FIG 7.

Rescue of replication-deficient rAd/AAV hybrid vectors by a replication-competent rAd in trans. (A) trans inhibition assay. PacI-linearized pAdEasy plasmid constructs pAdE-CMV-GFP as indicator DNA and pAdE-Rep1701/2186 as effector DNA were cotransfected into HEK-293 cells at different ratios, as indicated, and rAd particles containing the GFP genome were scored after first-round amplification of the primary freeze-thaw supernatants (obtained at 12 days posttransfection) of HEK-293 cells for 6 days. (B) Rescue assay. PacI-linearized pAdEasy plasmid constructs pAdE-CMV-GFP and pAdE-p40-Cap, which are shown schematically in panel D, were cotransfected into HEK-293 cells at different ratios, as indicated, and rAd particles containing either the GFP or the 3′ Rep sequences (AAV-2 nt 1782 to 2174, denoted AAV) were separately scored after transfection and amplification of the primary freeze-thaw supernatants as described in the legend to panel A. The apparent presence of rAd-CMV-GFP particles in the transfections devoid of the pAde-CMV-GFP plasmid is due to a relatively high background level of this specific real-time PCR product in combination with a high level of predilution of the probes. (C) For the rescue assay described in the legend to panel B, three further amplification rounds for 3, 5, and 4 days, respectively, were conducted, and the amounts of rAd-CMV-GFP and rAd-p40-Cap genomic particles were determined as described above. (D) Western analysis for AAV-2 Cap proteins in total cell extracts obtained after the fourth amplification round of the rescue assay described in the legends to panels B and C. LITR and RITR, left and right ITRs, respectively. Ψ, adenoviral packaging signal.