competence for transformation in V. cholerae, a key mode of horizontal gene transfer (HGT). Recent comparative genomic analyses were indicative of extensive HGT in this species. However, we can still expand our understanding of the complex regulatory network that drives competence in V. cholerae. Here, we present recent advances, including the elucidation of bipartite competence regulation mediated by QstR, the inclusion of the type VI secretion system in the competence regulon of pandemic O1 El Tor strains, and the identification of TfoS as a transcriptional regulator that links chitin to competence induction in V. cholerae. PMID:26615332

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed Central

    Yazaki, Yushin; Oyane, Ayako; Sogo, Yu; Ito, Atsuo; Yamazaki, Atsushi; Tsurushima, Hideo

    2015-01-01

    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. PMID:25874757

  5. Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

    PubMed

    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. PMID:23596105

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

    PubMed Central

    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

  7. N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae.

    PubMed

    Higashi, Dustin L; Biais, Nicolas; Weyand, Nathan J; Agellon, Al; Sisko, Jennifer L; Brown, Lewis M; So, Magdalene

    2011-01-01

    The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species. PMID:21731720

  8. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  9. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation

    PubMed Central

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi (“truffles”) with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

  10. MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

    PubMed Central

    2013-01-01

    Background The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. Results In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. Conclusions This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo. PMID:24295286

  11. Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

    PubMed

    Brenna, Andrea; Montanini, Barbara; Muggiano, Eleonora; Proietto, Marco; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites. PMID:24949275

  12. Coupling sperm mediated gene transfer and sperm sorting techniques: a new perspective for swine transgenesis.

    PubMed

    De Cecco, Marco; Spinaci, Marcella; Zannoni, Augusta; Bernardini, Chiara; Seren, Eraldo; Forni, Monica; Bacci, Maria Laura

    2010-09-15

    Flow cytometric separation of X and Y chromosome-bearing spermatozoa has been demonstrated to be effective in pigs, allowing the use of boar sexed semen in in vitro trials. Sperm Mediated Gene Transfer (SMGT) is a widely used and efficient technique for the creation of transgenic animals. The present research intended to prove that it is possible to associate sperm sexing with the SMGT technique in order to speed up the assessment of homozygous lines of transgenic pigs. In the first experiment, the sorting protocol was modified in order to obtain the highest DNA uptake by sorted spermatozoa. In the second experiment, spermatozoa that had undergone only sperm sorting, only SMGT, or both procedures (Sorted-SMGT) were used for in in vitro fertilization of in vitro matured oocytes. In the third experiment, transformed blastocysts of the desired gender (male) were obtained with Sorted-SMGT in an in vitro fertilization trial. The method we developed here allowed us to produce transgenic swine blastocysts of pre-determined gender, giving a positive answer at the aim to couple SMGT and sperm sorting in swine, obtaining fertile spermatozoa able to produce transgenic embryos of pre-determined gender. PMID:20537690

  13. A Preclinical Animal Model to Assess the Effect of Pre-existing Immunity on AAV-mediated Gene Transfer

    PubMed Central

    Li, Hua; Lin, Shih-Wen; Giles-Davis, Wynetta; Li, Yan; Zhou, Dongming; Xiang, Zhi Quan; High, Katherine A; Ertl, Hildegund CJ

    2009-01-01

    Hepatic adeno-associated virus (AAV)-serotype 2–mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid–specific CD8+ T cells. PMID:19367258

  14. In vitro functional correction of Hermansky-Pudlak Syndrome type-1 by lentiviral-mediated gene transfer.

    PubMed

    Ikawa, Yasuhiro; Hess, Richard; Dorward, Heidi; Cullinane, Andrew R; Huizing, Marjan; Gochuico, Bernadette R; Gahl, William A; Candotti, Fabio

    2015-01-01

    Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by oculocutaneous albinism, bleeding tendency and susceptibility to pulmonary fibrosis. No curative therapy is available. Genetic correction directed to the lungs, bone marrow and/or gastro-intestinal tract might provide alternative forms of treatment for the diseases multi-systemic complications. We demonstrate that lentiviral-mediated gene transfer corrects the expression and function of the HPS1 gene in patient dermal melanocytes, which opens the way to development of gene therapy for HPS. PMID:25468649

  15. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    SciTech Connect

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  16. Effect of Surface Chemistry on Gene Transfer Efficiency Mediated by Surface-induced DNA-doped Nanocomposites

    PubMed Central

    Sun, Bingbing; Yi, Minchang; Yacoob, Christina C.; Nguyen, Hai T.; Shen, Hong

    2011-01-01

    Surface-induced biomineralization represents an effective way to immobilize DNA molecules onto biomaterial surfaces for introducing DNA into cells in contact with or in an approximate distance to biomaterial surfaces. Our previous studies have investigated how the composition of mineralizing solutions affects the composition and pH responsiveness of nanocomposites and thus gene transfer efficiency in different cell types. In this study, we investigated how the functional groups of a biomaterial surface would affect the induction and crystallographic properties of nanocomposites and thus the gene transfer efficiency. Self-assembled monolayers (SAMs) with different terminus were used to control the functional groups of a surface. We demonstrated that the induction of DNA-doped nanocomposites depended on the surface functional groups, which is consistent with previous studies. The crystallographic properties did not vary significantly with the functional groups. DNA-doped nanocomposites induced by different surface functional groups resulted in different cellular uptake of DNA and thus gene transfer efficiency. The differential cellular uptake may be attributed to the interactions between nanocomposites and functional groups. The weaker inducer resulted in higher cellular uptake thus higher gene transfer efficiency. Together with others and our previous studies, our current results suggest that surface-mediated gene transfer by DNA-doped nanocomposites can be modulated through both mineralizing solutions and surface chemistries. PMID:22198137

  17. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  18. Evaluation of Vascular Delivery Methodologies to Enhance rAAV6-mediated Gene Transfer to Canine Striated Musculature

    PubMed Central

    Gregorevic, Paul; Schultz, Brian R; Allen, James M; Halldorson, Jeffrey B; Blankinship, Michael J; Meznarich, Norman A; Kuhr, Christian S; Doremus, Caitlin; Finn, Eric; Liggitt, Denny; Chamberlain, Jeffrey S

    2009-01-01

    A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of <20 minutes, markedly enhanced the transduction of limb muscles, compared with a simple bolus limb infusion of vector. A complementary method whereby vector was infused into the jugular vein led to efficient transduction of cardiomyocytes and to a lesser degree the diaphragm. Together these methods can be used to achieve transgene expression in heart, diaphragm, and limb muscles of juvenile dogs using rAAV6 vectors. These results establish that rAAV-mediated gene delivery is a viable approach to achieving systemic transduction of striated musculature in mammals approaching the dimensions of newborn humans. PMID:19471246

  19. Collective evolution of cyanobacteria and cyanophages mediated by horizontal gene transfer

    NASA Astrophysics Data System (ADS)

    Shih, Hong-Yan; Rogers, Tim; Goldenfeld, Nigel

    We describe a model for how antagonistic predator-prey coevolution can lead to mutualistic adaptation to an environment, as a result of horizontal gene transfer. Our model is a simple description of ecosystems such as marine cyanobacteria and their predator cyanophages, which carry photosynthesis genes. These genes evolve more rapidly in the virosphere than the bacterial pan-genome, and thus the bacterial population could potentially benefit from phage predation. By modeling both the barrier to predation and horizontal gene transfer, we study this balance between individual sacrifice and collective benefits. The outcome is an emergent mutualistic coevolution of improved photosynthesis capability, benefiting both bacteria and phage. This form of multi-level selection can contribute to niche stratification in the cyanobacteria-phage ecosystem. This work is supported in part by a cooperative agreement with NASA, Grant NNA13AA91A/A0018.

  20. Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

    PubMed Central

    Shen, Jian; Qin, Jinzhou; Bao, Jianqiang; Hu, Yuan; Zeng, Wenxian; Dong, Wuzi

    2014-01-01

    In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer. PMID:24651491

  1. Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer.

    PubMed

    Qu, Bin; Gu, Yihua; Shen, Jian; Qin, Jinzhou; Bao, Jianqiang; Hu, Yuan; Zeng, Wenxian; Dong, Wuzi

    2014-01-01

    In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120-240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epithelial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer. PMID:24651491

  2. Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery.

    PubMed

    Lu, Qin; Teng, Gao-Jun; Zhang, Yue; Niu, Huan-Zhang; Zhu, Guang-Yu; An, Yan-Li; Yu, Hui; Li, Guo-Zhao; Qiu, Ding-Hong; Wu, Chuan-Ging

    2008-02-01

    Transferrin-DNA complex mediated by transferrin receptor in combination with interventional trans-arterial injection into a target organ may be a duel-target-oriented delivery means to achieve an efficient gene therapy. In this study, transferrin receptor expression in normal human hepatocyte and two hepatocellular-carcinoma cells (Huh7/SK-Hep1) was determined. p53-LipofectAMINE with different amounts of transferrin was transfected into the cells and the gene transfection efficiency was evaluated. After VX2 rabbit hepatocarcinoma model was established, the transferrin-p53-LipofectAMINE complex was delivered into the hepatic artery via interventional techniques to analyze the therapeutic p53 gene transfer efficiency in vivo by Western blot, immunohistochemical/immunofluorescence staining analysis and survival time. The results were transferrin receptor expression in Huh7 and SK-Hep1 cells was higher than in normal hepatocyte. Transfection efficiency of p53 was increased in vitro in both Huh7 and SK-Hep1 cells with increasing transferrin in a dose-dependent manner. As compared to intravenous administration, interventional injection of p53-gene complex into hepatic tumor mediated by transferrin-receptor, could enhance the gene transfer efficiency in vivo as evaluated by Western blot, immunohistochemical/immunofluorenscence staining analyses and improved animal survival (H = 12.567, p = 0.0019). These findings show the transferrin-transferrin receptor system combined with interventional techniques enhanced p53-gene transfer to hepatic tumor and the duel-target-oriented gene delivery may be an effective approach for gene therapy. PMID:18347429

  3. Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo.

    PubMed

    Zhu, Aijun; Ren, Yu; Wang, Ning; Jin, Qiuyue; Zhang, Dongchang; Yang, Guangxiao; Wang, Quanying

    2015-11-01

    Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma. PMID:26143116

  4. Effect of ultrasound irradiation on bacterial internalization and bacteria-mediated gene transfer to cancer cells.

    PubMed

    Ninomiya, Kazuaki; Yamada, Ryuji; Meisaku, Hitomi; Shimizu, Nobuaki

    2014-05-01

    The present study demonstrates that ultrasound irradiation can facilitate bacteria-mediated gene delivery (bactofection). Escherichia coli modified with avidin were employed as a vehicle for delivery of the green fluorescent protein (GFP) gene, a model heterologous gene, into the breast cancer cell line MCF-7. Avidin-mediated binding of E. coli to MCF-7 cells enhanced the internalization of E. coli by approximately 17%, irrespective of the use of ultrasound irradiation. Furthermore, the use of ultrasound irradiation increased the internalization by approximately 5%, irrespective of the presence of avidin on the E. coli cell surface. The percentages of GFP-expressing MCF-7 cells at 24h after bactofection were below 0.5% and 2% for the case with only avidin-modification of E. coli cell surface and only ultrasound irradiation, respectively. However, combining avidin modification with the ultrasound treatment increased this value to 8%. Thus, the use of avidin-modified bacteria in conjunction with ultrasound irradiation has potential as an effective strategy for tumor-targeted bactofection. PMID:24373691

  5. Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids

    PubMed Central

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  6. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids. PMID:24695466

  7. A Heterotypic Bystander Effect for Tumor Cell Killing after AAVP-mediated Vascular-targeted Suicide Gene Transfer

    PubMed Central

    Trepel, Martin; Stoneham, Charlotte A.; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D.; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

    2009-01-01

    Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as the herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells via a novel targeted AAVP vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic bystander effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of co-cultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18α-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexin (Cx)43 and Cx26, which are expressed in both endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative non-mutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted AAVP, and further support the promising role of non-malignant tumor stromal cells as therapeutic targets. PMID:19671758

  8. Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver

    PubMed Central

    Mingozzi, Federico; Hasbrouck, Nicole C.; Basner-Tschakarjan, Etiena; Edmonson, Shyrie A.; Hui, Daniel J.; Sabatino, Denise E.; Zhou, Shangzhen; Wright, J. Fraser; Jiang, Haiyan; Pierce, Glenn F.; Arruda, Valder R.

    2007-01-01

    Adeno-associated virus (AAV)–mediated gene transfer of factor IX (F.IX) to the liver results in long-term expression of transgene in experimental animals, but only short-term expression in humans. Loss of F.IX expression is likely due to a cytotoxic immune response to the AAV capsid, which results in clearance of transduced hepatocytes. We used a nonhuman primate model to assess the safety of AAV gene transfer coupled with an anti–T-cell regimen designed to block this immune response. Administration of a 3-drug regimen consisting of mycophenolate mofetil (MMF), sirolimus, and the anti–IL-2 receptor antibody daclizumab consistently resulted in formation of inhibitory antibodies to human F.IX following hepatic artery administration of an AAV-hF.IX vector, whereas a 2-drug regimen consisting only of MMF and sirolimus did not. Administration of daclizumab was accompanied by a dramatic drop in the population of CD4+CD25+FoxP3+ regulatory T cells (Tregs). We conclude that choice of immunosuppression (IS) regimen can modulate immune responses to the transgene product upon hepatic gene transfer in subjects not fully tolerant; and that induction of transgene tolerance may depend on a population of antigen-specific Tregs. PMID:17609423

  9. Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer.

    PubMed Central

    Flowers, M E; Stockschlaeder, M A; Schuening, F G; Niederwieser, D; Hackman, R; Miller, A D; Storb, R

    1990-01-01

    We studied cultured canine keratinocytes to determine whether they could serve as targets for retrovirus-mediated gene transfer and whether infected cells could persist after transplantation into dogs, a large random-bred model for gene transfer studies. Canine keratinocytes obtained from skin biopsy samples were cultured in vitro with lethally irradiated NIH 3T3 cells used as a feeder layer. The keratinocyte colonies consisted of squamous epithelium with numerous desmosomes, tonofilaments, and keratohyalin granules. In addition, the cells were strongly reactive with monoclonal antibodies to cytokeratin intermediate filament proteins. For the infection studies, we grew the keratinocytes on a feeder layer of lethally irradiated PA317 retrovirus packaging cells, which produced a helper-free amphotropic retroviral vector containing the neomycin phosphotransferase (neo) gene. After cocultivation, 34% (range, 10-76%) of the keratinocytes were found to be resistant to the neomycin analogue G418. Infected keratinocytes were then transplanted into the dog of origin; 1% (range, less than 0.1-3%) of the keratinocytes obtained 27-130 days after transplantation from skin biopsy samples gave rise to G418-resistant colonies. We conclude that canine keratinocytes cultured in vitro can be infected efficiently with a neo gene-containing retroviral vector, and they show persistent G418 resistance for at least 130 days after transplantation into the skin donor. Images PMID:2315325

  10. Cell lineage study in the liver using retroviral mediated gene transfer. Evidence against the streaming of hepatocytes in normal liver.

    PubMed Central

    Bralet, M. P.; Branchereau, S.; Brechot, C.; Ferry, N.

