Sia Sin Fun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 105-121). / Abstracts in English and Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vi / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xv / LIST OF TABLES --- p.xvi / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- The interferon / Chapter 1.1.1 --- Classification of interferons --- p.1 / Chapter 1.1.1.1 --- Type IIFN --- p.2 / Chapter 1.1.1.2 --- Type II IFN --- p.3 / Chapter 1.1.2 --- Biosynthesis of IFN --- p.3 / Chapter 1.1.3 --- IFN-α/β receptor and signal transduction --- p.8 / Chapter 1.1.4 --- Functions induced by IFN / Chapter 1.1.4.1 --- Antiviral activity of IFN-α/β --- p.11 / Chapter 1.1.4.1.1 --- PKR (double-stranded RNA-dependent protein kinase) --- p.11 / Chapter 1.1.4.1.2 --- The 2-5A synthetase/RNase L system (The 2-5A system) --- p.16 / Chapter 1.1.4.1.3 --- Mx proteins --- p.17 / Chapter 1.1.4.2 --- Immunomodulatory function of IFN-α/β --- p.18 / Chapter 1.1.4.3 --- Inhibition of cell growth --- p.18 / Chapter 1.1.4.4 --- Control of apoptosis --- p.19 / Chapter 1.1.5 --- The significance of IFN system --- p.20 / Chapter 1.1.6 --- Subtype of murine IFN-α --- p.21 / Chapter 1.1.7 --- Production of IFN in response to infection --- p.23 / Chapter 1.1.8 --- Existing methods to determine the IFN-α subtypes productionin response to stimulus --- p.24 / Chapter 1.2 --- Influenza virus --- p.27 / Chapter 1.2.1 --- Classification --- p.27 / Chapter 1.2.2 --- The structure of influenza virus --- p.29 / Chapter 1.2.3 --- The viral genome and proteins --- p.29 / Chapter 1.2.4 --- Replicative cycle of influenza virus / Chapter 1.2.4.1 --- "Virus adsorption, entry and uncoating" --- p.31 / Chapter 1.2.4.2 --- Transcription and replication of vRNA --- p.31 / Chapter 1.2.4.3 --- Synthesis of viral proteins --- p.32 / Chapter 1.2.4.4 --- Packaging and budding of progeny virus --- p.33 / Chapter 1.2.5 --- Viral inhibition of the IFN response --- p.33 / Chapter 1.3 --- Aim of study --- p.35 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Overall procedures --- p.37 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- "Cell line, bacterial strain and vector" --- p.40 / Chapter 2.2.2 --- Chemicals --- p.40 / Chapter 2.2.3 --- "Culture media, buffer and other solutions" --- p.41 / Chapter 2.2.4 --- Reagents and nucleic acids --- p.41 / Chapter 2.2.5 --- Reaction kits --- p.42 / Chapter 2.2.6 --- Solutions --- p.42 / Chapter 2.2.7 --- Solutions of reaction kits --- p.43 / Chapter 2.2.8 --- Major equipments --- p.44 / Chapter 2.2.9 --- Primers --- p.44 / Chapter 2.2.10 --- Cell lysate --- p.45 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Design of IFN-α whole coding region and subtype specific primers using OLIGO´ёØ ver 50 --- p.46 / Chapter 2.3.2 --- Construction of plasmid DNA with the whole coding region of IFN-α gene / Chapter 2.3.2.1 --- Isolation of genomic DNA from L929 cells --- p.46 / Chapter 2.3.2.2 --- Amplification of whole coding region of IFN-α subtypes --- p.47 / Chapter 2.3.2.3 --- Preparation of plasmid DNA --- p.48 / Chapter 2.3.2.4 --- Restriction digestion of the vector pBluescript SKII (-) with SmaI --- p.48 / Chapter 2.3.2.5 --- Purification of DNA fragments from agarose gel --- p.49 / Chapter 2.3.2.6 --- Blunt end ligation --- p.49 / Chapter 2.3.2.7 --- Transformation --- p.49 / Chapter 2.3.2.8 --- Screening by polymerase chain reaction --- p.50 / Chapter 2.3.2.9 --- DNA sequencing --- p.50 / Chapter 2.3.3 --- Test on IFN-α subtype specific primers / Chapter 2.3.3.1 --- Determination of the specificity of IFN-α subtype specific primers by PCR --- p.51 / Chapter 2.3.3.2 --- Determination of the amplification of a particular subtype in the presence of excess of other templates --- p.51 / Chapter 2.3.4 --- Induction of expression of IFN in fibroblast L929 cells / Chapter 2.3.4.1 --- By chemical agents Poly(I) Poly(C) and DEAE --- p.52 / Chapter 2.3.4.2 --- By infection with influenza virus (A/NWS/33 and B/Lee/40) --- p.52 / Chapter 2.3.5 --- Detection of IFN-α subtypes expression / Chapter 2.3.5.1 --- Isolation of total RNA by guandidium thiocyanate - cesium