( tracentrifugation; this cannot be done ...)Safe Production of High-Titer Retrovirus

To produce stable cells, we chose antibiotic-resistant markers to complement those used with the neomycin- and hygromycin-resistant derivatives of Stratagenes MMLV-based replication-defective vector pFB. Thus, if desired, cell lines may be first selected with histidinol to obtain gag-pol expressing lines, then a double-stable cell line may be obtained by transfecting with a puromycin-resistant env vector (it should be noted here that although stable VSV-G-expressing lines have in the past been made, in general they are very poor due to the toxicity of VSV-G to cells in which it is stably expressed.¹ Therefore, we do not advise using the pVPack-VSV-G vector for stable puromycin-resistant cell line production). Finally, stable virus-producing lines may be constructed using a neo- or hyg-resistant viral vector or cotransfecting the viral vector with a pSV-Neo plasmid. The hisD and puro^r markers were included as downstream ORFs in IRES-containing bicistronic cassettes to ensure that selective maintenance of cells in antibiotic require expression of the gag-pol and env-encoding mRNAs.

Functional Testing of the pVPack Vectors

To test the pVPack vectors, each of the env-expressing vectors was individually transfected together with pVPack-GP and the green fluorescent protein (GFP)-expressing viral reporter vector pFB-GFP (unpublished); titers were determined by fluorescence- activated cell sorting (FACS). (Figure 3) and are calculated two ways. Titers calculated using percent-shifted values that are linear over two 10-fold dilutions (i.e., 1:10³ and 1:10⁴) are shown in bold. We previously determined titers by endpoint dilution (i.e., the smallest detectable shift that yields a percen
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