Nucleic Acids Research, Vol 25, Issue 14 2828-2834, Copyright © 1997 by Oxford University Press
B Bethke and B Sauer
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.
ARTICLES
Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single-copy transformants
National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Disease, Bethesda, MD 2089-1800, USA.
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