Figure 1.

Principle of RMCE and screening strategy for recombinants. The positive-negative selection marker HyTK that confers resistance to hygromycin (Hyg^R) and sensitivity to ganciclovir (Gan^S) is flanked by a pair of heterospecific LoxP sites placed in inverted orientation (inverted triangles) and integrated as a single copy into a genomic site. In the presence of Cre-recombinase and incoming DNA harboring the same pair of LoxP sites, exchange will replace the existing HyTK with DNA on the exchange plasmid. Cells that have successfully undergone RMCE can be selected based on resistance to ganciclovir. The tetracycline inducible LucGFP (TRE-LucGFP) is used as the reporter to characterize uniformity of gene expression between clones after integration into the genome. Also shown are the positions of BamHI site and Luc probe used for Southern blot and primer pairs (P1–P6) used for screening of recombinants.