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发表于 2004-8-24 23:53
Transient Infection of Woodchucks with WHV. Adult woodchucks (Marmota monax) were housed in the Laboratory Animal Facility of the Fox Chase Cancer Center. All experiments carried out with these woodchucks were reviewed and approved by the center''''s Institutional Animal Care and Use Committee. At 7–8 months of age, three woodchucks were subjected to liver wedge biopsy as described (9, 22), and, 4 weeks later, they were inoculated i.v. with 2 ml of WHV-positive serum from a chronically infected woodchuck (WHV titer, 109 per ml). A control woodchuck was inoculated with 2 ml of PBS. Serum was collected weekly, and liver biopsy samples were collected at 4, 8, 12, and 16 weeks postinoculation (p.i.). Aliquots were stored at –80°C until use. Portions of each liver biopsy were also fixed with formalin, for histological analysis after staining with hematoxylin and eosin or ethanol/acetic acid (3:1) (9) for immunohistochemistry and in situ hybridization.
Nucleic Acid Analyses of Woodchuck Serum. Viral titers were determined by Southern blot hybridization assays (23, 24). Fifty microliters of woodchuck serum was layered on a 10–20% sucrose step-gradient containing 0.15 M NaCl and 20 mM Tris·HCl (pH 7.5). Virus was collected by centrifugation for 3 h at 50,000 rpm and 4°C in a Beckman SW 60 rotor, and the pellet was digested with 30 µl of 0.01 M Tris·HCl (pH 7.4), 0.01 M EDTA, 0.2% (wt/vol) SDS, and 1 mg/ml Pronase for 1 h at 37°C. The mixture was then subjected to electrophoresis into a 1.5% agarose gel and subsequent Southern blotting to nitrocellulose sheets. A 32P-labeled DNA probe representing the complete WHV genome was used for hybridization. Signals were quantified by using a Fuji phosphorimager, and virus titers were estimated by reference to a WHV DNA standard.
Sorbital Dehydrogenase (SDH) Assay. SDH levels in woodchuck serum, an indicator of liver injury (25), were determined by Ani-lytics (Gaithersburg, MD). Concentrations are expressed in international units per liter.
Immunohistochemistry and in Situ Hybridization. Immunoperoxidase assays for detection of WHV core antigen, proliferating cell nuclear antigen (PCNA), and CD3 were carried out on acetic acid/ethanol-fixed and paraffin-embedded tissues as described (10). Woodchuck CD3 was detected by using a rabbit polyclonal anti-human CD3 epsilon chain antiserum (DAKO), and PCNA was detected with mouse monoclonal anti-PCNA antibodies (DAKO). WHV core antigen was detected by using an antiserum raised in rabbits to purified recombinant WHV core antigen (10). In situ hybridization for WHV nucleic acids was performed on ethanol/acetic acid-fixed tissues by use of a nonstrand-specific digoxygenin-dUTP-labeled DNA probe representing the complete WHV genome (26, 27).
Extraction of Viral and Cellular DNA from Liver. For analysis of replicating and cccDNAs, liver tissue (20–50 mg) was homogenized in 1.5 ml of TE (10 mM Tris·HCl, pH 7.5/1 mM NaEDTA). For extraction of replicative intermediates, half the homogenate was adjusted to a total volume of 6 ml with 0.025 M Tris·HCl (pH 7.4), 0.01 M EDTA, 0.25% (wt/vol) SDS, 0.05 M NaCl, and 2 mg/ml Pronase and incubated for 1 h at 37°C. Nucleic acids were then extracted with a 1:1 mixture of phenol/chloroform and collected by ethanol precipitation. cccDNA was extracted as described (28). Briefly, the other half of the homogenate was adjusted to a volume of 3 ml with 0.01 M Tris·HCl (pH 7.5) and 0.01 M EDTA. Two hundred microliters of 10% (wt/vol) SDS was added, the mixture was briefly vortex mixed, and 1 ml of 2.5 M KCl was added. After incubation at room temperature for 20 min, potassium dodecyl sulfate protein complexes were collected by centrifugation at 10,000 rpm for 20 min in a Sorvall SS34 rotor at 4°C. The supernatant was subjected to phenol/chloroform extraction. Nucleic acids were precipitated with ethanol overnight at room temperature. Southern blot hybridization analysis was then carried out as described (9). To quantify relative amounts of replicative intermediates, the amount of cell DNA was determined by fluorimetry (9). To quantify cccDNA, DNA extracted from a predetermined number of cells was assayed, as assessed by counting in a hemocytometer of fluorescent nuclei in the initial cell homogenates after staining with ethidium bromide.
