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

van Boxtel R, Toonen PW, Verheul M, van Roekel HS, Nijman IJ, Guryev V, Cuppen E - BMC Genomics (2008)

Bottom Line: In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by approximately 20%, resulting in an overall increase in efficiency of approximately 2.5 fold.This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research, Cancer Genomics Center, Royal Netherlands Academy of Sciences, Utrecht, The Netherlands. r.boxtel@niob.knaw.nl

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 approximately 20%, resulting in an overall increase in efficiency of approximately 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.

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Effect of ENU on the survival of msh6-/- males. Msh6-/- male rats show an increased mortality after treatment with different concentrations of ENU compared to untreated msh6-/- male rats. Survival of untreated and ENU-treated wild type rats is 100% in the indicated time-period (data not shown). Red arrowheads indicate the time points of the last ENU injection and the start of mating for F1 progeny that can be screened for mutations without risk for chimaeric progeny.
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Figure 1: Effect of ENU on the survival of msh6-/- males. Msh6-/- male rats show an increased mortality after treatment with different concentrations of ENU compared to untreated msh6-/- male rats. Survival of untreated and ENU-treated wild type rats is 100% in the indicated time-period (data not shown). Red arrowheads indicate the time points of the last ENU injection and the start of mating for F1 progeny that can be screened for mutations without risk for chimaeric progeny.

Mentions: We also found that ENU affects the survival of msh6-/- rats. Untreated msh6-/- rats show a median survival of 60 weeks [20], while ENU treatment decreased survival of mutant males to an average mean of 37 ± 3 weeks (Figure 1). This decrease in lifespan was found to be due to tumor development, mainly lymphomas (data not shown). It is crucial for performing ENU target-selected mutagenesis screens that animals are viable and healthy for at least 26 weeks to allow for mutagenesis and subsequent breeding. In previous studies, no reduction in lifespan was observed in both 40 mg/kg (10 out of 10 survived) and 35 mg/kg (9 out of 10 survived) ENU-treated wild type males until 1.5 years of age (E.C., unpublished data).


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

van Boxtel R, Toonen PW, Verheul M, van Roekel HS, Nijman IJ, Guryev V, Cuppen E - BMC Genomics (2008)

Effect of ENU on the survival of msh6-/- males. Msh6-/- male rats show an increased mortality after treatment with different concentrations of ENU compared to untreated msh6-/- male rats. Survival of untreated and ENU-treated wild type rats is 100% in the indicated time-period (data not shown). Red arrowheads indicate the time points of the last ENU injection and the start of mating for F1 progeny that can be screened for mutations without risk for chimaeric progeny.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567347&req=5

Figure 1: Effect of ENU on the survival of msh6-/- males. Msh6-/- male rats show an increased mortality after treatment with different concentrations of ENU compared to untreated msh6-/- male rats. Survival of untreated and ENU-treated wild type rats is 100% in the indicated time-period (data not shown). Red arrowheads indicate the time points of the last ENU injection and the start of mating for F1 progeny that can be screened for mutations without risk for chimaeric progeny.
Mentions: We also found that ENU affects the survival of msh6-/- rats. Untreated msh6-/- rats show a median survival of 60 weeks [20], while ENU treatment decreased survival of mutant males to an average mean of 37 ± 3 weeks (Figure 1). This decrease in lifespan was found to be due to tumor development, mainly lymphomas (data not shown). It is crucial for performing ENU target-selected mutagenesis screens that animals are viable and healthy for at least 26 weeks to allow for mutagenesis and subsequent breeding. In previous studies, no reduction in lifespan was observed in both 40 mg/kg (10 out of 10 survived) and 35 mg/kg (9 out of 10 survived) ENU-treated wild type males until 1.5 years of age (E.C., unpublished data).

Bottom Line: In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by approximately 20%, resulting in an overall increase in efficiency of approximately 2.5 fold.This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research, Cancer Genomics Center, Royal Netherlands Academy of Sciences, Utrecht, The Netherlands. r.boxtel@niob.knaw.nl

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 approximately 20%, resulting in an overall increase in efficiency of approximately 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.

Show MeSH
Related in: MedlinePlus