Using zinc finger nucleases for efficient and heritable gene disruption in zebrafish.
While the experimental tools developed for zebrafish have continued to advance the organism as a laboratory model, techniques for reverse genetics remain somewhat limited in scope. Zinc finger nucleases (ZFNs), chimeric fusions between DNA-binding zinc finger proteins and the non-specific cleavage domain of the FokI endonuclease, hold great promise for targeted mutagenesis in zebrafish, as demonstrated by two recent publications (Doyon et al., 2008, Nat Biotechnol. 26, 702-708; Meng et al., 2008, Nat Biotechnol. 26, 695-701). Because ZFNs can be designed to recognize a unique sequence in the genome, they can specifically bind and cleave a target locus, creating a double-strand break (DSB) that is repaired by one of two major DNA repair pathways. Repair by one of these pathways, non-homologous end joining, is often mutagenic, allowing one to screen for induced mutations in the target locus. By injecting into zebrafish embryos RNA encoding ZFNs that target three different loci, two groups have shown that ZFNs work efficiently to induce somatic and germline mutations (reviewed in (3)). We review here protocols for injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected fish for induced mutations, and subsequent recovery of the induced mutations.
McCammon
Jasmine M
JM
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
Amacher
Sharon L
SL
D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005838:Genotype; D016296:Mutagenesis / Q000235:genetics*; D015027:Zebrafish; D016335:Zinc Fingers / Q000235:genetics*
D004720:Endonucleases