Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals
<p>Abstract</p> <p>Background</p> <p>The laboratory rat <it>(Rattus norvegicus) </it>is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is li...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2008-10-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/9/460 |
| _version_ | 1818119854626963456 |
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| author | van Roekel Henk S Verheul Mark Toonen Pim W van Boxtel Ruben Nijman Isaac J Guryev Victor Cuppen Edwin |
| author_facet | van Roekel Henk S Verheul Mark Toonen Pim W van Boxtel Ruben Nijman Isaac J Guryev Victor Cuppen Edwin |
| author_sort | van Roekel Henk S |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The laboratory rat <it>(Rattus norvegicus) </it>is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. <it>N</it>-ethyl-<it>N</it>-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> |
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| format | Article |
| id | doaj.art-898c521c3254441d872383e80fed0789 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-11T05:16:49Z |
| publishDate | 2008-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-898c521c3254441d872383e80fed07892022-12-22T01:19:48ZengBMCBMC Genomics1471-21642008-10-019146010.1186/1471-2164-9-460Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animalsvan Roekel Henk SVerheul MarkToonen Pim Wvan Boxtel RubenNijman Isaac JGuryev VictorCuppen Edwin<p>Abstract</p> <p>Background</p> <p>The laboratory rat <it>(Rattus norvegicus) </it>is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. <it>N</it>-ethyl-<it>N</it>-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1471-2164/9/460 |
| spellingShingle | van Roekel Henk S Verheul Mark Toonen Pim W van Boxtel Ruben Nijman Isaac J Guryev Victor Cuppen Edwin Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals BMC Genomics |
| title | Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals |
| title_full | Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals |
| title_fullStr | Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals |
| title_full_unstemmed | Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals |
| title_short | Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals |
| title_sort | improved generation of rat gene knockouts by target selected mutagenesis in mismatch repair deficient animals |
| url | http://www.biomedcentral.com/1471-2164/9/460 |
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