Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish
Authors
Zhu, CongSmith, Tom
McNulty, Joseph C.
Rayla, Amy L.
Lakshmanan, Abirami
Siekmann, Arndt F.
Buffardi, Matthew
Meng, Xiangdong
Shin, Jimann
Padmanabhan, Arun
Cifuentes, Daniel
Giraldez, Antonio J.
Look, A. Thomas
Epstein, Jonathan A.
Lawson, Nathan D.
Wolfe, Scot A.
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyProgram in Gene Function and Expression
Document Type
Journal ArticlePublication Date
2011-10-15Keywords
ZebrafishZebrafish Proteins
Zinc Fingers
Gene Silencing
GATA2 Transcription Factor
Genetics and Genomics
Metadata
Show full item recordAbstract
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.Source
Development. 2011 Oct;138(20):4555-64. Link to article on publisher's siteDOI
10.1242/dev.066779Permanent Link to this Item
http://hdl.handle.net/20.500.14038/43962PubMed ID
21937602Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1242/dev.066779