Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer
Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer
复制标题
通过锌指核酸酶和体细胞核移植产生 GGTA1 双等位基因敲除猪
DOI：
10.1007/s11427-013-4601-2
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发表时间：
2014-02-01
影响因子：
9.1
通讯作者：
Fu ZhiXin
中科院分区：
文献类型：
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作者：
Bao Lei;Chen HaiDe;Fu ZhiXin
转基因猪是人类疾病的宝贵模型和异种移植器官的供体。传统的猪体细胞基因定位效率极低。锌指核酸酶（ZFN）技术已被证明是有效诱导基因组突变的有力工具。然而，zfn介导的猪靶向很少实现。在这里，我们使用ZFNs敲除猪α- 1,3 -半乳糖基转移酶（GGTA1）基因，该基因产生Gal表位，在猪向人移植中引发超急性免疫排斥反应。用靶向GGTA1编码区的ZFNs转染猪原代成纤维细胞。经药物筛选，获得18个单等位基因敲除细胞克隆和4个双等位基因敲除细胞克隆，效率分别为23.4%和5.2%。双等位细胞通过体细胞核移植（SCNT）用于克隆猪。3只GGTA1基因缺失仔猪出生，并从克隆胎儿中建立了1个敲除原代成纤维细胞系。GGTA1缺失猪细胞上的Gal表位完全从细胞膜上消失。功能上，GGTA1敲除细胞在与人血清孵育时免受补体介导的免疫攻击。该研究表明，ZFN是一种高效的基因修饰猪的工具。GGTA1缺失的猪和GGTA1缺失的胎儿成纤维细胞将有利于研究和猪到人的移植。
Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.