Journal
NATURE BIOTECHNOLOGY
Volume 26, Issue 6, Pages 702-708Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt1409
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Funding
- NICHD NIH HHS [P01 HD022486, HD22486] Funding Source: Medline
- NIGMS NIH HHS [R01 GM088041, R01 GM061952-02, R01 GM061952, R01 GM061952-03, R01 GM061952-06A2S1, R01 GM061952-06A2, R01 GM061952-01A2, R01 GM061952-04, 1-R01-GM061952, R01 GM061952-05] Funding Source: Medline
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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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