Improved multi-transgenic pig lines for xenotransplantation

该项目涉及多转基因猪的产生和表征，这些猪的器官抵抗异种移植后的超急性排斥反应（HAR）、急性血管排斥反应（AVR）和弥散性血管内凝血（DIC）。在第一阶段，现有的转基因hCD 59猪将与表达TNF α相关凋亡诱导配体（TRAIL）的猪交配-由该联盟的Wolf教授提供-以产生可培养双转基因核供体细胞系的胎儿。将用基于巨细胞病毒（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转染双转基因细胞系。在筛选和克隆选择之后，将使用体细胞核移植来产生多转基因后代。根据需要，将从这些原始供体细胞克隆中生产额外的动物，为联合体的其他成员提供同质的实验动物组。将通过已建立的方案在mRNA和蛋白质水平上测量转基因的表达，并将在体外评估生物活性。来自多转基因猪的心脏、肺和肾脏将由联盟中的其他合作伙伴使用其专门的灌注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.