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Mol Cell Biol, March 1998, p. 1444-1448, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

Michel Cohen-Tannoudji,¹ Sylvie Robine,² André Choulika,³ Daniel Pinto,² Fatima El Marjou,² Charles Babinet,¹ Daniel Louvard,² and Frédéric Jaisser^2,*

UMR 144 CNRS Laboratoire de Morphogenèse et Signalisation Cellulaires, Institut Curie, 75248 Paris Cedex 05,² and Unité de Biologie du Développement, CNRS URA 1960,¹ and Unité de Biologie Moléculaire du Développement,³ Institut Pasteur, 75015 Paris, France

Received 15 October 1997/Returned for modification 14 November 1997/Accepted 11 December 1997

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH₂-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10⁶) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

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^* Corresponding author. Present address: INSERM U246, Faculté de Médecine X. Bichat, 16 rue H. Huchard, 75018 Paris, France. Phone: 33 01 44856320. Fax: 33 01 42 34 63 77. E-mail: jaisser{at}bichat.inserm.fr.

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