A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.
We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.
Ory
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Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA.
Neugeboren
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Mulligan
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RC
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001769:Blood; D002460:Cell Line; D003470:Culture Media; D017931:DNA Primers; D006801:Humans; D007668:Kidney; D008562:Membrane Glycoproteins; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis
D003470:Culture Media; D017931:DNA Primers; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase