Advertisement

Generation of Genetically Modified Rodents Using Random ENU Mutagenesis

  • Ruben van Boxtel
  • Edwin Cuppen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 693)

Abstract

The generation of genetically modified animals using N-ethyl-N-nitrosourea (ENU) mutagenesis is a fast and highly effective method. The technique is based on treating male animals with the supermutagen ENU, which randomly introduces mutations in the spermatogonial stem cells. By breeding these animals with untreated females, an F1 population is generated in which each individual carries unique random ENU-induced mutations, which can be retrieved using either genotype-driven or phenotype-driven approaches. No complicated cell culturing techniques are required and since no foreign DNA is introduced, the mutant animals that are generated are not transgenic. Here, we describe the detailed protocols for ENU mutagenesis and for mutant retrieval.

Key words

ENU mutagenesis Mutation discovery Phenotype-driven screens Genotype-driven screens Mutant rodent models 

References

  1. 1.
    Russell, W. L., Kelly, E. M., Hunsicker, P. R., Bangham, J. W., Maddux, S. C., and Phipps, E. L. (1979) Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse, Proc Natl Acad Sci U S A 76, 5818–5819.PubMedCrossRefGoogle Scholar
  2. 2.
    Noveroske, J. K., Weber, J. S., and Justice, M. J. (2000) The mutagenic action of N-ethyl-N-nitrosourea in the mouse, Mamm Genome 11, 478–483.PubMedCrossRefGoogle Scholar
  3. 3.
    Rogers, D. C., Fisher, E. M., Brown, S. D., Peters, J., Hunter, A. J., and Martin, J. E. (1997) Behavioral and functional analysis of mouse phenotype: SHIRPA, a proposed protocol for comprehensive phenotype assessment, Mamm Genome 8, 711–713.PubMedCrossRefGoogle Scholar
  4. 4.
    Zan, Y., Haag, J. D., Chen, K. S., Shepel, L. A., Wigington, D., Wang, Y. R., Hu, R., Lopez-Guajardo, C. C., Brose, H. L., Porter, K. I., Leonard, R. A., Hitt, A. A., Schommer, S. L., Elegbede, A. F., and Gould, M. N. (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay, Nat Biotechnol 21, 645–651.PubMedCrossRefGoogle Scholar
  5. 5.
    Smits, B. M., Mudde, J., Plasterk, R. H., and Cuppen, E. (2004) Target-selected muta­genesis of the rat, Genomics 83, 332–334.PubMedCrossRefGoogle Scholar
  6. 6.
    Mashimo, T., Yanagihara, K., Tokuda, S., Voigt, B., Takizawa, A., Nakajima, R., Kato, M., Hirabayashi, M., Kuramoto, T., and Serikawa, T. (2008) An ENU-induced mutant archive for gene targeting in rats, Nat Genet 40, 514–515.PubMedCrossRefGoogle Scholar
  7. 7.
    Smits, B. M., Mudde, J. B., van de Belt, J., Verheul, M., Olivier, J., Homberg, J., Guryev, V., Cools, A. R., Ellenbroek, B. A., Plasterk, R. H., and Cuppen, E. (2006) Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis, Pharmacogenet Genomics 16, 159–169.PubMedGoogle Scholar
  8. 8.
    Justice, M. J., Carpenter, D. A., Favor, J., Neuhauser-Klaus, A., Hrabe de Angelis, M., Soewarto, D., Moser, A., Cordes, S., Miller, D., Chapman, V., Weber, J. S., Rinchik, E. M., Hunsicker, P. R., Russell, W. L., and Bode, V. C. (2000) Effects of ENU dosage on mouse strains, Mamm Genome 11, 484–488.PubMedCrossRefGoogle Scholar
  9. 9.