    1994-01-01

    The fate of normal hepatocytes in adult rat liver was studied after genetic labeling using the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal. The marker gene was introduced by direct in vivo retroviral-mediated gene transfer into hepatocytes 24 hours after partial hepatectomy. Analysis of beta-galactosidase expression in the liver at various time after gene transfer revealed that labeled hepatocytes were distributed throughout the entire lobule with a predominance in the periportal and mediolobular regions. Long-term experiments demonstrated that division of hepatocytes did occur as was revealed by the increasing number of beta-galactosidase-positive cells in isolated clusters. There was no evidence for the participation of stem cells in this process. Moreover, we found that after more than 1 year, the pattern of distribution of positive cells within the lobule was not modified. This suggests that hepatocytes do not migrate from the portal space to the perivenous region, as has been previously hypothesized. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8178942

  11. Therapeutic effects of viral vector-mediated antiangiogenic gene transfer in malignant ascites.

    PubMed

    Hampl, M; Tanaka, T; Albert, P S; Lee, J; Ferrari, N; Fine, H A

    2001-09-20

    Malignant ascites is a common complication of advanced intraabdominal neoplasms for which standard treatments are suboptimal. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the peritoneal wall due to high levels of vascular endothelial growth factor play a fundamental role in the pathogenesis of malignant ascites. To explore the advantage of viral vector-mediated "targeted antiangiogenic therapy" in ascites formation, we constructed and administered adenoviral vectors encoding several different antiangiogenic proteins (angiostatin, endostatin, platelet factor 4, and a fusion protein between angiostatin and endostatin) alone or in combination intraperitoneally in mice with peritoneal carcinomatosis from breast cancer (TA3 cells) and ovarian cancer (SKOV-3 i.p. and ES-2 cell lines) to explore the potential of additive or synergistic activity. Our data demonstrated statistically significant downregulation of ascites formation, tumor growth, vascularity, and prolongation of animal survival after intraperitoneal treatment with antiangiogenic adenoviral vectors in three different ascites tumor models. Combined treatment proved to be more effective than treatment with one vector alone. Reduced ascites formation was accompanied by decreased microvascular density in the peritoneal wall and increased apoptosis of tumor cells after administration of antiangiogenic vectors in vivo. Of interest was the observation that AdPF4 caused a significant decrease in the level of VEGF secreted by tumor cells both in vitro and in TA3 ascites tumor-bearing animals in vivo. These data suggest that adenoviral vector-mediated delivery of genes encoding antiangiogenic proteins may represent a potentially new treatment modality for malignant ascites. PMID:11560766

  12. Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer.

    PubMed

    Webster, Nicole L; Forni, Monica; Bacci, Maria Laura; Giovannoni, Roberto; Razzini, Riccardo; Fantinati, Paolo; Zannoni, Augusta; Fusetti, Lisa; Dalprà, Leda; Bianco, Maria Rosaria; Papa, Michele; Seren, Eraldo; Sandrin, Mauro S; Mc Kenzie, Ian F C; Lavitrano, Marialuisa

    2005-09-01

    Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins. PMID:15906394

  13. Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene

    PubMed Central

    De Kozak, Y; Thillaye-Goldenberg, B; Naud, M -C; Viana Da Costa, A; Auriault, C; Verwaerde, C

    2002-01-01

    Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-γ and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis. PMID:12390308

  14. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice.

    PubMed

    Ragot, T; Vincent, N; Chafey, P; Vigne, E; Gilgenkrantz, H; Couton, D; Cartaud, J; Briand, P; Kaplan, J C; Perricaudet, M

    1993-02-18

    Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle. PMID:8437625

  15. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    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. PMID:15474356

  16. Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative

    PubMed Central

    Wang, Yuqiang; Su, Jing; Cai, Wenwei; Lu, Ping; Yuan, Lifen; Jin, Tuo; Chen, Shuyan; Sheng, Jing

    2013-01-01

    Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79–100 nm and zeta potential was 6–15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53–65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery. PMID:23576866

  17. Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative.

    PubMed

    Wang, Yuqiang; Su, Jing; Cai, Wenwei; Lu, Ping; Yuan, Lifen; Jin, Tuo; Chen, Shuyan; Sheng, Jing

    2013-01-01

    Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79-100 nm and zeta potential was 6-15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53-65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery. PMID:23576866

  18. Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

    PubMed

    Li, Rongying; Oka, Kazuhiro; Yechoor, Vijay

    2012-01-01

    Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice. PMID:23093064

  19. Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract.

    PubMed

    Terao, R; Honda, K; Hatano, E; Uehara, T; Yamamoto, M; Yamaoka, Y

    1998-09-01

    Proliferative cholangitis (PC) associated with hepatolithiasis develops the stricture of main bile ducts, and is the main cause of residual and/or recurrent stones after repeated treatments for hepatolithiasis. The aim of this study was to inhibit PC using the cytostatic gene therapy with direct adenovirus-mediated retinoblastoma (Rb) gene transfer to the biliary tract. PC was induced by introducing a fine nylon thread into the bile duct in a rat model. The adenovirus vector encoding a nonphosphorylatable, constitutively active form of retinoblastoma gene product (AdRb) was administered directly into the biliary tract. The adenovirus vector encoding beta-galactosidase (AdlacZ) was also given as a control. The bile duct wall thickness and 5'-bromodeoxyuridine (BrdU) labeling index were compared among uninfected, AdlacZ-infected, and AdRb-infected PC rats. The Rb expression in the bile duct was detected using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical study. AdRb-infected bile ducts showed inhibition of the epithelial and fibrous tissue proliferation and the peribiliary gland hyperplasia, resulting in a significant reduction of wall thickness compared with uninfected and AdlacZ-infected ones. The BrdU labeling index was 4.87% +/- 3.06% in the AdRb-infected bile ducts, while those of uninfected and AdlacZ-infected ones were 15.48% +/- 4.61% and 11.72% +/- 1.23%, respectively (P < .05). In conclusion, our cytostatic gene therapy approach using direct Rb gene transfer into the biliary tract suppressed PC in a rat model and may offer an effective therapeutic option for reducing recurrences following treatments against hepatolithiasis. PMID:9731547

  20. Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

    PubMed

    Multhaup, Megan; Karlen, Andrea D; Swanson, Debra L; Wilber, Andrew; Somia, Nikunj V; Cowan, Morton J; McIvor, R Scott

    2010-07-01

    Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  1. Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

    PubMed Central

    Multhaup, Megan; Karlen, Andrea D.; Swanson, Debra L.; Wilber, Andrew; Somia, Nikunj V.; Cowan, Morton J.

    2010-01-01

    Abstract Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1α promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T−B− phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  2. A PiggyBac-mediated approach for muscle gene transfer or cell therapy.

    PubMed

    Ley, Déborah; Van Zwieten, Ruthger; Puttini, Stefania; Iyer, Pavithra; Cochard, Alessia; Mermod, Nicolas

    2014-11-01

    An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies. PMID:25310255

  3. Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene

    PubMed Central

    Zhao, Chen; Wu, Ningning; Deng, Fang; Zhang, Hongmei; Wang, Ning; Zhang, Wenwen; Chen, Xian; Wen, Sheng; Zhang, Junhui; Yin, Liangjun; Liao, Zhan; Zhang, Zhonglin; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Wu, Di; Ye, Jixing; Deng, Youlin; Zhou, Guolin; Luu, Hue H.; Haydon, Rex C.; Si, Weike; He, Tong-Chuan

    2014-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells. PMID:24658746

  4. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene

    SciTech Connect

    Ricciardone, M.D.

    1986-01-01

    Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.

  5. AAV-Mediated Gene Transfer of the Obesity-Associated Gene Etv5 in Rat Midbrain Does Not Affect Energy Balance or Motivated Behavior

    PubMed Central

    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. PMID:24710089

  6. scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

    PubMed Central

    Watson, Rachael S.; Broome, Ted A.; Levings, Padraic P.; Rice, Bret L.; Kay, Jesse D.; Smith, Andrew D.; Gouze, Elvire; Gouze, Jean-Noel; Dacanay, E. Anthony; Hauswirth, William W.; Nickerson, David M.; Dark, Michael J.; Colahan, Patrick T.; Ghivizzani, Steven C.

    2012-01-01

    With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA. PMID:23151520

  7. Ultrasound mediated gene transfection

    NASA Astrophysics Data System (ADS)

    Williamson, Rene G.; Apfel, Robert E.; Brandsma, Janet L.

    2002-05-01

    Gene therapy is a promising modality for the treatment of a variety of human diseases both inherited and acquired, such as cystic fibrosis and cancer. The lack of an effective, safe method for the delivery of foreign genes into the cells, a process known as transfection, limits this effort. Ultrasound mediated gene transfection is an attractive method for gene delivery since it is a noninvasive technique, does not introduce any viral particles into the host and can offer very good temporal and spatial control. Previous investigators have shown that sonication increases transfection efficiency with and without ultrasound contrast agents. The mechanism is believed to be via a cavitation process where collapsing bubble nuclei permeabilize the cell membrane leading to increased DNA transfer. The research is focused on the use of pulsed wave high frequency focused ultrasound to transfect DNA into mammalian cells in vitro and in vivo. A better understanding of the mechanism behind the transfection process is also sought. A summary of some in vitro results to date will be presented, which includes the design of a sonication chamber that allows us to model the in vivo case more accurately.

  8. Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

    PubMed Central

    Lawlor, Patricia A; Bland, Ross J; Das, Pritam; Price, Robert W; Holloway, Vallie; Smithson, Lisa; Dicker, Bridget L; During, Matthew J; Young, Deborah; Golde, Todd E

    2007-01-01

    Background Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. Results Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits. Conclusion The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that

  9. Retroviral Vector-Mediated Gene Transfer into the Chick Optic Vesicle by In Ovo Electroporation

    NASA Astrophysics Data System (ADS)

    Sakuta, Hiraki; Suzuki, Ryoko; Noda, Masaharu

    The chick embryo offers many advantages for developmental studies over other vertebrate embryos as it allows easy access for in ovo surgical manipulations, such as tissue transplantation and the implantation of cultured cells or chemically treated beads for the local release of humoral factors. In particular, owing to its external position in the embryo, the chick eye is a popular model for studying the patterning mechanism of the central nervous system (CNS). This patterning has a crucial role in shaping functional organization because it is the basis of the specific wiring in the CNS. Genetic analysis is not easy in the chick, as compared with the mouse for which transgene introduction or gene targeting techniques have been well established. However, because methods for the expression of exogenous genes and for gene silencing in the chick embryo have been recently developed, the functional analysis of genes has become possible in combination with classical techniques of developmental biology and neurobiology.

  10. Effect of SERCA2a overexpression in the pericardium mediated by the AAV1 gene transfer on rapid atrial pacing in rabbits.

    PubMed

    Kuken, B N; Aikemu, A N W E; Xiang, S Y; Wulasihan, M H Y T

    2015-01-01

    To study the effects of overexpression of the sarcoplasmic reticulum ATPase 2a (SERCA2a) gene on the activity and protein expression of SERCA2a after rapid atrial pacing (RAP) in New Zealand white rabbits. New Zealand white rabbits were randomly divided into a sham-operated group (group A), adeno-associated virus 1 (AAV1)/EGFP + atrial fibrillation (AF) model group (group B), or AVV1/SERCA2a + AF group (group C). The sham-operated group was used as a negative control. Each group consisted of 10 animals. Groups B and C were injected with 500 μL of the AAV1-EGFP reporter gene and 500 μL of the AAV1-SERCA2a target gene, respectively. Four weeks after AAV1-mediated gene transfer, the rabbits underwent 24 h of RAP to the right atrium. The animals were sacrificed and protein activity and protein expression in the myocardium were measured using the westernblot method. Four weeks after AAV1-mediated gene transfer, SERCA2a protein activity and expression were significantly higher in Group C than in Groups A and B (P < 0.05). RAP of the right atrium induced atrial fibrillation in rabbits, resulting in decreases in the activity and protein expression of SERCA2a. Pericardial AAV-1 mediated SERCA2a gene transfer resulted in the overexpression of SERCA2a, restoring SERCA2a activity and protein expression. PMID:26535677

  11. The role of helper lipids in cationic liposome-mediated gene transfer.

    PubMed Central

    Hui, S W; Langner, M; Zhao, Y L; Ross, P; Hurley, E; Chan, K

    1996-01-01

    In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton

  12. Effect of Host Modification and Age on Airway Epithelial Gene Transfer Mediated by a Murine Leukemia Virus-Derived Vector

    PubMed Central

    Johnson, Larry G.; Mewshaw, Jennifer P.; Ni, Hong; Friedmann, Theodore; Boucher, Richard C.; Olsen, John C.

    1998-01-01

    To study retroviral gene transfer to airway epithelia, we used a transient transfection technique to generate high titers (∼109 infectious units/ml after concentration) of murine leukemia virus (MuLV)-derived vectors pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G). Transformed (CFT1) and primary airway epithelial cells were efficiently transduced by a VSV-G-pseudotyped lacZ vector (HIT-LZ) in vitro. CFT1 cells and primary cystic fibrosis (CF) airway cell monolayers infected with a vector (HIT-LCFSN) containing human CF transmembrane conductance regulator (CFTR) in the absence of selection expressed CFTR, as assessed by Western blot analysis, and exhibited functional correction of CFTR-mediated Cl− secretion. In vitro studies of persistence suggested that pseudotransduction was not a significant problem with our vector preparations. In a sulfur dioxide (SO2) inhalational injury model, bromodeoxyuridine (BrdU) incorporation rates were measured and found to exceed 50% in SO2-injured murine tracheal epithelium. HIT-LZ vector (multiplicity of infection of ∼10) instilled into the SO2-injured tracheas of anesthetized mice transduced 6.1% ± 1.3% of superficial airway cells in tracheas of weanling mice (3 to 4 weeks old; n = 10), compared to 1.4 ± 0.9% in mice 5 weeks of age (n = 4) and 0.2% in mice older than 6 weeks (n = 15). No evidence for gene transfer following delivery of HIT-LZ to tracheas of either weanling or older mice not injured with SO2 was detected. Because only a small fraction of BrdU-labeled airway cells were transduced, we examined the stability of the vector. No significant loss of vector infectivity over intervals (2 h) paralleling those of in vivo protocols was detected in in vitro assays using CFT1 cells. In summary, high-titer vectors permitted complementation of defective CFTR-mediated Cl− transport in CF airway cells in vitro without selection and demonstrated that the age of the animal appeared to be a major

  13. Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

    PubMed Central

    Tran, P L; Weinbach, J; Opolon, P; Linares-Cruz, G; Reynes, J P; Grégoire, A; Kremer, E; Durand, H; Perricaudet, M

    1997-01-01

    A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis. PMID:9045862

  14. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer.

    PubMed

    Mostoslavsky, Gustavo; Fabian, Attila J; Rooney, Sean; Alt, Frederick W; Mulligan, Richard C

    2006-10-31

    Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic. PMID:17062750

  15. Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer

    PubMed Central

    Mostoslavsky, Gustavo; Fabian, Attila J.; Rooney, Sean; Alt, Frederick W.; Mulligan, Richard C.

