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Production of knockout rats using ENU mutagenesis and a yeast-based screening assay

Nature Biotechnology volume 21, pages 645–651 (2003)Cite this article

Abstract

The rat is a widely used model in biomedical research and is often the preferred rodent model in many areas of physiological and pathobiological research. Although many genetic tools are available for the rat, methods to produce gene-disrupted knockout rats are greatly needed. In this study, we developed protocols for creating N-ethyl-N-nitrosourea (ENU)-induced germline mutations in several rat strains. F1 preweanling pups from mutagenized Sprague Dawley (SD) male rats were then screened for functional mutations in Brca1 and Brca2 using a yeast gap-repair, ADE2-reporter truncation assay. We produced knockout rats for each of these two breast cancer suppressor genes.

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Figure 1: Heritable phenotypic mutant rats.
Figure 2: Brca1, Brca2 and agouti yeast cDNA/gDNA truncation assays.
Figure 3: Identification of a Brca2 knockout rat.
Figure 4: Screening for a Brca1 knockout rat.

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Acknowledgements

We thank Amy Moser for discussions and suggestions during the course of this work; R. Iggo, M. Tada, and T. Moriuchi for providing the pLSRP53 plasmid and the yIG397 yeast strain used in this paper; Henry Pitot for analysis of the histology sections; and Dinelli M. Monson and Millicent A. Shultz for technical assistance. This work has been supported by grants from the US National Institutes of Health (CA28954 and CA77494).

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  1. Yunhong Zan and Jill D Haag: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncology, McArdle Laboratory for Cancer Research, 1400 University Avenue, University of Wisconsin–Madison, Madison, 53706, Wisconsin, USA

    Yunhong Zan, Jill D Haag, Kai-Shun Chen, Laurie A Shepel, Don Wigington, Yu-Rong Wang, Rong Hu, Christine C Lopez-Guajardo, Heidi L Brose, Katherine I Porter, Rachel A Leonard, Andrew A Hitt, Stacy L Schommer, Anu F Elegbede & Michael N Gould

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  1. Yunhong Zan
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Correspondence to Michael N Gould.

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Zan, Y., Haag, J., Chen, KS. et al. Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21, 645–651 (2003). https://doi.org/10.1038/nbt830

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