title: Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single-copy transformants.
abstract: 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 'double lox' 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 more efficiently by double lox recombination than by Cre-mediated single crossover insertional recombination, and the absolute frequency of site-specific double lox 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 double lox strategy is generally applicable to Cre-mediated genomic 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.
authors.affiliation: National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Disease, Bethesda, MD 2089-1800, USA.
authors.forename: B
authors.initials: B
authors.lastname: Bethke
authors.forename: B
authors.initials: B
authors.lastname: Sauer
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D009153:Mutagens; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D016685:Mitomycin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; C045073:Cre recombinase; D019426:Integrases