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

Aim: We 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. Methods: The 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. Results: The 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. Conclusions: These results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.