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Targeted mutagenesis in zebrafish using customized zinc-finger nucleases

Nature Protocols volume 4, pages 1855–1868 (2009)Cite this article

Abstract

Zebrafish mutants have traditionally been obtained by using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc-finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc-Finger Consortium reagents for constructing engineered zinc-finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within 4 months.

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Figure 1: A diagram of zinc-finger nucleases (ZFN)-induced insertion or deletion (indel) mutations.
Figure 2
Figure 3: A representative gel of in vitro transcribed RNAs encoding zinc-finger nucleases (ZFNs).
Figure 4
Figure 5
Figure 6: Representative results of fluorescent PCR analysis of (a) a pool of wild-type embryos and (b) a pool of embryos from a founder.

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Acknowledgements

We thank Dr. A.J. Giraldez for valuable suggestions and D. Cotelle for technical help on the fluorescent PCR analysis, Drs. P. Schlueter and C. Sachidanandan for helpful suggestions on the manuscript. J.E.F., M.L.M. and J.K.J. are supported by the NIH (R01GM069906, R21RR024189, and R21HL091808) and the MGH Pathology Service. R.T.P. and J.-R.J.Y. are supported by the NIH (CA118498 and GM88040) and the Ned Sahin Fund. J.-R.J.Y. is also supported by the NIH (AG031300) and the Claflin Distinguished Scholar Award.

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Authors and Affiliations

  1. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Jonathan E Foley, Morgan L Maeder & J Keith Joung

  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jonathan E Foley, Morgan L Maeder & J Keith Joung

  3. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Joseph Pearlberg

  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    J Keith Joung

  6. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Randall T Peterson & Jing-Ruey J Yeh

  7. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Randall T Peterson & Jing-Ruey J Yeh

  8. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Randall T Peterson

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  1. Jonathan E Foley
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Contributions

J.P. provided the protocol for plasmid DNA isolation in a 96-well format. All the other authors contributed extensively to the protocol development and preparation of the manuscript.

Corresponding author

Correspondence to Jing-Ruey J Yeh.

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Competing interests

J.K.J. is an inventor on patent applications which describe the OPEN zinc finger engineering method. All other authors declare that they have no competing financial interests.

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Foley, J., Maeder, M., Pearlberg, J. et al. Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nat Protoc 4, 1855–1868 (2009). https://doi.org/10.1038/nprot.2009.209

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