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-VEGF(165) (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF(165)) 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 VEGF(165) to evaluate the feasibility of monitoring VEGF(165) 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 (131)I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1day 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 VEGF(165) mRNA (R(2)=0.93, P<0.05). The cellular uptake of (131)I-FIAU increased with increasing viral titers (R(2)=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 (131)I-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 VEGF(165) protein secretion was highly correlated with the uptake rate of (131)I-FIAU (R(2)=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.