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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (1) : 67-71     DOI: 10.1007/s11684-009-0010-0
RESEARCH ARTICLE |
Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus
Xixun DU*, Huamin XU*, Hong JIANG, Jun WANG, Lei WANG, Junxia XIE()
State Key Disciplines: Physiology(in incubation), Department of Physiology, Medical College of Qingdao University, Qingdao 266071, China
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Abstract  

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 pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene. Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escherichia 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 demonstrated 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 PacI. PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE, indicating the successful construction of recombinant adenovirus plasmid containing DMT1-IRE. GFP fluorescence further confirmed the generation of adenovirus. AdDMT1-IRE could efficiently infect C6 glioma cells. And cell viability decreased in Ad-DMT1-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.

Keywords divalent metal transporter 1      recombinant adenovirus      homologous recombination      iron     
Corresponding Authors: XIE Junxia,Email:jxiaxie@public.qd.sd.cn   
Issue Date: 05 March 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0010-0     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I1/67
Fig.1  Construction of recombinant adenovirus plasmid encoding DMT1-IRE. (a) RT-PCR for the human DMT1-IRE gene. Human full-length cDNA of DMT1-IRE was isolated by RT-PCR. (b) Double digestive maps of plasmid pMD18-T-DMT1-IRE with I and d III. (c) Double digestive maps of plasmid pAdCMV-DMT1-IRE with I and d III. (d) Restrictive enzyme analysis of pAdDMT1-IRE from clones with I. Candidate clone pAdDMT1-IRE yielded a large fragment, plus a smaller fragment of 4.5 kb. DMT1: divalent metal transporter 1; IRE: iron response element.
Fig.2  GFP fluorescence expression monitored by confocal. Linearized pAdEasy1-DMT1-IRE was transfected into HEK293 cells with lipofectamine 2000 and cells were harvested at the indicated time after transfection. Fluorescence microscopy of the infected cells was performed. (a) GFP fluorescence after 48 hours; (b) GFP fluorescence after 6 days.
Fig.3  The mRNA expression of DMT1-IRE in AdDMT1-IRE infected C6 cells. The mRNA levels of DMT1-IRE were observed by RT-PCR after infection for 36 hours. DMT1-IRE mRNA expression increased significantly after AdDMT1-IRE infection compared with the control in C6 cells. : <0.01 compared with control.
Fig.4  Cell viability detected by MTT. After exposure to ferrous iron for 24 hours, cells infected with AdDMT1-IRE induced a significant decrease in cell viability compared with the control after iron incubation. : <0.01 compared with control.
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