Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes

被引:76
作者
Friedel, RH
Plump, A
Lu, XW
Spilker, K
Jolicoeur, C
Wong, K
Venkatesh, TR
Yaron, A
Hynes, M
Chen, B
Okada, A
McConnell, SK
Rayburn, H
Tessier-Lavigne, M
机构
[1] Stanford Univ, Dept Biol Sci, Howard Hughes Med Inst, Stanford, CA 94305 USA
[2] CUNY City Coll, Dept Biol, New York, NY 10031 USA
关键词
ES cells; gene trapping;
D O I
10.1073/pnas.0505474102
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
A powerful tool for postgenomic analysis of mammalian gene function is gene targeting in mouse ES cells. We report that homologous recombination using a promoterless gene trap vector ("targeting trapping") yields targeting frequencies averaging above 50%, a significant increase compared with current approaches. These high frequencies appear to be due to the stringency of selection with promoterless constructs, because most random insertions are silent and eliminated by drug selection. The promoterless design requires that the targeted gene be expressed in ES cells at levels exceeding a certain threshold (which we estimate to be approximate to 1% of the transferrin receptor gene expression level, for the secretory trap vector used here). Analysis of 127 genes that had been trapped by random (nontargeted) gene trapping with the same vector shows that virtually all are expressed in ES cells above this threshold, suggesting that targeted and random trapping share similar requirements for expression levels. In a random sampling of 130 genes encoding secretory proteins, about half were expressed above threshold, suggesting that about half of all secretory genes are accessible by either targeted or random gene trapping. The simplicity and high efficiency of the method facilitate systematic targeting of a large fraction of the genome by individual investigators and large-scale consortia alike.
引用
收藏
页码:13188 / 13193
页数:6
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