Abstract：Under physiological conditions, thrombomodulin (TM) can combined with thrombin into a compound, which can prevent blood coagulation and promote fibrinolysis by activating protein C; and can be absorbed by endothelial cells, where the thrombin is decomposed by intracellular lysosomes. In addition, TM can be combined with coagulation factor Xa specificity, inhibit the activation of prothrombin, so that makes the generation of thrombin decreased significantly. When pig (Sus scrofa) blood vessels expose to the human (Homo sapiens) blood after xenotransplantation, the pig TM can't combine with human thrombin, which is the reason of coagulopathy. Wuzhishan Miniature pigs are best candidate for organ xenotransplantation donor with characteristics of highly similar anatomy and physiology to human. There are three aims of this research: produce transgenetic Wuzhishan Minipigs expressing human thrombomodulin (hTM), research hTM expression patten in endothelial cells of the transgenetic pigs, and provide practical basis for solve disorders in blood coagulation after xenotransplantation. Taking the Large White Pig’s TM bacterial arti?cial chromosome (BAC) as a template, constructed a PBR322-catch vector that had homologous short arm. A 7 kb promoter of porcine TM gene was sub-cloned from BAC of Large White Pig by gap-repair method mediated by red homologous recombination system in E. Coli., positive vector pBR322-catch-pProwas identified by enzyme. hTM amplified from human cDNA and bovine Growth Hormone PolyA (bGHpA) amplified by PCR, were linked to the pBlueScriptⅡ(SK-)vector. Vector pBR322-catch-pProwas was digested by enzyme to obtain the Large White Pig’s TM promoter, then connected it with vector pBlueScriptⅡ(SK-)to construct the specific expression pBS-hTM vector containing puromycin. The Xhol Ⅰ linearized pBS-hTM was integrated into the fetal fibroblasts cell lines (WPF167 and WPF169) by electroblot. Screening cells using culture solution contained 1.0 μg/mL puromycin for 10~15 days. Two cell clones with better growth characters were used as donor cells to conduct nuclear transfer to acquire reconstructed embryo. 972 reconstructed embryos were harvested, and then transferred into 5 Wuzhishan Miniature receptor pigs. New born piglets were identified by genomic DNA PCR, and umbilical cord tissue RT-PCR and Western blot. As results, positive catch vector pBR322-catch-pPro and expression vector pBS-hTM were successfully acquired. 354 cell clones were gained after 10~15 days screening, in which 339 cell clones were positive. The reconstructed embryo had no signi?-cant differences with the normal reconstructed embryos (P＞0.05). Two receptor pigs showed pregnant by B–ultrasonic determination after 30 days and 5 piglets were born after 120 days. 4 of new born piglets were identified positive and the expression of hTM specifically on the surface of endothelial cells. As a conclusion, transgenic cloned Wuzhishan Miniature pigs were successfully achieved, which specifically expressed hTM in vascular endothelial cells. This study could provide practical basis for further in-depth study in coagulation disorders after xenotransplantation.
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