Summary
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
Solnica-Krezel, L., Schier, A. F., and Driever, W. (1994). Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 136, 1401–1420.
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.
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.
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.
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.
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.
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.
Wienholds, E., and Plasterk, R. H. (2004). Target-selected gene inactivation in zebrafish. Methods Cell Biol. 77, 69–90.
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.
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.
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.
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.
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.
Wienholds, E., Schulte-Merker, S., Walderich, B., and Plasterk, R. H. (2002). Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99–102.
Acknowledgments
The authors would like to thank Freek van Eeden for details provided on the mutagenesis protocol. This work was supported by funds from the European Union-funded FP6 Integrated Project ZF-MODELS.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
de Bruijn, E., Cuppen, E., Feitsma, H. (2009). Highly Efficient ENU Mutagenesis in Zebrafish. In: Lieschke, G., Oates, A., Kawakami, K. (eds) Zebrafish. Methods in Molecular Biology, vol 546. Humana Press. https://doi.org/10.1007/978-1-60327-977-2_1
Download citation
DOI: https://doi.org/10.1007/978-1-60327-977-2_1
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-976-5
Online ISBN: 978-1-60327-977-2
eBook Packages: Springer Protocols