Kim Geon A, Lee Eun Mi, Jin Jun-Xue, Lee Sanghoon, Taweechaipaisankul Anukul, Hwang Jong Ik, Alam Zahid, Ahn Curie, Lee Byeong Chun
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Korea.
Center for Medical Innovation, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea.
Transgenic Res. 2017 Aug;26(4):435-445. doi: 10.1007/s11248-017-0021-6. Epub 2017 May 28.
As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.
作为人类移植器官的替代来源，猪因其生物学特征和器官大小的相似性而受到关注。然而，严重的免疫排斥反应阻碍了使用猪器官和组织进行成功的异种移植。为了克服免疫排斥，最近开发的基因工程系统，如与体细胞核移植（SCNT）相结合以制造胚胎的转录激活样效应因子核酸酶（TALEN），可用于生产与异种移植兼容的猪。我们使用TALEN系统靶向猪体内在人类中自然缺失的非Gal抗原胞苷单磷酸-N-乙酰神经氨酸羟化酶（CMAH）基因。将表达可溶性人肿瘤坏死因子受体I IgG-Fc（shTNFRI-Fc）和人血凝素标记的人血红素加氧酶-1（hHO-1）的Gal缺失细胞用针对CMAH的TALEN靶点进行转染。使用N-糖神经氨酸（Neu5Gc）/磁珠对缺乏CMAH的细胞进行阴性筛选，并通过荧光激活细胞分选（FACS）和DNA测序分析分离细胞的Neu5Gc表达水平。使用3种不同的基因修饰细胞克隆的克隆胚胎分别移植到3只受体中，55.6%（5/9）怀孕并产下3头克隆猪。通过测序证实CMAH基因成功发生基因破坏，显示克隆仔猪尾源成纤维细胞中CMAH无表达。除了通过体细胞核移植产生的仔猪中Neu5Gc表达降低外，与野生型动物相比，用于检测源自内皮细胞和成纤维细胞的四重基因修饰猪免疫反应性的抗体介导的补体依赖性细胞毒性试验和天然抗体结合显著降低。我们得出结论，通过将TALEN系统与转基因细胞相结合，靶向多个基因可能有助于生成用于异种移植的器官。我们生产了具有四重修饰基因CMAHKO/GTKO/shTNFRI-Fc/hHO-1的小型猪，通过克服超急性、急性和抗炎排斥反应，这些猪将适合用于异种移植。
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