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Transgenic Research

, Volume 26, Issue 2, pp 209–224 | Cite as

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

  • Kimyung Choi
  • Joohyun Shim
  • Nayoung Ko
  • Heejong Eom
  • Jiho Kim
  • Jeong-Woong Lee
  • Dong-Il Jin
  • Hyunil KimEmail author
Original Paper

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.

Keywords

Xenotransplantation Transgenic Pig α1,3-Galactosyltransferase Human CD39 

Notes

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.

Supplementary material

11248_2016_9996_MOESM1_ESM.docx (109 kb)
Supplementary material 1 (DOCX 108 kb)

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Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Kimyung Choi
    • 1
  • Joohyun Shim
    • 1
  • Nayoung Ko
    • 1
  • Heejong Eom
    • 1
  • Jiho Kim
    • 1
  • Jeong-Woong Lee
    • 2
  • Dong-Il Jin
    • 3
  • Hyunil Kim
    • 1
    Email author
  1. 1.Optipharm Inc.Cheongju-siRepublic of Korea
  2. 2.Functional Genomics Research CenterKorea Research Institute of Bioscience and BiotechnologyDaejeonRepublic of Korea
  3. 3.Department of Animal Science and BiotechnologyChungnam National UniversityDaejeonRepublic of Korea

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