Fujimura Tatsuya, Takahagi Yoichi, Shigehisa Tamotsu, Nagashima Hiroshi, Miyagawa Shuji, Shirakura Ryota, Murakami Hiroshi
The Animal Engineering Research Institute, Midorigahara, Tsukuba, Ibaraki, Japan.
Mol Reprod Dev. 2008 Sep;75(9):1372-8. doi: 10.1002/mrd.20890.
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 IB4 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 alphaGal antigens were not present in the cells of the cloned DKO pigs.
本研究的目的是使用一种新颖的简单细胞筛选方法分离α1,3-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基因单敲除（SKO）胎儿成纤维细胞培养3至9代，并使用附着在链霉亲和素包被磁珠上的生物素标记的IB4凝集素分离GalGT缺失细胞。经过15 - 17天的额外培养，从总共2.5×10⁷个GalGT-SKO细胞中获得了7个GalGT-DKO细胞集落。用DKO细胞重建的总共926个体细胞核移植胚胎被移植到8头受体猪中，产下4头仔猪，3头存活，6头死产。通过PCR和Southern印迹法证实克隆猪中不存在GalGT基因。流式细胞术分析显示克隆的DKO猪细胞中不存在αGal抗原。
