绵羊Nanog启动子克隆及其表达活性的检测

发布时间：2018-02-01 23:19

本文关键词： 绵羊 Nanog启动子载体构建表达活性分析　出处：《中国兽医学报》2017年07期 　论文类型：期刊论文

【摘要】：通过比对人、鼠、兔和牛的Nanog启动子保守区设计引物,PCR扩增绵羊Nanog基因启动子序列并分析其调控位点。将其连接到去掉自身CMV启动子的pAcGFP1-N1载体上,构建一个由Nanog启动子带动的真核表达载体(SNanog-pAcGFP1-N1),即构建一个能由该启动子序列带动并产生绿色荧光蛋白(GFP)的真核表达载体,并用脂质体法转染入终末分化细胞(小鼠成纤维细胞和绵羊成纤维细胞)、胚胎干细胞和小鼠胚胎,检测其表达活性。结果显示:成功克隆出1 836bp Nanog启动子序列,通过分析发现该序列含有SP1结合位点、CAAT框、TATA框等功能区;当转染SNanog-pAcGFP1-N1重组质粒后,小鼠胚胎和小鼠胚胎干细胞中能检测到绿色荧光,且在转染72h后达到最大荧光强度,而在小鼠成纤维细胞和绵羊成纤维细胞中不能观察到GFP。结果表明:本试验成功克隆获得绵羊Nanog启动子,所构建的真核表达载体具有在多能干细胞中特异性表达活性。
[Abstract]:Primers were designed by comparing the conserved region of Nanog promoter between human, mouse, rabbit and cow. The promoter sequence of sheep Nanog gene was amplified by PCR and its regulatory site was analyzed. The promoter was ligated to the pAcGFP1-N1 vector that removed its own CMV promoter. A Nanog promoter driven eukaryotic expression vector SNanog-pAcGFP1-N1) was constructed. A eukaryotic expression vector was constructed which was driven by the promoter sequence and produced green fluorescent protein (GFP) and transfected into terminal differentiation cells (mouse fibroblasts and sheep fibroblasts) by liposome method. The expression activity of embryonic stem cells and mouse embryos was detected. The results showed that 1 836 BP Nanog promoter sequence was cloned successfully and the sequence contained SP1 binding site. CAAT frame, Tata box and other functional areas; After transfection of SNanog-pAcGFP1-N1 recombinant plasmid, green fluorescence could be detected in mouse embryo and mouse embryonic stem cells, and the maximum fluorescence intensity was reached 72 hours after transfection. GFP was not observed in mouse fibroblasts and sheep fibroblasts. The results showed that sheep Nanog promoters were cloned successfully in this experiment. The constructed eukaryotic expression vector has specific expression activity in pluripotent stem cells.
【作者单位】：东北林业大学生命科学学院;
【基金】：东北林业大学大学生科研训练项目;东北林业大学生命科学学院大学生创新资助项目国家自然科学基金资助项目(31000990)
【分类号】：Q78;S826
【正文快照】： *Corresponding author,E-mail:wangchunsheng79@163.comNanog基因最早在小鼠囊胚期的内细胞团、胚胎干细胞以及原始生殖细胞中发现[1-2]。该基因编码的同源异型框蛋白,能绕过LIF/Stat3通路维持ES细胞的自我更新能力,在囊胚形成后的第2个胚胎命运的特化中发挥关键作用。随后的

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