Kolber-Simonds Donna, Lai Liangxue, Watt Steven R, Denaro Maria, Arn Scott, Augenstein Monica L, Betthauser Jeffery, Carter David B, Greenstein Julia L, Hao Yanhong, Im Gi-Sun, Liu Zhonghua, Mell Greg D, Murphy Clifton N, Park Kwang-Wook, Rieke August, Ryan David J J, Sachs David H, Forsberg Erik J, Prather Randall S, Hawley Robert J
Immerge Biotherapeutics, Inc., 300 Technology Square, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2004 May 11;101(19):7335-40. doi: 10.1073/pnas.0307819101. Epub 2004 May 3.
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 NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-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.
旧世界灵长类动物受体对猪器官的超急性排斥反应是由针对猪细胞表面表达的半乳糖基-α-1,3-半乳糖（Galα-1,3-Gal）表位的预先形成的抗体介导的。此前，我们通过用基因靶向的成纤维细胞进行核移植（NT），培育出了具有α-1,3-半乳糖基转移酶基因座（GGTA1）无效等位基因的近交小型猪。为了加快GGTA1基因敲除猪的培育，我们从杂合动物的成纤维细胞培养物中筛选出自发的无效突变细胞，用于另一轮核移植。观察到GGTA1功能的自发丧失率出乎意料地高，绝大多数无效细胞是由于野生型等位基因的丧失所致。通过分别使用经历了缺失和交叉/基因转换事件的成纤维细胞进行核移植，产生了基因靶向等位基因半合子和纯合子的健康仔猪。除了Galα-1,3-Gal表位的丧失外，这些基因敲除仔猪与来自同一近交系的野生型仔猪之间没有明显的表型差异。事实上，在杂合核移植动物中观察到的先天性异常在连续产生的基因敲除动物中并未再次出现。
