Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 37, Pages 13188-13193Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0505474102
Keywords
ES cells; gene trapping
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Funding
- NCRR NIH HHS [5G12RR03060, G12 RR003060] Funding Source: Medline
- NHLBI NIH HHS [U01HL66600, U01 HL066600] Funding Source: Medline
- NIGMS NIH HHS [S06 GM008168, S06GM08168] Funding Source: Medline
- NIMH NIH HHS [R01 MH060612, R01MH60612] Funding Source: Medline
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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.
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