Study Of Monitoring Therapeutic Gene Expression With Two Radionuclide Reporter Gene System

Objective Two recombinant adenovial vectors Ad5-VIE and Ad5-SIV which contain therapeutic gene and report gene were constructed to evaluate the feasibility of monitoring therapeutic gene vascular endothelial growth factor 165(VEGF165) gene with report gene GGCmotif sequence and mutant herpes simplex virus type 1 thymidine kinase(HSV1-sr39tk) gene.Methods1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system1) Construction and identification of recombinant adenovial vector Ad5-VIEGGC (diglycylcysteine) motifs were positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGCmotif-IRES-EGFP (Ad5-VIE) was constructed, carrying a CMV early promoter driving the expression of VEGF165 gene, GGC motifs and enhanced green fluoresce protein (EGFP), with the aid of an internal ribosomal entry site (IRES)。2) cellular uptake of99mTc-GHMSCs were infected with Ad5-VIE about 48h, then we perform the time-based (MOI=100, Incubation time:30min,60min,90min,120min)cellular uptake of 99mTc-GH and MOI based(MOI=0,10,25,50,100,incubation time:2h) cellular uptake study.3) Quantitative Real-time PCR (qRT-PCR) analysis for VEGF165 and EGFP mRNA.MSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, The total cellular RNA was extracted, then qRT-PCR was performed to detect the VEGF165 mRNA and EGFP mRNA.4) Semi-quantitative western-blot anlysis for VEGF165 proteinMSCs was infected with different infection units (MOI=0,10,25,50,100),48h later, the cytoplasmic protein was extracted from each well for Semi-quantitative western-blot study, The correlation analysis was performed between the cellular uptake of 99mTc-GH, which indirectly showed the expression of GGCmotifs and the expression of VEGF165 protein.5) Preliminary study of reporter gene imagingMSCs (1×107cells) was infected with 2.5×108 IU Ad5-VIE and Ad5-EGFP,24h after transfection, cells were collected after trypsin digestion, then suspended with 250μl saline. The cells suspension was multi-point injected in the SD rats lower limbs, then the SPECT imaging was peformed with the low-energy pinhole collimator 48h later.2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system1) Construction and identification of recombinant adenovial vector Ad5-SIVWith the aid of an internal ribosomal entry site (IRES) technique, a replication-defective adenovirus vector Ad5-SIV was constructed, carrying a CMV early promoter which driving the expression of HSV1-sr39tk gene and VEGF165 gene. PCR was performed to identify the adenovirus vector.2) Cellular uptake of131I-FIAUMSCs was infected with increasing multiplicities of infection (MOI= 0,10,25,50,75 and 100) of Ad5-SIV or Ad5-EGFP (used as a negative control). Forty-eight hours after infection, the culture medium was replaced by medium containg 131I-FIAU. After incubation for 3 hour, the radioactive medium was collected and the cells were then harvested and lysed with 1N NaOH in counting tube. Subsequently, the extracellular radioactivity in the medium and the intracellular radioactivity in cells lysate were determined by a gamma counter and corrected for decay. Meanwhile, the MSCs were infected with same MOI(MOI=50),and incubated with culture medium containing 131I-FIAU for 30min,60min,90min,120min,3h and 4h to determine the time course of131I-FIAU accumulation after adenovirus infection.3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.MSCs was infected with different MOI (MOI=0,10,25,50,75, 100IU/per cell),48h later, The total cellular RNA was extracted and qRT-PCR was performed to detect the HSV1-sr39tkmRNAandVEGF165 mRNA.4) Enzyme-Linked Immunosorbent Assay (ELISA) anlysis for VEGF165 proteinMSCs was infected with different infection units (MOI=0,10,25,50,75, 100IU/per cell),48h later, the culture medium was collected and centrifuged to remove all cellular fragments.ELISA was carried out according the manufacturer's introductions. Finally the absorbance of each well was determined within 30min using a microplate reader set to 450 nm. Finally, we compared the protein expression of VEGF165 and HSV1-sr39TK by analyzing the data obtained from ELISA and cellular uptake study.Results1. Study of monitoring the