Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

作     者：Xixun DU Huamin XU Hong JIANG Jun WANG Lei WANG Junxia XIE 

作者机构：State Key Disciplines:Physiology(in incubation)Department of PhysiologyMedical College of Qingdao UniversityQingdao 266071China 

出 版 物：《Frontiers of Medicine》 (医学前沿（英文版）)

年 卷 期：2009年第3卷第1期

页      面：67-71页

核心收录：

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

基　　金：National Natural Science Foundation of China, NSFC, (30400139, 30570649) Ministry of Education of the People's Republic of China, MOE, (20041065001) Ministry of Science and Technology of the People's Republic of China, MOST, (2006CB500704,2007CB516701) 

主　　题：divalent metal transporter 1 recombinant adenovirus homologous recombination iron 

摘      要：Divalent metal transporter 1(DMT1)is a ferrous iron import protein.The improper expression of DMT1 is involved in neurodegenerative diseases.In the present study,we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element(DMT1-IRE)and investigated its expression and function in the C6 glioma cell line.The DMT1-IRE gene,obtained by RT-PCR,was cloned into the shuttle plasmid-ing pAdTrack-CMV containing greenfluorescent protein(GFP)reporter gene.Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escher-ichia coli(E.coli)BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I.Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells(HEK293 cells),in which recombinant adenoviruses were generated within 7 to 10 days.The results demon-strated that we obtained the DMT1-IRE gene.pAdEasy1-DMT1-IRE yielded a large fragment,plus a smaller fragment of 4.5 kb after digestion with Pac I.PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE,indicating the successful construction of recombi-nant adenovirus plasmid containing DMT1-IRE.GFPfluorescence further confirmed the generation of recombi-nant AdDMT1-IRE adenovirus.AdDMT1-IRE could efficiently infect C6 glioma cells.And cell viability decreased in AdDMT1-IRE infected cells after iron overload compared to the control.These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.