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

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    Authors
    Zhu, Cong
    Smith, 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.
    Show allShow less
    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Program in Gene Function and Expression
    Document Type
    Journal Article
    Publication Date
    2011-10-15
    Keywords
    Zebrafish
    Zebrafish Proteins
    Zinc Fingers
    Gene Silencing
    GATA2 Transcription Factor
    Genetics and Genomics
    
    Metadata
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    Link to Full Text
    http://dx.doi.org/10.1242/dev.066779
    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.
    Source
    Development. 2011 Oct;138(20):4555-64. Link to article on publisher's site
    DOI
    10.1242/dev.066779
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/43962
    PubMed ID
    21937602
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1242/dev.066779
    Scopus Count
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