Nucleic Acids Research Advance Access originally published online on October 24, 2006
Nucleic Acids Research 2006 34(20):e139; doi:10.1093/nar/gkl728
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Nucleic Acids Research, 2006, Vol. 34, No. 20 e139
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Mutagenesis of diploid mammalian genes by gene entrapment
Department of Microbiology and Immunology, Room AA4206, Medical Center North, Vanderbilt University, School of Medicine 1161 21st Avenue South, AA4210, Nashville, TN 37232-2363, USA
*To whom correspondence should be addressed. Tel: +615 343 1379; Fax: +615 343 7392; Email: earl.ruley{at}vanderbilt.edu
Received June 17, 2006. Revised September 14, 2006. Accepted September 14, 2006.
The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 x 10–5 to 1.2 x 10–4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.
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