    2006-01-01

    Artemis gene mutations are responsible for the development of a severe combined immunodeficiency [radiation-sensitive (RS) SCID] characterized by a severe B and T cell deficiency and a normal natural killer cell population. To establish the feasibility of a gene therapy approach to the treatment of RS-SCID, we generated a series of lentiviral vectors expressing human Artemis from different promoters and used them to transduce highly purified hematopoietic stem cells (HSCs) from Artemis knockout mice. HSCs transduced by the different viruses were transplanted into either lethally irradiated Rag-1-deficient animals or Artemis knockout mice treated with a nonmyeloablative dose of Busulfan. In both models, transplantation of HSCs transduced by a vector that used a murine phosphoglycerate kinase (PGK) promoter led to a complete functional correction of the immunodeficiency. Corrected animals displayed rescue of mature B cells with normal levels of serum immunoglobulins, together with complete rescue of the T cell compartment as evidenced by the presence of mature T lymphocytes in peripheral blood as well as normal values of thymocytes in thymus. Those B and T cells were capable of activation, as shown both by in vitro stimulation responses and in vivo after immune challenge. Overall, the results indicate that a gene therapy approach for RS-SCID involving the transplantation of genetically modified HSCs is indeed feasible. Furthermore, our studies suggest the possibility that nonmyeloablative conditioning regimens might be effectively used to promote engraftment of genetically modified cells in the case of diseases where standard irradiation-based myeloablative bone marrow transplantation protocols may prove problematic. PMID:17062750

  16. A novel combination of promoter and enhancers increases transgene expression in vascular smooth muscle cells in vitro and coronary arteries in vivo after adenovirus-mediated gene transfer

    PubMed Central

    Appleby, CE; Kingston, PA; David, A; Gerdes, CA; Umaña, P; Castro, MG; Lowenstein, PR; Heagerty, AM

    2010-01-01

    Recombinant adenoviruses are employed widely for vascular gene transfer. Vascular smooth muscle cells (SMCs) are a relatively poor target for transgene expression after adenovirus-mediated gene delivery, however, even when expression is regulated by powerful, constitutive viral promoters. The major immediate-early murine cytomegalovirus enhancer/promoter (MIEmCMV) elicits substantially greater transgene expression than the human cytomegalovirus promoter (MIEhCMV) in all cell types in which they have been compared. The Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transgene expression in numerous cell lines, and fragments of the smooth muscle myosin heavy chain (SMMHC) promoter increase expression within SMC from heterologous promoters. We therefore, compared the expression of β-galactosidase after adenovirus-mediated gene transfer of lacZ under the transcriptional regulation of a variety of combinations of the promoters and enhancers described, in vitro and in porcine coronary arteries. We demonstrate that inclusion of WPRE and a fragment of the rabbit SMMHC promoter along with MIEmCMV increases β-galactosidase expression 90-fold in SMC in vitro and ≈40-fold in coronary arteries, compared with vectors in which expression is regulated by MIEhCMV alone. Expression cassette modification represents a simple method of improving adenovirus-mediated vascular gene transfer efficiency and has important implications for the development of efficient cardiovascular gene therapy strategies. PMID:12907954

  17. Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer.

    PubMed

    Tang, Shiue-Cheng; Sambanis, Athanassios

    2003-04-01

    Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD. PMID:12659868

  18. Complete genome sequence of Brachyspira intermedia reveals unique genomic features in Brachyspira species and phage-mediated horizontal gene transfer

    PubMed Central

    2011-01-01

    Background Brachyspira spp. colonize the intestines of some mammalian and avian species and show different degrees of enteropathogenicity. Brachyspira intermedia can cause production losses in chickens and strain PWS/AT now becomes the fourth genome to be completed in the genus Brachyspira. Results 15 classes of unique and shared genes were analyzed in B. intermedia, B. murdochii, B. hyodysenteriae and B. pilosicoli. The largest number of unique genes was found in B. intermedia and B. murdochii. This indicates the presence of larger pan-genomes. In general, hypothetical protein annotations are overrepresented among the unique genes. A 3.2 kb plasmid was found in B. intermedia strain PWS/AT. The plasmid was also present in the B. murdochii strain but not in nine other Brachyspira isolates. Within the Brachyspira genomes, genes had been translocated and also frequently switched between leading and lagging strands, a process that can be followed by different AT-skews in the third positions of synonymous codons. We also found evidence that bacteriophages were being remodeled and genes incorporated into them. Conclusions The accessory gene pool shapes species-specific traits. It is also influenced by reductive genome evolution and horizontal gene transfer. Gene-transfer events can cross both species and genus boundaries and bacteriophages appear to play an important role in this process. A mechanism for horizontal gene transfer appears to be gene translocations leading to remodeling of bacteriophages in combination with broad tropism. PMID:21816042

  19. Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells.

    PubMed

    Koirala, Adarsha; Makkia, Rasha S; Cooper, Mark J; Naash, Muna I

    2011-12-01

    Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ~3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases. PMID:21885113

  20. Lentivirus mediated HO-1 gene transfer enhances myogenic precursor cell survival after autologous transplantation in pig.

    PubMed

    Laumonier, Thomas; Yang, Sheng; Konig, Stephane; Chauveau, Christine; Anegon, Ignacio; Hoffmeyer, Pierre; Menetrey, Jacques

    2008-02-01

    Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation. PMID:18026170

  1. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

    PubMed Central

    2013-01-01

    Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

  2. Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo.

    PubMed

    Saito, Y; Swanson, X; Mhashilkar, A M; Oida, Y; Schrock, R; Branch, C D; Chada, S; Zumstein, L; Ramesh, R

    2003-11-01

    The tumor-suppressor gene PTEN encodes a multifunctional phosphatase that is mutated in a variety of human cancers. PTEN inhibits the phosphatidylinositol 3-kinase pathway and downstream functions, including activation of Akt/protein kinase B (PKB), cell survival, and cell proliferation in tumor cells carrying mutant- or deletion-type PTEN. In such tumor cells, enforced expression of PTEN decreases cell proliferation through cell-cycle arrest at G1 phase accompanied, in some cases, by induction of apoptosis. More recently, the tumor-suppressive effect of PTEN has been reported in ovarian and thyroid tumors that are wild type for PTEN. In the present study, we examined the tumor-suppressive effect of PTEN in human colorectal cancer cells that are wild type for PTEN. Adenoviral-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in colorectal cancer cells (DLD-1, HT29, and SW480) carrying wtPTEN than in normal colon fibroblast cells (CCD-18Co) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) and cell-cycle arrest at the G2/M phase, but not the G1 phase. Furthermore, treatment of human colorectal tumor xenografts (HT-29, and SW480) with Ad-PTEN resulted in significant (P=0.01) suppression of tumor growth. These results indicate that Ad-PTEN exerts its tumor-suppressive effect on colorectal cancer cells through inhibition of cell-cycle progression and induction of cell death. Thus Ad-PTEN may be a potential therapeutic for treatment of colorectal cancers. PMID:14528320

  3. [Plasmid pJP4 mediated gene horizontal transfer in a biofilm system and its effect on 2, 4-D degradation].

    PubMed

    Quan, Xiang-Chun; Tang, Hua; Hu, Li-Juan; Wang, Ran; Zhang, Ning

    2009-09-15

    With plasmid pJP4 (which contains functional gene cluster (tfd) encoding 2,4-D degradation) carrying genetic microorganism Pseudomonas putida SM1443:: gfp2x (pJP4:: dsRed) as the donor strain, events of plasmid mediated gene horizontal transfer and its effect on 2,4-D degradation was investigated in a biofilm system operated under fed-batch mode. The surviving status of the functional gene element in the gene-augmented system and effects of gene-augmentation on microbial community structure were also investigated. Results showed that introduction of pJP4 carrying strain to the biofilm system with 2, 4-D (initial concentration at 170 mg/L +/- 10 mg/L) as the sole carbon source could enhance the degradation of 2, 4-D. Enhancement was slight during the initial stage of operation, but it increased with increasing of fed batch runs. Difference in 2, 4-D average degradation rate between gene-augmented system and the control system achieved up to 13.3 mg/(L x h) at most. Through detecting functional gene tfdB and reporter gene gfp, pJP4 mediated gene horizontal transfer to the bacteria on biofilm was further approved. Effects of gene augmentation on microbial community structure was analyzed by PCR-DGGE analysis, and results showed that relatively higher stability of microbial community was maintained for the gene-augmented biofilm system compared to the control system when facing 2,4-D shock loadings. PMID:19927832

  4. BioShuttle-mediated Plasmid Transfer

    PubMed Central

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

  5. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    PubMed

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  6. Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

    PubMed Central

    Teramoto, S.; Bartlett, J. S.; McCarty, D.; Xiao, X.; Samulski, R. J.; Boucher, R. C.

    1998-01-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  7. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

    PubMed

    Teramoto, S; Bartlett, J S; McCarty, D; Xiao, X; Samulski, R J; Boucher, R C

    1998-11-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  8. The Skeletal Muscle Environment and Its Role in Immunity and Tolerance to AAV Vector-Mediated Gene Transfer

    PubMed Central

    Boisgérault, Florence; Mingozzi, Federico

    2015-01-01

    Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors. PMID:26122097

  9. Liposome-mediated in vivo E1A gene transfer suppressed dissemination of ovarian cancer cells that overexpress HER-2/neu.

    PubMed

    Yu, D; Matin, A; Xia, W; Sorgi, F; Huang, L; Hung, M C

    1995-10-01

    The HER-2/neu proto-oncogene is frequently amplified or overexpressed in many different types of human cancers, a phenomenon that has been shown to correlate with shorter survival time and lower survival rate in ovarian cancer patients. We previously reported that increased HER-2/neu expression led to more severe malignancy and increased metastatic potential in animal models and that the adenovirus 5 E1A gene repressed HER-2/neu gene expression at transcriptional level and was able to suppress tumor growth when stably transfected into human ovarian cancer SKOV-3 cells which overexpress HER-2/neu. To investigate whether the E1A gene may be used as a therapeutic agent for HER-2/neu-overexpressing human cancers in living hosts, we first developed tumor-bearing mice by injecting SKOV-3 cells that overexpress HER-2/neu intraperitonealy into female nu/nu mice. Five days later, we used cationic liposomes to directly deliver the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice that received the SKOV-3 cell injection. We found that liposome-mediated E1A gene transfer significantly inhibited growth and dissemination of ovarian cancer cells that overexpress HER-2/neu in the treated mice; about 70% of these mice survived at least 365 days, whereas all the control mice that did not receive the gene therapy developed severe tumor symptoms and died within 160 days. The results suggest that liposome-mediated E1A gene transfer may serve as an effective therapy for human ovarian cancers that overexpress HER-2/neu by directly targeting the HER-2/neu oncogene. PMID:7478560

  10. Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics.

    PubMed

    Grimaldi, Benedetto; de Raaf, Michiel A; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2005-07-01

    Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as "truffles". A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii. PMID:15868150

  11. In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition.

    PubMed

    Bire, Solenne; Ishac, Nicole; Rouleux-Bonnin, Florence

    2016-01-01

    Nowadays, nonviral gene transfer is currently of great importance for introducing exogenous genes into genomes and for ensuring that transgene expression is suitable for therapeutic and bioproduction purposes. The piggyBac transposon-based system is particularly interesting since it is easy to engineer and has a large cargo capacity, up to 100 kb. In its setup, the system requires only the piggyBac transposase protein and the transgene delineated by the two piggyBac-specific inverted terminal repeats. Usually the source of transposase is carried by a DNA plasmid. However, the principal drawback of this method is the lasting presence of the transposase, due to episomal persistence or possible integration of the transposase gene vector into the cell's genome. This can lead to genotoxic effects such as multiple genomic integration events and remobilization of the transposon vector once it has been integrated. One alternative to improve the safety of the system is to deliver the transposase as in vitro-synthesized messenger RNA in order to define a very narrow expression window during which a one-shot transposition process would occur. Issues that can be encountered when working on mRNA cell transfer are related to the quality of the synthetic mRNA, the system used to introduce mRNA into the cells and the bioavailability of the mRNA molecules. Here we describe a method to produce mRNA, verify its quality, determine which transfecting reagents can be used and how this mRNA is available to promote the transposition process in HeLa cells. Additionally, we illustrate this method in stromal mesenchymal cell lines in order to support hematopoiesis. PMID:27236801

  12. Enhancement of flap survival and changes in angiogenic gene expression after AAV2-mediated VEGF gene transfer to rat ischemic flaps.

    PubMed

    Wang, Xiao Tian; Avanessian, Bella; Ma, Qiangzhong; Durfee, Heather; Tang, Yu Qing; Liu, Paul Y

    2011-01-01

    Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno-associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2-GFP group, and one saline group. AAV2-VEGF or AAV2-GFP were injected intradermally into the rat dorsum in the AAV2-VEGF or AAV2-GFP group. The saline group received saline injection. A 3 × 10 cm flap was raised in each rat two weeks post-injection. One week after surgery, flap viability was evaluated. Angiogenesis real-time PCR array was performed to analyze the expression of angiogenesis-associated genes. The AAV2-VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2-VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis-associated genes in AAV2-VEGF treated flaps. Particularly, EGF, PDGF-A and VEGF-B genes were up-regulated in these flaps. In contrast, FGF2 gene expression was down-regulated. In conclusion, AAV2-VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival. PMID:21649787

  13. Using a Commercial Ultrasound Contrast Agent for Viral-Mediated Gene Transfer In Vitro and In Vivo

    NASA Astrophysics Data System (ADS)

    Howard, Candace M.; Forsberg, Flemming; Liu, Ji-Bin; Merton, Daniel A.; Minimo, Corrado; Claudio, Pier P.

    2007-05-01

    This study evaluated the feasibility of site-specific gene delivery mediated by diagnostic ultrasound using genes encapsulated in commercially available ultrasound contrast agents in vitro and in vivo. Five different commercially available contrast agents were tested in vitro for their ability to enclose an adenoviral vector carrying GFP. Prostate cancer cells (DU 145) or non small cell lung cancer cells (H23) were plated in 80 culture wells and insonified at 207 or 535 kPa peak negative pressure for 1 min after administration of 0.1 ml of bubbles reconstituted with the viral vector. Experiments were repeated with the delivery vehicle incubated with complement to inactivate unenclosed Adeno-GFP and with controls. After 24 hours transduction efficiency was demonstrated by fluorescent microscopy. In vivo 15 nude mice with 21 melanoma tumors (DB-1) implanted received 0.1 ml injections of contrast. Mice were split into 3 control and 4 active groups and ultrasound was performed for 4 min at 4 MHz using an Aplio scanner (Toshiba America Medical Systems, Tustin, CA). Tumors, heart, lungs and liver were harvested 48 hours later. Specimens underwent regular and fluorescent microscopy and were stained using an antibody against GFP. In vitro all contrast agents produced more fluorescence at 207 kPa than at 535 kPa. However, only Imagent (IMCOR Pharmaceuticals, San Diego, CA) was able to induce marked gene transduction with the inactivating agent. In vivo systemic delivery of Adeno-GFP carrying microbubbles following pre-treatment with the inactivating agent resulted in specific transduction of the tumor cells only with no uptake in heart, lungs or liver (unlike the controls). In conclusion, specific viral gene transduction has been obtained in vitro and in vivo through the use of ultrasound and Imagent microbubbles as delivery vehicles.

  14. Prevention of autoimmune recurrence and rejection by adenovirus-mediated CTLA4Ig gene transfer to the pancreatic graft in BB rat.

