Construction Of A Recombinant Lentivirus Expression Vector Carrying MAGA And Its Ferrin-Transporting Effect

Background:Recently,stem cell therapy and gene therapy using stem cells as vectors have been the focus in the medical research field. How to effectively track the transplanted cells'biological behavior in vivo is a serious problem to be solved before the clinical application of stem cells trerapy strategy. Due to its high temporal and spatial resolution, MRI is an ideal method in tracking cells in vivo. In the present studies, cells labeling with superparamagnetic iron oxide(SPIO) in vitro is most commonly used to track the cells after transplantation. One of the major shortcomings of this method is that the iron content in the cells decreases along with cell proliferation. MR reporter gene imaging is expected to overcome this problem. MagA, a gene fragment found in magnetotactic bacteria (Magnetospirillum magneticum AMB-1), plays an important role in the formation of magnetosome in the magnetotactic bacteria, and is expected to be an ideal reporter gene for MRI tracking of cells in vivo. The aim of this study is to construct a recombinant lentivirus expression vector carrying magA obtained by using artificial DNA synthesis, and to determine its ferrin-transfering effect in 293T cells, therefore lay a foundation for further study of in vivo tracking by using magA as a new MRI reporter gene.Methods:1 Synthesizing of magA and gene sequence analysis The gene magA was synthesized by synthetic DNA technology, and inserted into the plasmid pUC57 by recombinant DNA technique. The fragment of magA and puc57 were ligated by T4 DNA ligase. After transferring into the DH5a competent bacterium, the recombinant plasmid (pLenti/GFP-magA) were selected and indentified through blue and white screening and PCR with the primers. The recombinant plasmid DNA was extracted and digested by restriction endonuclease Xbaâ… and BamHâ… for further identification and sequence analysis.2 Construction of the lentivirus expression vector of magA The DNA of magA and Lentiviruus/GFP with restriction enzymes Kpnâ… and Smaâ… was digested and purified respectively, then the fragements with sticky ends of magA gene and lentivirus were obtained, and ligated with T4 DNA ligase and transferred them into the DH5a competent bacterium. The recombinant plasmid was inoculated into liquid LB(Amp+) culture medium, and the plasmid DNA(pLenti/GFP-magA) was extracted. The positive clones were identified with digestion and sequencing.3 Packing of recombinant lentivirus and target cells infecting According to the standard protocol of the company, the recombinant lentivirus of magA and GFP were packaged in the 293FT cells by co-transfecting with packaging plasmid, envelope plasmid, and lentiviral vector plasmid. The 293T cells were infected with the packaged lentivirus particles. Then, ampicillin was used for bolting quasi-cell line(293T/magA/GFP) until GFP expressed stably with high efficiency. At last, RT-PCR was used for testing the success of transferring of magA gene into 293T cells.4 Detecting the iron-transporting effect of magA in vitro and target cell's activty293T/magA/GFP cells were cultured in the medium containing 500uM ferric citrate for 4 generations. The cells of each generation were extracted and prepared for fluerescent observing, Prussian blue iron staining, electron microscopy detecting, and in vitro MR imaging. To determine the activity of the cells, trypan blue exclusion testing was performed .Results:The enzyme digestion and DNA sequence analysis confirmed that the sequence of magA was consistent with that in the genbank data. The magA were cloned into pLenti/EGFP in a right direction.The lentivirus of magA successfully transfected 293FT cells, which emitted green fluorescence under fluorescence microscope. The titer of resulting virus reached 108TU/ML and infected 293T cells successfully with an efficiency of more than 85%. RT-PCR confirmed that magA gene has been inserted into the genome of 293T cells.Prussian blue staining revealed numerous iron particles in the cytoplasm of 293T cells. Electron microscopy found lots of electron dense granules in the cytoplasm. T2*WI MR imaging revealed a signal decreasing with the amount of cells increasing. Cell viability rate was >90% , and no noteworthy difference found between the control group and the experimental group.Conclusion:The recombinant lentivirus expression vector of magA was successfully constructed and the iron transporting effect of magA in mammal 293T cells was verified, which provides a basis for further exploring MRI tracking of cells in vivo by using magA as a new ideal reporter gene.