title: Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.
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.
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Cong
authors.initials: C
authors.lastname: Zhu
authors.forename: Tom
authors.initials: T
authors.lastname: Smith
authors.forename: Joseph
authors.initials: J
authors.lastname: McNulty
authors.forename: Amy L
authors.initials: AL
authors.lastname: Rayla
authors.forename: Abirami
authors.initials: A
authors.lastname: Lakshmanan
authors.forename: Arndt F
authors.initials: AF
authors.lastname: Siekmann
authors.forename: Matthew
authors.initials: M
authors.lastname: Buffardi
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Meng
authors.forename: Jimann
authors.initials: J
authors.lastname: Shin
authors.forename: Arun
authors.initials: A
authors.lastname: Padmanabhan
authors.forename: Daniel
authors.initials: D
authors.lastname: Cifuentes
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Giraldez
authors.forename: A Thomas
authors.initials: AT
authors.lastname: Look
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Epstein
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Lawson
authors.forename: Scot A
authors.initials: SA
authors.lastname: Wolfe
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D030541:Databases, Genetic; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D050989:GATA2 Transcription Factor / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D050989:GATA2 Transcription Factor; C516059:Gata2a protein, zebrafish; D011993:Recombinant Fusion Proteins; D029961:Zebrafish Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific