Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: A novel selection method for Gal alpha 1,3-Gal antigen-deficient cells
Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: A novel selection method for Gal alpha 1,3-Gal antigen-deficient cells
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DOI：
10.1002/mrd.20890
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发表时间：
2008-09-01
影响因子：
2.5
通讯作者：
Murakami, Hiroshi
中科院分区：
文献类型：
--
作者：
Fujimura, Tatsuya;Takahagi, Yoichi;Murakami, Hiroshi
本研究的目的是用一种新的简单的细胞选择方法分离α-1，3-半乳糖基转移酶(GalGT)基因双敲除(DKO)细胞。为了获得GalGT-DKO细胞，将GalGT基因单基因敲除(SKO)的胎儿成纤维细胞培养3~9代，用生物素标记的1b4凝集素结合链霉亲和素包被的磁珠分离GalGT缺失细胞。经过15-17天的额外培养，从总共2.5×10(7)个GalGT-SKO细胞中获得了7个GalGT-DKO细胞克隆。将用DKO细胞构建的926个体细胞核移植胚胎移植到8头受体猪体内，产下4头产仔猪、3头活胎猪和6头死胎猪。经聚合酶链式反应和Southern印迹杂交证实克隆猪中不存在GalGT基因。流式细胞仪分析表明，克隆的DKO猪的细胞中不存在α-Gal抗原。
The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled 1B4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alpha Gal antigens were not present in the cells of the cloned DKO pigs.