(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors (1996) / Chapter Skim
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A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes
A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes
Pages 11400-11406
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From page 11400... ...
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 107 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 ~106 colony-forming units/ml.
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From page 11401... ...
The transfected cells were plated 48 h posttransfection by limiting dilution in media containing puromycin and tetracycline and independent clones were isolated. 293G cells were always grown in 293 growth medium supplemented with tetracycline and puromycin (293 G growth medium)
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From page 11402... ...
Viral supernatants harvested either from stable virus-producing cell lines or transiently transfected cells were concentrated by ultracentrifugation (8~. The stability of the GPGnlsLZ pseudotyped retrovirus and the ~CRIPLacZ amphotropic retrovirus was determined in normal human serum.
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From page 11403... ...
The high-level cell surface expression of VSV-G in our transient and stable cell lines may promote fusion of plasma membranes of adjacent cells in response to local pH changes (38)
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From page 11404... ...
Helper virus assays performed on unconcentrated viral supernatants as well as supernatants from transient transfections demonstrated that the retroviral stocks generated from the packaging cells were free of RCV (Table l J It was not possible to perform cocultivation-based helper virus assays on the 293GPGderived producer cell lines, since cocultivation of the cells with the Mus dunni indicator cells led to extensive cell fusion (data not shown)
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From page 11405... ...
virus critical for examining the potential utility of retroviral vectors for in vivo infection and the ability to transduce cells refractory to infection by standard vectors. While the virus produced from 293GPG cells is not fully resistant to human serum, it is significantly more resistant than amphotropic virus generated from murine cells.
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From page 11406... ...
Another issue relates to the potential of different packaging cell lines to give rise to helper virus due to the overlap of sequences between the particular vector used and the precise packaging sequences present in the packaging cell line (34, 35~. A more global inherent defect in the design of all murine and human derived packaging cell lines is the ability of retroviral RNAs which lack packaging sequences to be packaged, albeit at low efficiency, and transmitted to cells (40~.
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