Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations
Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations
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DOI：
10.1073/pnas.0307819101
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发表时间：
2004-05-11
影响因子：
11.1
通讯作者：
Hawley, RJ
中科院分区：
文献类型：
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作者：
Kolber-Simonds, D;Lai, LX;Hawley, RJ
旧大陆灵长类动物受体对猪器官的超急性排斥反应是通过表达在猪细胞表面的针对半乳糖-α-1，3-半乳糖(Galpha-1，3-Gal)表位的预制抗体来介导的。以前，我们用基因靶向的成纤维细胞进行核移植(NT)，获得了α-1，3-半乳糖基转移酶基因座(GGTA1)零等位基因的近交系小型猪。为了加速GGTA1缺失猪的产生，我们从杂合子动物的成纤维细胞培养中选择了自发的缺失突变细胞用于另一轮NT。GGTA1功能的自发损失率出人意料地高，绝大多数空细胞是由于WT等位基因的丢失造成的。NIT利用经过缺失和交叉/基因转换事件的成纤维细胞，分别产生了该基因靶向等位基因的半合子和纯合的健康仔猪。除Gala-1，3-Gal表位缺失外，这些零基因仔猪与同一近交系WT仔猪之间没有明显的表型差异。事实上，在杂合子NT动物中观察到的先天异常并没有在连续生产的无效动物中重现。
Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NIT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Gala-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.