Multimodality imaging of the new generation bi- and tri-fusion vectors in living mice.

<p>1a1 and 1a2. Fluorescence and Bioluminescence imaging. 10×10⁶ of 293T cells transfected with <i>CMV-ttk (A), CMV-fluc2-tdt-ttk (B), CMV-fluc2(C),</i> and <i>CMV-tdt (D)</i> plasmids were implanted subcutaneously in living mice (n = 3) and were imaged first for fluorescence and then for bioluminescence after injection of D- luciferin using IVIS imaging system. Signals were seen only from the cells expressing <i>CMV-tdt</i> and <i>CMV-fluc2-tdt-ttk</i> vector (for fluorescence) and from cells expressing <i>CMV- fluc2</i> and <i>CMV-fluc2-tdt-ttk</i> reporter for bioluminescence. Signals were recorded as max (pixel/sec/cm²/steradian). 1a3. microPET imaging. Mice described in 1a1 were injected with 200 µCi of 18F-FHBG and microPET imaging was performed after 1 hr for 10 minutes. Specific uptake of 18F-FHBG was seen in cells expressing the <i>CMV-ttk</i> and <i>CMV-fluc2-tdt-ttk</i> vectors. High nonspecific accumulation of 18F-FHBG was seen in the gastro-intestinal tract (GI). 1a4. Coronal section of the same microPET image described in 1a3. 1b. Graphical representation of the quantified fluorescence (1b1), bioluminescence (1b2) and microPET (1b3) signals. 1b1 and 1b2. Fluorescence (B and D) and bioluminescence (B and C) signals were calculated for the respective ROIs drawn over the sites of implanted cells. The SEM represents 3 experiments (ns.- statistically non-significant and *indicates p<0.05). 1b3. Percent injected dose (%ID/G) of 18F-FHBG uptakes were calculated for the respective ROIs drawn over the implanted cell (A and B) which showed similar uptake (ns). 1c. Comparative analysis of the fluorescence activity of the new bi and trifusion vectors in cell culture: 293T cells were transiently transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids and FACS analysis was done from equal number of cells after 24 hrs. All the experiments were performed in triplicate (*indicates p<0.05). 1d & 1e. Fluorescence and bioluminescence imaging of the new bi and triple fusion vector. 10×10⁶ of 293T cells transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids were implanted on the dorsal side of a nude mouse and imaged for fluorescence (1d) using Maestro system and bioluminescence (1e) as described above. Cells expressing <i>CMV-fluc2-tdt</i> clearly exhibited highest fluorescence and bioluminescence signals among all group of cells.