    PubMed

    Uchikoshi, F; Yang, Z D; Rostami, S; Yokoi, Y; Capocci, P; Barker, C F; Naji, A

    1999-03-01

    Type 1 diabetes is the result of a selective destruction of pancreatic islets by autoreactive T-cells. Therefore, in the context of islet or pancreas transplantation, newly transplanted beta-cells are threatened by both recurrent autoimmune and alloimmune responses in recipients with type 1 diabetes. In the present study, using spontaneously diabetic BB rats, we demonstrate that whereas isolated islets are susceptible to autoimmune recurrence and rejection, pancreaticoduodenal grafts are resistant to these biological processes. This resistance is mediated by lymphohematopoietic cells transplanted with the graft, since inactivation of these passenger cells by irradiation uniformly rendered the pancreaticoduodenal grafts susceptible to recurrent autoimmunity. We further studied the impact of local immunomodulation on autoimmune recurrence and rejection by ex vivo adenovirus-mediated CTLA4Ig gene transfer to pancreaticoduodenal grafts. Syngeneic DR-BB pancreaticoduodenal grafts transduced with AdmCTLA4Ig were rescued from recurrent autoimmunity. In fully histoincompatible LEW-->BB transplants, in which rejection and recurrence should be able to act synergistically, AdmCTLA4Ig transduced LEW-pancreaticoduodenal allografts enjoyed markedly prolonged survival in diabetic BB recipients. In situ reverse transcription-polymerase chain reaction revealed that transferred CTLA4Ig gene was strongly expressed in both endocrine and exocrine tissues on day 3. These results indicate the potential utility of local CD28-B7 costimulatory blockade for prevention of alloimmune and autoimmune destruction of pancreatic grafts in type 1 diabetic hosts. PMID:10078573

  15. Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates.

    PubMed

    Younan, Patrick M; Peterson, Christopher W; Polacino, Patricia; Kowalski, John P; Obenza, Willimark; Miller, Hannah W; Milless, Brian P; Gafken, Phil; DeRosa, Stephen C; Hu, Shiu-Lok; Kiem, Hans-Peter

    2015-05-01

    Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34(+) cells expressing the mC46 anti-HIV fusion protein. We show that SHIV(+), ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34(+) cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV(+), ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4(+) T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4(+)CCR5(+)T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial. PMID:25648264

  16. Lentivirus-mediated Gene Transfer in Hematopoietic Stem Cells Is Impaired in SHIV-infected, ART-treated Nonhuman Primates

    PubMed Central

    Younan, Patrick M; Peterson, Christopher W; Polacino, Patricia; Kowalski, John P; Obenza, Willimark; Miller, Hannah W; Milless, Brian P; Gafken, Phil; DeRosa, Stephen C; Hu, Shiu-Lok; Kiem, Hans-Peter

    2015-01-01

    Recent studies have demonstrated that genetically modified hematopoietic stem cells (HSCs) can reduce HIV viremia. We have developed an HIV/AIDS-patient model in Simian/human immunodeficiency virus (SHIV)-infected pigtailed macaques that are stably suppressed on antiretroviral therapy (ART: raltegravir, emtricitabine and tenofovir). Following SHIV infection and ART, animals undergo autologous HSC transplantation (HSCT) with lentivirally transduced cluster of differentiation (CD)34+ cells expressing the mC46 anti-HIV fusion protein. We show that SHIV+, ART-treated animals had very low gene marking levels after HSCT. Pretransduction CD34+ cells contained detectable levels of all three ART drugs, likely contributing to the low gene transfer efficiency. Following HSCT recovery and the cessation of ART, plasma viremia rebounded, indicating that myeloablative total body irradiation cannot completely eliminate viral reservoirs after autologous HSCT. The kinetics of recovery following autologous HSCT in SHIV+, ART-treated macaques paralleled those observed following transplantation of control animals. However, T-cell subset analyses demonstrated a high percentage of C-C chemokine receptor 5 (CCR5)-expressing CD4+ T-cells after HSCT. These data suggest that an extended ART interruption time may be required for more efficient lentiviral transduction. To avoid complications associated with ART interruption in the context of high percentages of CD4+CCR5+T-cells after HSCT, the use of vector systems not impaired by the presence of residual ART may also be beneficial. PMID:25648264

  17. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N.

    2013-01-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., “immunization against infarction”). PMID:21785893

  18. Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B

    SciTech Connect

    Thompson, A.R. Puget Sound Blood Center, Seattle, WA ); Darlington, G. ); Armentano, D.; Woo, S.L.C.

    1990-08-01

    Hemophilia B (Christmas disease) is a chromosome X-linked blood clotting disorder which results when factor IX is deficient or functionally defective. The enzyme is synthesized in the liver, and the existence of animal models for this genetic disease will permit the development of somatic gene therapy protocols aimed at transfer of the functional gene into the liver. The authors report the construction of an N2-based recombinant retroviral vector, NCMVFIX, for efficient transfer and expression of human factor IX cDNA in primary rabbit hepatocytes. In this construct the human cytomegalovirus immediate early promoter directs the expression of factor IX. Hepatocytes were isolated from 3-week-old New Zealand White rabbits, infected with the recombinant virus, and analyzed for secretion of active factor IX. The infected rabbit hepatocytes produced human factor IX that is indistinguishable from enzyme derived from normal human plasma. The recombinant protein is sufficiently {gamma}-carboxylated and is functionally active in clotting assays. These results establish the feasibility of using infected hepatocytes for the expression of this protein and are a step toward the goal of correcting hemophilia B by hepatic gene transfer.

  19. In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

    PubMed

    Vargiolu, A; Manzini, S; de Cecco, M; Bacci, M L; Forni, M; Galeati, G; Cerrito, M G; Busnelli, M; Lavitrano, M; Giovannoni, R

    2010-01-01

    Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic. PMID:20692428

  20. Life-Long Correction of Hyperbilirubinemia with a Neonatal Liver-Specific AAV-Mediated Gene Transfer in a Lethal Mouse Model of Crigler–Najjar Syndrome

    PubMed Central

    Bortolussi, Giulia; Zentillin, Lorena; Vaníkova, Jana; Bockor, Luka; Bellarosa, Cristina; Mancarella, Antonio; Vianello, Eleonora; Tiribelli, Claudio; Giacca, Mauro; Vitek, Libor

    2014-01-01

    Abstract Null mutations in the UGT1A1 gene result in Crigler–Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5–8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dl), despite 20–30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters. PMID:25072305

  1. A VSV-G Pseudotyped Last Generation Lentiviral Vector Mediates High Level and Persistent Gene Transfer in Models of Airway Epithelium In Vitro and In Vivo

    PubMed Central

    Copreni, Elena; Palmieri, Lucia; Castellani, Stefano; Conese, Massimo

    2010-01-01

    The aim of this work was to evaluate the efficiency and duration of gene expression mediated by a VSV-G pseudotyped last generation lentiviral (LV) vector. We studied LV efficiency in ex-vivo models of respiratory epithelial cells, obtained from bronchial biopsies and nasal polyps, by GFP epifluorescence and cytofluorimetry. In vivo efficiency and persistence of gene expression was investigated by GFP immunohistochemistry and luciferase activity in lung cryosections and homogenates, respectively, upon intranasal and intratracheal administration protocols in C57Bl/6 mice. Both primary bronchial and nasal epithelial cells were transduced up to 70–80% 72 hr after the LV infection. In vivo nasal luciferase expression was increased by lysophosphatidylcholine pre-treatment of the nose. Conversely, the bronchial epithelium was transduced in the absence of any pre-conditioning treatment and luciferase expression lasted for at least 6 months without any decline. We conclude that a last generation LV vector is a promising gene transfer agent in the target organ of genetic and acquired lung diseases, as in the case of cystic fibrosis. PMID:21994695

  2. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

    PubMed Central

    Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

    2012-01-01

    Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

  3. Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases.

    PubMed

    Koirala, Adarsha; Conley, Shannon M; Makkia, Rasha; Liu, Zhao; Cooper, Mark J; Sparrow, Janet R; Naash, Muna I

    2013-12-28

    Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy. PMID:24035979

  4. Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences

    PubMed Central

    Li, Qianhong; Guo, Yiru; Wu, Wen-Jian; Ou, Qinghui; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Chen, Ning; Dawn, Buddhadeb; Luo, Li; O’Brien, Erin

    2013-01-01

    The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy. PMID:21779912

  5. Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C--Staphylococcus A protein

    PubMed Central

    Cao, Haiyan; Zhang, Guo-rong; Geller, Alfred I.

    2010-01-01

    Because of the heterogeneous cellular composition of the brain, and especially the forebrain, cell type-specific expression will benefit many potential applications of direct gene transfer. The two prevalent approaches for achieving cell type-specific expression are using a cell type-specific promoter or targeting gene transfer to a specific cell type. Targeted gene transfer with Herpes Simplex Virus (HSV-1) vectors modifies glycoprotein C (gC) to replace the heparin binding domain, which binds to many cell types, with a binding activity for a specific cell surface protein. We previously reported targeted gene transfer to nigrostriatal neurons using chimeric gC--glial cell line-derived neurotrophic factor or gC--brain-derived neurotrophic factor protein. Unfortunately, this approach is limited to cells that express the cognate receptor for either neurotrophic factor. Thus, a general strategy for targeting gene transfer to many different types of neurons is desirable. Antibody-mediated targeted gene transfer has been developed for targeting specific virus vectors to specific peripheral cell types; a specific vector particle protein is modified to contain the Staphylococcus A protein ZZ domain, which binds immunoglobulin (Ig) G. Here, we report antibody-mediated targeted gene transfer of HSV-1 vectors to a specific type of forebrain neuron. We constructed a chimeric gC--ZZ protein, and showed this protein is incorporated into vector particles and binds Ig G. Complexes of these vector particles and an antibody to the NMDA receptor NR1 subunit supported targeted gene transfer to NR1-containing neocortical neurons in the rat brain, with long-term (2 months) expression. PMID:20599821

  6. Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia.

    PubMed

    Walters, R W; van't Hof, W; Yi, S M; Schroth, M K; Zabner, J; Crystal, R G; Welsh, M J

    2001-08-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  7. Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

    PubMed Central

    Walters, Robert W.; van't Hof, Wouter; Yi, Su Min P.; Schroth, Mary K.; Zabner, Joseph; Crystal, Ronald G.; Welsh, Michael J.

    2001-01-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl− transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  8. [Adeno-associated virus mediated T-bet gene transfer into SGC-7901 cell to regulate IFN-gamma production].

    PubMed

    Qiu, Gufeng; Wang, Suoying; Wang, Shengjun; Shao, Qixiang; Ma, Jie; Yang, Ming; Xu, Xiaopeng; Mao, Chaoming; Su, Zhaoliang; Huang, Xinxiang; Xu, Huaxi

    2009-06-01

    In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene. PMID:19634682

  9. Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.).

    PubMed

    Vermeulen, A; Vaucheret, H; Pautot, V; Chupeau, Y

    1992-06-01

    Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. PMID:24203132

  10. Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

    PubMed Central

    Shen, D W; Fojo, A; Roninson, I B; Chin, J E; Soffir, R; Pastan, I; Gottesman, M M

    1986-01-01

    Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells. Images PMID:3796599

  11. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

    PubMed

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C; Dandri, Maura; Pollok, Joerg-Matthias

    2016-05-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  12. Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.

    PubMed

    Cordier, L; Hack, A A; Scott, M O; Barton-Davis, E R; Gao, G; Wilson, J M; McNally, E M; Sweeney, H L

    2000-02-01

    In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process. PMID:10933922

  13. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer

    PubMed Central

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C.; Dandri, Maura

    2016-01-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  14. Generation of mouse induced pluripotent stem cells from different genetic backgrounds using Sleeping beauty transposon mediated gene transfer.

    PubMed

    Muenthaisong, Suchitra; Ujhelly, Olga; Polgar, Zsuzsanna; Varga, Eszter; Ivics, Zoltan; Pirity, Melinda K; Dinnyes, Andras

    2012-11-15

    Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR-outbred, (2) C57BL/6-inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers-e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds

  15. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular. PMID:15761667

  16. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    PubMed Central

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A.

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed. PMID:11914355

  17. Gene Transfer into Cardiac Myocytes

    PubMed Central

    Lang, Sarah E.; Westfall, Margaret V.

    2016-01-01

    Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function. PMID:25836585

  18. Adenovirus-mediated HIF-1α gene transfer promotes repair of mouse airway allograft microvasculature and attenuates chronic rejection.

    PubMed

    Jiang, Xinguo; Khan, Mohammad A; Tian, Wen; Beilke, Joshua; Natarajan, Ramesh; Kosek, Jon; Yoder, Mervin C; Semenza, Gregg L; Nicolls, Mark R

    2011-06-01

    Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2⁺ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2⁺ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection. PMID:21606594

  19. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    PubMed

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition. PMID:27565868

  20. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  1. Bridge mediated ultrafast heterogeneous electron transfer

    NASA Astrophysics Data System (ADS)

    Ramakrishna, S.; Willig, F.; May, V.

    2002-01-01

    Bridge mediated photoinduced ultrafast heterogeneous electron transfer (ET) from a molecularly anchored chromophore to a semiconductor surface is modelled theoretically. The continuum levels of the semiconductor substrate are taken into account in the numerical calculations via a polynomial expansion. Electron transfer for the direct injection case in the strong coupling limit is studied and compared with cases where intermediate bridging states are successively introduced to weaken the effective electronic coupling. The role of vibronic coherences in the strong electronic coupling limit as well as in off-resonant bridge mediated electron transfer is also discussed.

  2. Gene transfer for erythropoiesis enhancement.

    PubMed

    Naffakh, N; Danos, O

    1996-08-01

    The spectrum of anemias treated with recombinant human erythropoietin is rapidly broadening. Lifelong treatment with very high doses is now under evaluation for beta-thalassemia and sickle cell anemia. These indications make it worthwhile to search for methods that will allow a permanent systemic delivery of the hormone. Here, we review experimental gene-transfer-based procedures for erythropoietin delivery in vivo. In mice, both ex vivo and direct in vivo approaches for gene transfer have resulted in the long-term production of therapeutic levels of the hormone. Gene transfer of erythropoietin could become a viable alternative to the injection of the purified recombinant protein once reliable procedures for controlling transgene expression are available. PMID:8796920

  3. Gene transfer: transduction.

    PubMed

    Frangipani, Emanuela

    2014-01-01

    Bacteriophages able to propagate on Pseudomonas strains are very common and can be easily isolated from natural environments or lysogenic strains. The development of transducing systems has allowed bacterial geneticists to perform chromosome analyses and mutation mapping. Moreover, these systems have also been proved to be a successful tool for molecular microbiologists to introduce a foreign gene or a mutation into the chromosome of a bacterial cell. This chapter provides a description of the phage methodology illustrated by Adams in 1959 and applicable to strain PAO1 derivatives. PMID:24818891

  4. Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy.

    PubMed

    Gregory, L G; Waddington, S N; Holder, M V; Mitrophanous, K A; Buckley, S M K; Mosley, K L; Bigger, B W; Ellard, F M; Walmsley, L E; Lawrence, L; Al-Allaf, F; Kingsman, S; Coutelle, C; Themis, M

    2004-07-01

    Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic. PMID:15141156

  5. Gene transfer in intact animals

    NASA Astrophysics Data System (ADS)

    Cline, M. J.; Stang, H.; Mercola, K.; Morse, L.; Ruprecht, R.; Browne, J.; Salser, W.

    1980-04-01

    Resistance to methotrexate was induced in bone marrow cells of mice by transformation in vitro with DNA from a drug-resistant cell line. Transformed cells were injected in vivo and haematopoietic cells expressing resistance were selected by drug treatment of recipients. Transformed cells had elevated levels of dihydrofolate reductase and demonstrated a proliferative advantage over untransformed cells, indicating successful gene transfer.

  6. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice.

