FIG. 3.

Gene amplification and vector titer during Dox induction. (A) Southern analysis of genomic DNAs (4 μg/lane) prepared from TtetA2 packaging cells grown in Dox for the indicated number of days, digested with PstI (TtetA2), and probed for cap sequences to detect helper construct amplification. PstI-digested pAAVSoHD served as standards. Construct copy numbers per cell are indicated beneath the lanes. Standard fragment sizes (in kilobases) are shown on the left. (B) Southern analysis as for panel A except that genomic DNAs from TtetA2Rluc producer cells c.37 and c.49 were digested with XbaI and probed for luciferase sequences to detect vector amplification. XbaI-digested pA2RlucbSN served as standards. (C) Effect of Dox incubation time on the production of AAV2-Rluc from TtetA2Rluc c.37 and c.49. Cells (4 × 10⁶/10-cm-diameter dish) grown in Dox for the indicated number of days were infected with Ad at an MOI of 10, and vector stocks were harvested 3 days later. Stock titers were determined by luciferase transduction, and particle numbers were calculated as for Fig. 2A.