Objective      To construct a recombinant lentivirus expression vector encoding magA

a new MRI reporter gene

and to determine the iron-transporting effect of in vitro expressed magA.     Methods      The gene sequence of magA was synthesized by synthetic DNA technology

and then inserted into the lentivirus expression vector pLenti-EGFP by recombinant DNA technique. The recombinant lentivirus expression vector pLenti-EGFP/magA was verified by enzyme digestion and DNA sequencing. Then t

he recombinant lentivirus was packaged in the 293FT cells by co-transfecting with packaging plasmid

envelope plasmid

and lentiviral vector plasmid. The 293T cells were transfected with the packaged lentivirus particles

and then cultured in the medium containing 500 μmol/L ferric citrate. Prussian blue staining was performed to detect the iron particles in the 293T cells. Viability was assessed via Trypan blue exclusion testing.     Results      The enzyme digestion and DNA sequencing confirmed that the sequence of PCR-amplified magA was consistent with that in Genbank. The magA were cloned into pLenti-EGFP in a right direction. The three plasmids were transferred into 293T cells

which emitted green fluorescence under fluorescence microscope. The titer of resulting virus reached 10

8

TU/μl and infected 293T cells successfully with an efficiency of more than 80%. Prussian blue staining revealed numerous iron particles in the cytoplasm of 293T cells. Cell viability was 90% for all cell with no noteworthy difference between the control group and the experimental group.     Conclusion      The recombinant lentivirus expression vector of magA is successfully constructed and the iron transporting effect of magA in mammal 293T cells is verified

which provides a basis for further exploring MRI tracking in cells in vivo by using magA as a reporter gene.