1. Volume 125, Issue 1, Pages 9-18 (July 2003)
Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA 
C Klein, C.T Bock, H Wedemeyer, T Wüstefeld, S Locarnini, H.P Dienes, S Kubicka, M.P Manns, C Trautwein 
Gastroenterology 
Volume 125, Issue 1, Pages 9-18 (July 2003)
DOI: /S (03)

2. Figure 1
Injection of an HBV replication competent vector in mice results in HBV-specific protein expression. Twenty micrograms of an HBV replication competent vector (pHBV 1.5) were transferred in NMRI mice by high-volume injection via tail vein. (A and B) At the indicated time points after administration, serum levels of HBsAg (A ) and HBeAg (B) were determined. (C ) One hundred twenty days after injecting 20 μg of the pHBV 1.5 vector, HBsAg and HBeAg expression was determined compared with controls (pBS injected animals). (D) Three days after injection, immunohistochemistry for HBsAg and HBcAg was performed as indicated.
Gastroenterology  , 9-18DOI: ( /S (03) )

3. Figure 2
HBV virion and capsid formation after high-volume injection of a HBV replication competent vector in mice. (A ) Liver RNA was prepared, and, after extensive DNase digestion, a triplex RT-PCR was performed with primers detecting the pre-C/C and S RNA in comparison with the GAPDH signal. Time points are indicated before and after injection of the pHBV 1.5 vector. The positions of the GAPDH-, S-, and C-specific signals are indicated. (B) Three days after injection, whole liver extracts were prepared and viral capsids immunoprecipitated. Dot blot analysis using a P32-labeled HBV cDNA was performed to detect viral DNA. As a positive control, increasing amounts of a HBV plasmid were probed as indicated. (C ) Three days after injection, PEG precipitation of the serum of the animals was performed. After DNase and RNase digestion, an HBV-specific PCR for detection of viral DNA was performed. A representative example of animals injected with the pBS control vector (negative control) and the pHBV 1.5 construct are shown. Additionally, as a positive control, PCR was performed with 0.2 μg of the pHBV 1.5 vector. (D) Analysis of a quantitative real-time PCR of virions isolated from sera and capsids derived from liver is depicted at different days after injection of the pHBV 1.5 vector.
Gastroenterology  , 9-18DOI: ( /S (03) )

4. Figure 3
Antiviral treatment options can be tested after high-volume HBV-plasmid injection. (A ) Endogenous polymerase activity was studied in capsids derived from mouse liver at different time points after high-volume injection of the pHBV 1.5 construct. (B) Quantitative assessment of EPA activity by measuring relative radioactive incorporation. (C ) After high-volume injection of the pHBV 1.5 construct, animals were treated with carrier control or 2 different concentrations (33 and 100 mg/kg) of Lamivudine (3TC) and Adefovir (PMEA) each day by IP injection. Eleven days after injection, the livers of the mice were harvested, EPA assays were performed, and the radioactive incorporation was measured. The activity of the carrier control treated group was set to 100%, and the relative activity of treated animals was calculated accordingly.
Gastroenterology  , 9-18DOI: ( /S (03) )

5. Figure 4
An S-target siRNA blocks HBV gene expression in vivo. (A and B) The S-target or the scrambled siRNA (control) was coinjected with the pHBV 1.5 construct. At different days after injection, HBsAg (A ) or HBeAg (B) expression was measured in the serum of the animals. The results of at least 3 mice are shown per time point. Maximal serum HBsAg and HBeAg expression was set to 100%, and the relative changes were calculated accordingly. (C ) Liver RNA was prepared 1 and 3 days after injecting the control vector (pBS = negative control) and pHBV 1.5 with or without 6.5 nmol S-target. After extensive DNase digestion, a triplex RT-PCR was performed with primers detecting the pre-C/C and S RNA in comparison with the GAPDH signal. The positions of the GAPDH-, S-, and C-specific signals are indicated.
Gastroenterology  , 9-18DOI: ( /S (03) )

6. Figure 5
A C-target siRNA blocks HBV gene expression in vivo. (A and B) The C-target or the scrambled siRNA (control) was coinjected with the pHBV 1.5 construct. At different days after injection, HBsAg (A ) or HBeAg (B) expression was measured in the serum of the animals. The results of at least 3 mice are shown per time point. Maximal serum HBsAg and HBeAg expression was set to 100%, and the relative changes were calculated accordingly. (C ) Liver RNA was prepared 2 days after injecting the control vector (pBS = negative control) and pHBV 1.5 with or without 6.5 nmol C-target. After extensive DNase digestion, a triplex RT-PCR was performed with primers detecting the pre-C/C and S RNA in comparison with the GAPDH signal. The positions of the GAPDH-, S-, and C-specific signals are indicated.
Gastroenterology  , 9-18DOI: ( /S (03) )