Document Detail


Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.
MedLine Citation:
PMID:  21937602     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Cong Zhu; Tom Smith; Joseph McNulty; Amy L Rayla; Abirami Lakshmanan; Arndt F Siekmann; Matthew Buffardi; Xiangdong Meng; Jimann Shin; Arun Padmanabhan; Daniel Cifuentes; Antonio J Giraldez; A Thomas Look; Jonathan A Epstein; Nathan D Lawson; Scot A Wolfe
Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Development (Cambridge, England)     Volume:  138     ISSN:  1477-9129     ISO Abbreviation:  Development     Publication Date:  2011 Oct 
Date Detail:
Created Date:  2011-09-22     Completed Date:  2011-11-28     Revised Date:  2014-09-19    
Medline Journal Info:
Nlm Unique ID:  8701744     Medline TA:  Development     Country:  England    
Other Details:
Languages:  eng     Pagination:  4555-64     Citation Subset:  IM    
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Animals
Animals, Genetically Modified
Base Sequence
DNA / genetics,  metabolism
Databases, Genetic
Deoxyribonucleases, Type II Site-Specific / genetics*,  metabolism*
GATA2 Transcription Factor / genetics,  metabolism
Gene Targeting
Mutation
Neovascularization, Physiologic / genetics,  physiology
Recombinant Fusion Proteins / genetics,  metabolism
Zebrafish / embryology,  genetics*,  metabolism*
Zebrafish Proteins / genetics*,  metabolism*
Zinc Fingers / genetics*
Grant Support
ID/Acronym/Agency:
R01 GM081602/GM/NIGMS NIH HHS; R01 HL062974/HL/NHLBI NIH HHS; R01 HL079266/HL/NHLBI NIH HHS; R01 HL079266/HL/NHLBI NIH HHS; R01 HL093766/HL/NHLBI NIH HHS; R01 HL093766/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/GATA2 Transcription Factor; 0/GATA2 protein, zebrafish; 0/Recombinant Fusion Proteins; 0/Zebrafish Proteins; 9007-49-2/DNA; EC 3.1.21.-/endodeoxyribonuclease FokI; EC 3.1.21.4/Deoxyribonucleases, Type II Site-Specific
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish.
Next Document:  Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect ...