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Differential early gene expression in HBV X protein (HBx)-mediated hepatocarcinogenesis.

by Ray, Kit Ng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 112-121). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.iv / Abbreviations --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Hepatitis B Virus X Protein (HBx) --- p.5 / Chapter 1.2.1 --- The Genomic Structure of HBx --- p.5 / Chapter 1.2.2 --- The HBx Protein Structure --- p.6 / Chapter 1.2.3 --- Subcellular Localization of HBx --- p.7 / Chapter 1.2.4 --- Possible Functions of HBx --- p.8 / Chapter 1.3 --- Etiology of Hepatocellular Carcinoma (HCC) --- p.12 / Chapter 1.4 --- Relationship between HCC and HBx --- p.13 / Chapter 1.5 --- Aims of Study --- p.14 / Chapter 1.6 --- The Basis of Tet-On System --- p.15 / Chapter 1.7 --- The Basis of DNA Microarray --- p.18 / Chapter 1.8 --- The Basis of Two-Dimensional Electrophoresis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of a Tet-On HBx Expressing Cell Model --- p.22 / Chapter 2.1.1 --- Cloning of HBx Gene into pTRE2 Vector --- p.22 / Chapter 2.1.1.1 --- PCR of HBx Gene --- p.22 / Chapter 2.1.1.2 --- Purification of the PCR Product --- p.23 / Chapter 2.1.1.3 --- Restriction Enzyme Digestion --- p.23 / Chapter 2.1.1.4 --- Ligation of HBx into pTRE Vector --- p.24 / Chapter 2.1.1.5 --- Transformation of the Ligation Product into Competent Cells --- p.24 / Chapter 2.1.2 --- Preparation of the Plasmid DNA --- p.24 / Chapter 2.1.2.1 --- DNA Sequencing of the Cloned Plasmid DNA --- p.25 / Chapter 2.1.3 --- Cell Culture of AML12 Cell Line --- p.26 / Chapter 2.1.4 --- Transfection of pTet-On Vector into AML12 Cells --- p.26 / Chapter 2.1.5 --- Selection of the Transfected AML12 Cells by G418 --- p.27 / Chapter 2.1.6 --- Single Clone Isolation --- p.27 / Chapter 2.1.6.1 --- Luciferase Assay for Selection of Highly Inducible Clones --- p.28 / Chapter 2.1.7 --- Second Transfection of pTRE-HBx Plasmid --- p.28 / Chapter 2.1.8 --- Selection of the Transfected Cells by Hygromycin --- p.29 / Chapter 2.1.9 --- Second Single Clone Isolation --- p.29 / Chapter 2.1.10 --- Total RNA Isolation --- p.29 / Chapter 2.1.11 --- DNase I Digestion --- p.30 / Chapter 2.1.12 --- First-Strand cDNA Synthesis --- p.31 / Chapter 2.1.13 --- RT-PCR of HBx Gene --- p.31 / Chapter 2.1.14 --- Northern Blotting --- p.32 / Chapter 2.1.15 --- Preparation of the Probe --- p.33 / Chapter 2.1.16 --- Northern Blot Hybridization --- p.33 / Chapter 2.1.17 --- 3H-Thymidine Incorporation Assay --- p.34 / Chapter 2.1.18 --- Analysis of Cell Cycle by Flow Cytometry --- p.35 / Chapter 2.2 --- Microarray Analysis of Differential Gene Expression upon HBx Induction --- p.35 / Chapter 2.2.1 --- Sample Preparation for Microarray Analysis --- p.35 / Chapter 2.2.2 --- Probe Labelling --- p.36 / Chapter 2.2.3 --- Microarray Hybridization --- p.37 / Chapter 2.2.4 --- RT-PCR of the Candidate Genes --- p.38 / Chapter 2.2.5 --- Northern Blot Analysis of the Candidate Genes --- p.39 / Chapter 2.3 --- Two-Dimensional (2D) Gel Electrophoretic Analysis --- p.40 / Chapter 2.3.1 --- Protein Sample Preparation for 2D Gel Electrophoresis --- p.40 / Chapter 2.3.2 --- First-Dimension Isoelectric Focusing (IEF) --- p.40 / Chapter 2.3.3 --- Second-Dimension SDS-PAGE --- p.41 / Chapter 2.3.4 --- Silver Stain of 2D Gel --- p.42 / Chapter 2.3.5 --- Mass Spectroscopic Analysis --- p.43 / Chapter 2.4 --- Subcellular Localization of HBx --- p.44 / Chapter 2.4.1 --- Cloning of HBx into Green Fluorescent Protein (GFP) Expression Vector --- p.44 / Chapter 2.4.2 --- Transfection of GFP-HBx --- p.44 / Chapter 2.4.3 --- Propidium Iodide (PI) Staining --- p.45 / Chapter 2.4.4 --- Mitochondria Staining --- p.45 / Chapter 2.4.5 --- Subcellular Localization Study using Epi-Fluorescent Microscopy --- p.45 / Chapter 2.5 --- Analysis of Mitochondrial Transmembrane Potential --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of Tet-On AML12 Cell Line of HBx Gene --- p.47 / Chapter 3.2 --- Characterization of the HBx-Expressing Cell Model --- p.53 / Chapter 3.2.1 --- 3H-Thymidine Proliferation Assay --- p.53 / Chapter 3.2.2 --- Cell Cycle Analysis --- p.55 / Chapter 3.3 --- Microarray Analysis of Differential Gene Expression Pattern upon HBx Induction --- p.57 / Chapter 3.4 --- Northern Blot Analysis and RT-PCR of the Candidate Genes --- p.65 / Chapter 3.5 --- Differential Protein Expression Pattern under HBx Induction --- p.70 / Chapter 3.6 --- Subcellular Localization of HBx --- p.77 / Chapter 3.7 --- Analysis of Mitochondrial Transmembrane Potential --- p.83 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Conditional HBx-Expressing Cell Model --- p.84 / Chapter 4.2 --- The Effects of HBx in Clone X18 --- p.86 / Chapter 4.2.1 --- Proliferative Effect of HBx --- p.86 / Chapter 4.2.2 --- Deregulation of G2/M Checkpoint by HBx --- p.86 / Chapter 4.3 --- Early Differential Gene Expression due to HBx Induction --- p.88 / Chapter 4.4 --- The Relationship of the Potential Candidate Genes and Cancer Development --- p.90 / Chapter 4.5 --- The Protein Expression Pattern due to HBx Induction --- p.93 / Chapter 4.6 --- The Subcellular Localization of HBx --- p.96 / Chapter 4.7 --- The Possible Involvement of HBx in Mitochondrial Transmembrane Potential --- p.98 / Chapter 4.8 --- Conclusions --- p.101 / Chapter 4.9 --- Future Prospects --- p.104 / Appendix --- p.107 / References --- p.112

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323892
Date January 2002
ContributorsNg, Ray Kit., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiv, 121 leaves : ill. (chiefly col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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