Figure 2.

Generation of rAAV for gene targeting. Homology arms (HAs) are PCR-amplified from genomic DNA and fused to a selectable cassette via linker sequence homology. NotI restriction sites allow cloning of the fusion product into the AAV plasmid containing the ITR sequences necessary for viral packaging. Dual selection results in a population of plasmids containing the intended targeting cassettes flanked by ITRs. Co-transfection of packaging cells with the targeting plasmid and helper plasmids produces rAAV capable of genomic integration or deletion. A strategy to produce rAAV for the deletion of a generic exon is depicted. PCR primers used for amplification (P1, P2, P3 and P4) and fusion PCR (P1 and P4) are shown. Abbreviations are defined in Figure 1.