Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish
McNulty, Joseph C.
Rayla, Amy L.
Siekmann, Arndt F.
Giraldez, Antonio J.
Look, A. Thomas
Epstein, Jonathan A.
Lawson, Nathan D.
Wolfe, Scot A.
UMass Chan AffiliationsDepartment of Biochemistry and Molecular Pharmacology
Program in Gene Function and Expression
GATA2 Transcription Factor
Genetics and Genomics
MetadataShow full item record
AbstractZinc-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.
SourceDevelopment. 2011 Oct;138(20):4555-64. Link to article on publisher's site
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/43962
Related ResourcesLink to Article in PubMed