The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner mongolia University, Hohhot 010021, China.
Theriogenology. 2012 Sep 1;78(4):793-802. doi: 10.1016/j.theriogenology.2012.03.027. Epub 2012 Apr 26.
The fat-1 gene was isolated from roundworm Caenorhabditis elegans, and built into pIRES2-EGFP expression vectors driven by cytomegalovirus (CMV) promoter or cytomegalovirus enhancer and chickenβ-actin (CAG) promoter. Both CMV- and CAG-driven expression vectors were transfected to sheep fetal fibroblast cells. Positive transfected cells were used as donors for somatic cell nuclear transfer (SCNT) and the cloned embryos were transferred into the oviducts of synchronized recipient sheep. Two lambs derived from CMV vector and three lambs derived from CAG vector developed to term. Although Southern analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1 gene into the genome, fat-1 mRNAs were not detected by RT-PCR. However, there was fat-1 expression (detected by RT-PCR) in tissues from transgenic lambs driven by CAG vectors. To investigate potential mechanisms involved in the two transgene models, methylation state of the vector promoters were examined. In CMV-driven transgenics, CMV promoters had almost no methylation in transfected cells and the resultant cloned embryos, whereas high methylations were detected in tissues and organs in transgenic lambs. In the CAG-driven transgenics, there were almost no methylations in transgenic cells and transgenic cloned embryos, and cloned lambs expressed fat-1 mRNA (detected by RT-PCR). Moreover, although SV40 promoters which drove neo/kan marker gene in CMV vectors were highly methylated in tissues from transgenic lambs, they were without methylation in cells and embryos. Therefore, we concluded that highly methylated CMV promoters induced the silence of fat-1 transgene expression in sheep. Furthermore, CAG promoter, but not CMV promoter was suitable for generation of fat-1 transgenic sheep.
该 fat-1 基因是从秀丽隐杆线虫中分离出来的，构建到由巨细胞病毒（CMV）启动子或 CMV 增强子和鸡β-肌动蛋白（CAG）启动子驱动的 pIRES2-EGFP 表达载体中。CMV 和 CAG 驱动的表达载体均转染到绵羊胎儿成纤维细胞中。阳性转染细胞用作体细胞核移植（SCNT）的供体，克隆胚胎被转移到同步化的受体绵羊的输卵管中。两只源自 CMV 载体的羔羊和三只源自 CAG 载体的羔羊发育到足月。虽然使用源自 CMV 载体的两只羔羊的组织进行 Southern 分析表明 fat-1 基因已整合到基因组中，但 RT-PCR 未检测到 fat-1 mRNA。然而，源自 CAG 载体的转基因羔羊的组织中存在 fat-1 表达（通过 RT-PCR 检测）。为了研究两种转基因模型中涉及的潜在机制，检查了载体启动子的甲基化状态。在 CMV 驱动的转基因中，CMV 启动子在转染细胞和由此产生的克隆胚胎中几乎没有甲基化，而在转基因羔羊的组织和器官中检测到高甲基化。在 CAG 驱动的转基因中，转基因细胞和转基因克隆胚胎中几乎没有甲基化，克隆羔羊表达 fat-1 mRNA（通过 RT-PCR 检测）。此外，虽然 CMV 载体中驱动 neo/kan 标记基因的 SV40 启动子在转基因羔羊的组织中高度甲基化，但在细胞和胚胎中没有甲基化。因此，我们得出结论，高度甲基化的 CMV 启动子诱导绵羊中 fat-1 转基因表达沉默。此外，CAG 启动子而不是 CMV 启动子适合生成 fat-1 转基因绵羊。