    PubMed

    Kishida, T; Asada, H; Satoh, E; Tanaka, S; Shinya, M; Hirai, H; Iwai, M; Tahara, H; Imanishi, J; Mazda, O

    2001-08-01

    Direct intratumoral transfection of cytokine genes was performed by means of the in vivo electroporation as a novel therapeutic strategy for cancer. Plasmid vectors carrying the firefly luciferase, interleukin (IL)-12 and IL-18 genes were injected into established subcutaneous B16-derived melanomas followed by electric pulsation. When plasmid vectors with Epstein--Barr virus (EBV) nuclear antigen 1 (EBNA1) gene were employed, the expression levels of the transgenes were significantly higher in comparison with those obtained with conventional plasmid vectors. In consequence of the transfection with IL-12 and IL-18 genes, serum concentrations of the cytokines were significantly elevated, while interferon (IFN)-gamma also increased in the sera of the animals. The IL-12 gene transfection resulted in significant suppression of tumor growth, while the therapeutic effect was further improved by co-transfection with IL-12 and IL-18 genes. Repetitive co-transfection with IL-12 and IL-18 genes resulted in significant prolongation of survival of the animals. Natural killer (NK) and cytotoxic T lymphocyte (CTL) activities were markedly enhanced in the mice transfected with the cytokine genes. The present data suggest that the cytokine gene transfer can be successfully achieved by in vivo electroporation, leading to both specific and nonspecific antitumoral immune responses and significant therapeutic outcome. PMID:11509956

  7. Horizontal gene transfer in plants.

    PubMed

    Gao, Caihua; Ren, Xiaodong; Mason, Annaliese S; Liu, Honglei; Xiao, Meili; Li, Jiana; Fu, Donghui

    2014-03-01

    Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries. HGT often occurs in microbic and eukaryotic genomes. However, the pathways by which HGTs occur in multicellular eukaryotes, especially in plants, are not well understood. We systematically summarized more than ten possible pathways for HGT. The intimate contact which frequently occurs in parasitism, symbiosis, pathogen, epiphyte, entophyte, and grafting interactions could promote HGTs between two species. Besides these direct transfer methods, genes can be exchanged with a vector as a bridge: possible vectors include pollen, fungi, bacteria, viruses, viroids, plasmids, transposons, and insects. HGT, especially when involving horizontal transfer of transposable elements, is recognized as a significant force propelling genomic variation and biological innovation, playing an important functional and evolutionary role in both eukaryotic and prokaryotic genomes. We proposed possible mechanisms by which HGTs can occur, which is useful in understanding the genetic information exchange among distant species or distant cellular components. PMID:24132513

  8. Applications of Tol2 Transposon-Mediated Gene Transfer for Stable Integration and Conditional Expression of Electroporated Genes in Chicken Embryos

    NASA Astrophysics Data System (ADS)

    Sato, Yuki; Takahashi, Yoshiko

    Because of the high accessibility to developing embryos, avian embryos (chicken and quail) have long been used as a good model animal to study embryogenesis in vertebrates, especially amniotes (reviewed in Wolpert, 2004). The techniques used for “classical” avian embryology included tissue transplantations, tissue ablations, and cell-labeling by vital dye. At the end of the last century, the in ovo electropora tion technique was developed by Nakamura and his colleagues, and this modern method opened a way to study the roles of developmental genes directly in living embryos (Funahashi et al., 1999) reviewed in (Nakamura et al., 2004; Yasuda et al., 2000; Yasugi and Nakamura, 2000). This powerful technique allows us to introduce genes (DNA, RNA, morpholino) into embryos in a tissue-specific way by targeting a restricted area of embryonic tissues. Thus, the electroporation technique using chickens has provided numerous novel insights into the understanding of early development in vertebrates, making the chicken a unique model animal.

  9. Evaluation of All Nonsynonymous Single-Nucleotide Polymorphisms in the Gene Encoding Human Deoxyribonuclease I-Like 1, Possibly Implicated in the Blocking of Endocytosis-Mediated Foreign Gene Transfer

    PubMed Central

    Ueki, Misuzu; Kimura-Kataoka, Kaori; Fujihara, Junko; Iida, Reiko; Yasuda, Toshihiro

    2014-01-01

    Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution. PMID:24329527

  10. Evaluation of all nonsynonymous single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 1, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer.

    PubMed

    Ueki, Misuzu; Kimura-Kataoka, Kaori; Fujihara, Junko; Takeshita, Haruo; Iida, Reiko; Yasuda, Toshihiro

    2014-02-01

    Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution. PMID:24329527

  11. Plasmid-Mediated Quinolone Resistance Genes and Antibiotic Residues in Wastewater and Soil Adjacent to Swine Feedlots: Potential Transfer to Agricultural Lands

    PubMed Central

    Li, Juan; Wang, Thanh; Shao, Bing; Shen, Jianzhong; Wang, Shaochen

    2012-01-01

    Background: Inappropriate use of antibiotics in swine feed could cause accelerated emergence of antibiotic resistance genes, and agricultural application of swine waste could spread antibiotic resistance genes to the surrounding environment. Objectives: We investigated the distribution of plasmid-mediated quinolone resistance (PMQR) genes from swine feedlots and their surrounding environment. Methods: We used a culture-independent method to identify PMQR genes and estimate their levels in wastewater from seven swine feedlot operations and corresponding wastewater-irrigated farm fields. Concentrations of (fluoro)quinolones in wastewater and soil samples were determined by ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Results: The predominant PMQR genes in both the wastewater and soil samples were qnrD, qepA, and oqxB, whereas qnrS and oqxA were present only in wastewater samples. Absolute concentrations of all PMQR genes combined ranged from 1.66 × 107 to 4.06 × 108 copies/mL in wastewater and 4.06 × 106 to 9.52 × 107 copies/g in soil. Concentrations of (fluoro)quinolones ranged from 4.57 to 321 ng/mL in wastewater and below detection limit to 23.4 ng/g in soil. Significant correlations were found between the relative abundance of PMQR genes and (fluoro)quinolone concentrations (r = 0.71, p = 0.005) and the relative abundance of PMQR genes in paired wastewater and agricultural soil samples (r = 0.91, p = 0.005). Conclusions: Swine feedlot wastewater may be a source of PMQR genes that could facilitate the spread of antibiotic resistance. To our knowledge, this is the first study to examine the occurrence of PMQR genes in animal husbandry environments using a culture-independent method. PMID:22569244

  12. Foamy virus vectors for gene transfer

    PubMed Central

    Trobridge, Grant D.

    2009-01-01

    Foamy virus (FV) vectors are efficient gene delivery vehicles that have shown great promise for gene therapy in preclinical animal models. FVs or spumaretroviruses are not endemic in humans, but are prevalent in nonhuman primates and in other mammals. They have evolved means for efficient horizontal transmission in their host species without pathology. FV vectors have several unique properties that make them well-suited for therapeutic gene transfer including a desirable safety profile, a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. They mediate efficient and stable gene transfer to hematopoietic stem cells (HSCs) in mouse models, and in the canine large animal model. Analysis of FV vector integration sites in vitro and in hematopoietic repopulating cells shows they have a unique integration profile, and suggests they may be safer than gammaretroviruses or lentiviral vectors. Here properties of FVs relevant to the safety and efficacy of FV vectors are discussed. The development of FV vector systems is described, and studies evaluating their potential in vitro, and in small and large animal models is reviewed. PMID:19743892

  13. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    PubMed

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010

  14. Production of CFTR-null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer

    PubMed Central

    Rogers, Christopher S.; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A.; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W.; Kabel, Amanda C.; Yan, Ziying; Spate, Lee; Wax, David; Murphy, Clifton N.; Rieke, August; Whitworth, Kristin; Linville, Michael L.; Korte, Scott W.; Engelhardt, John F.; Welsh, Michael J.; Prather, Randall S.

    2008-01-01

    Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (ΔF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice. PMID:18324337

  15. Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer.

    PubMed

    Rogers, Christopher S; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W; Kabel, Amanda C; Yan, Ziying; Spate, Lee; Wax, David; Murphy, Clifton N; Rieke, August; Whitworth, Kristin; Linville, Michael L; Korte, Scott W; Engelhardt, John F; Welsh, Michael J; Prather, Randall S

    2008-04-01

    Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice. PMID:18324337

  16. Problems associated with gene transfer and opportunities for microgravity environments

    NASA Astrophysics Data System (ADS)

    Tennessen, Daniel J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

  17. Effects of Phosphorylatable Short Peptide-Conjugated Chitosan-Mediated IL-1Ra and igf-1 Gene Transfer on Articular Cartilage Defects in Rabbits

    PubMed Central

    Zhao, Ronglan; Peng, Xiaoxiang; Li, Qian; Song, Wei

    2014-01-01

    Previously, we reported an improvement in the transfection efficiency of the plasmid DNA-chitosan (pDNA/CS) complex by the utilization of phosphorylatable short peptide-conjugated chitosan (pSP-CS). In this study, we investigated the effects of pSP-CS-mediated gene transfection of interleukin-1 receptor antagonist protein (IL-1Ra) combined with insulin-like growth factor-1 (IGF-1) in rabbit chondrocytes and in a rabbit model of cartilage defects. pBudCE4.1-IL-1Ra+igf-1, pBudCE4.1-IL-1Ra and pBudCE4.1-igf-1 were constructed and combined with pSP-CS to form pDNA/pSP-CS complexes. These complexes were transfected into rabbit primary chondrocytes or injected into the joint cavity. Seven weeks after treatment, all rabbits were sacrificed and analyzed. High levels of IL-1Ra and igf-1 expression were detected both in the cell culture supernatant and in the synovial fluid. In vitro, the transgenic complexes caused significant proliferation of chondrocytes, promotion of glycosaminoglycan (GAG) and collagen II synthesis, and inhibition of chondrocyte apoptosis and nitric oxide (NO) synthesis. In vivo, the exogenous genes resulted in increased collagen II synthesis and reduced NO and GAG concentrations in the synovial fluid; histological studies revealed that pDNA/pSP-CS treatment resulted in varying degrees of hyaline-like cartilage repair and Mankin score decrease. The co-expression of both genes produced greater effects than each single gene alone both in vitro and in vivo. The results suggest that pSP-CS is a good candidate for use in gene therapy for the treatment of cartilage defects and that igf-1 and IL-1Ra co-expression produces promising biologic effects on cartilage defects. PMID:25390659

  18. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

    NASA Astrophysics Data System (ADS)

    Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

    2000-04-01

    Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

  19. Plasmid-mediated gene transfer between insect-resident bacteria, Enterobacter cloacae, and plant-epiphytic bacteria, Erwinia herbicola, in guts of silkworm larvae.

    PubMed

    Watanabe, K; Sato, M

    1998-11-01

    Five strains of Enterobacter cloacae isolated from several species of plants and insects were able to grow in the guts of silkworm larvae. A much larger population of Ent. cloacae strains was detected in the insect guts and feces collected 3 and 6 days than in samples collected 1 day after feeding artificial diets contaminating these bacteria. Furthermore, insect-origin strains of Ent. cloacae were mated with a donor strain, epiphytic Erwinia herbicola, harboring RSF1010 and pBPW1::Tn7 plasmids in the insect guts by introducing these bacteria through separate artificial diets administered at different times. A number of transconjugants, Ent. cloacae strains which had acquired RSF1010 plasmid, were detected from guts and fecal samples at transfer frequencies of 10(-2) to 10(-3) per recipient. Thus, gene transfer between epiphytic Er. herbicola and insect-resident Ent. cloacae strains in the insect guts was confirmed. These findings may provide significant information about the role of "in insecta mating" in the evolution of these bacteria. PMID:9767717

  20. Charge transfer-mediated singlet fission.

    PubMed

    Monahan, N; Zhu, X-Y

    2015-04-01

    Singlet fission, the splitting of a singlet exciton into two triplet excitons in molecular materials, is interesting not only as a model many-electron problem, but also as a process with potential applications in solar energy conversion. Here we discuss limitations of the conventional four-electron and molecular dimer model in describing singlet fission in crystalline organic semiconductors, such as pentacene and tetracene. We emphasize the need to consider electronic delocalization, which is responsible for the decisive role played by the Mott-Wannier exciton, also called the charge transfer (CT) exciton, in mediating singlet fission. At the strong electronic coupling limit, the initial excitation creates a quantum superposition of singlet, CT, and triplet-pair states, and we present experimental evidence for this interpretation. We also discuss the most recent attempts at translating this mechanistic understanding into design principles for CT state-mediated intramolecular singlet fission in oligomers and polymers. PMID:25648486

  1. Charge Transfer-Mediated Singlet Fission

    NASA Astrophysics Data System (ADS)

    Monahan, N.; Zhu, X.-Y.

    2015-04-01

    Singlet fission, the splitting of a singlet exciton into two triplet excitons in molecular materials, is interesting not only as a model many-electron problem, but also as a process with potential applications in solar energy conversion. Here we discuss limitations of the conventional four-electron and molecular dimer model in describing singlet fission in crystalline organic semiconductors, such as pentacene and tetracene. We emphasize the need to consider electronic delocalization, which is responsible for the decisive role played by the Mott-Wannier exciton, also called the charge transfer (CT) exciton, in mediating singlet fission. At the strong electronic coupling limit, the initial excitation creates a quantum superposition of singlet, CT, and triplet-pair states, and we present experimental evidence for this interpretation. We also discuss the most recent attempts at translating this mechanistic understanding into design principles for CT state-mediated intramolecular singlet fission in oligomers and polymers.

  2. Rhodobacter capsulatus DprA is essential for RecA-mediated gene transfer agent (RcGTA) recipient capability regulated by quorum-sensing and the CtrA response regulator.

    PubMed

    Brimacombe, Cedric A; Ding, Hao; Beatty, J Thomas

    2014-06-01

    Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation. PMID:24784901

  3. Horizontal gene transfer from Agrobacterium to plants

    PubMed Central

    Matveeva, Tatiana V.; Lutova, Ludmila A.

