PUBLICATION
Targeted mutagenesis in zebrafish using customized zinc-finger nucleases
- Authors
- Foley, J.E., Maeder, M.L., Pearlberg, J., Joung, J.K., Peterson, R.T., and Yeh, J.R.
- ID
- ZDB-PUB-091221-14
- 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.
Citation
Foley, J.E., Maeder, M.L., Pearlberg, J., Joung, J.K., Peterson, R.T., and Yeh, J.R. (2009) Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Nature Protocols. 4(12):1855-1867.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping