Targeted mutagenesis in zebrafish using customized zinc-finger nucleases - PubMed (original) (raw)

Targeted mutagenesis in zebrafish using customized zinc-finger nucleases

Jonathan E Foley et al. Nat Protoc. 2009.

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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Figures

Figure 1

Figure 1

A diagram of ZFN-induced indel mutations. A full ZFN target site consists of two “half-sites” separated by a 5–6 bp spacer. Each half-site contains a 9 bp sequence that can be recognized by a 3-finger zinc finger array. A ZFN consists of a zinc finger array fused to a nuclease domain. A heterodimeric pair of ZFNs binds to the left and right half sites and induces a double strand DNA break (DSB) in the spacer. Cells utilize non-homologous end joining (NHEJ) machinery to repair the DSB, an error-prone process that can lead to random insertions or deletions (indels) at the target site.

Figure 2

Figure 2

Flowchart outlining the experimental procedures described in this protocol and expected timing for each step.

Figure 3

Figure 3

A representative gel of in vitro transcribed RNAs encoding ZFNs. The first lane is DNA size standard. Lane 2–3, 4–5 & 6–7 contain 3 sets of ZFN RNAs (−, before poly(A) tailing reaction; +, after poly(A) tailing reaction).

Figure 4

Figure 4

Representative results of normal-looking embryos (a) and deformed embryos (b) after injections of ZFN-encoding RNAs.

Figure 5

Figure 5

Representative results of sequencing traces of a wild-type (a) and a mutant (b) embryo.

Figure 6

Figure 6

Representative results of fluorescent PCR analysis of a pool of wild-type embryos (a) and a pool of embryos from a founder (b). The PCR products from the wild-type embryos sometimes contain more than one peak due to PCR stutter or non-specific amplification. However, the additional peak (b, double asterisks) that is not present in the wild-type control sample indicates the presence of ZFN-induced indel mutation.

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