Abstract
Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.
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Acknowledgements
We would like to thank Dr. Hiroshi Nagashima, Dr. Gorge Arnord, Tuna Guengoer, Ingrid Kola and Tatiana Schroeter for their invaluable technical support. This work was financially supported by the German Research Council (TRR 127 ‘Biology of xenogeneic cell, tissue and organ transplantation—from bench to bedside’) and by EXIST Forschungstransfer 03FABY064. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors are members of COST Action BM1308 “Sharing Advances on Large Animal Models—SALAAM”.
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MK, SL, BF and EW conceived and designed the study. MK, SL, BK, EK, EJ, NK, VZ performed the experiments and MK and BK drafted the manuscript. All authors read and approved the final manuscript.
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Kurome, M., Leuchs, S., Kessler, B. et al. Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs. Transgenic Res 26, 309–318 (2017). https://doi.org/10.1007/s11248-016-0004-z
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DOI: https://doi.org/10.1007/s11248-016-0004-z