敲除GGTA1同时表达人白细胞抗原G5的基因修饰猪的构建

Generation of genetically modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5

摘要:

由于猪的诸多生理和遗传特征与人类近似，猪一直被认为是理想的异种器官移植候选供体动物。但是物种之间严重的免疫不相容是影响猪到灵长类异种器官移植的最关键因素。为了克服物种之间的免疫排斥反应，诸多研究对猪进行了各种各样的基因修饰，消除或抑制造成异种器官移植的免疫原，以获得可应用于灵长类移植的供体器官。本研究利用转录激活因子样效应物核酸酶(transcription activator-like effector nuclease,TALEN)对猪的α-1,3-半乳糖苷转移酶(α-1,3-galactosyltransferase,GGTA1)基因进行敲除，该基因编码的酶催化合成α-1,3-半乳糖,这是导致猪到灵长类异种器官移植中产生超急性免疫排斥的关键因子。同时本研究也通过转基因方法导入一种重要的免疫抑制因子，人类白细胞抗原G5(human leukocyte antigen-G5,HLA-G5)，来进一步增加猪器官在异种器官移植中的免疫耐受。本研究筛选了GGTA1双等位基因敲除(GGTA1 knockout,GTKO)且HLA-G5转基因导入的猪成纤维细胞，并通过体细胞核移植(somatic cell nuclear transfer,SCNT)构建相应的基因修饰猪模型。本研究获得了20头GGTA1双等位基因敲除的克隆猪，其中10头同时表达HLA-G5蛋白。经检测，α-1,3-半乳糖在GTKO/HLA-G5克隆猪的组织和细胞上完全缺失，Western blotting和免疫荧光染色显示，HLA-G5在克隆猪的组织和细胞上成功表达。功能学分析显示，从GTKO/HLA-G5克隆猪分离的成纤维细胞具有更好的免疫耐受，相比于单一的GTKO猪和未经基因修饰的野生型猪，GTKO/HLA-G5克隆猪的细胞对补体介导的细胞裂解反应具有更强的耐受。GTKO/HLA-G5克隆猪可以为异种器官移植提供一种有用的动物模型。

Abstract:

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings.However,antibody-mediated immunity,which includes both natural and induced antibody responses,is a major challenge for the success of pig-to-primate xenotransplantation.Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation.In this study,we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1,3-galactosyltransferase gene GGTA1,which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation.Meanwhile,human leukocyte antigen-G5 gene HLA-G5,which acts as an immunosuppressive factor,was co-transfected with TALEN into porcine fetal fibroblasts.The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer.As a result,we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein.Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent.Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets.Functionally,the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs.These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.