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Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.

http://www.ncbi.nlm.nih.gov/pubmed/21937602

Zinc-finger nucleases (ZFNs) allow targeted gene inactivation in a wide range of model organisms. However, construction of target-specific ZFNs is technically challenging. Here, we evaluate a straightforward modular assembly-based approach for ZFN construction and gene inactivation in zebrafish. From an archive of 27 different zinc-finger modules, we assembled more than 70 different zinc-finger cassettes and evaluated their specificity using a bacterial one-hybrid assay. In parallel, we constructed ZFNs from these cassettes and tested their ability to induce lesions in zebrafish embryos. We found that the majority of zinc-finger proteins assembled from these modules have favorable specificities and nearly one-third of modular ZFNs generated lesions at their targets in the zebrafish genome. To facilitate the application of ZFNs within the zebrafish community we constructed a public database of sites in the zebrafish genome that can be targeted using this archive. Importantly, we generated new germline mutations in eight different genes, confirming that this is a viable platform for heritable gene inactivation in vertebrates. Characterization of one of these mutants, gata2a, revealed an unexpected role for this transcription factor in vascular development. This work provides a resource to allow targeted germline gene inactivation in zebrafish and highlights the benefit of a definitive reverse genetic strategy to reveal gene function.

Pubmed ID: 21937602 RIS Download

Mesh terms: Animals | Animals, Genetically Modified | Base Sequence | DNA | Databases, Genetic | Deoxyribonucleases, Type II Site-Specific | GATA2 Transcription Factor | Gene Targeting | Mutation | Neovascularization, Physiologic | Recombinant Fusion Proteins | Zebrafish | Zebrafish Proteins | Zinc Fingers

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Associated grants

  • Agency: NIGMS NIH HHS, Id: R01 GM081602
  • Agency: NHLBI NIH HHS, Id: R01 HL062974
  • Agency: NHLBI NIH HHS, Id: R01 HL079266
  • Agency: NHLBI NIH HHS, Id: R01 HL079266
  • Agency: NHLBI NIH HHS, Id: R01 HL093766
  • Agency: NHLBI NIH HHS, Id: R01 HL093766

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