    2014-01-01

    Most genetic engineering of plants uses Agrobacterium mediated transformation to introduce novel gene content. In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana and Linaria. In these natural examples of horizontal gene transfer from Agrobacterium to plants, the T-DNA donor is assumed to be a mikimopine strain of A. rhizogenes. A sequence homologous to the T-DNA of the Ri plasmid of Agrobacterium rhizogenes was found in the genome of untransformed Nicotiana glauca about 30 years ago, and was named “cellular T-DNA” (cT-DNA). It represents an imperfect inverted repeat and contains homologs of several T-DNA oncogenes (NgrolB, NgrolC, NgORF13, NgORF14) and an opine synthesis gene (Ngmis). A similar cT-DNA has also been found in other species of the genus Nicotiana. These presumably ancient homologs of T-DNA genes are still expressed, indicating that they may play a role in the evolution of these plants. Recently T-DNA has been detected and characterized in Linaria vulgaris and L. dalmatica. In Linaria vulgaris the cT-DNA is present in two copies and organized as a tandem imperfect direct repeat, containing LvORF2, LvORF3, LvORF8, LvrolA, LvrolB, LvrolC, LvORF13, LvORF14, and the Lvmis genes. All L. vulgaris and L. dalmatica plants screened contained the same T-DNA oncogenes and the mis gene. Evidence suggests that there were several independent T-DNA integration events into the genomes of these plant genera. We speculate that ancient plants transformed by A. rhizogenes might have acquired a selective advantage in competition with the parental species. Thus, the events of T-DNA insertion in the plant genome might have affected their evolution, resulting in the creation of new plant species. In this review we focus on the structure and functions of cT-DNA in Linaria and Nicotiana and discuss their possible evolutionary role. PMID:25157257

  4. Lateral Gene Transfer from the Dead

    PubMed Central

    Szöllősi, Gergely J.; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

    2013-01-01

    In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria. [Gene tree reconciliation; lateral gene transfer; macroevolution; phylogeny.] PMID:23355531

  5. Horizontal gene transfer between bacteria and animals.

    PubMed

    Dunning Hotopp, Julie C

    2011-04-01

    Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria-to-animal transfer occur as part of intimate relationships such as those of endosymbionts and their invertebrate hosts, particularly insects and nematodes, while numerous transfers are also found in asexual animals. Both of these observations are consistent with modern evolutionary theory, in particular the serial endosymbiotic theory and Muller's ratchet. Although it is tempting to suggest that these particular lifestyles promote horizontal gene transfer, it is difficult to ascertain given the nonrandom sampling of animal genome sequencing projects and the lack of a systematic analysis of animal genomes for such transfers. PMID:21334091

  6. High expression hampers horizontal gene transfer.

    PubMed

    Park, Chungoo; Zhang, Jianzhi

    2012-01-01

    Horizontal gene transfer (HGT), the movement of genetic material from one species to another, is a common phenomenon in prokaryotic evolution. Although the rate of HGT is known to vary among genes, our understanding of the cause of this variation, currently summarized by two rules, is far from complete. The first rule states that informational genes, which are involved in DNA replication, transcription, and translation, have lower transferabilities than operational genes. The second rule asserts that protein interactivity negatively impacts gene transferability. Here, we hypothesize that high expression hampers HGT, because the fitness cost of an HGT to the recipient, arising from the 1) energy expenditure in transcription and translation, 2) cytotoxic protein misfolding, 3) reduction in cellular translational efficiency, 4) detrimental protein misinteraction, and 5) disturbance of the optimal protein concentration or cell physiology, increases with the expression level of the transferred gene. To test this hypothesis, we examined laboratory and natural HGTs to Escherichia coli. We observed lower transferabilities of more highly expressed genes, even after controlling the confounding factors from the two established rules and the genic GC content. Furthermore, expression level predicts gene transferability better than all other factors examined. We also confirmed the significant negative impact of gene expression on the rate of HGTs to 127 of 133 genomes of eubacteria and archaebacteria. Together, these findings establish the gene expression level as a major determinant of horizontal gene transferability. They also suggest that most successful HGTs are initially slightly deleterious, fixed because of their negligibly low costs rather than high benefits to the recipient. PMID:22436996

  7. Horizontal gene transfer, genome innovation and evolution.

    PubMed

    Gogarten, J Peter; Townsend, Jeffrey P

    2005-09-01

    To what extent is the tree of life the best representation of the evolutionary history of microorganisms? Recent work has shown that, among sets of prokaryotic genomes in which most homologous genes show extremely low sequence divergence, gene content can vary enormously, implying that those genes that are variably present or absent are frequently horizontally transferred. Traditionally, successful horizontal gene transfer was assumed to provide a selective advantage to either the host or the gene itself, but could horizontally transferred genes be neutral or nearly neutral? We suggest that for many prokaryotes, the boundaries between species are fuzzy, and therefore the principles of population genetics must be broadened so that they can be applied to higher taxonomic categories. PMID:16138096

  8. Gene Transfers Between Distantly Related Organisms

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.

    2003-01-01

    With the completion of numerous microbial genome sequences, reports of individual gene transfers between distantly related prokaryotes have become commonplace. On the other hand, transfers between prokaryotes and eukaryotes still excite the imagination. Many of these claims may be premature, but some are certainly valid. In this chapter, the kinds of supporting data needed to propose transfers between distantly related organisms and cite some interesting examples are considered.

  9. Horizontal gene transfer of stress resistance genes through plasmid transport.

    PubMed

    Shoeb, Erum; Badar, Uzma; Akhter, Jameela; Shams, Hina; Sultana, Maria; Ansari, Maqsood A

    2012-03-01

    The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature. PMID:22805823

  10. Decrease in neuroimmune activation by HSV-mediated gene transfer of TNFα soluble receptor alleviates pain in rats with diabetic neuropathy.

    PubMed

    Ortmann, Kathryn L Maier; Chattopadhyay, Munmun

    2014-10-01

    The mechanisms of diabetic painful neuropathy are complicated and comprise of peripheral and central pathophysiological phenomena. A number of proinflammatory cytokines are involved in this process. Tumor necrosis factor α (TNF-α) is considered to be one of the major contributors of neuropathic pain. In order to explore the potential role of inflammation in the peripheral nervous system of Type 1 diabetic animals with painful neuropathy, we investigated whether TNF-α is a key inflammatory mediator to the diabetic neuropathic pain and whether continuous delivery of TNFα soluble receptor from damaged axons achieved by HSV vector mediated transduction of DRG would block or alter the pain perception in animals with diabetic neuropathy. Diabetic animals exhibited changes in threshold of mechanical and thermal pain perception compared to control rats and also demonstrated increases in TNFα in the DRG, spinal cord dorsal horn, sciatic nerve and in the foot skin, 6 weeks after the onset of diabetes. Therapeutic approaches by HSV mediated expression of p55 TNF soluble receptor significantly attenuated the diabetes-induced hyperalgesia and decreased the expression of TNFα with reduction in the phosphorylation of p38MAPK in the spinal cord dorsal horn and DRG. The overall outcome of this study suggests that neuroinflammatory activation in the peripheral nervous system may be involved in the pathogenesis of painful neuropathy in Type 1 diabetes which can be alleviated by local expression of HSV vector expressing p55 TNF soluble receptor. PMID:24880032

  11. Immunotherapy of Malignancy by in vivo Gene Transfer into Tumors

    NASA Astrophysics Data System (ADS)

    Plautz, Gregory E.; Yang, Zhi-Yong; Wu, Bei-Yue; Gao, Xiang; Huang, Leaf; Nabel, Gary J.

    1993-05-01

    The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2K^s gene within the CT26 mouse colon adenocarcinoma (H-2K^d) or the MCA 106 fibrosarcoma (H-2K^b) induced a cytotoxic T-cell response to H-2K^s and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

  12. In vitro gene transfer by electrosonoporation.

    PubMed

    Escoffre, J M; Kaddur, K; Rols, M P; Bouakaz, A

    2010-10-01

    Among the nonviral methods for gene delivery in vitro, electroporation is simple, inexpensive and safe. To upregulate the expression level of transfected gene, we investigated the applicability of electrosonoporation. This approach consists of a combination of electric pulses and ultrasound assisted with gas microbubbles. Cells were first electroporated with plasmid DNA encoding-enhanced green fluorescent protein and then sonoporated in presence of contrast microbubbles. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Sonoporation can improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells. These results demonstrated that in vitro gene transfer by electrosonoporation could provide a new potent method for gene transfer. PMID:20850028

  13. Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells.

    PubMed

    Liu, Ping; Sun, Liang; Chen, Hui; Sun, Shui; Zhou, Dongsheng; Pang, Bo; Wang, Jian

    2015-11-01

    The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF‑β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue. PMID:26328747

  14. Experiments on gene transferring to primary hematopoietic cells by liposome.

    PubMed

    Hu, L; Zhang, B

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by X-gal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33 +/- 2.68)% in human and about (16.28 +/- 2.95)% in mouse without G418 selection. After G418 selection, the blue cell rate was (46.06 +/- 3.47)% in human and (43.45 +/- 4.1)% in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia. PMID:12840913

  15. Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.

    PubMed

    Ishihara, Akikazu; Shields, Kathleen M; Litsky, Alan S; Mattoon, John S; Weisbrode, Steven E; Bartlett, Jeffrey S; Bertone, Alicia L

    2008-06-01

    This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites. PMID:18241059

  16. Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

    PubMed Central

    Stacey, A; Mulligan, R; Jaenisch, R

    1987-01-01

    A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal. Images PMID:3599181

  17. Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells.

    PubMed

    Majdoul, Saliha; Seye, Ababacar K; Kichler, Antoine; Holic, Nathalie; Galy, Anne; Bechinger, Burkhard; Fenard, David

    2016-01-29

    Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and γ-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140° formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy. PMID:26668323

  18. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  19. The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

    PubMed

    Alton, Eric W F W; Baker, Alison; Baker, Eilidh; Boyd, A Christopher; Cheng, Seng H; Coles, Rebecca L; Collie, D David S; Davidson, Heather; Davies, Jane C; Gill, Deborah R; Gordon, Catherine; Griesenbach, Uta; Higgins, Tracy; Hyde, Stephen C; Innes, J Alastair; McCormick, Dominique; McGovern, Michael; McLachlan, Gerry; Porteous, David J; Pringle, Ian; Scheule, Ronald K; Shaw, Darren J; Smith, Sionagh; Sumner-Jones, Stephanie G; Tennant, Peter; Vrettou, Christina

    2013-12-01

    Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients. PMID:24090839

  20. Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X-Linked Muscular Dystrophy and May Induce Immune Tolerance

    PubMed Central

    Hayashita-Kinoh, Hiromi; Yugeta, Naoko; Okada, Hironori; Nitahara-Kasahara, Yuko; Chiyo, Tomoko; Okada, Takashi; Takeda, Shin'ichi

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype. PMID:25586688

  1. Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

    PubMed

    Le Guiner, Caroline; Stieger, Knut; Toromanoff, Alice; Guilbaud, Mickaël; Mendes-Madeira, Alexandra; Devaux, Marie; Guigand, Lydie; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne; Adjali, Oumeya

    2014-01-01

    Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed. PMID:25248159

  2. AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

    PubMed Central

    Elsworth, JD; Redmond, DE; Leranth, C; Bjugstad, KB; Sladek, JR; Collier, TJ; Foti, SB; Samulski, RJ; Vives, KP; Roth, RH

    2009-01-01

    Neural transplantation offers the potential of treating Parkinson’s disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson’s disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced over-expression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson’s disease. PMID:18346734

  3. Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury

    PubMed Central

    Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren

    2014-01-01

    Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good

  4. Transferred interbacterial antagonism genes augment eukaryotic innate immune function

    PubMed Central

    Chou, Seemay; Daugherty, Matthew D.; Peterson, S. Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L.; Fritz-Laylin, Lillian K.; Ferrin, Michael A.; Harding, Brittany N.; Jacobs-Wagner, Christine; Yang, X. Frank; Vollmer, Waldemar; Malik, Harmit S.

    2015-01-01

    Horizontal gene transfer (HGT) allows organisms to rapidly acquire adaptive traits1. Though documented instances of HGT from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option2. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce anti-bacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system (T6SS)3. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years via purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the etiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for facile co-option by eukaryotic innate immune systems. PMID:25470067

  5. Repeated, recent and diverse transfers of a mitochondrial gene to the nucleus in flowering plants.

    PubMed

    Adams, K L; Daley, D O; Qiu, Y L; Whelan, J; Palmer, J D

    2000-11-16

    A central component of the endosymbiotic theory for the bacterial origin of the mitochondrion is that many of its genes were transferred to the nucleus. Most of this transfer occurred early in mitochondrial evolution; functional transfer of mitochondrial genes has ceased in animals. Although mitochondrial gene transfer continues to occur in plants, no comprehensive study of the frequency and timing of transfers during plant evolution has been conducted. Here we report frequent loss (26 times) and transfer to the nucleus of the mitochondrial gene rps10 among 277 diverse angiosperms. Characterization of nuclear rps10 genes from 16 out of 26 loss lineages implies that many independent, RNA-mediated rps10 transfers occurred during recent angiosperm evolution; each of the genes may represent a separate functional gene transfer. Thus, rps10 has been transferred to the nucleus at a surprisingly high rate during angiosperm evolution. The structures of several nuclear rps10 genes reveal diverse mechanisms by which transferred genes become activated, including parasitism of pre-existing nuclear genes for mitochondrial or cytoplasmic proteins, and activation without gain of a mitochondrial targeting sequence. PMID:11099041

  6. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

    PubMed

    Khalil, Rowaida K S; Skinner, Craig; Patfield, Stephanie; He, Xiaohua

    2016-07-01

    Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health. PMID:27109772

  7. Viral Vectors for in Vivo Gene Transfer

    NASA Astrophysics Data System (ADS)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  8. [Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

    PubMed

    Wang, Guozhong; Hu, Shenjiang; Zheng, Zhelan; Sun, Jian; Zheng, Xia; Zhu, Zhaohui; Li, Jiang; Yao, Yumei

    2006-08-01

    To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle. PMID:17002125

  9. Gene transfer from a parasitic flowering plant to a fern

    PubMed Central

    Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

    2005-01-01

    The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts. PMID:16191635

  10. Unsupervised learning in detection of gene transfer.

    PubMed

    Hamel, L; Nahar, N; Poptsova, M S; Zhaxybayeva, O; Gogarten, J P

    2008-01-01

    The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events. PMID:18509479

  11. Syntrophic growth via quinone-mediated interspecies electron transfer

    PubMed Central

    Smith, Jessica A.; Nevin, Kelly P.; Lovley, Derek R.

    2015-01-01

    The mechanisms by which microbial species exchange electrons are of interest because interspecies electron transfer can expand the metabolic capabilities of microbial communities. Previous studies with the humic substance analog anthraquinone-2,6-disulfonate (AQDS) suggested that quinone-mediated interspecies electron transfer (QUIET) is feasible, but it was not determined if sufficient energy is available from QUIET to support the growth of both species. Furthermore, there have been no previous studies on the mechanisms for the oxidation of anthrahydroquinone-2,6-disulfonate (AHQDS). A co-culture of Geobacter metallireducens and G. sulfurreducens metabolized ethanol with the reduction of fumarate much faster in the presence of AQDS, and there was an increase in cell protein. G. sulfurreducens was more abundant, consistent with G. sulfurreducens obtaining electrons from acetate that G. metallireducens produced from ethanol, as well as from AHQDS. Co-cultures initiated with a citrate synthase-deficient strain of G. sulfurreducens that was unable to use acetate as an electron donor also metabolized ethanol with the reduction of fumarate and cell growth, but acetate accumulated over time. G. sulfurreducens and G. metallireducens were equally abundant in these co-cultures reflecting the inability of the citrate synthase-deficient strain of G. sulfurreducens to metabolize acetate. Evaluation of the mechanisms by which G. sulfurreducens accepts electrons from AHQDS demonstrated that a strain deficient in outer-surface c-type cytochromes that are required for AQDS reduction was as effective at QUIET as the wild-type strain. Deletion of additional genes previously implicated in extracellular electron transfer also had no impact on QUIET. These results demonstrate that QUIET can yield sufficient energy to support the growth of both syntrophic partners, but that the mechanisms by which electrons are derived from extracellular hydroquinones require further investigation. PMID

  12. Lateral gene transfer in the subsurface

    SciTech Connect

    Barkay, Tamar; Sobecky, Patricia

    2007-08-27

    Lateral gene transfer (LGT) is an important adaptive mechanism among prokaryotic organisms. This mechanism is particularly important for the response of microorganisms to changing environmental conditions because it facilitates the transfer of a large number of genes and their rapid expression. Together the transferred genes promote rapid genetic and metabolic changes that may enhance survival to newly established and sometimes hostile environmental conditions. The goal of our project was to examine if and how LGT enhances microbial adaptation to toxic heavy metals in subsurface environments that had been contaminated by mixed wastes due to activities associated with the production of nuclear energy and weapons. This task has been accomplished by dividing the project to several sub-tasks. Thus, we: (1) Determined the level of resistance of subsurface bacterial isolates to several toxic metals, all identified as pollutants of concern in subsurface environments; (2) Designed, tested, and applied, a molecular approach that determined whether metal resistance genes had evolved by LGT among subsurface bacteria; and (3) Developed a DNA hybridization array for the identification of broad host range plasmids and of metal resistance plasmids. The results are briefly summarized below with references to published papers and manuscripts in preparation where details about our research can be found. Additional information may be found in copies of our published manuscripts and conference proceedings, and our yearly reports that were submitted through the RIMS system.

