Improved multi-transgenic pig lines for xenotransplantation

本项目涉及异种移植后具有抵抗超急性排斥反应(HAR)、急性血管排斥反应(AVR)和弥散性血管内凝血(DIC)的器官的多重转基因猪的产生和鉴定。在第一阶段，现有的转基因hCD59猪将与该联盟的沃尔夫教授提供的表达TNFa相关凋亡诱导配体(TRAIL)的猪交配，以产生胚胎，从中可以培养出双转基因核供体细胞系。双转基因细胞系将被导入基于巨细胞病毒(CMV)启动子和人血红素加氧酶1(HHO-1)和人血栓调节蛋白(HTM)编码序列的基因构建体。经过筛选和克隆选择后，将利用体细胞核移植产生多基因转基因后代。根据需要，将从这些原始供体细胞克隆中生产出额外的动物，为该联盟的其他成员提供同质的实验动物组。转基因的表达将通过已建立的方案在mRNA和蛋白质水平上进行测量，并将在体外进行生物活性评估。来自多重转基因猪的心脏、肺和肾脏将由财团中的其他合作伙伴使用他们专门的灌流和移植方案进行评估。

This project involves the generation and characterization of multi-transgenic pigs with organs which resist hyperacute rejection (HAR), acute vascular rejection (AVR) and disseminated intravascular coagulation (DIC) following xeno-transplantation. In the first phase, existing transgenic hCD59 pigs will be mated with pigs expressing TNFa-related apoptosis inducing ligand (TRAIL) - provided by Prof. Wolf of this consortium - to produce fetuses from which double-transgenic nuclear donor cell lines can be cultured. The double-transgenic cell lines will be transfected with gene constructs based on the cytomegalovirus (CMV) promoter and the coding sequences for human heme-oxygenase 1 (hHO-1) and human thrombomodulin (hTM). After screening and clone selection, somatic nuclear transfer will be used to produce poly-transgenic offspring. Additional animals will be produced as required from these original donor cell clones to give homogeneous groups of experimental animals for other members of the consortium. Expression of transgenes will be measured at the mRNA and protein level by established protocols and biological activity will be assessed in vitro. Hearts, lungs and kidneys from the multi-transgenic pigs will be evaluated by other partners in the consortium using their specialized perfusion and transplantation protocols.