    Augustin, M., Sedlmeier, R., Peters, T., Huffstadt, U., Kochmann, E., Simon, D., Schoniger, M., Garke-Mayerthaler, S., Laufs, J., Mayhaus, M., Franke, S., Klose, M., Graupner, A., Kurzmann, M., Zinser, C., Wolf, A., Voelkel, M., Kellner, M., Kilian, M., Seelig, S., Koppius, A., Teubner, A., Korthaus, D., Nehls, M., and Wattler, S. (2005) Efficient and fast targeted production of murine models based on ENU mutagenesis, Mamm Genome 16, 405–413.PubMedCrossRefGoogle Scholar
  10. 10.
    Concepcion, D., Seburn, K. L., Wen, G., Frankel, W. N., and Hamilton, B. A. (2004) Mutation rate and predicted phenotypic target sizes in ethylnitrosourea-treated mice, Genetics 168, 953–959.PubMedCrossRefGoogle Scholar
  11. 11.
    Michaud, E. J., Culiat, C. T., Klebig, M. L., Barker, P. E., Cain, K. T., Carpenter, D. J., Easter, L. L., Foster, C. M., Gardner, A. W., Guo, Z. Y., Houser, K. J., Hughes, L. A., Kerley, M. K., Liu, Z., Olszewski, R. E., Pinn, I., Shaw, G. D., Shinpock, S. G., Wymore, A. M., Rinchik, E. M., and Johnson, D. K. (2005) Efficient gene-driven germ-line point mutagenesis of C57BL/6 J mice, BMC Genomics 6, 164.PubMedCrossRefGoogle Scholar
  12. 12.
    Quwailid, M. M., Hugill, A., Dear, N., Vizor, L., Wells, S., Horner, E., Fuller, S., Weedon, J., McMath, H., Woodman, P., Edwards, D., Campbell, D., Rodger, S., Carey, J., Roberts, A., Glenister, P., Lalanne, Z., Parkinson, N., Coghill, E. L., McKeone, R., Cox, S., Willan, J., Greenfield, A., Keays, D., Brady, S., Spurr, N., Gray, I., Hunter, J., Brown, S. D., and Cox, R. D. (2004) A gene-driven ENU-based approach to generating an allelic series in any gene, Mamm Genome 15, 585–591.PubMedCrossRefGoogle Scholar
  13. 13.
    Sakuraba, Y., Sezutsu, H., Takahasi, K. R., Tsuchihashi, K., Ichikawa, R., Fujimoto, N., Kaneko, S., Nakai, Y., Uchiyama, M., Goda, N., Motoi, R., Ikeda, A., Karashima, Y., Inoue, M., Kaneda, H., Masuya, H., Minowa, O., Noguchi, H., Toyoda, A., Sakaki, Y., Wakana, S., Noda, T., Shiroishi, T., and Gondo, Y. (2005) Molecular characterization of ENU mouse mutagenesis and archives, Biochem Biophys Res Commun 336, 609–616.PubMedCrossRefGoogle Scholar
  14. 14.
    van Boxtel, R., Toonen, P. W., van Roekel, H. S., Verheul, M., Smits, B. M., Korving, J., de Bruin, A., and Cuppen, E. (2008) Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis, Carcinogenesis 29, 1290–1297.PubMedCrossRefGoogle Scholar
  15. 15.
    Claij, N., van der Wal, A., Dekker, M., Jansen, L., and te Riele, H. (2003) DNA mismatch repair deficiency stimulates N-ethyl-N-nitrosourea-induced mutagenesis and lymphomagenesis, Cancer Res 63, 2062–2066.PubMedGoogle Scholar
  16. 16.
    van Boxtel, R., Toonen, P. W., Verheul, M., van Roekel, H. S., Nijman, I. J., Guryev, V., and Cuppen, E. (2008) Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals, BMC Genomics 9, 460.PubMedCrossRefGoogle Scholar
  17. 17.
    Smits, B. M., Haag, J. D., Cuppen, E., and Gould, M. N. (2007) Rat Knockout and Mutant Models, in Sourcebook of Models for Biomedical Research (Conn, P. M., Ed.), pp. 171–178, Humana Press Inc., Totowa NJ.Google Scholar
  18. 18.
    Coghill, E. L., Hugill, A., Parkinson, N., Davison, C., Glenister, P., Clements, S., Hunter, J., Cox, R. D., and Brown, S. D. (2002) A gene-driven approach to the identification of ENU mutants in the mouse, Nat Genet 30, 255–256.PubMedCrossRefGoogle Scholar
