Construction of pGL3-EPO-HRE reporter gene vector and detection of its function
- VernacularTitle:pGL3-EPO-HRE报告基因质粒的构建及功能鉴定
- Author:
Yan SU
;
Hongwei LI
;
Jing ZHANG
;
Ruijuan XIU
- Publication Type:Journal Article
- Keywords:
hypoxia inducible factor-1?;
hypoxia responsible element;
plasmid construction;
gene expression
- From:
Basic & Clinical Medicine
2006;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct pGL3-EPO-HRE reporter gene vector and to detect its function.Methods To synthesize two single-strand DNA fragments which had overlap sequence at the 3'end,then mixed them to construct the double-strand DNA fragment which include three copies of(57 bp) human erythropoietin(EPO) HRE.There was MluⅠor BglⅡ digestive site at the end of this DNA fragment.After inserting the(87 bp) fragment into pMD18-T for sequencing,the correct sequence was inserted into pGL3-promoter to construct pGL3-EPO-HRE.The recombinant vector was co-transfected into MCF-7 cells with pcDNA3-HIF-1?or pcDNA3 respectively,(12 h) later cells were treated with(100 ?mol/L) cobalt chloride(CoCl_(2)) for(24 h), then the expression of pGL3-EPO-HRE was observed by dual-luciferase reporter gene assay method.Results The sequence of pGL3-EPO-HRE was identical with human EPO HRE sequence.pGL3-EPO-HRE luciferase activity increased about 10 fold(P
