Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Production of porcine fibroblasts carrying a vector enforced specific expression of CD73 to endothelial cells

돼지 혈관내피세포 특이적 CD73 발현 벡터가 도입된 돼지 섬유아세포 생산

  • Oh, Keon Bong (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Haesun (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Hwang, Seongsoo (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Ock, Sun-A (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Chung, Hak-Jae (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Byun, Sung June (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Poongyeon (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Im, Gi-Sun (Animal Biotechnology Division, National Institute of Animal Science, RDA)
  • 오건봉 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 이해선 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황성수 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 옥선아 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 정학재 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 변승준 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 이풍연 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 임기순 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Received : 2016.07.22
  • Accepted : 2016.09.12
  • Published : 2016.09.30
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Abstract

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

Keywords

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