Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish
- Authors
- 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.
- ID
- ZDB-PUB-111011-4
- 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
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.