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Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the
context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral
stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is
stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures
that the foreign genetic material will be faithfully transmitted to the cells’ progeny. However, oncoretroviral vectors derived
from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral
modiated gene transfer efficiency in primary T-lymphocytes, in particular T cell activation status, viral receptor expression,
and culture conditions. 相似文献
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Abad JL Serrano F San Román AL Delgado R Bernad A González MA 《The journal of gene medicine》2002,4(1):27-37
Background
Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells.Methods
Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T‐cell line (MT‐2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double‐ and triple‐transduced cells were isolated by fluorescence cell sorting.Results
Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti‐CD3. The transient production of viral particles encoding EGFP, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.Conclusions
This study describes new experimental conditions for efficient single‐step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches. Copyright © 2002 John Wiley & Sons, Ltd.5.
Ozturk F Park PJ Tellez J Colletti E Eiden MV Almeida-Porada G Porada CD 《The journal of gene medicine》2012,14(3):169-181
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Suzuki A Obi K Urabe T Hayakawa H Yamada M Kaneko S Onodera M Mizuno Y Mochizuki H 《Journal of neurochemistry》2002,82(4):953-960
Neuronal progenitor cells (NPC) are particularly suited as the target population for genetic and cellular therapy of neurological disorders such as Parkinson's disease or stroke. However, genetic modification of these cells using retroviral vectors remains a great challenge because of the low transduction rate and the need for fetal calf serum (FCS) during the transduction process that induces the cell differentiation to mature neurons. To overcome these problems, we developed a new retrovirus production system in which the simplified retroviral vector GCDNsap engineered to be resistant to denovo methylation was packaged in the vesicular stomatitis virus G protein (VSV-G), concentrated by centrifugation, and resuspended in serum-free medium (StemPro-34 SFM). In transduction experiments using enhanced green fluorescent protein (EGFP) as a marker, the concentrated FCS-free virus supernatant infected NPC at a high rate, while maintaining the ability of these cells to self-renew and differentiate in vitro. When such cells were grafted into mouse brains, EGFP-expressing NPC were detected in the region around the injection site at 8 weeks post transplantation. These findings suggest that the gene transfer system described here may provide a useful tool to genetically modify NPC for treatments of neurological disorders. 相似文献
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Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This
implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development
of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression
is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of
cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective
expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the
potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections. 相似文献
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基因治疗研究中脂质体介导的基因转移技术 总被引:2,自引:0,他引:2
对于脂质体的深入研究特别是阳离子脂质体的研制使其逐步成为重要的基因转移载体之一,并且初步应用于基因治疗研究,同时多种靶向脂质体的研制也为体内靶向基因转移和表达奠定了基础。本文就脂质体的结构、功能、在基因治疗研究中的应用以及各种靶向脂质体的研制进行了介绍。 相似文献
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BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period. 相似文献
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Lishuang Zhang Huayan Zhang Xin Gong Lan Yang Liang Miao Yuhua Shi Yan Zhang Wei Kong Chuntao Zhang Yaming Shan 《Journal of peptide science》2014,20(1):46-54
Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39‐residue semen‐derived infection‐enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13‐residue peptide P13 (Ac‐671NWFDITNWLWYIK683) derived from the membrane‐proximal external region of the human immunodeficiency virus type 1 (HIV‐1) gp41 transmembrane protein, together with its 16‐residue peptide derivative (P16) were found to enhance HIV‐1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV‐1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV‐1 infection by these two active peptides. P16 could more effectively augment HIV‐1 YU‐2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
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Hot topics in adeno-associated virus as a gene transfer vector 总被引:4,自引:0,他引:4
Adeno-associated virus (AAV) is a promising viral vector in treating many kinds of hereditary diseases. The broad host range,
low level of immune response, and longevity of gene expression observed with this vector have enabled the initiation of a
number of clinical trials using this gene delivery system. Another potential benefit of AAV vectors is their ability to integrate
site-specifically in the presence of Rep proteins. However, this virus is not well characterized. To obtain high level, persistent
expression of the foreign gene, some problems should be solved. In this article, we will describe the advances in some fields
of recombinant AAV technology that overcome certain limitations of the vector as a gene delivery system, such as the transduction
efficiency, the production, the package capacity, and elimination of immune responses, as well as the applications involving
these recombinant vectors for the treatment of some diseases. 相似文献
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Fresnay S Chalmers DE Ferrand C Colombain C Newton I Yerly-Motta V Lienard A Darodes de Tailly P Hervé P Tiberghien P Saas P 《The journal of gene medicine》2002,4(6):601-612
Background
Gene transfer using retroviral transduction offers the advantage of long‐term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential.Methods
Immature DCs were generated from murine bone marrow (BM) using either GM‐CSF alone or GM‐CSF plus IL‐4. The cells were transduced directly with retroviral supernatants or by co‐culture with the GP + E‐86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed.Results
Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL‐4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM‐CSF plus IL‐4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down‐regulated transgene expression in the co‐cultured packaging cell line in a promoter‐dependent manner.Conclusions
We have defined optimal conditions to generate and transduce murine BM‐derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL‐4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.19.
Kaspar BK Roth DM Lai NC Drumm JD Erickson DA McKirnan MD Hammond HK 《The journal of gene medicine》2005,7(3):316-324
Adeno-associated viral vectors (AAV) can direct long-term gene expression in post-mitotic cells. Previous studies have established that long-term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 10(11) DNase resistant particles (drp; high-performance liquid chromatography (HPLC)-purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20-32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64-5.28 x 10(13) drp; HPLC-purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long-term expression of therapeutic genes. 相似文献