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Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT

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Abstract

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

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Acknowledgements

This work was supported by grants from Major Program on Basic Research Projects of Yunnan Province (Grant No. 2014FC006), the National Genetically Modified Organisms Breeding Major Projects (Grant No. 2016ZX08009-003-006) and the National Natural Science Foundation of China (Grant No. 31560637).

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  1. Fengjuan Liu and Jinji Liu have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, China

    Fengjuan Liu, Jinji Liu, Yubo Qing, Honghui Li, Kaixiang Xu, Wanyun Zhu, Heng Zhao, Baoyu Jia, Jianxiong Guo, Hong-Ye Zhao & Hong-Jiang Wei

  2. College of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China

    Fengjuan Liu, Jinji Liu, Zaimei Yuan, Yubo Qing, Honghui Li, Heng Zhao, Baoyu Jia, Weirong Pan & Wenmin Cheng

  3. College of Veterinary Medicine, Yunnan Agricultural University, Kunming, 650201, China

    Xuezeng Zhang & Hong-Jiang Wei

  4. Hunan Xeno Life Science Co., Ltd, Changsha, 410600, China

    Wei Wang

  5. Institute for Cell Transplantation and Gene Therapy, The Third Xiangya Hospital Central-South University, Changsha, 410013, China

    Wei Wang

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  1. Fengjuan Liu
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Liu, F., Liu, J., Yuan, Z. et al. Generation of GTKO Diannan Miniature Pig Expressing Human Complementary Regulator Proteins hCD55 and hCD59 via T2A Peptide-Based Bicistronic Vectors and SCNT. Mol Biotechnol 60, 550–562 (2018). https://doi.org/10.1007/s12033-018-0091-6

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