FIG. 5.
Assays for rcAAV. (A) RCA. 293T cells were infected with 1.6 × 104 particles of wtAAV2 (filter 1), 9.5 × 108 particles of AAV2-Rluc vector prepared from TtetA2Rluc c.49 (filter 2), or 3 × 105 particles of AAV2-LAPSN vector made by transfecting 293 cells (filter 3) in the presence of Ad (vector and virus particle numbers were determined by Southern analysis of purified virion DNAs on alkaline gels); 28 h later, cells were aspirated onto nylon membrane filters, lysed, and the DNA bound to the filters was hybridized to a fragment containing the entire rep and cap genes. The circle marks the position of the circular filter exposure on the film. Radioactive spots represent individual foci of rcAAV. (B) Sequential amplification assay. Southern blot of DNA prepared from cells after one round (1×) or two rounds (2×) of rcAAV amplification on 293 cells in the presence of Ad. For the lanes with two rounds of amplification, 108 vector particles (lanes 1 to 6), no vector (lane 7), or 10 to 1,000 particles of wtAAV2 (lanes 8 to 10) were used for the first round of amplification, and 0.5-ml aliquots of the resulting lysates were used for the second round of amplification. For the lanes with one round of amplification, uninfected cells (lane 11) or cells infected with 103 or 104 particles of wtAAV2 (lanes 12 and 13) were analyzed. A DNA fragment containing the rep and cap genes was used as a probe. AAV2-LAPSN stocks were made by transfection of 293 cells (lane 1) or by transfection of TtetA2 cells (lane 2). The AAV2-RAP stock (lane 3) was made by two sequential rounds of infection of TtetA2 cells after generating the original seed stock by transfection of TtetA2 cells. AAV2-Rluc stocks were made by infection of TtetA2 cells with a seed stock obtained by transfection (lane 4) or were purified from the indicated producer cell lines (lanes 5 and 6).