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

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

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Acknowledgements

This study was supported by grants from the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (Nos. 2006CB500704, 2007CB516701), Science Foundation of the Ministry of Education of China (No. 20041065001) and the National Natural Science Foundation of China (Grant Nos. 30400139 and 30570649).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg