Figure 1.
Schematic diagram of plasmid and processing of the Core-Fluc fusion protein.
A.Schematic representation of the plasmids used in the present study. B. Schematic diagram of Core-Fluc fusion protein processing. pGL3-attB-CoreFluc plasmid encoded a precursor protein which was composed of the entire core protein (aa 1–191) and Fluc protein and was further processed into two proteins (the mature core protein (p21) and Fluc) within the signal sequence by host SPPase [15].Thus the expression level of core protein was reflected by the activity of Fluc which could be detected by the IVIS camera in whole animals.
Figure 2.
HCV core protein and Fluc expression in Huh 7 cells.
A. Fluc expression was detected by Luciferase Assay. The pGL3- attB-Core, pGL3-attB-Fluc, pGL3-attB-Core-Fluc vectors were transfected into Huh-7 cells. After 48 h, the cells were harvested and luciferase activity measured. The transfection efficiency was normalized with Renilla luciferase activity and the normalized luciferase activity was plotted. B. Western blot analysis of HCV core protein and Fluc expression. Total cell lysates were prepared from Huh7 cells, and transferred to membranes, followed by incubation with monoclonal antibodies specific for the HCV core (top), Fluc (middle) or β-actin (bottom) that serves as a control for protein loading.
Figure 3.
HCV core protein and Fluc expression in the liver of C57BL/6 mice.
A. Bioluminescent Imaging analysis of the Fluc activity. C57BL/6 mice were injected with 10 µg pGL3-attB-Core, pGL3-attB-Fluc, pGL3-attB-CoreFluc respectively and monitored by bioluminescent imaging. Fluc activity was measured 24 hours post-injection. B. Evaluation of Fluc activity in vivo over the course of the experiment.Values represent means±S.D. (n = 5). C. Western blot analysis of the HCV core protein and Fluc. Mice were transfected and sacrified at 24 hr. Fluc and core protein detection was performed as described above.
Figure 4.
Efficient suppression of the HCV core protein and Fluc expression by pU6- shRNA expression vector in vitro.
A.Inhibition of Fluc activity by pU6-mediated shRNA expression vector. shRNA-Scramble vector, shRNA–Fluc vector, shRNA-452 vector, shRNA-479 vector, shRNA-523 vector or pU6-vector (2 µg) were co-transfected with 0.5 µg pGL3-attB-CoreFluc vector and pRL-TK into Huh-7 cells. Luciferase reporter assay was performed 48 h post-transfection. The transfection efficiency was normalized with Renilla luciferase activity and the normalized luciferase activity was plotted taking the control (pU6-vector) as 100%. The presence of statistical difference has been indicated as follows: * = p<0.05; ** = p<0.01. B.Western blot analysis to establish the effects of various shRNA expression vectors on the levels of HCV core protein. Total cell lysates were electrophoresed on 12% SDS polyacrylamide gels, and transferred to membranes, followed by incubation with polyclonal antibodies for the core protein. And β-actin serves as a control for protein loading.
Figure 5.
Efficient suppression of the HCV core protein expression by pU6- shRNA vector in the mouse liver.
pU6-Vector, shRNA-Scramble vector, shRNA–Fluc vector, shRNA-452 vector or shRNA-523 vector (10 µg) were co-transfected with 10 µg pGL3-attB-CoreFluc vector into the mouse liver. Real-time in vivo imaging of FLuc activity in mice 8, 24, 36 and 48 hrs after hydrodynamic injection. The presence of statistical difference has been indicated as follows: * = p<0.05; ** = p<0.01. The area used for calculation of light-intensities has been boxed. All the experiments shown have been repeated at least thrice.
Figure 6.
Integrase mediates increased and prolonged expression of HCV core protein and Fluc in the mouse liver.
A.B. Bioluminescence imaging Fluc activity in mice. Two groups of mice received a large-volume tail vein injection of 10 µg of pGL3-attB-CoreFluc with 10 µg pCS(squares) or with 10 µg pCMV-Int(diamonds). FLuc activity was monitored by bioluminescence imaging in mice throughout the experiment. C. Western blot analysis of HCV core protein expression. Proteins were extracted from liver tissues and transferred to membranes, followed by incubation with monoclonal antibodies specific for the HCV core (top) and β-actin (bottom) that serves as a control for protein loading.
Figure 7.
Detection of integrase-mediated site-specific recombination at the DNA level.
A. Nested PCR was performed, using primers that detect the junction between the attB site of pGL3-attB-CoreFluc and mpsL1, a preferred pseudo attP site in the mouse genome. B. The sequences depicted the crossover region between the mouse pseudo attP site and the attB site. Shot lines represent bases missing in the novel joint. The 25 bp nearest the crossover of each genomic P arm was compared to wild-type attP [13].
Figure 8.
Efficient suppression of the HCV core protein and Fluc expression by pU6- shRNA expression vector in stable mouse model.
shRNA-Scramble vector, or shRNA-523 vector (10 µg) were transfected into the stable mouse model. Real-time in vivo imaging of FLuc expression in mice 6, 12, 24 and 48 hrs after injection. The luciferase activity was plotted taking the control (before injection) as 100%.The presence of statistical difference has been indicated as follows: * = p<0.05; ** = p<0.01.
Figure 9.
Hydrodynamic delivery shRNA effects on plasma ALT levels and cytokine IL-6/IL-1β levels in mice.
C57BL/6 mice (n = 5 per group) were hydrodynamically transfected with 10 µg of shRNA-Scramble vector, or 10 µg shRNA–523 vector, or 10 µg empty control plasmid pU6-Vector. A. Plasma ALT and, B. plasma IL-6 and, C. plasma IL-1β levels (means ± s.d.) were determined at different time after transfection.
Figure 10.
Effects of hydrodynamic injection shRNA on hepatic histology in mice.
C57BL/6 mice were treated as described in the legend to Fig. 9. Liver sections were stained with hemotoxylin/eosin. Livers were examined histologically from normal, uninjected animals(A) or from animals sacrificed 8 h (B-D), 24 h (E–G), and 48 h (H–J) after hydrodynamic injection. Magnification, x 40.