2.1.2 Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line

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1 i 1 INTRODUCTION 1.1 Human Hepatitis B virus (HBV) Pathogenesis of Hepatitis B Genome organization of HBV Structure of HBV virion HBV life cycle Experimental models for the study and therapy of HBV infection Cytokines as mediator immune modulation and antiviral responses Large family of interferons Type I interferon Type II interferon Induction of interferon expression Interferon-inducible intracellular mechanisms as antiviral response Adenovirus as vector for gene transfer Pathogenesis of adenoviral infection Structure and genome organization of Adenovirus Adenovirus life cycle Recombinant Adenovirus type 5 as vector for gene transfer Tetracycline-based inducible system: the Tet-off and Tet-on system Aims of study 19 2 RESULTS Suppression of HBV replication following cytokine gene transfer Expression and Secretion of cytokine following adenoviral transduction Detection of secretion of mifnγ using mifnγ-specific ELISA Indirect detection of secretion of mifnα and mifnβ by induction of 23 2,5 -oligoadenylate synthetase (2 5 OAS) transcription Indirect measurement of secretion of interferon by detection of induction of IP10 transcription in vitro and in vivo Characterization of antiviral effect of cytokine expression on HBV replication in transduced mouse hepatocytes line Characterization of the antiviral effect of cytokine expression on HBV replication in HBV transgenic mice Antiviral effect on HBV replication dependent on the dose of the adenoviral vector

2 ii Kinetics of an antiviral effect on HBV replication Time course of constitutive expression of cytokine in vivo Effect on HBV replication following gene transfer for tetregulatable and liver specific cytokine expression Establishment of a system allowing regulatable, liver specifc gene expression using adenoviral vectors Establishment of the Adbiluc system in vitro Basal expression of luciferase by Adbiluc in primary human 43 hepapotocytes Gene expression in hepatoma HuH7 cells expressing the reverse 44 tet-transactivator Adenovirus mediated rtta expression Tet-inducible luciferase expression using an one-component 48 vector system Establishment of the Adbiluc system in vivo Induction of luciferase expression in the liver of tta transgenic 49 mice Kinetics of luciferase activity in living tta or rtta transgenic 51 mice Co-expression of two marker genes in liver cells Time course of cytokine and luciferase expression following 55 gene transfer in rtta transgenic mice Antiviral effect of tet-inducible cytokine gene transfer on HBV replication in HBV transgenic mice, a model for chronic infection H1.3/rtTA double transgenic mouse as model for chronic 57 infection Effect on HBV replication in TetH1.3 transgenic mice Tet-inducible cytokine expression in rtta transgenic mice infected with AdGH1.3, a model for acute HBV infection DISCUSSION Establishment of experimental mouse models In vitro and in vivo models for HBV replication Efficiency of adenoviral-mediated gene transfer into the liver of mice 67

3 iii 3.2 A liver specific tetracycline-regulated gene expression via adenoviral gene transfer in mice Novel option to study hepatitis B virus infection Is the cytokine expression a good strategy for antiviral approach on HBV 73 infection? Antiviral effects of each cytokine on HBV replication Immune response to the adenoviral vector in vivo Futures perspectives 83 4 MATERIALS and METHODS Material Instruments Important chemicals, solutions and buffers Molecular weight markers Cell types and characteristics Bacteria strains Mice types Antibodies Oligonucleotids DNA probes Methods Methods for molecular analysis Quick plasmid DNA mini-preparation Preparation of large amounts of plasmid DNA Ethanol and Isopropanol DNA precipitation Cleavage of DNA using restriction enzymes Quick isolation or purification of DNA-fragments Remove the 5 end phosphate of DNA-fragment by alkaline 93 phosphatase The polymerase chain reaction (PCR) Real-time PCR by Light Cycler Ligation of DNA fragments into vectors Generation of blunt ends in digested plasmid 95

4 iv Rubidium Chloride method for preparing transformation 95 competent E. coli Bacteria transformation by heath stock Production of transformation electro-competent bacteria Transformation of electro-competent bacteria Biological methods Cytotoxicity staining assay by crystal violet staining In vivo monitoring of luciferase activity Preparation of avertin narcose solution Calcium phosphate transfection of plasmid in cell culture Methods for biochemical analysis Electrophoretic separation of DNA on agarose gel DNA extraction with cell lysate or tissue by phenol/chloroform 98 extraction DNA extraction with tail of transgenic mouse for PCR screening DNA extraction from blood or serum of mouse for real-time 99 PCR Preparation of nuclease-free water Extraction of total RNA from cell lysate or tissue by 99 TRIZOL LS methods Extraction of mrna from total RNA by Oligotex TM kit Western Blot analysis Southern Blot analysis Dot Blot analysis Northern Blot analysis Luciferase activity assay Protein determination of Bradford methods Murine IFNγ specific ELISA HBsAg and HBeAg specific ELISA Titration of alanine transaminase (ALT) Cell culture Cell culture medium Passage of cells Infection of cells with adenovirus Preparation of primary mouse hepatocytes Adenovirus generation and production Constructing recombinant Shuttle plasmid Generation of recombinant adenoviral DNA Production recombinant Adenovirus Production of high titer recombinant Adenovirus stocks 110

5 v Titration of adenovirus stocks by GFP expression and/or 110 cytopathic effect Titration of adenovirus stocks by immunofluorescence staining Titration of adenovirus stocks by OD measure at 260nm reading Cloning and generation of adenoviral vectors AdShuttle AdmIFNγ or AdvIL AdGmIFNβ AdGFP AdTTR-rtTA Adbiluc and Adbiluc Adbiluc-GFP Adbiluc-mIFNγ Adbiluc-mIFNα Adbiluc-mIFNβ Adbiluc-rtTA and Adbiluc2-rtTA SUMMARY APPENDIX Abbreviations References Curriculum vitae Acknowledgement 140

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