[Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis].
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.
Shi
Qin-dong
QD
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.
Zhang
Peng-bo
PB
Kang
Qian-yan
QY
Chen
Xin-lin
XL
Tian
Yu-mei
YM
Liu
Jian-xin
JX
Liu
Yong
Y
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
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins