Segmental genomic replacement by Cre-mediated recombination: Genotoxic stress activation of the p53 promoter in single-copy transformants

Genotoxic stress results in transcriptional activation of the p53 promoter. To gain more detailed information on genotoxic induction of the p53 promoter at a uniform genomic locus, we have developed an efficient strategy for replacing a defined genomic segment in mouse NIH 3T3 cells with exogenous transfected DNA using a 'doublelox' targeting strategy mediated by Cre DNA recombinase, The strategy utilizes a pair of heterospecific lox sites engineered both into the genome and onto the targeting DNA. This allows direct replacement of genomic DNA by a Cre-catalyzed double crossover event, p53-CAT reporter constructs were site-specifically placed into the genomic target 20-fold move efficiently by doublelox recombination than by Cre-mediated single crossover insertional recombination, and the absolute frequency of site-specific doublelox targeting exceeded the frequency of transformation due to random illegitimate recombination of transfected DNA into the genome. Resulting targeted single-copy integrants of the p53-CAT reporter show strong genotoxic induction by mitomycin C, and a dynamic range of induction that exceeds that seen in transient transfection assays, The doublelox strategy is generally applicable to Cre-mediated genome targeting in any cell and should be of particular utility in the site-specific targeting of DNA into embryonic stem (ES) cells for the production of gene-modified mice.