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Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation
Published on: June 27, 2017
[Activity analysis of human beta β2-microglobulin gene promoter in P815 cells]
Xing-qian Zhang1, Wen-han Mei, Guan-xiang Qian
1Department of Biochemistry and Laboratory of Molecular Biology, Research Center for Human Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China. zxqsh2000@yahoo.com
Aim:
To clone the human beta2-microglobulin(beta2m) gene promoter and study its activity in P815 cells.
Methods:
PCR amplification was performed using primers based on human beta2m gene sequence from GenBank and human genomic DNA as a template. The PCR product was directedly ligated into pBluescript II vector for sequencing. The promoter fragment was subcloned into a pcDNA3-EGFP plasmid after it had been identified correctly. A mouse mastocytoma cell line P815 was transiently and stably transfected with the plasmid containing human beta2m gene promoter and enhanced green fluorescence protein(EGFP) gene. The mRNA expression of EGFP in transiently transfected cells was quantified by RT-PCR and that in stably transfected cells was detected by fluorescence microscope and flow cytometry(FCM).
Results:
A 302 bp DNA fragment was amplified and cloned into the pcDNA3-EGFP vector. RT-PCR analysis showed that EGFP mRNA expression was induced by IFN-gamma in a dose-dependent manner. There was no difference in the fluorescence positive cell rate between the IFN-gamma-treated group and the control group. But the fluorescence intensity of the 5 x 10(5) U/L IFN-gamma-treated group increased about 2 folds compared with that of the control group.
Conclusion:
Human beta2m gene promoter is active in mouse mastocytoma P815 cells. It can regulate the expression of reporter gene under the control of IFN-gamma.
