title: [Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis].
abstract: OBJECTIVE
  To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers.
  
  
  METHODS
  EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy.
  
  
  RESULTS
  After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture.
  
  
  CONCLUSIONS
  we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.
authors.affiliation: Research Center for Neuroscience, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Xi'an Jiaotong University College of Medicine, Xi'an 710061, China. shiqindong@163.com.
authors.forename: Qin-dong
authors.initials: QD
authors.lastname: Shi
authors.forename: Peng-bo
authors.initials: PB
authors.lastname: Zhang
authors.forename: Qian-yan
authors.initials: QY
authors.lastname: Kang
authors.forename: Xin-lin
authors.initials: XL
authors.lastname: Chen
authors.forename: Yu-mei
authors.initials: YM
authors.lastname: Tian
authors.forename: Jian-xin
authors.initials: JX
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
mesh_terms: D015687:Cell Hypoxia; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000096:biosynthesis*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000096:biosynthesis; D014162:Transfection
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins