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Open Access Methodology article

Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

Ruben van Boxtel12, Pim W Toonen12, Mark Verheul12, Henk S van Roekel12, Isaac J Nijman12, Victor Guryev12 and Edwin Cuppen12*

Author Affiliations

1 Hubrecht Institute for Developmental Biology and Stem Cell Research, Cancer Genomics Center, Royal Netherlands Academy of Sciences, Utrecht, The Netherlands

2 University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

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BMC Genomics 2008, 9:460  doi:10.1186/1471-2164-9-460

Published: 7 October 2008

Abstract

Background

The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive.

Results

As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes.

Conclusion

Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.