Production of porcine fibroblasts carrying a vector enforced specific expression of CD73 to endothelial cells

• Published in: Journal of animal reproduction & biotechnology (Online) Vol. 31; no. 3; pp. 161 - 168
• Main Authors: Oh, Keon Bong, Lee, Haesun, Hwang, Seongsoo, Ock, Sun-A, Chung, Hak-Jae, Byun, Sung June, Lee, Poongyeon, Im, Gi-Sun
• Format: Journal Article
• Language: English
• Published: The Korean Society of Animal Reproduction and Biotechnology 30.09.2016
• Subjects: cd73; coagulation; porcine fibroblasts; porcine icam2 promoter
• ISSN: 2671-4639; 2671-4663
• DOI: 10.12750/JET.2016.31.3.161

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha1,3-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 CD73in 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.