Establishment of a T-Ag and NF-kappaB binding elements gene co-transfected stable HUVECs cell line.
To set up a T-Ag gene transfected stable human umbilical venous endothelial cells (HUVECs) cell line and a T-Ag, nuclear transcriptional factor kappa B (NF-kappaB) binding elements linked with luciferase reporter gene co-transfected stable HUVECs cell line. Cultured HUVECs were transfected with pCI-neo-T-Ag and pRSV-luc-3XkappaB by lipofectin. The G418 selected monoclones were subcultured. The expression of marker protein, vWF and the characteristic of uptake of lipids were compared by Western blotting and immunocytochemistry in non-transfected and transfected HUVECs. The reporter gene assay was done in the presence of TNF-alpha. A T-Ag gene transfected stable HUVECs cell line and a T-Ag and NF-kappaB binding elements linked with luciferase reporter gene co-transfected stable HUVECs cell lines were set up. The expression of vWF of these cell lines was similar with those in non-transformed HUVECs. The function of uptaking of lipids was preserved as well in transfected cell lines. Furthermore, TNF-alpha, a typical cytokine increasing the activity of NF-kappaB was used to treat the transfected cells O/N. The higher luciferase reporter gene activity was seen. A pCI-neo-T-Ag and pRSV-luc-3X kappaB co-transfected stable HUVECs cell line might be used to check reporter gene activity directly. It might be a useful tool to screen drugs acting on transcription level.
Liu
Y X
YX
Department of Pharmacology, School of Basic Medical Sciences, Health Science Center, Peking University, Beijing, 100083, China.
Cao
W S
WS
Irani
K
K
Li
X J
XJ
D000957:Antigens, Viral, Tumor / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D014841:von Willebrand Factor / Q000096:biosynthesis
D000957:Antigens, Viral, Tumor; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D014841:von Willebrand Factor; D008156:Luciferases