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Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39

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Abstract

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

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Acknowledgements

We thank Jun Bum Lee for technical support and animal husbandry. This work was supported by a Grant from the Next-Generation BioGreen 21 Program (No. PJ01119602), Rural Development Administration and Bio-industry Technology Development Program (No. IPET312060-05-4-HD060) Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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  1. Optipharm Inc., 63, Osongsaengmyeong 6-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea

    Kimyung Choi, Joohyun Shim, Nayoung Ko, Heejong Eom, Jiho Kim & Hyunil Kim

  2. Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

    Jeong-Woong Lee

  3. Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, Republic of Korea

    Dong-Il Jin

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  1. Kimyung Choi
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  2. Joohyun Shim
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  5. Jiho Kim
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  6. Jeong-Woong Lee
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  8. Hyunil Kim
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Correspondence to Hyunil Kim.

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Choi, K., Shim, J., Ko, N. et al. Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39 . Transgenic Res 26, 209–224 (2017). https://doi.org/10.1007/s11248-016-9996-7

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