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

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases 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. The zebrafish has proven to be an excellent model for vertebrate development and disease due to its rapid development, transparent embryos and its relatively facile forward genetics. Techniques for reverse genetic approaches in zebrafish, however, are limited to mRNA knockdown strategies using modified antisense oligomers (morpholinos) and TILLING for http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

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Publisher
Springer Journals
Copyright
Copyright © 2008 Nature Publishing Group
ISSN
1087-0156
eISSN
1087-0156
D.O.I.
10.1038/nbt1409
Publisher site
See Article on Publisher Site

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. The zebrafish has proven to be an excellent model for vertebrate development and disease due to its rapid development, transparent embryos and its relatively facile forward genetics. Techniques for reverse genetic approaches in zebrafish, however, are limited to mRNA knockdown strategies using modified antisense oligomers (morpholinos) and TILLING for

Journal

Nature BiotechnologySpringer Journals

Published: May 25, 2008

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