  13. Perinatal Gene Transfer to the Liver

    PubMed Central

    McKay, Tristan R; Rahim, Ahad A; Buckley, Suzanne M.K; Ward, Natalie J; Chan, Jerry K.Y; Howe, Steven J; Waddington, Simon N

    2011-01-01

    The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer. PMID:21774770

  14. Clinical Applications Involving CNS Gene Transfer

    PubMed Central

    Kantor, Boris; McCown, Thomas; Leone, Paola; Gray, Steven J.

    2015-01-01

    Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood–brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this be a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective. This chapter will explore the past, current, and future status of CNS gene therapy, focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors. PMID:25311921

  15. Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall

    NASA Astrophysics Data System (ADS)

    Nabel, Elizabeth G.; Plautz, Gregory; Nabel, Gary J.

    1990-09-01

    A recombinant β-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.

  16. In vivo Cytokine Gene Transfer by Gene Gun Reduces Tumor Growth in Mice

    NASA Astrophysics Data System (ADS)

    Sun, Wenn H.; Burkholder, Joseph K.; Sun, Jian; Culp, Jerilyn; Turner, Joel; Lu, Xing G.; Pugh, Thomas D.; Ershler, William B.; Yang, Ning-Sun

    1995-03-01

    Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor α and interferon γ genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon γ genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon γ were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

  17. Modulation of Treg function improves adenovirus vector-mediated gene expression in the airway.

    PubMed

    Nagai, Y; Limberis, M P; Zhang, H

    2014-02-01

    Virus vector-mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the cystic fibrosis transmembrane conductance regulator gene. The pathological proinflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limits the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (T(reg)) and to improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway. PMID:24385144

  18. Direct Gene Transfer into Human Cultured Cells Facilitated by Laser Micropuncture of the Cell Membrane

    NASA Astrophysics Data System (ADS)

    Tao, Wen; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  19. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  20. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    PubMed Central

    Papke, R. Thane; Corral, Paulina; Ram-Mohan, Nikhil; de la Haba, Rafael R.; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  1. Non-Viral Gene Transfer as a Tool for Studying Transcription Regulation of Xenobiotic Metabolizing Enzymes

    PubMed Central

    Bonamassa, Barbara; Liu, Dexi

    2010-01-01

    Numerous xenobiotic metabolizing enzymes are regulated by nuclear receptors at transcriptional level. The challenge we currently face is to understand how a given nuclear receptor interacts with its xenobiotics, migrates into nucleus, binds to the xenobiotic response element of a target gene, and regulates transcription. Toward this end, new methods have been developed to introduce the nuclear receptor gene into appropriate cells and study its activity in activating reporter gene expression under the control of a promoter containing xenobiotic response elements. The goal of this review is to critically examine the gene transfer methods currently available. We concentrate on the gene transfer mechanism, advantages and limitations of each method when employed for nuclear receptor-mediated gene regulation studies. It is our hope that the information provided highlights the importance of gene transfer in studying the mechanisms by which our body eliminates the potentially harmful substances and maintains the homeostasis. PMID:20713102

  2. Torsion-Mediated Interaction between Adjacent Genes

    PubMed Central

    Meyer, Sam; Beslon, Guillaume

    2014-01-01

    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 (co)regulation in eukaryotes. PMID:25188032

  3. Enhanced effect of microdystrophin gene transfection by HSV-VP22 mediated intercellular protein transport

    PubMed Central

    Xiong, Fu; Xiao, Shaobo; Yu, Meijuan; Li, Wanyi; Zheng, Hui; Shang, Yanchang; Peng, Funing; Zhao, Cuiping; Zhou, Wenliang; Chen, Huanchun; Fang, Liurong; Chamberlain, Jeffrey S; Zhang, Cheng

    2007-01-01

    Background Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo. Results Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice. Conclusion These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD. PMID:17617925

  4. Simple rapid method for gene transfer

    SciTech Connect

    Cockburn, A.F.; Meier, H.

    1990-01-30

    The object of the present invention is to provide methods for gene transfer that reduce or eliminate cellular pretreatment steps, e.g., the removal of cell wall by chemical or enzymatic methods, is rapid and can be practiced without the need of additional expensive equipment. Cells, embryos or tissues selected for genetic manipulation are suspended in an Eppendorf tube in an aliquot of the desired genetic material to be transferred to which the resulting mixture is added and is agitated by vortexing from about 30 to about 90 seconds. The cells, embryos or tissue are sedimented and the DNA supernatant removed. After sedimentation, the injected material is resuspended in or on a growth medium to assay for expression.

  5. Reducible cationic lipids for gene transfer.

    PubMed Central

    Wetzer, B; Byk, G; Frederic, M; Airiau, M; Blanche, F; Pitard, B; Scherman, D

    2001-01-01

    One of the main challenges of gene therapy remains the increase of gene delivery into eukaryotic cells. We tested whether intracellular DNA release, an essential step for gene transfer, could be facilitated by using reducible cationic DNA-delivery vectors. For this purpose, plasmid DNA was complexed with cationic lipids bearing a disulphide bond. This reduction-sensitive linker is expected to be reduced and cleaved in the reducing milieu of the cytoplasm, thus potentially improving DNA release and consequently transfection. The DNA--disulphide-lipid complexation was monitored by ethidium bromide exclusion, and the size of complexes was determined by dynamic light scattering. It was found that the reduction kinetics of disulphide groups in DNA--lipid complexes depended on the position of the disulphide linker within the lipid molecule. Furthermore, the internal structure of DNA--lipid particles was examined by small-angle X-ray scattering before and after lipid reduction. DNA release from lipid complexes was observed after the reduction of disulphide bonds of several lipids. Cell-transfection experiments suggested that complexes formed with selected reducible lipids resulted in up to 1000-fold higher reporter-gene activity, when compared with their analogues without disulphide bonds. In conclusion, reduction-sensitive groups introduced into cationic lipid backbones potentially allow enhanced DNA release from DNA--lipid complexes after intracellular reduction and represent a tool for improved vectorization. PMID:11389682

  6. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

    PubMed

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I

    2016-03-01

    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer. PMID:26560080

  7. Proton Transfer in Nucleobases is Mediated by Water

    SciTech Connect

    Khistyaev, Kirill; Golan, Amir; Bravaya, Ksenia B.; Orms, Natalie; Krylov, Anna I.; Ahmed, Musahid

    2013-08-08

    Water plays a central role in chemistry and biology by mediating the interactions between molecules, altering energy levels of solvated species, modifying potential energy proles along reaction coordinates, and facilitating ecient proton transport through ion channels and interfaces. This study investigates proton transfer in a model system comprising dry and microhydrated clusters of nucleobases. With mass spectrometry and tunable vacuum ultraviolet synchrotron radiation, we show that water shuts down ionization-induced proton transfer between nucleobases, which is very ecient in dry clusters. Instead, a new pathway opens up in which protonated nucleo bases are generated by proton transfer from the ionized water molecule and elimination of a hydroxyl radical. Electronic structure calculations reveal that the shape of the potential energy prole along the proton transfer coordinate depends strongly on the character of the molecular orbital from which the electron is removed, i.e., the proton transfer from water to nucleobases is barrierless when an ionized state localized on water is accessed. The computed energetics of proton transfer is in excellent agreement with the experimental appearance energies. Possible adiabatic passage on the ground electronic state of the ionized system, while energetically accessible at lower energies, is not ecient. Thus, proton transfer is controlled electronically, by the character of the ionized state, rather than statistically, by simple energy considerations.

  8. Gene therapy: Biological pacemaker created by gene transfer

    NASA Astrophysics Data System (ADS)

    Miake, Junichiro; Marbán, Eduardo; Nuss, H. Bradley

    2002-09-01

    The pacemaker cells of the heart initiate the heartbeat, sustain the circulation, and dictate the rate and rhythm of cardiac contraction. Circulatory collapse ensues when these specialized cells are damaged by disease, a situation that currently necessitates the implantation of an electronic pacemaker. Here we report the use of viral gene transfer to convert quiescent heart-muscle cells into pacemaker cells, and the successful generation of spontaneous, rhythmic electrical activity in the ventricle in vivo. Our results indicate that genetically engineered pacemakers could be developed as a possible alternative to implantable electronic devices.

  9. Gene duplication and transfer events in plant mitochondria genome

    SciTech Connect

    Xiong Aisheng Peng Rihe; Zhuang Jing; Gao Feng; Zhu Bo; Fu Xiaoyan; Xue Yong; Jin Xiaofen; Tian Yongsheng; Zhao Wei; Yao Quanhong

    2008-11-07

    Gene or genome duplication events increase the amount of genetic material available to increase the genomic, and thereby phenotypic, complexity of organisms during evolution. Gene duplication and transfer events have been important to molecular evolution in all three domains of life, and may be the first step in the emergence of new gene functions. Gene transfer events have been proposed as another accelerator of evolution. The duplicated gene or genome, mainly nuclear, has been the subject of several recent reviews. In addition to the nuclear genome, organisms have organelle genomes, including mitochondrial genome. In this review, we briefly summarize gene duplication and transfer events in the plant mitochondrial genome.

  10. An XMRV Derived Retroviral Vector as a Tool for Gene Transfer

    PubMed Central

    2011-01-01

    Background Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. Findings We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. Conclusions We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies. PMID:21651801

  11. Optical gene transfer by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Konig, Karsten; Riemann, Iris; Tirlapur, Uday K.

    2003-07-01

    Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany; focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.

  12. Lentiviral vector gene transfer to porcine airways.

    PubMed

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1-based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF).Molecular Therapy - Nucleic Acids (2012) 1, e56; doi:10.1038/mtna.2012.47; published online 27 November 2012. PMID:23187455

  13. CXCR4 gene transfer prevents pressure overload induced heart failure

    PubMed Central

    LaRocca, Thomas J.; Jeong, Dongtak; Kohlbrenner, Erik; Lee, Ahyoung; Chen, JiQiu; Hajjar, Roger J.; Tarzami, Sima T.

    2012-01-01

    Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adenoreceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure. PMID:22668785

  14. Improved retroviral suicide gene transfer in colon cancer cell lines after cell synchronization with methotrexate

    PubMed Central

    2011-01-01

    Background Cancer gene therapy by retroviral vectors is mainly limited by the level of transduction. Retroviral gene transfer requires target cell division. Cell synchronization, obtained by drugs inducing a reversible inhibition of DNA synthesis, could therefore be proposed to precondition target cells to retroviral gene transfer. We tested whether drug-mediated cell synchronization could enhance the transfer efficiency of a retroviral-mediated gene encoding herpes simplex virus thymidine kinase (HSV-tk) in two colon cancer cell lines, DHDK12 and HT29. Methods Synchronization was induced by methotrexate (MTX), aracytin (ara-C) or aphidicolin. Gene transfer efficiency was assessed by the level of HSV-TK expression. Transduced cells were driven by ganciclovir (GCV) towards apoptosis that was assessed using annexin V labeling by quantitative flow cytometry. Results DHDK12 and HT29 cells were synchronized in S phase with MTX but not ara-C or aphidicolin. In synchronized DHDK12 and HT29 cells, the HSV-TK transduction rates were 2 and 1.5-fold higher than those obtained in control cells, respectively. Furthermore, the rate of apoptosis was increased two-fold in MTX-treated DHDK12 cells after treatment with GCV. Conclusions Our findings indicate that MTX-mediated synchronization of target cells allowed a significant improvement of retroviral HSV-tk gene transfer, resulting in an increased cell apoptosis in response to GCV. Pharmacological control of cell cycle may thus be a useful strategy to optimize the efficiency of retroviral-mediated cancer gene therapy. PMID:21970612

  15. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    SciTech Connect

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  16. In vivo expression of adenovirus-mediated lacZ gene in murine nasal mucosa.

    PubMed

    Arimoto, Yukiko; Nagata, Hiroshi; Isegawa, Naohisa; Kumahara, Keiichiro; Isoyama, Kyoko; Konno, Akiyoshi; Shirasawa, Hiroshi

    2002-09-01

    Adenovirus is a good tool for transferring exogenous genes into various organs because the virus has a wide spectrum of infection. In this report, we demonstrate that a recombinant adenovirus, Ax1CAlacZ, can transfer an exogenous lacZ gene into murine nasal mucosa in vivo. The efficiency of the exogenous gene expression varied for different cell types and was improved by optimizing the method of administration. In the olfactory region, the olfactory epithelia, sustentacular cells and olfactory nerve efficiently expressed lacZ gene transferred by Ax1CAlacZ using either of two administration methods, dripping or injecting. In contrast, in the respiratory region, the respiratory epithelia but not the subepithelial tissues expressed lacZ gene transferred by Ax1CAlacZ, and the efficiency of the gene transfer, which was low when the virus was administered by nasal drops, was improved when the virus was administered by injection. Our study demonstrated that gene transfer mediated by adenovirus is more efficient in the olfactory epithelia than in the respiratory epithelia, and may be applicable to nasal or paranasal diseases such as olfactory epithelial disturbances. PMID:12403125

  17. Impact of plant and environmental factors on ALS-resistant gene transfer rate from ClearfieldTM rice to red rice biotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pollen-mediated transfer of ALS-resistant gene from ClearfieldTM (CL) rice to red rice can affect the population dynamics and long-term management of red rice. To mitigate gene flow, it is important to understand the plant and environmental factors which affect gene transfer rate. This experiment ai...

