[Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system].
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.
Department of Hematology, Xuzhou Medical College Affiliated Hospital, Jiangsu Province, China.
Wei
W
Chen
Chong
C
Chen
Huan-Xin
HX
Zhang
Jiang
J
Cao
Wei
W
Sang
Qing-Yun
QY
Wu
Kai
K
Zhao
Yu
Y
Zang
Ling-Yu
LY
Zeng
Kai-Lin
KL
Xu
D051722:ADAM Proteins / Q000235:genetics*; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D019976:Cloning, Organism; D017930:Genes, Reporter; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011401:Promoter Regions, Genetic
D008565:Membrane Proteins; D008156:Luciferases; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072197:ADAM10 Protein; C491234:Adam10 protein, mouse