Mice conditionally expressing HCV core gene were generated as described [9
]. Briefly, the HCV core gene (genotype 1b) was cloned into PUGH10-3 downstream of the tetracycline response element (TRE) [19
]. Fertilized ova from FVB/N mice were injected with the construct. Founder mice were crossed with a second transgenic FVB/N line that is homozygous for the tetracycline transactivator (tTA) under control of the liver-enriched activating protein (LAP) promoter [18
]. Unless otherwise indicated, mating pairs were maintained on doxycycline (DOX)-containing chow (200 mg/kg; Bio-Serve, Frenchtown, NJ) for suppression of HCV-core during development and through weaning. At approximately one month of age, DOX was withdrawn. Core expression was evaluated by liver biopsies taken at two months after birth. Five rearing designs (five mice for each rearing design) were used for the control mice to specify the HCV core protein effect: 1) double transgenic mice (DTM) that express both the HCV core and tTA were always fed DOX-containing chow; 2) other DTM were maintained on normal chow (i
., no DOX); 3) single transgenic mice (STM) that express tTA only were fed DOX-containing chow until 1 month of age; 4), STM were maintained on DOX-containing chow all the time; and 5), other STM were maintained on normal chow. All mice were kept in specific pathogen-free rooms with regular quarantine. Only those that were serum-negative for common pathogen and viral markers were used in the experiments. Those with infectious agents, such as mouse hepatitis virus, mouse parvovirus, minute virus of mice, Reovirus-3, pneumonia virus of mice, epizootic diarrhea of infant mice, Theiler's murine encephalomyelitis virus, lymphocytic choriomeningitis virus, ectromelia, Sendai virus, sialodacryoadenitis virus, mycoplasma pulmonis, pinworms and fur mites, were excluded. The protocol was approved by Animal Care and Use Committee at Chang Gung Memorial Medical Center.
Evaluation of the in situ HCV core protein level
The HCV core protein level was examined in situ in a wedge biopsy of liver, kidney, thymus, spleen, omentum, skin, heart, muscle, intestine, and lung. All biopsy specimens were obtained with the 2-month-old mice under isoflurane anesthesia. A portion of each organ was used for protein extraction and Western blot analysis, while the remainder was processed for histological study. HeLa cells carrying the same LAP-tTA (Clontech, Mountain View, CA) were transiently transfected with the TRE-HCV core construct in the absence of DOX. HeLa cell or mouse liver protein extracts were analyzed by Western blotting. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and incubated with a 1:250 dilution of monoclonal mouse anti-HCV core antibody (Anogen, Mississauga, Ontario, Canada). After washes, membranes were incubated with secondary antibody (Bio-Rad), and developed using an ECL kit (Amersham, Buckinghamshire, UK). HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, UK) was used as an internal control.
For immunohistochemical analysis, tissue samples were harvested, fixed in 4% paraformaldehyde, treated with 0.1% Triton X-100, incubated with HCV core protein antibody, and subsequently incubated with secondary antibody (Caltag, Carlsbad, CA). The background staining was eliminated by a mouse-on-mouse kit (Vector, Burlingame, CA).
Evaluation of fatty liver
The oil-red-O stain for hepatic fat visualization was done with a commercial kit (Biogenex, San Francisco, CA).
Evaluation of hepatic injury
For immunohistology, liver tissue samples were harvested and immediately fixed as described above for 12–24 h. Antibodies for fibronectin (Labvision, Fremont, CA), collagen (Biogenesis, Raleigh, NC), and alpha-smooth muscle actin (α-SMA) (Dako, Glostrup, Denmark) were applied followed by the respective second antibodies. The nuclei were counter-stained with methylene blue (Dako).
ALT was measured in tail blood using a Vitros DT60 II chemistry system (Johnson&Johnson, Rochester, NY). Frozen liver samples were homogenized and used to detect MDA by the thiobarbituric acid method (ZeptoMetrix, Buffalo, NY).
Paired liver samples were studied in three experiments. In the first experiment (I), three DTM with high core expression were compared to three STM on the same diet regimen (DOX-containing chow until 1 month of age and then the permissive diet). In the second experiment (II), three DTM with intermediate core expression were compared to three STM on the aforementioned diet. In the third experiment (III), three DTM with intermediate core expression were compared to three DTM with high core expression using the same diet regimen. For all the experiments, the RNA were extracted from the livers of five pairs of 2-month-old female DTM versus 2-month-old female STM; and five pairs of 2-month-old male DTM versus 2-month-old male STM.
Total RNA was extracted and quantified as described previously [17
]. 0.5 μg of total RNA was amplified by a fluorescent linear amplification kit (Agilent Technology) and labeled with Cy3-CTP or Cy5-CTP (CyDye; PerkinElmer, Waltham, MA) during the in vitro transcription process. RNA from the experimental mice was labeled with Cy5, and RNA from control mice was labeled with Cy3. Microarray analysis was performed according to a previously described protocol [17
]. The microarray data have been deposited in the following website: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16403
(accession number: GSE16403).
The 30 genes with a known function and significantly increased or decreased expression from both experiments II and III (i.e., more than 1.8-fold in comparison with the control groups) were analyzed by Q-RT-PCR using the same RNA used for the microarray analyses. To prepare a cDNA pool from each RNA sample, total RNA (5 μg) was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega, Madison, WI). Specific oligonucleotide primer pairs (appendix 1) selected from the Roche Universal Probe Library (Roche Corp, Castle Hill, Australia) were used for Q-RT-PCR. The specificity of each primer pair was tested using common reference RNA (Stratagene, La Jolla, CA) as the DNA template to perform Q-RT-PCR, followed by a DNA 500 chip run on a Bioanalyzer 2100 (Agilent Technology) to check the size of the PCR product.
Q-RT-PCR was performed on a Roche LightCycler 1.5 using the LightCycler®
FastStart DNA MasterPLUS SYBR Green I kit (Roche) as described previously [17
Injection of CD55 in transgenic mice with hepatic inflammation
Since the half-life of recombinant CD55 in vivo
is around 24 hours and the recommended dose for rodents is 10 mg/kg [20
], 250 μg of CD55 (R&D Systems, Minneapolis, MN) in 2.5 mL of phosphate-buffered saline were injected daily into the tail veins of five 2-month-old DTM with intermediate core expression. In our pilot study, serum ALT significantly decreased in the DTM after three daily injections (unpublished data). ALT assays and liver biopsies were performed before and 3 days after injection. Controls consisted of five 2-month-old DTM with intermediate core expression injected with the phosphate-buffered saline vehicle.
Repeated measure analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons were used to examine the time trends and group differences. Normalized microarray data were further filtered for missing genes and for genes exhibiting low expression levels. Significantly regulated genes (P < 0.05) represented expression levels that differed by at least 1.8. Considering multiple comparisons, the adjusted P values for the Benjamini and Hochberg method (false discovery rates; FDR) were also calculated for the selected genes, and an adjusted P value of less than 0.05 was chosen for subsequent functional and pathway exploration. Analyses were accomplished using the SAS 8.0 statistical package (SAS Institute, Cary, NC), and P < 0.05 was considered statistically significance.
Sex bias analysis was performed using the SAM [21
], and P < 0.05 represented statistical significance.
Generic slim in GeneSpring version 7.3.1 (Agilent Technologies) was used for the functional category classification (Student's t
-test was used in gene ontology (GO)). Potential pathways involved in fibrosing steatohepatitis were investigated using a web-based service (ArrayXPath; http://www.snubi.org/software/ArrayXPath/
), where Fisher's exact test and the FDR following Storey's scheme were applied to evaluate the statistical significance.