Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single-copy transformants.
AUTOR(ES)
Bethke, B
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=146822Documentos Relacionados
- Cre-mediated somatic site-specific recombination in mice.
- Cre-mediated gene deletion in the mammary gland.
- Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.
- Cell Depletion Due to Diphtheria Toxin Fragment A after Cre-Mediated Recombination
- B lymphocyte-specific, Cre-mediated mutagenesis in mice.