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Using Zinc Finger Nucleases for Efficient and Heritable Gene Disruption in Zebrafish

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Engineered Zinc Finger Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 649))

Abstract

While the experimental tools developed for zebrafish have continued to advance the organism as a laboratory model, techniques for reverse genetics remain somewhat limited in scope. Zinc finger nucleases (ZFNs), chimeric fusions between DNA-binding zinc finger proteins and the non-specific cleavage domain of the FokI endonuclease, hold great promise for targeted mutagenesis in zebrafish, as demonstrated by two recent publications (Doyon et al., 2008, Nat Biotechnol. 26, 702–708; Meng et al., 2008, Nat Biotechnol. 26, 695–701). Because ZFNs can be designed to recognize a unique sequence in the genome, they can specifically bind and cleave a target locus, creating a double-strand break (DSB) that is repaired by one of two major DNA repair pathways. Repair by one of these pathways, non-homologous end joining, is often mutagenic, allowing one to screen for induced mutations in the target locus. By injecting into zebrafish embryos RNA encoding ZFNs that target three different loci, two groups have shown that ZFNs work efficiently to induce somatic and germline mutations (reviewed in (3)). We review here protocols for injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected fish for induced mutations, and subsequent recovery of the induced mutations.

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Note added in proof

After this manuscript was reviewed, another group published the successful application of ZFN-mediated mutagenesis in zebrafish (26).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

    Jasmine M. McCammon & Sharon L. Amacher

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  1. Jasmine M. McCammon
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  2. Sharon L. Amacher
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Editors and Affiliations

  1. School of Molecular &, Microbial Biosciences, University of Sydney, Cnr. Butlin Ave. & Maze Cres., Sydney, 2006, New South Wales, Australia

    Joel P. Mackay

  2. Dept. Pharmacology & Genome Center, University of California, Davis, E. Health Sciences Drive 451, Davis, 95616, California, USA

    David J. Segal

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McCammon, J.M., Amacher, S.L. (2010). Using Zinc Finger Nucleases for Efficient and Heritable Gene Disruption in Zebrafish. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_18

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  • DOI: https://doi.org/10.1007/978-1-60761-753-2_18

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-752-5

  • Online ISBN: 978-1-60761-753-2

  • eBook Packages: Springer Protocols

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