For quantification of integrated viral DNA, the potassium dodecyl sulfate pellet was suspended in 0.4 ml of TE (10 mM Tris·HCl, pH 7.5/1 mM NaEDTA) and dissolved by incubation at 55°C with frequent vortex mixing. The solution was adjusted to 50 mM with NaCl and extracted two times with an equal volume of phenol and precipitated with ethanol. DNA concentrations were determined by fluorimetry with SYBR Green dye.
For the isolation of cellular DNA from small liver fragments, frozen liver (1 mg) was homogenized in 0.5 ml of TE at 0°C, and 0.01 ml was removed for counting the total number of nuclei. The remaining sample was adjusted to 0.2 M NaCl by the addition of 0.4 ml of 0.4 M NaCl and incubation at 0°C for 15 min. Nuclei were pelleted by microfuge centrifugation, and the supernatant fluid was discarded. The pellet was resuspended in TE, adjusted to 0.2% Triton X-100, 0.2% SDS, and 500 µg/ml proteinase K. The sample was incubated for 2 h at 55°C with frequent vortex mixing and then overnight at 37°C, adjusted to 50 mM NaCl, extracted twice with phenol/chloroform (1:1 vol/vol), ethanol-precipitated, and dissolved in TE.
PCR Primers and Linkers. A nonphosphorylated linker was designed to ligate unidirectionally to blunt ends of cellular DNA generated by PvuII so that the recognition site would be regenerated. The strand intended to ligate to the 5'''' phosphate of the PvuII site, LNK-D, consisted of the sequence 5''''-TGCCCTCGTCCCTAATGCAG, and this oligonucleotide was also used as the linker-specific primer in amplification of viral cell junctions. The complementary strand of the linker was 5''''-CTGCATTAGGGA. The resulting partial duplex of the double-stranded linker contained one blunt end with a PvuII half site (.CAG-3'''') capable of ligating to the 5''''-P-CTG. of the PvuII blunt ends of cellular DNA. At the other end of the linker, the 3'''' OH was recessed and unable to ligate to a PvuII blunt end.
The viral-specific primer, WHV2250–2224, was used with the linker-specific primer, LNK-D, to amplify viral cell junctions. This oligonucleotide primed DNA synthesis in the minus direction on the WHV genome, toward the left end limits of viral cell junctions, into cellular DNA toward the ligated linker. The S1 primer set consisted of primers WHV2185–2167 and WHV2195–2216, and the nested S2 primer set consisted of primers WHV2165–2145 and WHV2217–2245. All viral-specific primers are designated by WHV followed by the inclusive nucleotides (5''''-3'''' direction) numbered according to the sequence of Galibert et al. (29).
Assays for Integrated DNA. The standard quantitative assay for integrated DNA measured the detection of viral cell junctions from a known number of cells, calculated from the amount of cellular DNA used in the assay. Specifically, we detected the left end junctions, which are those in which cellular sequences were joined to the left end of a viral DNA sequence oriented left to right by increasing nucleotide number. Left end junctions are clustered downstream of nucleotide position 1935 (see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org). Viral cell junctions were detected by a procedure that involved sequential application of ligation-mediated PCR and inverse nested PCR. The steps are outlined in Fig. 1.
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