Xing Feiyue, Zhao Kesen, Qin Qinghe, Wang Jingzhen, Deng Peng, Jiang Yong
Key Lab for Shock and Microcirculation of the People's Liberation Army, First Military Medical University, Guangzhou 510515, China.
Zhonghua Yi Xue Za Zhi. 2002 Aug 25;82(16):1093-6.
To construct the red fluorescent protein reporter gene vector containing human eNOS promoter sequence to study the mechanism of regulating the expression of eNOS gene by vessel wall shear stress.
The genomic DNA of endothelial cells from fetal umbilical vein was drawn. The gene sequence of eNOS promoter gene therein was cloned by PCR technique and constructed into the red fluorescent protein vector, pDsRed-1. The recombinant vector, pDseNOSRed was then transfected into 293 cells, human fetal renal epithelial cells. Blank vector, pDsRed-1, was transfected into 293 cells as controls. The expression and distribution of the reporter gene were observed by fluorescent microscopy.
PCR and double restriction enzyme digestion showed that the recombinant vector, pDseNOSRed, was constructed correctly. This vector was highly expressed in the 293 cells. Expression of red fluorescence, evenly distributed in whole cells, occurred since 12 hours after transfection, reached the peak concentration 3648 h after transfection, and dissappeared almost completely 120 h after. No red fluorescence was observed in the control cells.
A red fluorescent protein reporter gene vector containing human eNOS promoter sequence and expressed highly in mammalian cells has been constructed successfully, thus providing an important and convenient tool to study the mechanism mechanism of regulating the expression of eNOS gene by vessel wall shear stress.
构建含人内皮型一氧化氮合酶（eNOS）启动子序列的红色荧光蛋白报告基因载体，以研究血管壁切应力对eNOS基因表达的调控机制。
提取人胎儿脐静脉内皮细胞基因组DNA，采用聚合酶链反应（PCR）技术克隆其中eNOS启动子基因序列，并构建至红色荧光蛋白载体pDsRed-1中，构建重组载体pDseNOSRed。将重组载体转染人胚肾上皮细胞293细胞，以转染空载体pDsRed-1的293细胞作为对照。采用荧光显微镜观察报告基因的表达及分布情况。
PCR及双酶切鉴定显示重组载体pDseNOSRed构建正确，该载体在293细胞中高表达。转染后12小时开始出现均匀分布于整个细胞的红色荧光表达，转染后36~48小时荧光强度达到峰值，120小时后几乎完全消失。对照细胞未见红色荧光。
成功构建了含人eNOS启动子序列且在哺乳动物细胞中高表达的红色荧光蛋白报告基因载体，为研究血管壁切应力调控eNOS基因表达的机制提供了重要且便捷的工具。
