Zebrafish pp 3-12

Part of the Methods in Molecular Biology book series (MIMB, volume 546)

Highly Efficient ENU Mutagenesis in Zebrafish

  • Ewart de Bruijn
  • Edwin Cuppen
  • Harma Feitsma


ENU (N-ethyl-N-nitrosourea) mutagenesis is a widely accepted and proven method to introduce random point mutations in the genome. Because there are no targeted knockout strategies available for zebrafish so far, random mutagenesis is currently the preferred method in both forward and reverse genetic approaches. To obtain high-density mutagenized zebrafish, six consecutive ENU treatments are applied at weekly intervals to adult male zebrafish by bathing them in ENU solution. With this procedure an average germ line mutation load of one mutation every 1.0 × 105–1.5 × 105 basepairs is reached routinely in our lab.

Key words

Zebrafish ENU Mutagenesis TILLING Method Mutant Knockout 


  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    Mullins, M. C., Hammerschmidt, M., Haffter, P., and Nusslein-Volhard, C. (1994). Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr. Biol. 4, 189–202.CrossRefPubMedGoogle Scholar
  3. 3.
    Solnica-Krezel, L., Schier, A. F., and Driever, W. (1994). Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1401–1420.PubMedGoogle Scholar
  4. 4.
    Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., and Nusslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36.PubMedGoogle Scholar
  5. 5.
    Baraban, S. C., Dinday, M. T., Castro, P. A., Chege, S., Guyenet, S., and Taylor, M. R. (2007). A large-scale mutagenesis screen to identify seizure-resistant zebrafish. Epilepsia 48, 1151–1157.CrossRefPubMedGoogle Scholar
  6. 6.
    Moore, J. L., Rush, L. M., Breneman, C., Mohideen, M. A., and Cheng, K. C. (2006). Zebrafish genomic instability mutants and cancer susceptibility. Genetics 174, 585–600.CrossRefPubMedGoogle Scholar
  7. 7.
    Dosch, R., Wagner, D. S., Mintzer, K. A., Runke, G., Wiemelt, A. P., and Mullins, M. C. (2004). Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev. Cell 6, 771–780.CrossRefPubMedGoogle Scholar
  8. 8.
    Wagner, D. S., Dosch, R., Mintzer, K. A., Wiemelt, A. P., and Mullins, M. C. (2004). Maternal control of development at the midblastula transition and beyond: mutants from the zebrafish II. Dev. Cell 6, 781–790.CrossRefPubMedGoogle Scholar
  9. 9.
    Cheng, K. C., and Moore, J. L. (1997). Genetic dissection of vertebrate processes in the zebrafish: a comparison of uniparental and two-generation screens. Biochem. Cell Biol. 75, 525–533.CrossRefPubMedGoogle Scholar
  10. 10.
    Wienholds, E., and Plasterk, R. H. (2004). Target-selected gene inactivation in zebrafish. Methods Cell Biol. 77, 69–90.CrossRefPubMedGoogle Scholar
  11. 11.
    Wienholds, E., van Eeden, F., Kosters, M., Mudde, J., Plasterk, R. H., and Cuppen, E. (2003). Efficient target-selected mutagenesis in zebrafish. Genome Res. 13, 2700–2707.CrossRefPubMedGoogle Scholar
  12. 12.
    Berghmans, S., Murphey, R. D., Wienholds, E., Neuberg, D., Kutok, J. L., Fletcher, C. D.,Morris, J. P., Liu, T. X., Schulte-Merker, S., Kanki, J. P., Plasterk, R., Zon, L. I., and Look, A. T. (2005) tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc. Natl Acad. Sci. U S A 102, 407–412.CrossRefPubMedGoogle Scholar
  13. 13.
    Feitsma, H., Leal, M. C., Moens, P. B., ­Cuppen, E., and Schulz, R. W. (2007). Mlh1 deficiency in zebrafish results in male sterility and aneuploid as well as triploid progeny in females. Genetics 175, 1561–1569.CrossRefPubMedGoogle Scholar
  14. 14.
    Hurlstone, A. F., Haramis, A. P., Wienholds, E., Begthel, H., Korving, J., Van Eeden, F., Cuppen, E., Zivkovic, D., Plasterk, R. H., and Clevers, H. (2003). The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature 425, 633–637.CrossRefPubMedGoogle Scholar
  15. 15.
    Wienholds, E., Koudijs, M. J., van Eeden, F. J., Cuppen, E., and Plasterk, R. H. (2003). The microRNA-producing enzyme Dicer1 is essential for zebrafish development. Nat. Genet. 35, 217–218.CrossRefPubMedGoogle Scholar
  16. 16.
    Wienholds, E., Schulte-Merker, S., Walderich, B., and Plasterk, R. H. (2002). Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99–102.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Ewart de Bruijn
    • 1
  • Edwin Cuppen
    • 1
  • Harma Feitsma
    • 1
  1. 1.Hubrecht Institute for Developmental Biology and Stem Cell ResearchUtrechtThe Netherlands

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