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© Research
Publication : Molecular and cellular biology

I-SceI-induced gene replacement at a natural locus in embryonic stem cells

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Molecular and cellular biology - 01 Mar 1998

Cohen-Tannoudji M, Robine S, Choulika A, Pinto D, El Marjou F, Babinet C, Louvard D, Jaisser F

Link to Pubmed [PMID] – 9488460

Mol. Cell. Biol. 1998 Mar;18(3):1444-8

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 x 10[-6]) 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.

http://www.ncbi.nlm.nih.gov/pubmed/9488460