Figure 6

Transduction of 293T-EpoR cells using lentiviral vector pseudotypes preloaded with TVA-Epo or TVB-Epo during vector production. (A) Open bars: ALV-A Env-pseudotyped lentiviral vectors prepared in 6-well plates using different amounts of the pTVA-Epo plasmid ranging from 0 to 1.0 μg. Striped bars: In vivo-preloaded vectors were subjected to a subsequent preloading step in vitro using the TVA-Epo protein (40 V5 units of TVA-Epo per 50 μl of virus stock). (B) Open bars: ALV-B Env-pseudotyped lentiviral vectors prepared in 6-well plates using different amounts of pTVB-Epo ranging from 0 to 3.5 μg. Striped bars: In vivo-preloaded vectors were subjected to a subsequent preloading step in vitro using the TVB-Epo protein (14 V5 units of TVB-Epo per 50 μl of virus stock). The titers shown in panels A and B represent the mean ± SD from three independent experiments. (C) Western blot analysis of ALV-A-pseudotyped lentiviral vectors prepared by co-transfection with different amounts of pTVA-Epo. Lanes 1-5: Two-μl aliquots of vector stocks prepared by co-transfection using 0.5 μg (lane 1), 0.1 μg (lane 2), 0.04 μg (lane 3), 0.02 μg (lane 4) and 0 μg (lane 5) of the pTVA-Epo plasmid DNA and concentrated ~35-fold by ultracentrifugation were analyzed. Lane 6: Unconcentrated vectors prepared using 0.4 μg of the pTVA-Epo plasmid DNA. The molecular weights of the protein markers used are indicated.