Institute of Farm Animal Genetics, Department of Biotechnology, Mariensee, Neustadt, Germany.
Xenotransplantation. 2009 Nov-Dec;16(6):486-95. doi: 10.1111/j.1399-3089.2009.00537.x.
The inability of porcine thrombomodulin (TM) to activate human anticoagulant protein C after pig-to-human xenotransplantation may lead to an aberrant activation of coagulation with microthrombosis and ultimately failure of the transplanted organ. Here, we describe the production of triple-transgenic pigs expressing hCD59/DAF and human thrombomodulin (hTM) and tested hTM-transgenic fibroblasts obtained from these pigs for their ability to activate human protein C in a new in vitro assay.
Fibroblast cell cultures were established from a hCD59/DAF transgenic pig and transfected with a vector coding for hTM under transcriptional control of the CMV promoter. Transfected cells were analyzed for integration and expression of the hTM vector by PCR and RT-PCR. One cell clone was used as donor for somatic cell nuclear transfer to produce triple transgenic (CD59/DAF/hTM) pigs. Pigs were characterized in detail with regard to hTM integration and expression by PCR, RT-PCR, Northern blot, Western blot, immunostaining, and FACS analysis. Fibroblasts from hTM-transgenic pigs were analyzed in a new in vitro hTM coactivity assay to assess the production of activated protein C (aPC) and results were compared to those from wild-type controls.
In total, 1040 cloned transgenic embryos were transferred to eight recipients. Five recipients remained pregnant and delivered 22 piglets. Expression of hTM was detected in all xenorelavant organs including heart, liver, kidney, lung, and pancreas. The lowest levels of expression were found in lung and liver while all animals showed a strong, but frequently patchy expression pattern of hTM in heart, kidney, and pancreas. The hTM cofactor activity (ranging on a scale from 5-18 U/10(5) cells/2h) was significantly higher in fibroblasts of hTM-transgenic clones compared to wild-type porcine fibroblasts (1.7 U/10(5) cells/2h).
For the first time, healthy hTM-transgenic pigs could be successfully generated by somatic cell nuclear transfer. hTM can be expressed in porcine organs without perturbation of the porcine coagulation system. hTM-transgenic porcine fibroblasts showed elevated aPC production in an in vitro hTM coactivity assay. These findings warrant further work on the control of the xenogenic activation of coagulation by transgenic approaches.
猪源血栓调节蛋白（TM）在猪到人的异种移植后不能激活人抗凝蛋白 C，可能导致凝血的异常激活和微血栓形成，并最终导致移植器官的衰竭。在这里，我们描述了生产表达 hCD59/DAF 和人血栓调节蛋白（hTM）的三重转基因猪，并测试了从这些猪中获得的 hTM 转基因成纤维细胞在新的体外测定中激活人蛋白 C 的能力。
从 hCD59/DAF 转基因猪中建立成纤维细胞培养物，并在 CMV 启动子转录控制下用编码 hTM 的载体转染。通过 PCR 和 RT-PCR 分析转染细胞中 hTM 载体的整合和表达。选择一个细胞克隆作为供体进行体细胞核移植，以产生三重转基因（CD59/DAF/hTM）猪。通过 PCR、RT-PCR、Northern blot、Western blot、免疫染色和 FACS 分析详细描述猪 hTM 整合和表达的特征。在新的体外 hTM 协同活性测定中分析 hTM 转基因猪的成纤维细胞，以评估激活蛋白 C（aPC）的产生，并将结果与野生型对照进行比较。
总共将 1040 个克隆的转基因胚胎转移到 8 个受体中。5 个受体怀孕并分娩了 22 头小猪。hTM 的表达在所有与异种相关的器官中均有检测到，包括心脏、肝脏、肾脏、肺和胰腺。肺和肝脏中的表达水平最低，而所有动物的心脏、肾脏和胰腺中均表现出强烈但常呈斑片状的 hTM 表达模式。hTM 辅因子活性（范围在 5-18 U/105 细胞/2h 之间）在 hTM 转基因克隆的成纤维细胞中明显高于野生型猪成纤维细胞（1.7 U/105 细胞/2h）。
首次通过体细胞核移植成功生成了健康的 hTM 转基因猪。hTM 可以在不干扰猪凝血系统的情况下在猪器官中表达。在体外 hTM 协同活性测定中，hTM 转基因猪成纤维细胞显示出升高的 aPC 产生。这些发现证明了通过转基因方法控制异种凝血激活的进一步研究是合理的。
