Figure 1.
The eGFP expression cassette was digested using AseI and Afl II from pEGFP-N3 plasmid. After the ligation of DNA fragment and ODN caps, the mixture was used as temple for PCR amplification. The eGFP-MiLV was purified by PCR cleanup kits and used for further in vitro and in vivo experiments.
Figure 2.
Expression of GFP in eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP-MiLV.
Cells were seeded in to six-well plates and transfected with MiLV or plasmid DNA (0.1 µM DNA/cm2 per plasmid). The transfection efficiency (expression of GFP) was evaluated 48 h after the transfection.
Figure 3.
Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP-MiLV.
A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm2 pEGFP-N3 plasmid or eGFP-MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm2 pEGFP-N3 plasmid or eGFP-MiLV. Data are representative of at least three experiments. *p<0.05.
Figure 4.
Expression of transgene of eGFP-MiLV and pEGFP-N3 plasmid in mice muscle.
After intramuscular injection, at least one mouse of each group was killed per week to detect the GFP expression. The muscle sections (5 µm) were observed by fluorescence microscope. The control group was injected with PBS. 1, 2, 4, 8 represents the weeks after intramuscular injection. The fluorescence of eGFP-MiLV lasted more than two months in mouse muscle.
Figure 5.
Mean ELISA values of GFP from equal weight (40 µg ) eGFP-MiLV and pEGFP-N3 plasmid immunized mice.
The ELISA was performed with mouse sera from individual animals of different vaccination groups. Blood samples were taken and prepared at the indicated time-points before each eGFP-MiLV or pEGFP-N3 plasmid vaccination, and analyzed for reactivity against the GFP (the antigen). * p<0.05; ** p<0.01.
Figure 6.
Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP-MiLV and pEGFP-N3 plasmid.
Mice received an intravenous injection of 40 µg eGFP-MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p<0.05 compared to the pEGFP-N3 group.
Figure 7.
Expression of TK in B95-8 cell transfected by use of MEKK1-MiLV or pCMV/MEKK1 plasmid.
A: The structure of MEKK1-MiLV. The pLMP1 resulted in MEKK1 being expressed only in EBV positive cells. B: B95-8 cells were transfected by equal molar of MEKK1-MiLV or pCMV/MEKK1 plasmid for 24 h, and then treated with 1 mM NaB for 18 h, followed by treatment with 100 µg/ml GCV for 3 days. The TK expression was detected by immunoblot analysis with a patient’s EBV-TK serum. The β-actin protein levels served as the loading controls.
Figure 8.
Relative viability of B95-8 cells transfected by MEKK1-MiLV or pCMV/MEKK1 plasmid then incubated with GCV/NaB.
Control was treated with GCV/NaB; MiLV group was transfected with MEKK1-MiLV and then treated with GCV/NaB; Plasmid group was transfected with pCMV/MEKK1 plasmid and then treated with GCV/NaB. (A): The phenotypic change of B95-8 cells. Twenty four hours after transfection, cells were treated with 1 mM NaB for 18 h followed by 100 µg/ml GCV for 3 days. (B): MTT detected relative viability of B95-8 cells. The values are the mean of 3 separate experiments with error bars representing the standard deviations. The MEKK1 transfection enhanced the sensitivity of B95-8 cells to GCV/NaB. The MEKK1-MiLV transfected B95-8 cells were more sensitive (p<0.05, paired t test) to GCV/NaB than that of pCMV/MEKK1 plasmid. * p<0.05.
Figure 9.
Inhibition of nasopharyngeal carcinoma growth in athymic nude mice with NaB/GCV treatment combined with MEKK1-MiLV or pCMV/MEKK1 plasmid.
The mice (8 mice per group) were inoculated with B95-8 cells transfected with equal mole of MEKK1-MiLV or pCMV/MEKK1 plasmid. Then they were treated with single intraperitoneal injection of NaB (500 µl of 50 mM sodium butyrate in PBS) and intraperitoneal injection of GCV (100 mg/kg twice a day for 5 days). The cancer growth was monitored every 2 days using calipers. * p<0.05; ** p<0.01.