用于异种移植的新型基因改造猪的培育

目的培育用于异种移植的新型基因改造猪，在α-Gal基因敲除以及膜辅蛋白CD46、血栓调节蛋白（TM）基因转入的基础上，进一步将Ⅱ类反式激活因子基因N端缺失显性负向（CⅡTA-DN）基因转入猪胎儿成纤维细胞，以抑制猪白细胞抗原（SLA）Ⅱ类分子的表达。方法设计合成博来霉素（Zeocin）抗性基因片段，并将其与含有CⅡTA-DN基因的pST205载体连接，构建置于人Tie2增强子和CMVB—actin启动子下游的pNMU105／CⅡTA-DN表达载体，将线性化的pNMU105通过脂质体转染法转入已经敲除了α-Gal并转入CD46、TM基因的猪胎儿成纤维细胞181B（DKO／CD46／TM）。Zeocin抗性筛选转染pNMU105质粒的181B细胞，PCR方法检测CⅡTA-DN基因在181B细胞中的转入，获得阳性单克隆并进行核移植，得到DKO／CD46／TM／CⅡTA-DN转基因猪。取仔猪的耳缘组织裂解后进行基因鉴定。结果成功设计Zeocin抗性基因片段并构建pNMU105／CⅡTA-DN表达载体，经过质粒转染和Zeocin抗性药物筛选获得多株阳性单克隆，顺利通过核移植得到转基因猪。仔猪耳缘组织经PCR鉴定，在592bp位置检测到预期条带，证明均为CⅡTA-DN转基因阳性。结论通过新型基因改造猪的培育，在抑制超急性排斥反应和补体反应、降低凝血和人CD4＋T细胞免疫反应的理论基础上进行了转基因模型猪的构建，为异种移植中更加有效地减少免疫损伤、提高移植器官的存活做出了新的尝试。

Production of a new generation of genetically modified pigs for xenotransplantation

Objective To produce new generation of genetically modified pigs as a better donor for xenotransplantation, C Ⅱ TA- DN expression vector was transfected into DKO/CD46/TM porcine fetal fibroblast cells to obtain DKO/CIM6/TM/CⅡTA-DN transgenie pigs. We expect the C Ⅱ TA-DN will suppress porcine SLA Ⅱ expression. Methods Designed the Zeoein resistant gene fragments and linked them to pST205 vectors. A CⅡ TA-DN transgene under a CMV β-actin promoter with an endothelium-specific Tie2 enhancer was constructed and named as pNMU105. A porcine fetal fibroblast cell line 181B （DKO/CD46/TM）were transfected with pNMUI05, and the Zeoein resistant colonies were isolated. PCR confirmed positive cells were used for nuclear transfer to clone the pigs. Results The Zeocin resistant gene fragments were designed and the pNMU105/C Ⅱ TA- DN expression plasmids were constructed. After transfection and antibiotic screening, we got dozens of Zeocin resistantcolonies. The DKO/CD46/TM/C Ⅱ TA-DN transgenic pigs were produced. The expected bands of 592 bp were visible in the results of PCR. Conclusions Through the cultivation of a new generation of genetically modified pigs, the clone pig model was constructed that would theoretically suppress hyperacute rejection and complement reaction, reduce coagulation and the human CD4＋ T cell immunity reaction. Thus we have made a new attempt to greatly improve the survival rate of transplanted xeno-organs in xenotransplantation.