Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.
Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.
复制标题
DOI：
10.1371/journal.pone.0063241
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Hwang WS
中科院分区：
文献类型：
--
作者：
Jeong YH;Park CH;Jang GH;Jeong YI;Hwang IS;Jeong YW;Kim YK;Shin T;Kim NH;Hyun SH;Jeung EB;Hwang WS
本研究利用ires介导的多顺反子载体，制备了共表达人CD55、CD59和h -转移酶（HT）的转基因猪。当考虑将临床异种移植作为人体器官移植的替代来源时，研究的重点是超急性排斥反应（HAR）。利用体细胞核移植（SCNT）技术培育了35头转基因仔猪，并通过PCR分析证实了来自脐带样本的转基因基因的基因组整合性。从仔猪新鲜脐带静脉中分离18个猪脐静脉内皮细胞（SUVEC）。与人脐静脉内皮细胞（HUVEC）相比，我们观察到转基因SUVEC （Tg SUVEC）中转基因基因的表达更高。这些基因中，HT和hCD59在Tg检测器官中的表达水平高于非Tg控制器官，而hCD55在它们之间的表达水平无差异。Tg克隆在各器官中的转基因表现出器官特异性和空间表达模式。使用0 - 50%的人血清溶液，我们进行了人补体介导的细胞溶解试验。结果表明，总体而言，Tg SUVEC比非Tg SUVEC具有更高的存活率，HT表达水平较高的Tg SUVEC往往具有更多的α-Gal表位下调，从而具有更强的细胞毒性保护作用。相比之下，一些CD55低表达的Tg SUVEC对细胞溶解的抗性反应降低。这些结果表明，HT表达水平与α-Gal表位表达水平呈负相关，hCD55、hCD59和HT蛋白在SUVECs中的联合表达显著增强了对人血清介导的细胞溶解的保护反应。综上所述，这些结果表明，将多顺反子载体系统与SCNT方法相结合，为产生具有多种遗传修饰的转基因大型动物提供了一种快速有效的选择。
The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.
登录
查看更多内容
影响因子：
6.2
作者：
Cowan, PJ;Somerville, CA;D'Apice, AJF
通讯作者：
D'Apice, AJF
影响因子：
3.7
作者：
Kong Q;Wu M;Huan Y;Zhang L;Liu H;Bou G;Luo Y;Mu Y;Liu Z
通讯作者：
Liu Z
影响因子：
3.7
作者：
Deng W;Yang D;Zhao B;Ouyang Z;Song J;Fan N;Liu Z;Zhao Y;Wu Q;Nashun B;Tang J;Wu Z;Gu W;Lai L
通讯作者：
Lai L
影响因子：
14.9
作者：
Bouabe H;Fässler R;Heesemann J
通讯作者：
Heesemann J
影响因子：
14.8
作者：
Baudin, Bruno;Bruneel, Arnaud;Vaubourdolle, Michel
通讯作者：
Vaubourdolle, Michel