Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc-Finger Nucleases

Nat Biotechnol. 2008 Jun;26(6):702-8. doi: 10.1038/nbt1409. Epub 2008 May 25.

Abstract

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Genetically Modified / physiology*
  • Deoxyribonucleases / genetics
  • Gene Targeting / methods*
  • Genetic Engineering / methods*
  • Mutagenesis, Site-Directed / methods*
  • Protein Engineering / methods
  • Zebrafish / genetics*
  • Zebrafish Proteins / genetics*
  • Zinc Fingers / genetics*

Substances

  • Zebrafish Proteins
  • Deoxyribonucleases