Volume 5 Supplement 1

24th Congress of the International Association for Breast Cancer Research. Advances in human breast cancer research: preclinical models

Open Access

Modeling breast cancer in the rat: a method to produce knockout rats

  • MN Gould1
Breast Cancer Research20035(Suppl 1):29

https://doi.org/10.1186/bcr688

Published: 1 October 2003

The rat is an important murine model for biomedical research. It is a key model for many diseases including cardiovascular diseases, diabetes, neurological diseases, and certain cancers such as breast cancer. Through much work the rat genomic toolbox is almost full and includes dense genetic maps, an approximately sevenfold genomic sequence and transgenic technologies. A key missing tool was the ability to produce knockout rats. This was in part due to the fact that despite over a decade of work embryonic stem cells could not be made to go germline. In order to approach this problem we developed a unique technology that combined the use of germline mutagenesis with a yeast truncation gap repair assay to select gene-specific knockout rats. We first developed the protocols to efficiently mutagenize the rat genome with ethyl nitroso urea. In order to establish knockout rats for specifically chosen genes, we bred ethyl nitroso urea mutagenized males with untreated females to produce F1 pups. These pre-weanling pups were screened for functional mutations (e.g. nonsense mutations, out of frame-shifts mutations, etc.). This was accomplished using a highly efficient and economical yeast gap-repair truncation assay. For this assay either genomic DNA or cDNA is produced from tail clips of rat pups. This DNA is then used as a PCR template to amplify the targeted gene sequence. This unpurified PCR product together with our reporter vector is cotransformed into yeast that does not express ADE2. The yeast through homologous recombination clones the PCR product into the non-integrated ADE2 reporter plasmid. In scoring the yeast plates, a negative plate has mostly white large colonies and a plate positive for a knockout allele has one-half red colonies (rat) and one-half white colonies. We have used this technology to knockout two breast cancer suppressor genes, Brca1 and Brca2. The technology described can be applied to most rapidly breeding species ranging from zebra fish to mice.

Authors’ Affiliations

(1)
McArdle Laboratory, University of Wisconsin

Copyright

© BioMed Central 2002

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