Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors.
HIV-derived vectors are of potential clinical relevance due to their ability to transduce nondividing cells in vitro and in vivo. However, the generation of cell lines stably and reproducibly expressing high amounts of defined subviral particles, capable of packaging and transducing HIV-derived vectors, has been hampered by the cytotoxicity of some of the required gene products, in particular of the HIV-1 protease. The successful use of regulatable gene expression systems to overcome this problem requires that the remaining basally expressed gene product activity is below the threshold for cytotoxicity. To try to achieve this, we have consecutively introduced appropriate plasmids, encoding HIV rev and HIV gag/pol gene products, each under the control of separate ecdysone-inducible promoters, into human 293 cells. Using a protocol in which a specific HIV protease inhibitor, Saquinavir, was continuously present in the culture medium during selection, we could generate stable cell lines inducibly expressing high amounts of subviral particles. A cell line, termed 293-Rev/Gag/Pol(i), which has been characterized in more detail, inducibly releases, within 48 h postinduction, high amounts of HIV Gag/Pol particles (about 10 microg CA/ml). These HIV Gag/Pol particles can package and transduce third-generation HIV vectors to high titers. Thus, in addition to other applications, the 293-Rev/Gag/Pol(i) cell line represents a "founder" packaging cell line which, depending on the requirement, can be further modified to include specific transgene-encoding vector and targeting glycoprotein genes.
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Forschungsschwerpunkt Angewandte Tumorvirologie, F0200, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, Heidelberg, 69120, Germany.
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Schaal
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Bosch
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D002460:Cell Line; D002469:Cell Separation; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015685:Fusion Proteins, gag-pol / Q000096:biosynthesis* / Q000235:genetics*; D015694:Gene Products, rev / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D019258:Saquinavir / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes; D054321:rev Gene Products, Human Immunodeficiency Virus
D015685:Fusion Proteins, gag-pol; D015694:Gene Products, rev; D017320:HIV Protease Inhibitors; D054321:rev Gene Products, Human Immunodeficiency Virus; D019258:Saquinavir