Parameters and efficiency of direct gene disruption by zinc finger nucleases in medaka embryos.
Mar Biotechnol (NY)
; 16(2): 125-34, 2014 Apr.
Article
em En
| MEDLINE
| ID: mdl-24149659
ABSTRACT
Zinc finger nucleases (ZFNs) can generate targeted gene disruption (GD) directly in developing embryos of zebrafish, mouse and human. In the fish medaka, ZFNs have been attempted on a transgene. Here, we developed procedures and parameters for ZFN-mediated direct GD on the gonad-specifically expressed gsdf locus in medaka. A pair of ZFNs was designed to target the first exon of gsdf and their synthetic mRNAs were microinjected into 1-cell stage embryos. We reveal dose-dependent survival rate and GD efficiency. In fry, ZFN mRNA injection at 10 ng/µl led to a GD efficiency of 30 %. This value increased up to nearly 100 % when the dose was enhanced to 40 ng/µl. In a typical series of experiments of ZFN mRNA injection at 10 ng/µl, 420 injected embryos developed into 94 adults, 4 of which had altered gsdf alleles. This leads to a GD efficacy of â¼4 % in the adulthood. Sequencing revealed a wide variety of subtle allelic alterations including additions and deletions of 1â¼18 bp in length in ZFN-injected samples. Most importantly, one of the 4 adults examined was capable of germline transmission to 15.2 % of its F1 progeny. Interestingly, ontogenic analyses of the allelic profile revealed that GD commenced early in development, continued during subsequent stages of development and in primordia for different adult organs of the three germ layers. These results demonstrate the feasibility and--for the first time to our knowledge--the efficacy of ZFN-mediated direct GD on a chromosomal gene in medaka embryos.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oryzias
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Fator de Crescimento Transformador beta
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Dedos de Zinco
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Marcação de Genes
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Proteínas de Peixe-Zebra
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MicroRNAs
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Endonucleases
Limite:
Animals
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article