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. 1997 Jul 15;25(14):2828–2834. doi: 10.1093/nar/25.14.2828

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

B Bethke 1, B Sauer 1
PMCID: PMC146822  PMID: 9207031

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.

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Selected References

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