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Gene Replacement by Zinc Finger Nucleases in Medaka Embryos

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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.

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Abbreviations

GFP:

Green fluorescent protein

gfp:

Gene for GFP

gsdf:

Gene for gonadal soma-derived factor

RFP:

Red fluorescent protein

rfp:

Gene for RFP

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Acknowledgments

We thank Jiaorong Deng for breeding fish, Veronica Wong and Choy Mei Foong for laboratory management. This work was supported by grant from the National Research Foundation Singapore (NRF-CRP7-2010-03) to YH, by Young Scientists research fellowship from the Japan Society for Promotion of Science (No. 0773) to GG and research scholarship from the National University of Singapore to XZ.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Correspondence to Yoshitaka Nagahama or Yunhan Hong.

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Fig. S1

Map (top) and sequence (bottom) of pGRgsdf vector. Dark blue, sequences of gsdf homologous arms; red, rfp sequence; light blue, CV sequence; green, pf sequence. (DOC 71 kb)

Table S1

Primers use for PCR analysis and amplification of Southern probes (DOC 112 kb)

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Guan, G., Zhang, X., Naruse, K. et al. Gene Replacement by Zinc Finger Nucleases in Medaka Embryos. Mar Biotechnol 16, 739–747 (2014). https://doi.org/10.1007/s10126-014-9587-7

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  • DOI: https://doi.org/10.1007/s10126-014-9587-7

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