FIG. 5.

Passaging stability analysis of ITR-containing transgene cassette (BacGFP). (A) Analysis of rescued rAAV cassette. Sf9 cells were infected with BacGFP from consecutive passage stocks (m.o.i. of 5 each) in addition to BacRep (P2, m.o.i. of 5). Forty-eight hours postinfection, DNA was prepared by Hirt DNA extraction, resolved using a 1.2% agarose gel, transferred to a nylon filter, and hybridized with a ³²P-labeled GFP probe. (B) Analysis of rAAV2-GFP titers of vector stocks prepared using BacGFP P2 through P5 helpers. Sf9 cells were co-infected with BacVP and BacRep (P2, m.o.i. of 5 each). In addition, cells were co-infected with BacGFP at the indicated passages (m.o.i. 5 of each). Seventy-two hours postinfection, cells were harvested and rAAV infectious titers in crude cell lysates were calculated using GFP fluorescence assay using C12 cells co-infected with Ad5 (m.o.i. of 10) [18].