Abstract
Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.
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Acknowledgements
We thank the “National Key R&D Program of China” and “Yunnan Provincial Science and Technology Department” for the support provided for this study.
Funding
This work was supported by Grants from National Key R&D Program of China (Grant No. 2019YFA0110700) and Yunling Scholars Programme of Yunnan Province and Innovative Research Team of Science and Technology in Yunnan Province.
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HJW and HYZ conceived and designed the experiments; HZ, YL, TW, ZY, YQ, HL, JG, BJ, XZ, WC, YS, WL, JW, DZ, KK and HJW performed the experiments; HJW, HYZ, HZ and HL analyzed the data; HJW, HYZ and KX wrote the paper. All authors reviewed the manuscript.
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All animal experiments were performed with approval from the Animal Care and Use Committee of Yunnan Agricultural University in China.
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Heng Zhao, Yuying Li and Thanapa Wiriyahdamrong are co-first authors.
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Zhao, H., Li, Y., Wiriyahdamrong, T. et al. Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning. Transgenic Res 29, 369–379 (2020). https://doi.org/10.1007/s11248-020-00201-2
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DOI: https://doi.org/10.1007/s11248-020-00201-2