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

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.

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  • 1Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, California 94804, USA.

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.

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PMID:
18500334
[PubMed - indexed for MEDLINE]
PMCID:
PMC2674762
Free PMC Article

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