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Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells

Abstract

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles1. Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes2,3. In fact, several genome-wide4,5,6,7 and region-specific8,9,10 screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics-from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

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Figure 1: The effect of ENU on ES cell viability.
Figure 2: Expression and activity of Agt in ES cells and primary fibroblasts.

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Acknowledgements

We thank L. Liu and A. Hanson for technical assistance on the Agt assay; and S. Bultman, P. Green and J. Schimenti for helpful discussions and critical review of the manuscript. This work was supported by an NIH grant to T.M. The transgenic facility infrastructure was supported by funds from the Keck Foundation, University Hospitals of Cleveland and Case Western Reserve University School of Medicine.

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Correspondence to Terry Magnuson.

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Chen, Y., Yee, D., Dains, K. et al. Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nat Genet 24, 314–317 (2000). https://doi.org/10.1038/73557

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