[Construction and expression of red fluorescent protein reporter gene vector containing human eNOS promoter].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xing
Feiyue
F
Key Lab for Shock and Microcirculation of the People's Liberation Army, First Military Medical University, Guangzhou 510515, China.
Zhao
Kesen
K
Qin
Qinghe
Q
Wang
Jingzhen
J
Deng
Peng
P
Jiang
Yong
Y
D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C496314:NOS3 protein, human