I-SceI-induced gene replacement at a natural locus in embryonic stem cells.
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.
Cohen-Tannoudji
M
M
Unité de Biologie du Développement, CNRS URA 1960, Institut Pasteur, Paris, France.
Robine
S
S
Choulika
A
A
Pinto
D
D
El Marjou
F
F
Babinet
C
C
Louvard
D
D
Jaisser
F
F
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D002874:Chromosome Mapping; D003598:Cytoskeletal Proteins; D004249:DNA Damage; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D000099149:Ezrin
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; D008840:Microfilament Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D000099149:Ezrin; C026615:villin; C037725:SCEI protein, S cerevisiae