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Safe Production of High-Titer Retrovirus

tage above the No plasmid control; these values are also shown where applicable). All four of the vectors yielded titers greater than or equal to107 cfu/ml using the more conservative method and approached 108 cfu/ml by using the endpoint dilution method titers. These titers are higher than those reported for any other commercially available packaging system.

Fig.3

We also carried out experiments to show that the vectors can be selected and maintained in stable transfection experiments (data not shown). Stable CHO cell lines transfected with pVPack-GP were selected in histidine-free medium using a range of histidinol from 75 to 500 M and gave no significant variation in colony formation across this range. Likewise, each of the vectors (pVPack-Ampho, pVPack-Eco, and pVPack-10A1) were selected with 5 to 7 g/ml of puromycin. They showed a high level of colony formation above untransfected CHO cells, which showed no colony formation at all with the puromycin concentrations tested. The pVPack-VSV-G vector was not tested for stable cell formation due to reported toxicity of the VSV-G envelope protein to the host cell. As stated above, we do not recommend the use of this vector for stable cell production.

Conclusions

The pVPack vector system allows high-titer virus to be produced for most applications, providing that a high titer retroviral expression vector such as pFB is used, and very high transfection efficiencies are attained when producing virus. For the latter, we found that 293 cells and their derivatives work especially well when used with Stratagenes Transfection MBS transfection kit, modified according to Pear and colleagues6 (see legend to Figure 3). For certain low-titer vectors [e.g., vectors wit
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