Targeted mutagenesis in zebrafish using customized zinc-finger nucleases.
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.
Foley
Jonathan E
JE
Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Maeder
Morgan L
ML
Pearlberg
Joseph
J
Joung
J Keith
JK
Peterson
Randall T
RT
Yeh
Jing-Ruey J
JR
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
D003851:Deoxyribonucleases