Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene.
OBJECTIVE
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.
Department of Nuclear Medicine, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, 430022, China.
Guopeng
G
Zhang
Xiaoli
X
Lan
Tzu-Chen
TC
Yen
Quan
Q
Chen
Zhijun
Z
Pei
Chunxia
C
Qin
Yongxue
Y
Zhang
D000256:Adenoviridae / Q000235:genetics / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D003561:Cytarabine / Q000031:analogs & derivatives; D004274:DNA, Recombinant / Q000235:genetics; D005240:Feasibility Studies; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000000981:diagnostic imaging / Q000378:metabolism* / Q000821:virology*; D064847:Multimodal Imaging; D049268:Positron-Emission Tomography; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014057:Tomography, X-Ray Computed; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D019562:Viral Load
D004274:DNA, Recombinant; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003561:Cytarabine; C026023:fiacitabine; D013937:Thymidine Kinase