expression of therapy gene VEGF165 via GGC/99mTc-GH report gene/probe system1) Construction and identification of recombinant adenovial vector Ad5-VIEPlasmid PDC316-VEGFGGCmotif-IRES-EGFP was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-VIE was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 4.5×109IU/ml (classical TCID50 method)2) Cellular uptake of99mTc-GHTime dependent uptake showed that in Ad5-VIE infected MSCs, uptake rate of 99mTc-GH increased graduately over time, and the highest uptake occurred at 120min with a peak uptake rate of 7.72±0.22%. But in the control groups, the uptake of Ad5-EGFP infected cells at a low level all the time. The titers dependent uptake showed that uptake rate of 99mTc-GH in Ad5-VIE infected MSCs increased with the increasing viral titer (r2=0.86, P<0.05).3) qRT-PCR analysis for VEGF165 and EGFP mRNA.The qRT-PCR showed that VEGF165 mRNA and EGFP mRNA expression increased with increasing virus titers (r2=0.91 and r2=0.90, p<0.05). Furthermore there was a good correlation between VEGF165 mRNA and EGFP mRNA (r2=0.99,P<0.05).4) Semi-quantitative western-blot anlysis for VEGF165 proteinSemi-quantitative western-blot results showed that in the Ad5-VIE groups, expression of cytoplasmic VEGF165 protein increased graduately; while in the Ad5-EGFP group and uninfected control group, VEGF165 protein did not express. The relative amount of VEGF165 and the cell uptake rate increased with the titer gradually increased, a good correlation exited between expression of VEGF165 protein and GGC motifs (r2=0.90, P <0.05).5) Preliminary study of reporter gene imagingIn vivo imaging shows that after intravenous injection of 99mTc-GH, the bilateral renal and left lower extremity (infection with Ad5-VIE cell group) could be imaging clearly and immediately. With the imaging agent discharge, bladder imaging progressively clearly, the left lower limbs imaging gradually blurred,2h or so, the imaging almost disappeared. In cells transfected with Ad5-EGFP group, MSCs cells group, no significant imaging emerged.2. Study of monitoring the expression of therapy gene VEGF165 via HSVl-sr39tk/FIAU report gene/probe system1) Construction and identification of recombinant adenovial vector Ad5-SIVPlasmid PDC316-sr39tk-IRES-VEGF165 was identified by PCR, corresponding endonucleases digestion and sequencing. The recombinant adenovirus Ad5-SIV was identified by PCR, and the results showed the specific target band. The titer of purified adenovirus stock was 2×1011VP/ml (OD260 method) and 7.9×109IU/ml (classical TCID50 method)2) Cellular uptake of131I-FIAUThe time dependent cellular uptake studies showed that uptake rates increased rapidly between 30min and 150min and reached a plateau after 150min,at 150min point, the uptake rates were 20.06±1.33%. The uptake rates of 131I-FIAU with Ad5-SIV-infected cells were significantly higher than those with Ad5-EGFP-transfected groups at all time points (t=12.978-38.253).The MOI dependent cellular uptake studies showed that the cellular uptake of 131I-FIAU increased with increasing virus titer (R2=0.89, P<0.05)3) qRT-PCR analysis for HSV1-sr39tk and VEGF165 mRNA.HSV1-sr39tk mRNA and VEGF165 mRNA could express successfully after MSCs infected with Ad5-SIV at a series MOI (0,10,25,50,75,100 IU/per cell). Additionally, HSV1-sr39tk mRNA and VEGF165 mRNA expression increased with increasing virus titer (R2=0.97 and R2=0.96, p<0.05). Moreover, the real-time PCR results showed a good correlation between HSV1-Sr39tk mRNA and VEGF165 mRNA (R2=0.93,P<0.05)4) ELISA anlysis for VEGF165 proteinThe data obtained from ELISA showed a good correlation between VEGF protein and adenovirus titer (R2=0.99, P<0.05) after the MSCs was infected with different MOI (0,10, 25,50,75,100 IU/per cell) for 48h., furthermore, VEGF protein secretion was correlated well with the uptake rate of 131I-FIAU (R2=0.84, P<0.05)Conclusion:The recombinant adenovirus Ad5-VIE and Ad5-SIV was successfully constructed, and the in vitro study confirmed that expression of therapeutic gene VEGF165 correlated significantly with the report gene GGC motifs sequence and HSV1-sr39tk gene. This provided a theoretical basis for monitoring therapeutic gene with nuclear reporter gene imaging.