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http://dx.doi.org/10.5352/JLS.2017.27.8.857

Cloning and Activity Analysis of the FosB Promoter Region from Human Genomic DNA  

Na, Han-Heom (Department of Biological Sciences, College of Natural Sciences, Kangwon National University)
Kang, Yoonsung (Institute for Diagnostic Markers, Eudipia Inc)
Kim, Keun-Cheol (Department of Biological Sciences, College of Natural Sciences, Kangwon National University)
Publication Information
Journal of Life Science / v.27, no.8, 2017 , pp. 857-863 More about this Journal
Abstract
The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.
Keywords
Activity; biomarkers; cloning; FosB; promoter;
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