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. 2013 May 15;41(13):6609–6617. doi: 10.1093/nar/gkt404

Figure 2.

Figure 2.

Vaccinia helper carrier-supported AAV helper gene expression and rAAV production. (a) Rep and Cap expression profile of vaccinia carrier: panels marked ‘Cap/vv’ and ‘Rep/vv’, respectively. VVs (vv-Rep78, vv-Rep52, vv-VP1, vv-VP2 and vv-VP3) were used to infect HeLa S3 cells at MOI of 1 along with Ad-AAV-CMV-EGFP infection (MOI 5) at 16 h earlier. The cells were collected at the time identified, and the expression of rep and cap was analyzed by western blot. The expression of rep and cap in the triple plasmid method is also shown: panels marked ‘Cap/tri’ and ‘Rep/tri’, respectively. (b) Vector yield comparison using different combinations of vaccinia carriers. HeLa S3 cells were infected by Ad-AAV-CMV-EGFP at MOI 5 followed by infection of 5VVs (vv-Rep78, vv-Rep52, vv-VP1, vv-VP2 and vv-VP3), 4VVs (vv-Rep78, vv-Rep52, vv-VP1 and vv-VP2) or 2VVs (vv-w5 and vv-w8). AAV-CMV-GFP vectors were harvested 36 h later. After heat inactivation, cell lysate derived from 1 × 104 cells was used to infect 5 × 105 16 095 cells. The y-axis shows the yield measured by flow cytometry. Each GFP positive cell represents 1 × 103 AAV viral particles.