Constructing the dual luciferase reporter vector containing human DRD1 promoter region / 中国组织工程研究

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.