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

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.