FIG. 1.

Generation of OneBac: AAV1–12 rep/cap carrying Sf9 cell lines and their baculovirus-induced expression profiles. (A) Plasmid pIR-rep78-hr2-RBE expressing rep of AAV2 (AAV-Rep construct) and the derivatives for AAV4 or AAV12 rep are depicted with the expressed proteins Rep78 and Rep52. Plasmid pIR-VP-hr2-RBE expressing cap of AAV2 (AAV-Cap construct) and the derived plasmids for cap of AAV serotypes 1–12 are depicted. The expressed proteins VP1 and VP2 start with an ACG codon and VP3 starts from an internal ATG. AcMNPVhr2, Autographa californica multiple nuclear polyhedrosis virus homologous region 2; bsd, blasticidin S deaminase; polh, baculovirus polyhedrin promoter; RBE, Rep-binding element (AAV2). (B) AAV serotype-dependent Rep- and Cap expression was analyzed by Western blot analysis of cell extracts harvested in parallel to rAAV production. Cell extracts from 293 cells were analyzed 72 hr posttransfection of the packaging plasmids. Equivalents of 5×10⁴ 293 cells were loaded per well. Cell extracts from rep/cap-expressing Sf9 cells were prepared 72 hr p.i. with the recombinant Bac-rAAV at an MOI=5. For Sf9 cells, an equivalent of 1–2×10⁴ cells were loaded per well. Rep proteins were detected by mAb 303.9, initially generated for AAV2 Rep, but also detecting Rep of AAV4 and AAV12. VP proteins expressed from AAV serotype-specific cap genes were reacted with mAb B1, which detects all three capsid proteins of most AAV serotypes with the exception of AAV4, -11, and -12. mAb A1 binds to VP1 of all serotypes. (C) Cell extracts from three independent rAAV preparations for each serotype were analyzed by quantitative Western blot analysis to compare the ratios of VP1, VP2, and VP3 expressed in 293 or Sf9 cells, respectively. VP proteins expressed in either cell type were analyzed by mAb B1 and a secondary antibody emitting near-infrared fluorescence as quantified by an Odyssey imager (Licor). 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; mAb, monoclonal antibody; MOI, multiplicity of infection; p.i., postinfection; rAAV, recombinant AAV.