title: Targeted mutagenesis in zebrafish using customized zinc-finger nucleases.
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.
authors.affiliation: Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Foley
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: Joseph
authors.initials: J
authors.lastname: Pearlberg
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.forename: Randall T
authors.initials: RT
authors.lastname: Peterson
authors.forename: Jing-Ruey J
authors.initials: JR
authors.lastname: Yeh
mesh_terms: D000818:Animals; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D016368:Frameshift Mutation; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D015183:Restriction Mapping; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D003851:Deoxyribonucleases