Construction of a reporter gene vector and methylation of perforin promoter in vitro
- VernacularTitle:穿孔素基因启动子的克隆及体外区域性高甲基化
- Author:
Juan YUAN
;
Rong XIAO
;
Qianjin LU
;
Yaping LI
;
Xinjie YANG
;
Feng WANG
;
Yi ZHAN
;
Guiying ZHANG
;
Jing ZHANG
;
Ying ZHOU
- Publication Type:Journal Article
- Keywords:
Membrane glycopreteins/BI;
Cloning,molecular;
DNA methylation
- From:
Journal of Chinese Physician
2009;11(7):871-874
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a luciferase reporter gene vector of perforin promoter and methylate it in vitro. Methods The promoter of the human peffofin was amplified by PCR, cloned into pMD18-T vector and subcloned into pGL3-Basie vector, and then con-finned by restriction mapping and DNA sequencing. The regions of interest were excised with the appropriate restriction endonucleases, then it were methylated with methylase Sss I(M. SssI)and S-adenosymethioine(SAM), and methylation was confirmed by digestion with appropri-ate methylation sensitive enzyme AciI and agrose gel electrophoresis, and then the fragment was relegated back into the promoter-reporter constructor pGL3-Basic. Results The result of DNA sequencing showed that the sequence of cloned promoter was right. The result of diges-tion methylation with appropriate methylation sensitive enzyme showed that perforin promoter was completely methylated. Conclusion The promoter of perforin was successfully cloned and completely methylated in vitro, which provided an important basis for the study of transfec-tion.
