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ZFIN ID: ZDB-PUB-091221-14
Targeted mutagenesis in zebrafish using customized zinc-finger nucleases
Foley, J.E., Maeder, M.L., Pearlberg, J., Joung, J.K., Peterson, R.T., and Yeh, J.R.
Date: 2009
Source: Nature Protocols   4(12): 1855-1867 (Journal)
Registered Authors: Yeh, Jing-Ruey (Joanna)
Keywords: none
MeSH Terms:
  • Animals
  • Deoxyribonucleases/chemistry
  • Deoxyribonucleases/metabolism*
  • Embryo, Nonmammalian
  • Frameshift Mutation*
  • Genetic Vectors
  • Mutagenesis, Site-Directed/methods*
  • Polymerase Chain Reaction
  • Protein Engineering
  • Restriction Mapping
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zinc Fingers*
PubMed: 20010934 Full text @ Nat. Protoc.
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.