Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes.
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 approximately 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.
Friedel
Roland H
RH
Department of Biological Sciences, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
Plump
Andrew
A
Lu
Xiaowei
X
Spilker
Kerri
K
Jolicoeur
Christine
C
Wong
Karen
K
Venkatesh
Tadmiri R
TR
Yaron
Avraham
A
Hynes
Mary
M
Chen
Bin
B
Okada
Ami
A
McConnell
Susan K
SK
Rayburn
Helen
H
Tessier-Lavigne
Marc
M
D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology; D020869:Gene Expression Profiling; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D008722:Methods; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D011995:Recombination, Genetic; D013234:Stem Cells / Q000378:metabolism