Published online 26 July 2006
Nucleic Acids Research, 2006, Vol. 34, No. 13 e92
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses
1 The Salk Institute for Biological Studies, Gene expression Laboratory 10010 N. Torrey Pines Rd. La Jolla, CA 92037, USA 2 Institut Pasteur, Unité d'Expression Génétique et Maladies 25, rue du Dr. Roux, 75724 Paris Cedex 15, France
*To whom correspondence should be addressed. Tel: 33 1 40 61 34 80; Fax: 33 1 40 61 30 33; Email: ftoledo{at}pasteur.fr
Received June 4, 2006. Revised July 6, 2006. Accepted July 6, 2006.
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.
Correspondence may also be addressed to Geoffrey M. Wahl. Tel: 1 858 453 4100, ext. 1255; Fax: 1 858 535 1871; Email: wahl{at}salk.edu
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