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[Construction of a luciferase reporter vector containing response element of activator protein 2α and its application
Mengjia Gong1, Jianwu Zhou, Yang Bi
1Stem Cell Biology and Therapy Laboratory, Children's Hospital of Chongqing Medical University; Ministry of Education Key Laboratory of Child Development and Disorders; Chongqing Municipal Stem Cell Therapy Engineering Technical Center, Chongqing 400014, China.E-mail: gmj.0710@aliyun.com.
Objective:
To construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α.
Methods:
Four tandem-linked response elements of AP2α were 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-AP2α and 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 AP2α transcriptional 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-δbHLH significantly lowered while Ad-dnAP2α-δTAD 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 AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.
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