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.

Abstract
 서 론
 재료 및 방법
  1. 사람 CD39 및 CD73 동시발현 벡터의 제작
  2. 세포의 분리 및 배양
  3. 면역 염색 (Immuno-fluorescence assay)
  4. 관 형성 분석 (Tube formation assay)
  5. 세포에 유전자 도입
  6. 유세포 (Flow cytometry) 분석
  7. 통계 분석 (Statistical analysis)
 결과 및 고찰
 결론
 REFERENCES

• Jin-Gu No(Animal Biotechnology Division, National Institute of Animal Science) | 노진구
• Sung-June Byun(Animal Biotechnology Division, National Institute of Animal Science) | 변승준
• Hyeon Yang(Animal Biotechnology Division, National Institute of Animal Science) | 양현
• Sun A Ock(Animal Biotechnology Division, National Institute of Animal Science) | 옥선아
• Jae-Seok Woo(Animal Biotechnology Division, National Institute of Animal Science) | 우제석
• Hwi-Cheul Lee(Animal Biotechnology Division, National Institute of Animal Science) | 이휘철
• In-sul Hwang(Animal Biotechnology Division, National Institute of Animal Science) | 황인설
• Ji-Youn Kim(Animal Biotechnology Division, National Institute of Animal Science) | 김지윤
• Sang Hyoun Park(Animal Biotechnology Division, National Institute of Animal Science) | 박상현
• Joo Young Lee(Animal Biotechnology Division, National Institute of Animal Science) | 이주영
• Keon Bong Oh(Animal Biotechnology Division, National Institute of Animal Science) | 오건봉 Corresponding Author