Evaluation of Transcriptional Efficiency of Hepatitis B Virus Covalently Closed Circular DNA by Reverse Transcription-PCR Combined with the Restriction Enzyme Digestion Method

Chou, Yi-Chun; Jeng, King‐Song; Chen, Mong Liang; Liu, Hsiao Hui; Liu, Tzu Ling; Chen, Ya Ling; Liu, Yu Chih; Hu, Cheng; Chang, Chung-Ming

doi:10.1128/jvi.79.3.1813-1823.2005

Cited by 49 publications

(59 citation statements)

References 45 publications

(45 reference statements)

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“…36 In our previous studies (reference 37 and unpublished data), we examined a number of siRNAs and shRNAs that target various HBV transcripts ( Figure 1a) for their abilities to suppress HBV replication and gene expression in cultured cells and in mice that were co-transfected with the plasmid pHBV1.3 containing a 1.3-fold HBV genome. 38 Three of the shRNAs (HBV-B4, -C, -S1) showed better HBV inhibition effect than the others. To examine whether the three shRNAs are effective in intervening more clinically relevant hepatitis B infection, we investigated their effects in HBV transgenic mice, which produce 1-10 Â 10 8 HBV virions/ml of serum, a level comparable to or higher than that found in the serum of patients with chronic active hepatitis B infection.…”

Section: Hbv Inhibition By Shrna In Cultured Cellsmentioning

confidence: 99%

“…HepG2 cells were co-transfected with 2 mg of pHBV1.3 plasmid containing a 1.3-fold overlength HBV genome (ayw subtype) 38 and 2 mg of various pAAVEMBL plasmids using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in 24-well plates. The amount of hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) in the supernatant was analyzed 3 days later by enzyme-linked immunosorbent assay.…”

Section: Co-transfection In Vitromentioning

confidence: 99%

“…Total DNA and RNA were extracted from liver tissues and examined for the presence of HBV DNA and RNA by Southern and Northern blotting, respectively. 38 The signals were quantified using ImageQuant software (Amersham Biosciences, Piscataway, NJ, USA). HBcAg was detected by immunohistochemical staining of paraffin-embedded tissues.…”

Section: Hbv Transgenic Mice and Viral Vector Deliverymentioning

confidence: 99%

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Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

Chen

et al. 2006

Gene Ther

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RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAibased therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log 10 decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.

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Section: Hbv Inhibition By Shrna In Cultured Cellsmentioning

confidence: 99%

Section: Co-transfection In Vitromentioning

confidence: 99%

Section: Hbv Transgenic Mice and Viral Vector Deliverymentioning

confidence: 99%

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Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

Chen

et al. 2006

Gene Ther

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“…The membranes were cross-linked using an ultraviolet cross-linker (Stratagene) and hybridized with a-32 P-radiolabeled DNA probes (2 Â 10 8 counts/min/AL) produced by a random prime labeling system (rediprime II) (Amersham Biosciences). Hybridization and washing conditions were the same as previously (29) and the membranes were then exposed to X-ray film at -70jC. To confirm the amounts of total RNA loaded in each lane, the membranes were hybridized afterward with an 18S ribosomal RNA gene, which served as an internal control.…”

Section: Cancer Epidemiology Biomarkers and Prevention Cancer Epidemiomentioning

confidence: 99%

Decreased Expression of UK114 Is Related to the Differentiation Status of Human Hepatocellular Carcinoma

Chong

Huang²,

et al. 2008

Cancer Epidemiology, Biomarkers &Amp; Prevention

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Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples. (Cancer Epidemiol Biomarkers Prev 2008;17(3):535 -42)

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“…13 These mice contain a 1.3-fold HBV genome (genotype D, serotype ayw) expressing HBV pregenomic and messenger RNAs under the control of the promoter and enhancer regulatory elements of the virus. 40 The Tg[HBV1.3]24-3 line was used in this study, as it produces the highest and most stable HBV titers. ICR/HBV mice were maintained in the hemizygous state by mating with wild-type ICR mice purchased from BioLASCO Taiwan Co. (Ilan, Taiwan).…”

Section: Materials and Methods Animalsmentioning

confidence: 99%

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus

Chang

Sun

et al. 2011

Gene Ther

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Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

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Evaluation of Transcriptional Efficiency of Hepatitis B Virus Covalently Closed Circular DNA by Reverse Transcription-PCR Combined with the Restriction Enzyme Digestion Method

Cited by 49 publications

References 45 publications

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA

Decreased Expression of UK114 Is Related to the Differentiation Status of Human Hepatocellular Carcinoma

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus

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