Figure 8.

RMCE in Cre-expressing HEK293 cells using incompatible lox sites. (A) A schematic diagram depicting RMCE between a shuttle plasmid containing a floxed kan^r marker (pGEM7zf+loxm2/66 kan^rlox71) and a parental adenoviral chromosome containing a floxed LacZ gene (AdCMVloxm2/71LacZlox66). A unique BstBI restriction site in the parental chromosome allows sizing of the left terminal restriction fragment. (B) Restriction digestion of viral DNA isolated from plaque-purified virus resulting from co-transfection of HEK293Cre cells with shuttle plasmid and viral DNA depicted in (A). X-Gal staining for the production of β-galactosidase distinguishes parental viral plaques (blue) from putative recombinant plaques (clear). Restriction analysis of representative blue and clear plaques confirms the predicted size for parental (5.1 kb) and recombinant viruses (2.8 kb), respectively.