Construction of pGL3-EPO-HRE reporter gene vector and detection of its function / 基础医学与临床
Basic & Clinical Medicine
; (12)2006.
Article
em Zh
| WPRIM
| ID: wpr-587887
Biblioteca responsável:
WPRO
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
Texto completo:
1
Índice:
WPRIM
Tipo de estudo:
Diagnostic_studies
Idioma:
Zh
Revista:
Basic & Clinical Medicine
Ano de publicação:
2006
Tipo de documento:
Article