Production Of Transgenic Pigs Specifically Expressing Human TBM And CD39

Xenotransplantation is considered to be one of the most effective way to solve the organ shortage. Pig is an ideal source to provide organs because of the similarly physical structure with humans. Humanized genetically modified pigs to nonhuman primates xenotransplantation research has made significant progress. However, clotting disorders by molecular mismatch is one of the remaining critical issues. Our attention has been directed toward thrombomodulin(TBM) and CD39(ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1), both of which play an important role in overcome the inflammation and coagulation disorders. TBM can inhibit coagulation by way of activation of protein C, CD39 can inhibit inflammation and platelet aggregation by way of degradation of ATP. In this study, we aim to generation of transgenic pig specifically expressing human TBM and CD39 and provide model to research clotting disorders within xenograft.The 7kb and 9kb TBM promoter of pig were obtained by gene trapping and two espression vectors of h TBM(p7K(p TBM)-h TBM, p9K(p TBM)-h TBM) were constructed. To validate and compare the expression specificity, two vectors were transferred into pig endothelial cells, ear fibroblast and kidney cells. Transfection cells were cultured for 48 h and harvested for RT-PCR and Western Blot. The results of RT-PCR have shown that 7kb and 9kb promoter can specifically regulate the expression of h TBM in vascular endothelial; Western Blot analysis have shown that the activity of 9kb promoter is higher than 7kb promoter(P<0.05).The expression vector of p9K(p TBM)-h TBM was selected and transferred into pig ear fibroblasts. After hygromycin selection, three cell colonies expressing a high level of h TBM were selected as donors for SCNT. A total of 1203 embryos were transferred into five surrogate sows, four of whom became pregnant and give birth to 10 piglets of which expressing h TBM positively. Immunohistochemical study of three organization(heart, liver and kidney) from one dead cloned pig(22#) have shown that the h TBM was highly expressive in the vascular endothelial cells.1.5kb human CD39 c DNA expression vector which regulated by the 1.5kb pig insulin promoter(PIP, contain exon 1 and intron 1) was constructed. To validate the expression specificity in virto, the vector was transfected into mice MIN-6 islet tumor β-cells. Cells were cultured for 48 h and harvested for RT-PCR and Western Blot. The RT-PCR results have shown that the PIP protmoter can specificity regulated the h CD39 expression in inselt cells, but the protein was not detected by Western Blot.In brief, we successfully constructed the h TBM expressing vector and producted cloned pig expressing h TBM in vascular endothelial cells. This work provides us a basis for solving the clotting disorders caused by a molecular mismatch in xenotransplantation.