ZFIN ID: ZDB-PUB-111011-4
Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish
Zhu, C., Smith, T., McNulty, J., Rayla, A.L., Lakshmanan, A., Siekmann, A.F., Buffardi, M., Meng, X., Shin, J., Padmanabhan, A., Cifuentes, D., Giraldez, A.J., Look, A.T., Epstein, J.A., Lawson, N.D., and Wolfe, S.A.
Date: 2011
Source: Development (Cambridge, England) 138(20): 4555-4564 (Journal)
Registered Authors: Cifuentes, Daniel, Epstein, Jonathan A., Giraldez, Antonio, Lakshmanan, Abirami, Lawson, Nathan, Look, A. Thomas, McNulty, Joseph, Shin, Jimann, Siekmann, Arndt Friedrich, Smith, Tom, Wolfe, Scot A.
Keywords: gata2, vascular development, zebrafish, zinc-finger nuclease
MeSH Terms: Animals; Animals, Genetically Modified; Base Sequence; DNA/genetics; DNA/metabolism; Databases, Genetic; Deoxyribonucleases, Type II Site-Specific/genetics*; Deoxyribonucleases, Type II Site-Specific/metabolism*; GATA2 Transcription Factor/genetics; GATA2 Transcription Factor/metabolism; Gene Targeting; Mutation; Neovascularization, Physiologic/genetics; Neovascularization, Physiologic/physiology; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Zebrafish/embryology; Zebrafish/genetics*; Zebrafish/metabolism*; Zebrafish Proteins/genetics*; Zebrafish Proteins/metabolism*; Zinc Fingers/genetics*
PubMed: 21937602 Full text @ Development
FIGURES   (current status)
ABSTRACT

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.

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