Targeted mutagenesis in zebrafish using customized zinc-finger nucleases

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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.