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 prom oter. To gain more detailed information on genotoxic induction of the p53 promoter at a uniform genomic locus, we have developed an efficien t strategy for replacing a defined genomic segment in mouse NIH 3T3 ce lls with exogenous transfected DNA using a 'doublelox' targeting strat egy mediated by Cre DNA recombinase, The strategy utilizes a pair of h eterospecific lox sites engineered both into the genome and onto the t argeting DNA. This allows direct replacement of genomic DNA by a Cre-c atalyzed 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 inser tional recombination, and the absolute frequency of site-specific doub lelox targeting exceeded the frequency of transformation due to random illegitimate recombination of transfected DNA into the genome. Result ing targeted single-copy integrants of the p53-CAT reporter show stron g genotoxic induction by mitomycin C, and a dynamic range of induction that exceeds that seen in transient transfection assays, The doublelo x strategy is generally applicable to Cre-mediated genome targeting in any cell and should be of particular utility in the site-specific tar geting of DNA into embryonic stem (ES) cells for the production of gen e-modified mice.