Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene

AimWe constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF165 (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF165) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF165 to evaluate the feasibility of monitoring VEGF165 expression using reporter gene expression.MethodsThe MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe 131I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1 day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control.ResultsThe real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF165 mRNA (R2 = 0.93, P < 0.05). The cellular uptake of 131I-FIAU increased with increasing viral titers (R2 = 0.89; P < 0.05), and in the group that received an MOI of 100, a peak value of 30.15% ± 1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of 131I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t = 18.43-54.83, P < 0.05). Moreover, the rate of VEGF165 protein secretion was highly correlated with the uptake rate of 131I-FIAU (R2 = 0.84, P < 0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg.ConclusionsThese results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.