Constructing the dual luciferase reporter vector containing human DRD1 promoter region
10.3969/j.issn.2095-4344.2016.40.020
- VernacularTitle:人DRD1基因启动子区克隆和双荧光素酶报告基因系统的构建
- Author:
Chunhong WANG
;
Zhe LI
;
Minghan WANG
;
Lili DENG
;
Huan WANG
- Publication Type:Journal Article
- Keywords:
Receptors,Dopamine;
Genes,Reporter;
Transcription Initiation Site;
Tissue Engineering
- From:
Chinese Journal of Tissue Engineering Research
2016;20(40):6060-6066
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:The polymorphisms of dopamine receptor in promoter region wil affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, final y lead to related diseases.
OBJECTIVE:To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene.
METHODS:DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pGM-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pGL3-Basic. The cloned pGL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with pGL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence.
RESULTS AND CONCLUSION:(1) Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing. (2) Compared with the negative control group, the HEK293 cel s transfected by recombinant vectors presented transcriptional activity. (3) In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successful y constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.
