ZFIN ID: ZDB-PUB-110803-50
Inducing High Rates of Targeted Mutagenesis in Zebrafish Using Zinc Finger Nucleases (ZFNs)
McCammon, J.M., Doyon, Y., and Amacher, S.L.
Date: 2011
Source: Methods in molecular biology (Clifton, N.J.)   770: 505-527 (Chapter)
Registered Authors: Amacher, Sharon, McCammon, Jasmine
Keywords: none
MeSH Terms:
  • Animal Husbandry
  • Animals
  • Deoxyribonucleases/chemistry*
  • Deoxyribonucleases/metabolism*
  • Embryo, Nonmammalian/metabolism
  • Female
  • Genes, Reporter/genetics
  • Genetic Loci/genetics
  • Genetic Techniques*
  • Genotype
  • Germ Cells/cytology
  • Germ Cells/metabolism
  • Male
  • Microinjections
  • Mutagenesis*
  • Phenotype
  • RNA/biosynthesis
  • Saccharomyces cerevisiae/genetics
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zinc Fingers*
PubMed: 21805278 Full text @ Meth. Mol. Biol.
ABSTRACT
Animal models, including the zebrafish, without a reliable embryonic stem cell system are not easily amenable to targeted mutagenesis for studying gene function. Three recent publications have shown that zinc finger nucleases (ZFNs) have circumvented this shortcoming in zebrafish. Similar to restriction enzymes, ZFNs can introduce site-specific double-strand breaks (DSBs); moreover, they can be designed to recognize virtually any target sequence. Because the preferred DSB repair pathway in zebrafish embryos, non-homologous end joining, is error-prone, ZFNs can be used to create mutations in a gene of interest. Here we review the protocols for a yeast-based assay to detect effective ZFNs. Additionally, we detail the procedures for synthesis and injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected embryos for induced mutations in the soma, and recovery of germline mutations.
ADDITIONAL INFORMATION No data available