RRC ID 30922
Author Guan G, Zhang X, Naruse K, Nagahama Y, Hong Y.
Title Gene replacement by zinc finger nucleases in medaka embryos.
Journal Mar. Biotechnol.
Abstract Gene replacement (GR) via homologous recombination is a powerful tool for genome editing. Recently, direct GR is achieved successfully by coinjection of mRNAs for engineered endonucleases such as zinc finger nucleases (ZFNs) and donor DNA in developing embryos of diverse organisms. Here, we report the procedures and efficiency for direct GR by using ZFNs in the fish medaka. Upon zygotic coinjection of mRNAs encoding ZFNs that target the gonad-specifically expressed gsdf locus, linear DNA of GR vector pGRgsdf containing the red fluorescent protein (rfp) gene flanked by two homology arms of ~1-kb each underwent GR via homologous recombination. Specifically, 15 of 231 adults from manipulated embryos contained a GR allele in the caudal fin, producing an efficiency of ~7 % for somatic GR. Progeny test revealed that two out of nine fertile fish containing the GR allele in the fin were capable of transmitting the GR allele to ~6 % of F1 generation at adulthood, generating an efficiency of ~22 % for germline transmission. Sequencing and Southern blotting validated precise GR. We show that the GR allele expressed a chimeric gsdf:rfp RNA between gsdf and cointegrated rfp specifically in the gonad, demonstrating recapitulation of endogenous RNA expression as predicted for the defined GR allele. Most importantly, RFP expression coincides faithfully with the gonad-specific gsdf expression in developing embryos and adults. These results demonstrate, for the first time, the feasibility and efficiency of ZFN-mediated precise GR directly in the developing embryo of medaka as a lower vertebrate model.
Volume 16(6)
Pages 739-47
Published 2014-12
DOI 10.1007/s10126-014-9587-7
PMID 25097139
MeSH Animals Base Sequence Blotting, Southern DNA Primers / genetics Deoxyribonucleases / genetics* Embryo, Nonmammalian / metabolism* Gene Transfer Techniques* Homologous Recombination / genetics* Molecular Sequence Data Oryzias / genetics* Polymerase Chain Reaction Sequence Analysis, DNA Zinc Fingers / genetics*
IF 2.748
Times Cited 2
Medaka HdrR-II1(IB178)