Construction Of A Reporter Gene Vector And In Vitro Methylation Of Perforin Promoter

Objective: To construct a luciferase reporter gene vector of perforin promoter and methylate it in vitro.Methods: (1)Amplify the promoter of the human perforin promoter by PCR, and then clone into pMD18-T vector and subclone into pGL3-Basic vector. The recombinant plasmid was confirmed by restriction mapping and DNA sequencing. (2) Excise the regions of intrest using the appropriate restriction endonucleases, methlate excised fragment with methylase SssI (M.SssI) and S-adenosymethioine (SAM), comfirm methylation by digestion with appropriate methylation sensitive enzyme AciI and agrose gel electrophresis, and then relegate fragment back into the promoter-reporter construct pGL3-Basic.Results:(1)The result of DNA sequencing showed that the sequence of cloned promoter is right. (2) The result of digestion methylation with appropriate methylation sensitive enzyme showed that perforin promoter was methylated completely.Conclusion: The promoter of perforin was cloned successfully, and methylated in vitro completely. It will be a important basis for the study of transfection.