转基因小鼠支持细胞体外重编程为诱导多能性干细胞的研究  

Study on Induced Pluripotent Stem Cells Derived from Sertoli Cells of Transgenic Mice in vitro

作　　者：杜军慧[1] 曹文广[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2014年第11期184-190,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家转基因重大专项"优质转基因肉羊新品种培育"(2013ZX08008-003;2014ZX08008-003)

摘　　要：试验旨在研究利用4种特定转录因子将转基因小鼠支持细胞体外重编程为诱导多能性干细胞(induced pluripotent stem cells,iPS)。通过逆转录病毒载体将4种特定转录因子Oct4、Sox2、c-Myc及Klf4导入转基因小鼠支持细胞,同时以绿色荧光蛋白(green fluorescence protein,GFP)空载体检测病毒感染情况。感染的支持细胞3d左右表达GFP蛋白,同时细胞开始缩小聚集,失去原有支持细胞形态,逐步形成突起样克隆,16d左右感染的支持细胞不表达GFP蛋白,形成具有典型干细胞特征的iPS细胞。碱性磷酸酶染色表明iPS细胞处于未分化状态;反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)检测iPS细胞可表达胚胎干细胞(embryonic stem cells,ESC)特异性基因;体外悬滴培养可形成拟胚体(embryoid body,EBs),可分化为三胚层来源的多种细胞。This experiment was conducted to use four defined transcription factors that induced sertoli cells of transgenic mice to induced pluripotent stem （iPS） cells. Sertoli cells of transgenic mice were infected with retrovirus vectors that lead to the expression of Oct4, Sox2, c-Myc and Klf4, green fluorescence protein （GFP） was also transduced to monitor the infection efficiency as well as an indicator of exogenous gene in part of induction experiments. As early as 3 days post-infection, the cells started to express GFP and morphologically differ from uninfected sertoli cells. As the culture prolonged, the single cell was getting smaller and more cells aggregating to form colonies. At 16 days, the GFP were silenced and compact and round-shaped colonies formed. Alkaline phosphatase staining showed that iPS cells in the undifferentiated condition, reverse transcription-polymerase chain reaction （RT-PCR） showed that iPS cells could express the specific genes of embryonic stem cells, and could form embryoid body （EBs） which could differentiate into myriad cell types of three layer.

关 键 词：转红色荧光蛋白基因小鼠 支持细胞 体外诱导重编程 诱导多能性干细胞 

分 类 号：Q813.5[生物学—生物工程]