Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system.
- Author:
Wei CHEN
1
;
Chong CHEN
;
Huan-Xin ZHANG
;
Jiang CAO
;
Wei SANG
;
Qing-Yun WU
;
Kai ZHAO
;
Yu ZANG
;
Ling-Yu ZENG
;
Kai-Lin XU
Author Information
1. Department of Hematology, Xuzhou Medical College Affiliated Hospital, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
ADAM Proteins;
genetics;
ADAM10 Protein;
Amyloid Precursor Protein Secretases;
genetics;
Animals;
Cell Line;
Cloning, Organism;
Genes, Reporter;
Genetic Vectors;
Luciferases;
genetics;
Membrane Proteins;
genetics;
Mice;
Mice, Inbred BALB C;
Plasmids;
Promoter Regions, Genetic
- From:
Journal of Experimental Hematology
2012;20(3):740-743
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.
