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pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins

om the 5 LTR; spurious activation/transcription of 3-flanking endogenous genes from the promoter within the 3 LTR will not occur in the SIN vector; and inactivation of the proviral LTRs protects against mobilization of proviral pFB-ERV derivatives by endogenous retroviral structural proteins.

Titer Determination

Vector titer was determined by G418-resistant colony formation. Amphotropic virus was produced by transient transfection using the producer line HW293-A (unpublished data), and viral supernatants were used to infect NIH3T3 cells. In Table 1, the titers for both experiments are on the order of 105 colony forming units (cfu)/ml. Even at high dilutions of supernatant (1:103), which likely give rise to single-copy infected cells in accordance with the Poisson kinetics of viral transduction, colonies are resistant to as high as 1 mg/ml G418 without substantial loss of titer, indicating efficient expression of the Neo gene from the third ORF.

Performance of pFB-ERV in Mass Populations of Infected Cells

Fig.2

NIH3T3 cells were infected with various dilutions of viral supernatant, and one day following infection the cells were transfected with the ecdysone-inducible reporter vector pEGSH-luc. The following day, cells were induced for 20 hours with ponA, then assayed for luciferase activity. In Figure 2 (at an MOI of 1.0), a strong induction is achieved, which is reproducible in separate infected populations.

Analysis of Clonal Isolates of Individual Cells Infected with pFB-ERV

Fig.3

NIH3T3 cells were infected with pFB-ERV supernatants and selected with 600 g/ml G418. Resistant colonies were picked and expanded. In an initial screen, 24 colonies were transfected with reporter vector, and induced
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