Construction of recombinant plasmid pCGN-HAM-N1-wcAPC and this plasmid inhibition of activation of T-cell factor/lymphocyte enhancer factor promoter

Abstract

Objective To construct an eukaryotic expression recombinant plasmid named pCGN-HAM-N1-wcAPC and transfected it into cell line to simply analysis it function.Methods Insert a big one fragment 9000 bp Adenomatous polyposis coli (APC) gene into pCGN-HAM-N1,which is a eukaryotic expression vector with a HA epitope tag by Cloning polymerase chain reaction (PCR) products into pMD-18T and cutting one fragment insert into another plasmid.Then the recombinant vector was identified by incision enzyme and DNA sequence.Transfected 293T cell with the indicated amounts of vectors,including β-catenin,topflash,and Renilla luciferase,by Lipofectamine 2000.Analyzed luciferase activity and checked the function of pCGN-HAM-N1-wcAPC.Results 9000 bp APC gene insets into 5.2 kb vector pCGN-HAM-N1,DNA sequence was identical with APC cDNA in NCBI.The transfected pCGN-HAM-N1-wcAPC group luciferase activity was significantly lower than empty vector group.Conclusion The eukaryotic expression recombinant plasmid and transfection of it were successfully constructed,which will be used further study.pCGN-HAM-N1-wcAPC inhibits of Activation of T-cell factor/lymphocyte enhancer factor (Tcf/Lef) Promoter. Key words: Competent cells; Adenomatous polyposis coli gene; Clone; Recombinant plasmid ; Promoter