Fig 1.
Schematic overview of the created conditional inducible triple-transgenic mouse model for rapid real-time detection of HCV NS3/4A protease activity.
(A) The pBI-NS3/4A transgenic vector. (B) The pBI-NS3/4A vector was digested with Bam HI and Sal I and submitted to gel electrophoresis to confirm the correct construction. M: 500–12,000 bp. (C) Construction of the pTet-On-rtTA, pBI-Cre and pBI-NS3/4A plasmids. rtTA is expressed under the Lap promoter, but without Dox induction, rtTA is unable to bind to Ptetbi-1 to provide temporal control of gene expression. (D) Schematic overview of the created triple-transgenic mouse model for rapid real-time assessment of HCV NS3/4A protease activity. In the presence of Dox, rtTA binds to the Ptetbi-1 sequence on the pBI-Cre and pBI-NS3/4A vectors and activates transcription of the Cre and Fluc genes in pBI-Cre. Cre-mediated removal of the Fluc-BGH-PolyA STOP cassette leads to NS3/4A expression from the pBI-NS3/4A plasmid. The NS3/4A protease then cleaves at the 4B5A junction, resulting in the secretion of Gluc into either culture medium or blood. TM, C-terminal transmembrane domain; BGH, bovine growth hormone.
Fig 2.
Verification of the functionality of the pBI-NS3/4A plasmid in vitro.
(A) The pBI-NS3/4A plasmid was co-transfected into CHO cells with pTet-On-rtTA and pBI-Cre or pBI-NS3/4A alone as a control. The cells were treated with Dox (l μg/mL) at 6 h after transfection and harvested 48 h after transfection. Cell lysates were measured for Fluc activity. (B) Gluc activity was measured in the culture medium. (C) BLI was used to assess Fluc signals in the co-transfected cells. To accomplish this, 100 μL D-luciferin (1 mg/mL in PBS) was added to the cells, and they were then scanned using an IVIS Imaging System. (D) BLI was used to assess Gluc signals in the culture medium of the co-transfected cells. To accomplish this, 100 μL coelenterazine-SOL (100 μM) was added to the culture medium, and the mixture was then scanned using an IVIS Imaging System. (E) A representative western blot. Samples of culture medium from the co-transfected cells were concentrated and subsequently analyzed using a rabbit anti-Gluc antibody. Cell lysates from the co-transfected cells were analyzed using a mouse anti-NS3/4A antibody. (F) The co-transfected cells were induced with various concentrations of Dox (0.l, 0.5, l, and 2 μg/mL). NS3/4A protease expression in the co-transfected cells was analyzed via western blotting. (G) Samples of the culture medium from the co-transfected cells were assessed using a Gluc activity assay.
Fig 3.
Generation and verification of the functionality of NS3/4A/Lap/LC-1 triple-transgenic mice.
(A) PCR analysis of NS3/4A transgenic mice. M: DL 500–12,000; WT: wild type. (B) PCR analysis of triple-transgenic mice that contained the NS3/4A, rtTA and Cre transgenes. The results are representative of a single triple-transgenic mouse. M: DL 5000. (C) Representative BLI of Fluc signal. NS3/4A transgenic mice and triple-transgenic mice were induced with Dox (1 mg/mL Dox and 50 g/L sugar were dissolved in their drinking water) for 3 days. Then, the mice were intraperitoneally injected with D-luciferin and scanned using an IVIS imaging system. Non-Dox-induced NS3/4A transgenic mice and triple-transgenic mice were used as negative controls. (D) Gluc activity (shown in RLU) in the plasma of NS3/4A transgenic mice and triple-transgenic mice prior to and after Dox induction for 3 days (n = 20 mice/group). Each symbol represents an individual animal. The horizontal bars indicate group medians. *** = p<0.001 compared to the negative control. (E) Representative western blot analysis. Liver homogenates from NS3/4A transgenic mice and triple-transgenic mice induced with Dox for 3 days were analyzed via western blotting using anti-NS3 and anti-β-actin antibodies.
Fig 4.
Liver damage caused by NS3/4A protease expression in the triple-transgenic mice.
(A) Representative image of immunohistochemical staining of the NS3/4A protease in liver tissue sections from a representative WT mouse (left column), NS3/4A transgenic mouse (middle column) and triple-transgenic mouse (right column). The mice were induced with Dox for 3 days (1 mg/mL Dox and 50 g/L sugar were dissolved in their drinking water). The slides were counterstained with H&E. The upper images were taken at 10X magnification (scale bars, 200 μm). The lower images were taken at 40 X magnification (scale bars, 50 μm). (B) Histological examination of tissue damage. Liver sections from a representative WT mouse (left column), NS3/4A transgenic mouse (middle column) and triple-transgenic mouse (right column) following induction with Dox for 3 days. The sections were stained with H&E. The upper images were taken at 10 X magnification (scale bars, 200 μm). The lower images were taken at 40 X magnification (scale bars, 50 μm). (C) Analysis of serum ALT and AST levels in WT mice, NS3/4A transgenic mice and triple-transgenic mice following induction with Dox for 3 days (n = 10).
Fig 5.
Utilizing the triple-transgenic mouse model to evaluate the effects of NS3/4A protease inhibitors.
(A) Two groups of triple-transgenic mice (n = 5 per group) were treated with telaprevir (200 mg/kg) or DMSO via oral gavage twice daily for 10 days. During the treatment period, the mice were continuously induced with Dox (1 mg/mL Dox and 50 g/L sugar were dissolved in their drinking water). (B) Triple-transgenic mice were induced with Dox for 10 days (n = 5 per group). At the third day after Dox induction, the mice were treated with boceprevir (100 mg/kg) or DMSO via oral gavage twice daily for 7 days. Blood samples were collected daily from the caudal vein to measure plasma Gluc activity. Each data point represents the mean ± SD. *** = p<0.001 compared with the vehicle control.