Nucleic Acids Research Advance Access published online on September 23, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn603
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Methods Online |
Enhanced gene trapping in mouse embryonic stem cells
1Department of Molecular Hematology, University of Frankfurt Medical School, Frankfurt am Main, 2Institute of Developmental Genetics, Helmholtz Zentrum München – German Research Center for Environmental Health, Technical University, Munich, 3Department of Vertebrate Genomics, Max-Planck Institute for Molecular Genetics, Berlin and 4Center for Cardiovascular Research, Charité – Universitätsmedizin Berlin, Germany
*To whom correspondence should be addressed. Tel: +49 6301 6696; Fax: +49 6301 6390; Email: melchner{at}em.uni-frankfurt.de
Received July 10, 2008. Revised August 15, 2008. Accepted September 6, 2008.
Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.