Abstract
Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genomewide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)–mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.
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Acknowledgments
The authors wish to thank Lucy Rowe and Mary Barter for genetic mapping services, and Timothy O’Brien, Melissa Berry, and Karen Svenson for critical reading of the manuscript. This work was supported by an NIH grant (GM45415) to JCS and a Cancer Center grant (CA34196) to The Jackson Laboratory.
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Munroe, R.J., Ackerman, S.L. & Schimenti, J.C. Genomewide two-generation screens for recessive mutations by ES cell mutagenesis. Mamm Genome 15, 960–965 (2004). https://doi.org/10.1007/s00335-004-2406-0
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DOI: https://doi.org/10.1007/s00335-004-2406-0