Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.
Development
; 138(20): 4555-64, 2011 Oct.
Article
en En
| MEDLINE
| ID: mdl-21937602
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Pez Cebra
/
Desoxirribonucleasas de Localización Especificada Tipo II
/
Dedos de Zinc
/
Proteínas de Pez Cebra
Tipo de estudio:
Evaluation_studies
/
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Development
Asunto de la revista:
BIOLOGIA
/
EMBRIOLOGIA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Estados Unidos