Figure 1.

AAV5 capsid expression pattern in stable Sf9 cell lines in comparison to transiently transfected 293 cells. (A) Depiction of different cap expression systems for production of rAAV5. (Left panel) Regulation of capsid protein expression in AAV wt. (Center) Capsid protein expression strategy in the OneBac system with an ACG start codon for VP1. (Right) Capsid protein expression in OneBac 2.0 with an artificial intron containing a second polh promoter inserted into the cap gene. The promoters and their transcripts are displayed. The start codons for the translation of the individual VP proteins are indicated. (B) AAV5 Cap expression was analyzed by Western blot analysis of cell extracts from three independent rAAV5 packaging experiments (I–III). Cell extracts from 293 cells were analyzed 72 hr posttransfection. Cell extracts from rep/cap-expressing Sf9 cells were prepared 72 hr postinfection with recombinant Bac-rAAV at MOI=5. VP proteins expressed were detected with mAb B1. (C) Quantitative analysis of the Western blots shown in (B) to compare the relative ratios of VP1, VP2, and VP3 expression in 293 or Sf9 cells, respectively. A secondary antibody emitting near-infrared fluorescence was used and its fluorescence was quantified by an Odyssey imager. Expression levels of VP3 were arbitrarily set to 10 and expression levels of VP2 and VP1 were calculated as relative values thereof. The results of three experiments are given as mean±standard deviation. Please note the logarithmic scale of the y axis. AAV, adeno-associated virus vectors; rAAV, recombinant AAV.