Abstract:
: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.
journal_name
Mol Cell Bioljournal_title
Molecular and cellular biologyauthors
Cohen-Tannoudji M,Robine S,Choulika A,Pinto D,El Marjou F,Babinet C,Louvard D,Jaisser Fdoi
10.1128/mcb.18.3.1444subject
Has Abstractpub_date
1998-03-01 00:00:00pages
1444-8issue
3eissn
0270-7306issn
1098-5549journal_volume
18pub_type
杂志文章abstract::Transfection of C3H/10T1/2 cells with a c-myc gene resulted in the acquisition of responsiveness to transforming growth factor type beta. Cells transfected with an activated H-ras gene or an H-ras and c-myc gene, however, exhibited a transformed morphology and spontaneous soft-agar growth, a phenotype induced reversib...
journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.7.7.2649
更新日期:1987-07-01 00:00:00
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journal_title:Molecular and cellular biology
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journal_title:Molecular and cellular biology
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1128/mcb.20.7.2358-2366.2000
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pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1128/MCB.00808-08
更新日期:2008-12-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.9.9.4113
更新日期:1989-09-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/mcb.5.7.1743
更新日期:1985-07-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.15.6.3430
更新日期:1995-06-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/MCB.00999-09
更新日期:2010-03-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.17.8.4633
更新日期:1997-08-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.23.17.6187-6199.2003
更新日期:2003-09-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.12.4.1872
更新日期:1992-04-01 00:00:00
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pub_type: 杂志文章
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更新日期:1997-05-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.15.6.3344
更新日期:1995-06-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.9.12.5726
更新日期:1989-12-01 00:00:00
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pub_type: 杂志文章
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更新日期:1981-10-01 00:00:00
abstract::The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constituti...
journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.12.4.1568
更新日期:1992-04-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.15.9.4921
更新日期:1995-09-01 00:00:00
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pub_type: 杂志文章
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更新日期:1988-12-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/mcb.8.8.3043
更新日期:1988-08-01 00:00:00
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journal_title:Molecular and cellular biology
pub_type: 杂志文章
doi:10.1128/MCB.21.15.5262-5275.2001
更新日期:2001-08-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/MCB.26.5.1806-1816.2006
更新日期:2006-03-01 00:00:00