  18. Methods for Gene Transfer to the Central Nervous System

    PubMed Central

    Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

    2015-01-01

    Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

  19. Gene transfer system for Rhodopseudomonas viridis.

    PubMed Central

    Lang, F S; Oesterhelt, D

    1989-01-01

    A gene transfer system for Rhodopseudomonas viridis was established which uses conjugation with Escherichia coli S17-I as the donor and mobilizable plasmids as vectors. Initially, plasmids of the incompatibility group P1 (pRK290 and pRK404) were used. The more effective shuttle vectors between E. coli and R. viridis, pKV1 and pKVS1, were derived from plasmid pBR322 and showed the highest conjugation frequency (10(-2] thus far demonstrated in purple bacteria. It was also demonstrated that Rhizobium meliloti can be used as a donor for conjugation with R. viridis. From a genomic cosmid library of R. viridis constructed in the vector pHC79, clones that coded for subunits H (puh operon), L, M and cytochrome c (puf operon) of the photosynthetic reaction center were isolated and characterized. For linkage of the two operons on the genome, cosmids that overlapped with the operon-carrying clones were identified. The relative positions of the two operons could not be determined, but the operons must be more than 100 kilobase pairs apart. Thus, the genomic organization of the reaction center in R. viridis is different from that of Rhodobacter capsulatus, for which a distance of about 39 kilobase pairs was determined. From a spontaneous mutant of R. viridis that is resistant to the herbicide terbutryn, the puf operon was cloned in pKVS1 and transferred by conjugation into R. viridis wild-type cells. The resulting exconjugants were resistant to the herbicide, which demonstrated that the puf operon on pKVS1 constructions was functionally expressed in R. viridis. Images PMID:2666398

  20. Moving toward a higher efficiency of microcell-mediated chromosome transfer.

    PubMed

    Liskovykh, Mikhail; Lee, Nicholas Co; Larionov, Vladimir; Kouprina, Natalay

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. PMID:27382603

  1. Moving toward a higher efficiency of microcell-mediated chromosome transfer

    PubMed Central

    Liskovykh, Mikhail; Lee, Nicholas CO; Larionov, Vladimir; Kouprina, Natalay

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. PMID:27382603

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

    PubMed

    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. PMID:23821951

  3. Recent Trends of Polymer Mediated Liposomal Gene Delivery System

    PubMed Central

    Lee, Sang-Soo; George Priya Doss, C.; Yagihara, Shin; Kim, Do-Young

    2014-01-01

    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. PMID:25250340

  4. Horizontal gene transfer of a Chlamydial tRNA-guanine transglycosylase gene to eukaryotic microbes.

    PubMed

    Manna, Sam; Harman, Ashley

    2016-01-01

    tRNA-guanine transglycosylases are found in all domains of life and mediate the base exchange of guanine with queuine in the anticodon loop of tRNAs. They can also regulate virulence in bacteria such as Shigella flexneri, which has prompted the development of drugs that inhibit the function of these enzymes. Here we report a group of tRNA-guanine transglycosylases in eukaryotic microbes (algae and protozoa) which are more similar to their bacterial counterparts than previously characterized eukaryotic tRNA-guanine transglycosylases. We provide evidence demonstrating that the genes encoding these enzymes were acquired by these eukaryotic lineages via horizontal gene transfer from the Chlamydiae group of bacteria. Given that the S. flexneri tRNA-guanine transglycosylase can be targeted by drugs, we propose that the bacterial-like tRNA-guanine transglycosylases could potentially be targeted in a similar fashion in pathogenic amoebae that possess these enzymes such as Acanthamoeba castellanii. This work also presents ancient prokaryote-to-eukaryote horizontal gene transfer events as an untapped resource of potential drug target identification in pathogenic eukaryotes. PMID:26435002

  5. Development of second- and third-generation bovine immunodeficiency virus-based gene transfer systems.

    PubMed

    Matukonis, Meghan; Li, Mengtao; Molina, Rene P; Paszkiet, Brian; Kaleko, Michael; Luo, Tianci

    2002-07-20

    Lentivirus-based gene transfer systems have demonstrated their utility in mediating gene transfer to dividing and nondividing cells both in vitro and in vivo. An early-generation gene transfer system developed from bovine immunodeficiency virus (BIV) has been described (Berkowitz et al., J. Virol. 2001;75:3371-3382). In this paper, we describe the development of second-generation (three-plasmid) and third-generation (four-plasmid) BIV-based systems. All accessory genes (vif, vpw, vpy, and tmx) and the regulatory gene tat were deleted or largely truncated from the packaging construct. Furthermore, we split the packaging function into two constructs by expressing Rev in a separate plasmid. Together with our minimal BIV transfer vector construct and a vesicular stomatitis virus G glycoprotein-expressing plasmid, the BIV vectors were generated. The vectors produced by the three- and four-plasmid systems had titers greater than 1 x 10(6) transducing units per milliliter and were fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These results suggest that the accessory genes vif, vpw, vpy, and tmx are dispensable for functional BIV vector development. The modifications made to the packaging constructs improve the safety profile of the vector system. Finally, BIV vectors provide an alternative to human immunodeficiency virus-based gene transfer systems. PMID:12162812

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

    PubMed Central

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

    2015-01-01

    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. PMID:26379286

  7. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

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

    PubMed

    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. PMID:26379286

  9. Photoregulation of a phytochrome gene promoter from oat transferred into rice by particle bombardment.

    PubMed Central

    Bruce, W B; Christensen, A H; Klein, T; Fromm, M; Quail, P H

    1989-01-01

    The regulatory photoreceptor phytochrome controls the transcription of its own phy genes in a negative feedback fashion. We have exploited microprojectile-mediated gene transfer to develop a rapid transient expression assay system for the study of DNA sequences involved in the phytochrome-regulated expression of these genes. The 5'-flanking sequence and part of the structural region of an oat phy gene have been fused to a reporter coding sequence (chloramphenicol acetyltransferase, CAT) and introduced into intact darkgrown seedlings by using high-velocity microprojectiles. Expression is assayable in less than 24 hr from bombardment. The introduced oat phy-CAT fusion gene is expressed and down-regulated by white light in barley, rice, and oat, whereas no expression is detected in three dicots tested, tobacco, cucumber, and Arabidopsis thaliana. In bombarded rice shoots, red/far-red light-reversible repression of expression of the heterologous oat phy-CAT gene shows that it is regulated by phytochrome in a manner parallel to that of the endogenous rice phy genes. These data indicate that the transduction pathway components and promoter sequences involved in autoregulation of phy expression have been evolutionarily conserved between oat and rice. The experiments show the feasibility of using high-velocity microprojectile-mediated gene transfer for the rapid analysis of light-controlled monocot gene promoters in monocot tissues that until now have been recalcitrant to such studies. Images PMID:2602370

  10. Passive Immunization against HIV/AIDS by Antibody Gene Transfer

    PubMed Central

    Yang, Lili; Wang, Pin

    2014-01-01

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics. PMID:24473340

  11. Passive immunization against HIV/AIDS by antibody gene transfer.

    PubMed

    Yang, Lili; Wang, Pin

    2014-02-01

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics. PMID:24473340

  12. A recently transferred cluster of bacterial genes in Trichomonas vaginalis - lateral gene transfer and the fate of acquired genes

    PubMed Central

    2014-01-01

    Background Lateral Gene Transfer (LGT) has recently gained recognition as an important contributor to some eukaryote proteomes, but the mechanisms of acquisition and fixation in eukaryotic genomes are still uncertain. A previously defined norm for LGTs in microbial eukaryotes states that the majority are genes involved in metabolism, the LGTs are typically localized one by one, surrounded by vertically inherited genes on the chromosome, and phylogenetics shows that a broad collection of bacterial lineages have contributed to the transferome. Results A unique 34 kbp long fragment with 27 clustered genes (TvLF) of prokaryote origin was identified in the sequenced genome of the protozoan parasite Trichomonas vaginalis. Using a PCR based approach we confirmed the presence of the orthologous fragment in four additional T. vaginalis strains. Detailed sequence analyses unambiguously suggest that TvLF is the result of one single, recent LGT event. The proposed donor is a close relative to the firmicute bacterium Peptoniphilus harei. High nucleotide sequence similarity between T. vaginalis strains, as well as to P. harei, and the absence of homologs in other Trichomonas species, suggests that the transfer event took place after the radiation of the genus Trichomonas. Some genes have undergone pseudogenization and degradation, indicating that they may not be retained in the future. Functional annotations reveal that genes involved in informational processes are particularly prone to degradation. Conclusions We conclude that, although the majority of eukaryote LGTs are single gene occurrences, they may be acquired in clusters of several genes that are subsequently cleansed of evolutionarily less advantageous genes. PMID:24898731

  13. Different gene transfer methods at the very early, early, late and whole embryonic stages in chicken.

    PubMed

    Gong, Ping; Yang, Y P; Yang, Y; Feng, Yan P; Li, S J; Peng, Xiu L; Gong, Y Z

    2012-12-01

    New technologies in gene transfer combined with experimental embryology make the chicken embryo an excellent model system for gene function studies. The techniques of in ovo electroporation, in vitro culture for ex ovo electroporation and retrovirus-mediated gene transfer have already been fully developed in chicken. Yet to our knowledge, there are no definite descriptions on the features and application scopes of these techniques. The survival rates of different in vitro culture methods were compared and the EGFP expression areas of different gene transfer techniques were explored. It was that the optimal timings of removing embryo for EC culture and Petri dish system was at E1.5 and E2.5, respectively; and optimal timing of injecting retrovirus is at E0. Results indicated that the EC culture, in ovo electroporation, the Petri dish system and retrovirus-mediated method are, respectively, suitable for the very early, early, late and whole embryonic stages in chicken. Comparison of different gene transfer methods and establishment of optimal timings are expected to provide a better choice of the efficient method for a particular experiment. PMID:23134602

  14. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

    PubMed

    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes. PMID:26414246

  15. Limitations of the murine nose in the development of nonviral airway gene transfer.

    PubMed

    Griesenbach, Uta; Sumner-Jones, Stephanie G; Holder, Emma; Munkonge, Felix M; Wodehouse, Theresa; Smith, Stephen N; Wasowicz, Marguerite Y; Pringle, Ian; Casamayor, Isabel; Chan, Mario; Coles, Rebecca; Cornish, Nikki; Dewar, Ann; Doherty, Ann; Farley, Raymond; Green, Anne-Marie; Jones, Bryony L; Larsen, Mia D B; Lawton, Anna E; Manvell, Michelle; Painter, Hazel; Singh, Charanjit; Somerton, Lucinda; Stevenson, Barbara; Varathalingam, Anusha; Siegel, Craig; Scheule, Ronald K; Cheng, Seng H; Davies, Jane C; Porteous, David J; Gill, Deborah R; Boyd, A Christopher; Hyde, Steve C; Alton, Eric W F W

    2010-07-01

    A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials. PMID:19648474

  16. Direct transfer of IL-12 gene into growing Renca tumors.

    PubMed

    Budryk, M; Wilczyńska, U; Szary, J; Szala, S

    2000-01-01

    We investigated the feasibility of transferring naked plasmid DNA containing a therapeutic gene (IL-12) into mice harboring growing Renca tumors. We found that naked DNA transferred into growing Renca and B16(F10) tumors gives higher expression level of reporter gene than complexes of DNA with DDAB/DOPE or DC-Chol/DOPE. Transfer of naked DNA carrying the IL-12 gene into growing Renca tumors causes a distinct therapeutic effect that depends on the time span between inoculation of mice with cancer cells and the beginning of the therapy. Therapy started on day 3 resulted in total cure (100%) of mice. PMID:11051203

  17. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    SciTech Connect

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  18. The Effect of Adenovirus-Mediated Gene Expression of FHIT in Small Cell Lung Cancer Cells

    PubMed Central

    Zandi, Roza; Xu, Kai; Poulsen, Hans S.; Roth, Jack A.; Ji, Lin

    2012-01-01

    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-1Met/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-1Met/APR-246, a synergistic cell growth inhibition was achieved. PMID:22085272

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

    PubMed

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

    2015-01-01

    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. PMID:25729995

  20. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  1. Improved efficiency of the walnut somatic embryo gene transfer system.

    PubMed

    McGranahan, G H; Leslie, C A; Uratsu, S L; Dandekar, A M

    1990-01-01

    AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. PMID:24226275

  2. Dust mediated transfer of phosphorus to alpine lake ecosystems

    NASA Astrophysics Data System (ADS)

    Brahney, J.; Ballantyne, A. P.; Kociolek, P.; Neff, J. C.

    2013-05-01

    Alpine lakes receive a large fraction of their nutrients from atmospheric sources; thus they are potentially sensitive to variations in atmospheric dust loading. Dust generation in western USA is thought to be increasing due to climate and land-use practices; however, the dust-mediated transfer of nutrients to alpine lakes and the potential for ecological consequences has not been extensively investigated in this region. Here, we explore the spatial changes in lake water chemistry and biology across a gradient of dust deposition in the Wind River Range, Wyoming, USA. Areas that receive more dust have altered lake and sediment water chemistries as well as altered planktonic communities. In particular, we found that phosphorus concentrations and primary productivity in dust-impacted alpine lakes were significantly greater than non-dust impacted lakes. The data illustrate the degree to which dust deposition may influence lake water chemistry and planktonic species composition, and the potential influence of human activities on remote alpine ecosystems.

  3. [Establishment of lentivirus-mediated system of double suicide genes and its killing effects on K562 cells].

    PubMed

    Jiang, Yi-Rong; Liu, Chun-Sheng; Chen, Xue-Liang; Ma, Dao-Xin

    2004-02-01

    To establish lentivirus-mediated system of double suicide genes and explore its killing effects on K562 cells, lentivirus transfer vector for double suicide genes was constructed using molecular methods, three plasmids of lentivirus gene transfer vector system were transferred into packaging cell line 293T using lipofectine method, the transfer effect was observed through fluorescence microscopy, the lentivirus particles were observed by means of electron microscopy. High titer of lentivirus was harvested from the supernatant of virus-producing cell culture and concentrated by high-speed centrifugation with Poly-L-Lysine (PLL). The K562 cells were infected with the concentrated supernatant containing the virus with the double suicide genes. Fluorescence microscopy and RT- PCR confirmed the integration and expression of extraneous gene. The cytotoxicity to these transgenic cells treated with 5-FC and GCV was measured by MTT assays. The growth inhibition ratio (GIR) of cells and inhibition concentration 50 (IC(50)) were counted. After administration of GCV and 5-FC, the changes of those cells were observed through scanning electron microscope. The results showed that lentivirus transfer vector with double suicide genes was constructed successfully. The above-mentioned plasmids were effectively transferred into 293T cells. So much green fluorescence was observed through fluorescence microscope. A lot of lentivirus particles were observed through transmission electron microscope. Double suicide genes mediated by lentivirus were stably integrated and expressed in K562 cells after infection with the concentrated virus using fluorescence microscopy and RT-PCR. The GIR of K562 cells using GCV or 5-FC was 48.73% or 50.69% respectively and it was apparently higher than that of untransfected cells (P < 0.01). When using GCV and 5-FC together, the GIR was 87.69%, which was apparently higher than that of group using GCV or 5-FC alone (P < 0.01). In conclusion, lentivirus-mediated

  4. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion.

    PubMed

    Nikolaidis, Nikolas; Doran, Nicole; Cosgrove, Daniel J

    2014-02-01

    Horizontal gene transfer (HGT) has been described as a common mechanism of transferring genetic material between prokaryotes, whereas genetic transfers from eukaryotes to prokaryotes have been rarely documented. Here we report a rare case of HGT in which plant expansin genes that code for plant cell-wall loosening proteins were transferred from plants to bacteria, fungi, and amoebozoa. In several cases, the species in which the expansin gene was found is either in intimate association with plants or is a known plant pathogen. Our analyses suggest that at least two independent genetic transfers occurred from plants to bacteria and fungi. These events were followed by multiple HGT events within bacteria and fungi. We have also observed that in bacteria expansin genes have been independently fused to DNA fragments that code for an endoglucanase domain or for a carbohydrate binding module, pointing to functional convergence at the molecular level. Furthermore, the functional similarities between microbial expansins and their plant xenologs suggest that these proteins mediate microbial-plant interactions by altering the plant cell wall and therefore may provide adaptive advantages to these species. The evolution of these nonplant expansins represents a unique case in which bacteria and fungi have found innovative and adaptive ways to interact with and infect plants by acquiring genes from their host. This evolutionary paradigm suggests that despite their low frequency such HGT events may have significantly contributed to the evolution of prokaryotic and eukaryotic species. PMID:24150040

  5. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer With Recombinant Adenovirus in Nonhuman Primates

    PubMed Central

    Fontanellas, Antonio; Hervás-Stubbs, Sandra; Mauleón, Itsaso; Dubrot, Juan; Mancheño, Uxua; Collantes, María; Sampedro, Ana; Unzu, Carmen; Alfaro, Carlos; Palazón, Asis; Smerdou, Cristian; Benito, Alberto; Prieto, Jesús; Peñuelas, Iván; Melero, Ignacio

    2010-01-01

    Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector. PMID:20087317

  6. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. The authors report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 x 10 /sup -4/-3 x 10/sup -3/. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  7. Targeted gene knockout in chickens mediated by TALENs.

    PubMed

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

    2014-09-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  8. Targeted gene knockout in chickens mediated by TALENs

    PubMed Central

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

    2014-01-01

    Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications. PMID:25139993

  9. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium ve...

  10. Baculovirus-mediated Gene Delivery and RNAi Applications

    PubMed Central

    Makkonen, Kaisa-Emilia; Airenne, Kari; Ylä-Herttulala, Seppo

    2015-01-01

    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. PMID:25912715