Reconstruction Of The Lentivirus Vector Encoding FTH1and The Expression Of FTH1in SK-N-SH Cells

Background and objectiveIn recent years, with the development of molecular imaging in genetherapy, magnetic resonance(MR) reporter gene imaging has become apopular study point. Numerous studies have confirmed the feasibility andadvantages of MR reporter gene imaging. Unlike the magnetic probe whichdecrease along with the cell proliferation, MR reporter gene expresssteadily in the target cells and can be used in the dynamic long-termmonitoring of the gene therapy. Choosing a safe, stable and effective reportgene is the precondition of reporter gene imaging. According to the reports,among the current MRI report genes, only two genes including magA andferritin can use endogenous iron for MR imaging. In the previous study, weused magA as the MRI report gene and transduced it into the293FT cells.The results showed that the magA gene can transfer iron, but the ironcontent would not be enough to change the MR signal. In this study,lentivirus vector carrying FTH1gene will be reconstructed and transfectedinto neuroblastoma cells, and the expression of FTH1gene will be dectected and identified. Our aim is to establish an ideal reporter gene forfurther study of induciable MR reporter gene imaging.Methods:1PCR amplification and identification of the FTH1geneThe FTH1gene was amplified from cloning template (BC000857) byPCR with the primers, the FTH1-F:5′-GGAATTCATGACGACCGCGTCCACC-3′and the FTH1-R:5′-TTTGCGGCCGCTTAGCTTTCATTAT-3′. The target gene wasidentified by enzyme digests and sequence analysis.2Construction of the lentivirus vector encoding FTH1The DNA of FTH1and lentivirus vector were digested by DoubleDigests with EcoR I、Not I. The fragments of FTH1gene and lentivirusvector were connected with DNA ligase, then transfered them into theE.coli DH5α, finally obtained the recombinant lentiviral vector. Therecombinant plasmid was identified with digestion and gene sequencing.3Packing the recombinant lentiviral vector with GFPThe recombinant plasmid were packaged by packaging plasmid,envelope plasmid and lentivirus vector in293T cell. Fluorescencemicroscope was used for observing the Green fluorescence expression in293T cells, then the viral titers was calculated with flow cytometry.4Detecting the expression of FTH1gene in tumor cellsThe pLenO-GFP-FTH1was transfected into SK-N-SH cells. The expression of GFP was observed under a fluorescence microscopy at24and48h after transfection respectively. The cells with high efficientexpression of FTH1gene were singled out with Flow cytometry screening.pLenO-GFP-FTH1/SK-N-SH and the contrast SK-N-SH were cultured inculture containing500μmol/L ferric ammonium citrate for4generations.The expression of target gene of the different generations was detected withprussian blue iron staining and celectron microscopy. According to trypanblue exclusion testing, the activity of the cell was determined.Results:1Identification of reconstructed pLenO-GFP-FTH1The digests and sequence analysis demonstrated that the DNA sequenceof FTH1was in accordance with that in the genbank, which testified thatthe lentivirus vector encoding FTH1was constructed successfully.2FTH1encoded lentivirus vector packaging and viral titeringLarge amount of fluorescece was observed in the293T pakage cells.The flow cytometery detected the fluorescence expression efficiency in the293T cells transfected with0.1uL GFP-FTH1was high up to85%. Theviral titers reached8.9×108TU/ml.The SK-N-SH cells transfected with pLenO-GFP-FTH1expressedFTH1protein efficiently, which were cultured in a medium supplementedwith FAC. According to trypan blue exclusion testing, There was nosignificant difference on the survival rate of two groups of cells. Stable expression of FTH1was confirmed by fluorescence microscope.3Transfection of SK-N-SH with FTH1The SK-N-SH cells transfected with pLenO-GFP-FTH1were detectedwith large amount of green fluorescence under a fluorescence microscopy.After4generation, the fluorescence was still in the cells, suggesting thatthe FTH1was successfully transfected in the tumor cells and expressedsteadily.4Expression of FTH1in the SK-N-SHComparing with the control group, prussian blue iron staining andcelectron microscopy found a lot of iron particles in the cytoplasm ofSK-N-SH cells. Upon continuously passaging，there were still a lot of ironparticles in the cytoplasm of SK-N-SH cells with FTH1gene. Trypan bluetest showed no difference in the number of vive cells between experimentaland contrast groups.Conclusion:The lentivirus vector encoding FTH1was Constructed successfully,and the expression of FTH1was identified in sk-n-sh cell, which provides abasis for further gene regulation by using FTH1as an MR reporter gene.