FIG.4.

Effect of AAV rep gene expression on the ability to rescue AAV/Ad.EGFP hybrid vectors in PER.tTA.Cre76 cells. (A) pHV.EGFP was transfected into PER.tTA.Cre76 cells together with the Ad E2a expression construct pCMV.E2a alone (−) or with pCMV.E2a plus the AAV rep expression plasmid pKS.P5.Rep (+). Subsequently, the PER.tTA.Cre76 cells were infected with Ad.floxedΨ at an MOI of 2.5 IU/cell (n = 4). Upon the emergence of complete CPE, the cells were harvested and lysed by three cycles of freezing and thawing. Cellular debris was removed by low-speed centrifugation, and the recovered supernatants were filtered and used to infect HeLa indicator cells. Forty-eight hours p.i., the number of EGFP-positive cells was determined by FACS analysis. AAV/Ad.EGFP hybrid vector titers are expressed in GTU per milliliter. The data are represented as means with standard deviations. (B) Representative FACS histograms of HeLa indicator cells receiving fivefold-diluted cell lysates derived from the PER.tTA.Cre76 cell cultures described for panel A. The presence (+) and absence (−) of pKS.P5.Rep are indicated at the right. The results are presented as EGFP intensities versus cell counts. The percentage of EGFP-positive cells is given in each histogram. Dashed lines, corresponding to mock-transduced HeLa cells, were used to set the EGFP background signal at 0.05%.