Construction of a luciferase reporter vector containing response element of activator protein 2αand its application in study of osteogenetic differentiation
- VernacularTitle:AP2α荧光素酶报告基因的构建及在BMPs诱导成骨分化研究中的应用
- Author:
Mengjia GONG
1
;
Jianwu ZHOU
;
Yang BI
Author Information
1. 重庆医科大学附属儿童医院儿研所干细胞实验室//儿童发育疾病研究教育部重点实验室//重庆市干细胞治疗工程技术研究中心
- Keywords:
activator protein 2α;
luciferase reporter gene;
dominant negative mutants;
bone morphogenetic protein
- From:
Journal of Southern Medical University
2013;(11):1571-1576
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a luciferase reporter vector containing the response element of transcription protein AP2αfor screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α. Methods Four tandem-linked response elements of AP2αwere cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2αand its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2αtranscriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity. Results The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-△bHLH significantly lowered while Ad-dnAP2α-△TAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection. Conclusions The luciferase reporter vector containing the response element of AP2αwe constructed allows detection of AP2αtranscriptional activity. BMP9 can significantly enhance AP2αtranscriptional activity.
