Safe Production of High-Titer Retrovirus ( h exceptionally large insert...)

Safe Production of High-Titer Retrovirus

h exceptionally large inserts or some self-inactivating (SIN) vectors], use of the VSV-G envelope protein allows the viral supernatants to reach a concentration of several thousand-fold by simple ultacentrifugation without a substantial decrease in the stability of the virion particles.⁵

As an addendum, we recently used the pVPack-GP and pVPack-VSV-G vectors together with a set of ten ViraPort retroviral plasmid cDNA expression libraries⁷ for the large scale production of viral supernatants (see below for available supernatants). The high titers attainable with the vectors allow a consistent, high-throughput production of large numbers of frozen aliquots for each of the libraries, with each aliquot containing greater than or equal to 2 x 10⁶ cDNA-harboring infectious virions when thawed. The ViraPort cDNA library retroviral supernatants are ready for immediate transduction and eliminate the need for plasmid library amplification, plasmid DNA preparation, and producer cell transfection for virus production.

Methods

Virus production was carried out by cotransfecting a 293-cell derivative with 3 g each of pFB-GFP, pVPack-GP, and one of the four env-expressing vectors (pVPack-VSV-G, pVPack-Ampho, pVPack-Eco or pVPack-10A1), using the Transfection MBS transfection protocol (Stratagene) modified according to Pear et al.⁶ Filtered viral supernatants were serially diluted in DMEM + 10% CS to a final volume of 1 ml/sample and supplemented with DEAE-Dextran (Sigma) to a final concentration of 10 g/ml. Culture media was removed from six-well tissue culture plates containing 2 x 10⁵ NIH3T3 cells/well (plated the previous day) and replaced with 1 ml of viral dilution. Each diluted viral sample was applied to a well containing the NIH3T3 cells, and incubated for 3 hours; 1 ml of prewarmed DMEM + 10% CS was then added
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( h exceptionally large insert...)Safe Production of High-Titer Retrovirus

Methods