Construction of a reporter gene vector and methylation of perforin promoter in vitro / 中国医师杂志
Journal of Chinese Physician
;
(12): 871-874, 2009.
Artigo
em Chinês
| WPRIM
| ID: wpr-393500
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Journal of Chinese Physician
Ano de publicação:
2009
Tipo de documento:
Artigo
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