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. 2015 Jul 2;26(10):688–697. doi: 10.1089/hum.2015.050

Figure 3.

Figure 3.

Comparative analysis of the RBE+ and ΔRBE rAAV5 producer cell lines. (A) Depiction of the constructs for the expression of rep and cap. Rep and cap expression is regulated by a polyhedrin promoter (polh) and the enhancer element hr2-0.9. Further elements on these construct include an ampicillin resistance gene (ampR), the blasticidin S deaminase gene (bsd), and the RBE with the terminal resolution site (trs). (B) Number of rep, cap, and bsd gene or hr2-0.9 copies in uninfected or baculovirus-infected AAV5 cell lines E5 (RBE+) or clone 7 (ΔRBE). In case of infected cells, total DNA was isolated 72 hr after baculovirus infection. Gene copy numbers were determined by quantitative Light Cycler PCR with gene-specific primers. (C) Analysis of total genomic DNA by agarose gel (0.7%) electrophoresis. An amount of 10 μg of total DNA from two different rAAV5 producer cell lines (clone 7 and E5) was loaded per lane. For baculovirus-infected cell lines, DNA was additionally digested with different sets of restriction enzymes as indicated. SwaI represents a single cutter for the cap expression constructs that were transfected in Sf9 cells for the generation of the cell lines. AvrII, Bsu36I, and MfeI were chosen as noncutting enzymes (3× noncut) for the cap expression constructs. The restriction enzymes AseI and XbaI flank the AAV5 cap gene resulting in a 2650 bp fragment. (D) For the Southern blot a hybridization probe was used that detects the AAV5 cap gene (AAV5 genome: nt 2232–3603). (E) Analysis of copackaging of rep, cap, bsd, and hr2-0.9 sequences into AAV5 capsids during vector production. Aliquots from AVB sepharose-purified AAV vector preparation were digested with proteinase K to release the packaged DNA from the capsids. The samples were analyzed by quantitative Light Cycler PCR with primers specific for the vector genome or the indicated genes, respectively. The titers of rAAV5 particles containing the vector genome are shown as absolute values, whereas the portions of rep-, cap-, or bsd-gene and hr2-0.9 element-containing particles are displayed as percentages thereof.