Abstract：Clotting disorders by molecular mismatch is one of the remaining critical issues in pig-to-human xenotransplantation. Thrombomodulin (TBM) is an endothelial transmembrane glycoprotein that plays a critical role in the control of coagulation. Studies demonstrated that pig (Sus scrofa) TBM can't effectively regulate blood clotting and thrombosis if the molecules do not match. Thus, the expression of exogenous human TBM in pig vascular endothelial cells may be helpful for overcoming the molecular incompatibilities. In this study, the 7 and 9 kb promoter of pig TBM gene were obtained by gap-repair method, and correspondingly 2 espression vectors of hTBM (p7K(pTBM)-hTBM, p9K(pTBM)-hTBM) were constructed. To validate and compare the specificity and effciency of the 2 vectors, both of them were transfected into endothelial cells, ear fibroblast and kidney cells of Wuzhishan miniature pig. The transfected cells were cultured for 48 h and then harvested for RT-PCR and Western blot analysis. The results of RT-PCR indicated that both the 7 and 9 kb promoters could specifically regulate the expression of hTBM in vascular endothelium; qRT-PCR and Western blotting analysis showed that the activity of 9 kb promoter was higher than the that of 7 kb counterpart (P＜0.05). Altogether, the results in vitro demonstrated that the 9 kb TBM promoter of pig was a more suitable candidate for endothelial cell specific expression, based on the fact that it could drive the expression of hTBM at higher efficiency . This work lays the foundation for the future transgenic pig efficiently express anti-coagulation factor in its vascular endothelium, which will solve the clotting disorders caused by a molecular mismatch in xenotransplantation.
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