  19. 19.
    Justice, M. J., Noveroske, J. K., Weber, J. S., Zheng, B., and Bradley, A. (1999) Mouse ENU mutagenesis, Hum Mol Genet 8, 1955–1963.PubMedCrossRefGoogle Scholar
  20. 20.
    Hrabe de Angelis, M. H., Flaswinkel, H., Fuchs, H., Rathkolb, B., Soewarto, D., Marschall, S., Heffner, S., Pargent, W., Wuensch, K., Jung, M., Reis, A., Richter, T., Alessandrini, F., Jakob, T., Fuchs, E., Kolb, H., Kremmer, E., Schaeble, K., Rollinski, B., Roscher, A., Peters, C., Meitinger, T., Strom, T., Steckler, T., Holsboer, F., Klopstock, T., Gekeler, F., Schindewolf, C., Jung, T., Avraham, K., Behrendt, H., Ring, J., Zimmer, A., Schughart, K., Pfeffer, K., Wolf, E., and Balling, R. (2000) Genome-wide, large-scale production of mutant mice by ENU muta­genesis, Nat Genet 25, 444–447.PubMedCrossRefGoogle Scholar
  21. 21.
    Kile, B. T., Hentges, K. E., Clark, A. T., Nakamura, H., Salinger, A. P., Liu, B., Box, N., Stockton, D. W., Johnson, R. L., Behringer, R. R., Bradley, A., and Justice, M. J. (2003) Functional genetic analysis of mouse chromosome 11, Nature 425, 81–86.PubMedCrossRefGoogle Scholar
  22. 22.
    Lindblad-Toh, K., Winchester, E., Daly, M. J., Wang, D. G., Hirschhorn, J. N., Laviolette, J. P., Ardlie, K., Reich, D. E., Robinson, E., Sklar, P., Shah, N., Thomas, D., Fan, J. B., Gingeras, T., Warrington, J., Patil, N., Hudson, T. J., and Lander, E. S. (2000) Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse, Nat Genet 24, 381–386.PubMedCrossRefGoogle Scholar
  23. 23.
    Saar, K., Beck, A., Bihoreau, M. T., Birney, E., Brocklebank, D., Chen, Y., Cuppen, E., Demonchy, S., Dopazo, J., Flicek, P., Foglio, M., Fujiyama, A., Gut, I. G., Gauguier, D., Guigo, R., Guryev, V., Heinig, M., Hummel, O., Jahn, N., Klages, S., Kren, V., Kube, M., Kuhl, H., Kuramoto, T., Kuroki, Y., Lechner, D., Lee, Y. A., Lopez-Bigas, N., Lathrop, G. M., Mashimo, T., Medina, I., Mott, R., Patone, G., Perrier-Cornet, J. A., Platzer, M., Pravenec, M., Reinhardt, R., Sakaki, Y., Schilhabel, M., Schulz, H., Serikawa, T., Shikhagaie, M., Tatsumoto, S., Taudien, S., Toyoda, A., Voigt, B., Zelenika, D., Zimdahl, H., and Hubner, N. (2008) SNP and haplotype mapping for genetic analysis in the rat, Nat Genet 40, 560–566.PubMedCrossRefGoogle Scholar
  24. 24.
    Nelms, K. A. and Goodnow, C. C. (2001) Genome-wide ENU mutagenesis to reveal immune regulators, Immunity 15, 409–418.PubMedCrossRefGoogle Scholar
  25. 25.
    Smits, B. M., Peters, T. A., Mul, J. D., Croes, H. J., Fransen, J. A., Beynon, A. J., Guryev, V., Plasterk, R. H., and Cuppen, E. (2005) Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics, Genetics 170, 1887–1896.PubMedCrossRefGoogle Scholar
  26. 26.
    Keays, D. A., Clark, T. G., and Flint, J. (2006) Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens, Mamm Genome 17, 230–238.PubMedCrossRefGoogle Scholar
  27. 27.
    Nickerson, D. A., Tobe, V. O., and Taylor, S. L. (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing, Nucleic Acids Res 25, 2745–2751.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ruben van Boxtel
    • 1
  • Edwin Cuppen
    • 1
  1. 1.Royal Netherlands Academy of SciencesHubrecht Institute and University Medical Center UtrechtUtrechtThe Netherlands

Personalised recommendations