Cre-mediated recombination of the pVII gene from the Ad5-VII-loxP viral genome.

A) Schematic of the Ad5 L2 coding region. Open reading frames that encode the proteins penton (III), pVII, V, and X/μ are depicted in colors. LoxP sites are indicated by upward red arrows. The downward arrow indicates infection of a Cre recombinase-expressing cell line with Ad5-VII-loxP. Cre-mediated recombination leads to excision of the pVII gene with the maintenance of a single lox site. (B) Southern blot analysis of total cell DNA isolated from infections of either 293 cells (–, lanes 2 and 3) or 293 cells expressing Cre recombinase (+, lanes 4 and 5) with the Ad5-VII-loxP virus. Cells were harvested at 48 (lanes 2 and 4) or 72 (lanes 3 and 5) hr post-infection (hpi) and DNAs were analyzed by Southern blot. U represents intact (unfloxed) pVII gene and F represents the recombined (floxed) pVII gene. DNA size markers are indicated on the left (lane 1). (C) Western blot analyses of lysates from 293 cells (–) and 293 cells expressing Cre (+) infected with the Ad5-VII-loxP virus. Ad5 L2 proteins were detected with specific antibodies as described in Supplemental Information. Cellular alpha-tubulin (α-tubulin) was used as a loading control.