Department of Surgery, Transplant Surgery Section, Indiana University School of Medicine, Indianapolis, Indiana.
J Surg Res. 2013 Oct;184(2):e37-42. doi: 10.1016/j.jss.2012.11.054. Epub 2012 Dec 21.
The process of selecting transgenic cells has been one of the bottlenecks in the generation of transgenic animals by somatic cell nuclear transfer (SCNT). In particular, selection for the Gal double-knockout (Gal-DKO) genotype has been time consuming and inefficient. The objective of this work was to generate a highly efficient system to select Gal-DKO cells to be used in SCNT without affecting the efficiency in production of Gal-null pigs.
Fetal liver-derived cells deficient in Gal-expression were initially selected by fluorescence-activated cell sorting (FACS) using IB4 conjugated to a fluorescent dye. Cells recovered by FACS were cultured and expanded, followed by a second round of selection using streptavidin magnetic beads and IB4 lectin biotin.
Recovery efficiency of target cells was 0.04% for the first selection using FACS and 0.3% for the second round by magnetic beads. Full reprogramming was obtained on selected Gal-DKO cells after FACS and magnetic beads selection, when used for SCNT to produce the Gal-null piglets. Cells obtained from magnetic beads developed 48 colonies; the Gal-null genotype was found in 44 of them (91.7%). Three of these colonies were used to generate piglets by SCNT. From three recipients receiving embryos, two became pregnant and produced 17 piglets, all of them DKO.
Sequential selection of Gal-DKO cells by FACS/magnetic beads is a highly efficient system to generate null cells. Selected cells were successfully used to generate healthy double-knockout piglets by SCNT.
通过体细胞核移植（SCNT）生成转基因动物的过程中，选择转基因细胞一直是一个瓶颈。特别是，Gal 双敲除（Gal-DKO）基因型的选择既耗时又低效。本研究的目的是开发一种高效的系统，在不影响 Gal 缺失型猪生产效率的情况下，用于 SCNT 的 Gal-DKO 细胞选择。
首先使用与荧光染料偶联的 IB4 通过荧光激活细胞分选（FACS）对缺乏 Gal 表达的胎肝来源细胞进行初始选择。FACS 回收的细胞进行培养和扩增，然后使用链霉亲和素磁珠和 IB4 凝集素生物素进行第二轮选择。
第一轮 FACS 选择的靶细胞回收效率为 0.04%，第二轮磁珠选择的效率为 0.3%。经 FACS 和磁珠选择后，用于 SCNT 生产 Gal 缺失型仔猪的 Gal-DKO 细胞可获得完全重编程。从磁珠获得的细胞形成了 48 个集落；其中 44 个（91.7%）为 Gal 缺失型。其中 3 个集落用于通过 SCNT 生成仔猪。在 3 个接受胚胎的受体中，有 2 个怀孕并产下了 17 头仔猪，均为 DKO。
通过 FACS/磁珠对 Gal-DKO 细胞进行连续选择是一种高效的系统，可生成缺失型细胞。选择的细胞成功用于通过 SCNT 生成健康的双敲除仔猪。
