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Mouse mutants from chemically mutagenized embryonic stem cells

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Abstract

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain1 and interlocus2 variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

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Figure 1: Breeding strategy for recovery of recessive mutations.
Figure 2: Phenotypes of mutants.

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Acknowledgements

We thank T. Magnuson for CT129 ES cells; T. Gridley for CJ7 ES cells; A. Planchart for input on design of culture experiments; J. Sztein for assistance with sperm examination; C. O'Neill for animal care; and T. O'Brien, G. Cox, T. Magnuson and M.A. Handel for critical evaluation of the manuscript. This work was supported by NIH grant GM45415 to J.C.S. and a Cancer Center Grant CA34196 to The Jackson Laboratory V.L.B. was supported by an NIH training grant (HD07065) and fellowship NIH (HD08441). S.W.M.J. is an Assistant Investigator of The Howard Hughes Medical Institute. Hearing tests were performed under a contract (DC62108) to K. Johnson from the National Institute on Deafness and Other Communicative Disorders.

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Authors and Affiliations

  1. The Jackson Laboratory, Bar Harbor, Maine, USA

    Robert J. Munroe, Rebecca A. Bergstrom, Qing Y Zheng, Brian Libby, Richard Smith, Simon W.M. John, Kerry J. Schimenti, Victoria L. Browning & John C. Schimenti

  2. The Howard Hughes Medical Institute, Bar Harbor, Maine, USA

    Richard Smith & Simon W.M. John

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  1. Robert J. Munroe
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  2. Rebecca A. Bergstrom
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  3. Qing Y Zheng
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  4. Brian Libby
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  7. Kerry J. Schimenti
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  8. Victoria L. Browning
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  9. John C. Schimenti
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Correspondence to John C. Schimenti.

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Munroe, R., Bergstrom, R., Y Zheng, Q. et al. Mouse mutants from chemically mutagenized embryonic stem cells. Nat Genet 24, 318–321 (2000). https://doi.org/10.1038/73563

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