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Nat Protoc. 2009;4(12):1855-67. doi: 10.1038/nprot.2009.209.

Targeted mutagenesis in zebrafish using customized zinc-finger nucleases.

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

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.

PMID:
20010934
PMCID:
PMC2814337
DOI:
10.1038/nprot.2009.209
[Indexed for MEDLINE]
Free PMC Article
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