RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses.
In recent years, tremendous insight has been gained on p53 regulation by targeting mutations at the p53 locus using homologous recombination in ES cells to generate mutant mice. Although informative, this approach is inefficient, slow and expensive. To facilitate targeting at the p53 locus, we developed an improved Recombinase-Mediated Cassette Exchange (RMCE) method. Our approach enables efficient targeting in ES cells to facilitate the production of mutant mice. But more importantly, the approach was Adapted for targeting in Somatic cells to Accelerate Phenotyping (RMCE-ASAP). We provide proof-of-concept for this at the p53 locus, by showing efficient targeting in fibroblasts, and rapid phenotypic read-out of a recessive mutation after a single exchange. RMCE-ASAP combines inverted heterologous recombinase target sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics. These general principles should make RMCE-ASAP applicable to any locus.
Toledo
Franck
F
The Salk Institute for Biological Studies, Gene expression Laboratory, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA. ftoledo@pasteur.fr
Liu
Chung-Wen
CW
Lee
Crystal J
CJ
Wahl
Geoffrey M
GM
D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D018390:Gene Targeting / Q000379:methods*; D016158:Genes, p53; D019426:Integrases; D051379:Mice / Q000235:genetics*; D010641:Phenotype; D011995:Recombination, Genetic; D013234:Stem Cells; D014764:Viral Proteins
D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases