Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Development of aortic endothelial cells to express CD37 and CD73 isolated from alpha 1,3-galactosyltransferase knock-out and MCP expressing pig

alpha 1,3-galactosyltransferase 기능 제거 및 MCP 발현 형질전환 돼지의 대동맥 혈관내피세포에 CD37/CD73 발현 세포주 개발

  • No, Jin-Gu (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Byun, Sung-June (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Yang, Hyeon (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Ock, Sun A (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Woo, Jae-Seok (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Hwi-Cheul (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Hwang, In-sul (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Kim, Ji-Youn (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Park, Sang Hyoun (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Joo Young (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Oh, Keon Bong (Animal Biotechnology Division, National Institute of Animal Science, RDA)
  • 노진구 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 변승준 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 양현 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 옥선아 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 우제석 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 이휘철 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황인설 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 김지윤 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 박상현 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 이주영 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 오건봉 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Received : 2018.08.23
  • Accepted : 2018.09.27
  • Published : 2018.09.30
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Abstract

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

Keywords

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