Figure 3.

Vacccinia carriers support rAAV production in HEK293 cells with integrated rAAV genomes. The HEK293 cells with integrated pscAAV-CB-EGFP were selected for testing the vaccinia carrier helper function. The cells were infected with Ad-T7 (MOI of 5) for 6 h before infection by vv-w5 and vv-w8, at MOI of 1. In parallel, the cells were also transfected with pH22 + pfΔ6. The infected cells were harvested at 24 h after infection, whereas the transfected cells were collected at 48 h after transfection. (a) Profile of rAAV DNA replication in AAV cell line induced by vaccinia carrier infection (lane ‘VV’) or transfected AAV helper plasmids (lane ‘pH22’). Dpn I-digested Hirt DNA (10 µg) was used in each lane for Southern blotting with GFP-specific probe. m, monomer; d, dimer. The first lane is monomer-sized pscAAV-CB-EGFP as a standard (10 ng). (b) Comparison of rAAV yield using plasmid transfection or vaccinia helper carrier. Cell lysates derived from 10⁴ cells were used to infect 5 × 10⁵ 16 095 cells. EGFP positive cells were measured by flow cytometry. Each GFP positive cell represents 1 × 10³ AAV viral particles.