[Activity analysis of human beta β2-microglobulin gene promoter in P815 cells].
OBJECTIVE
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.


CONCLUSIONS
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.
Zhang
Xing-qian
XQ
Department of Biochemistry and Laboratory of Molecular Biology, Research Center for Human Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China. zxqsh2000@yahoo.com
Mei
Wen-han
WH
Qian
Guan-xiang
GX
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D034801:Mastocytoma / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D001613:beta 2-Microglobulin / Q000235:genetics* / Q000378:metabolism*
D001613:beta 2-Microglobulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma