title: [Construction and identification of human p-selectin promotor luciferase reporter gene vector].
abstract: OBJECTIVE
  To construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter.
  
  
  METHODS
  Primers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with Kpn I and Xho I, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-α and As2O3, and the transcriptional activity of p-selectin promoter were assessed.
  
  
  RESULTS
  pGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573±0.4703 vs 0.03955±0.05894). pGL3- 1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter.
  
  
  CONCLUSIONS
  pGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.
authors.affiliation: Guangdong Provincial Key Laboratory for Shock and Microcirculation Research, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China.E-mail: 331523317@qq.com.
authors.forename: Ruo-Ting
authors.initials: RT
authors.lastname: Xu
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Wei-Lu
authors.initials: WL
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Xian-Yan
authors.initials: XY
authors.lastname: Liu
authors.forename: Wen-Qiang
authors.initials: WQ
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Tan
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
mesh_terms: D017930:Genes, Reporter; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases; D019007:P-Selectin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D019007:P-Selectin; C546937:SELP protein, human; D008156:Luciferases