chloride ultracentrifugation --- p.53 / Chapter 2.3.5.2 --- Synthesis of first strand cDNA --- p.54 / Chapter 2.3.5.3 --- Normalization of RNA samples --- p.54 / Chapter 2.3.5.4 --- RT-PCR amplification of the IFN-α subtypes --- p.54 / Chapter 2.3.5.5 --- "RT-PCR amplification of IFN-γ, IFN-α receptor 1，IFN-α receptor 2 (transmembrane and soluble form) and IFN-(3" --- p.55 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Designing of primers for IFN-α genes --- p.56 / Chapter 3.1.1 --- Design of IFN-α primers for amplification of whole coding region --- p.56 / Chapter 3.1.2 --- Design of IFN-α subtype specific primers --- p.58 / Chapter 3.2 --- Cloning of the IFN-α subtype genes / Chapter 3.2.1 --- Purification of genomic DNA --- p.59 / Chapter 3.2.2 --- PCR conditions used for amplification of whole coding region of mIFN-α subtypes --- p.61 / Chapter 3.2.3 --- Subcloning the whole coding region of IFN-α genes from amplified fragments --- p.63 / Chapter 3.3 --- Optimization of specific amplification condition for subtype specific primers by cross-PCR --- p.64 / Chapter 3.4 --- Determination of the amplification of a particular subtype in the excess of other templates --- p.67 / Chapter 3.5 --- Determination of the gene expression in poly (I) poly (C) treated L929 cells / Chapter 3.5.1 --- Spectrophotometric analysis of total RNA --- p.70 / Chapter 3.5.2 --- Agarose gel electrophoresis of RNA --- p.72 / Chapter 3.5.3 --- Normalization of RNA sample --- p.73 / Chapter 3.5.4 --- Detection of IFN-α subtypes mRNA in L929 cell induced with poly(I) poly(C)-DEAE dextran --- p.74 / Chapter 3.5.5 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expressionin Poly(I) Poly(C)-DEAE dextran or DEAE dextran treated L929 cells" --- p.70 / Chapter 3.6 --- Measurement of gene expression in L929 cells infected with influenza virus / Chapter 3.6.1 --- Spectrophotometric analysis of total RNA --- p.83 / Chapter 3.6.2 --- Agarose gel electrophoresis of RNA samples --- p.84 / Chapter 3.6.3 --- Normalization of RNA samples --- p.86 / Chapter 3.6.4 --- Detection of IFN-α subtypes mRNA in L929 cell infected with influenza A/NWS/33 virus --- p.87 / Chapter 3.6.5 --- Detection of IFN-α subtypes mRNA in L929 cells infected with influenza B/Lee/40 virus --- p.90 / Chapter 3.6.6 --- "Detection of IFN-γ, IFN-α/β receptor and IFN-β expression in L929 cells infected with A/NWS/33 and B/Lee/40" --- p.93 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.97 / REFERENCES --- p.105 / APPENDIX --- p.122

Tsang Sai Leong. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Abstract (in Chinese) --- p.(i) / Abstract --- p.(iii) / Table of contents --- p.(v) / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The interferon --- p.1 / Chapter 1.1.1 --- About type I IFN --- p.1 / Chapter 1.1.2 --- IFN α/β receptor and signal transduction --- p.3 / Chapter 1.1.3 --- IFN induction --- p.3 / Chapter 1.1.4 --- Functions --- p.4 / Chapter 1.1.5 --- MuIFN α subtypes --- p.8 / Chapter 1.1.6 --- Gene expression --- p.9 / Chapter 1.2 --- Aim of study: Functions and gene expression --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- "Cell line, bacterial strain, virus strain and plasmid vector" --- p.15 / Chapter 2.1.2 --- Chemicals --- p.20 / Chapter 2.1.3 --- "Culture media, buffer and other solutions" --- p.20 / Chapter 2.1.4 --- Reagents and nucleic acids --- p.21 / Chapter 2.1.5 --- Reaction kits --- p.22 / Chapter 2.1.6 --- Solutions --- p.22 / Chapter 2.1.7 --- Major equipments --- p.24 / Chapter 2.1.8 --- Primers used --- p.24 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- "Cloning of MuIFN αl2, MuIFN αl and MuIFN α4 from L929 genomic DNA and their subcloning into pEGFP-Nl mammalian expression vector" --- p.26 / Chapter 2.2.1.1 --- PCR of MuIFN αl2 --- p.26 / Chapter 2.2.1.2 --- Gel purification of MuIFN αl2 PCR product --- p.26 / Chapter 2.2.1.3 --- Ligation of MuIFN αl2 PCR product into pGEM-T vector --- p.26 / Chapter 2.2.1.4 --- Sequencing of clones which were positive in PCR screening --- p.26 / Chapter 2.2.1.5 --- Subcloning of the gene from pGEM-T vector to pEGFP-Nl --- p.28 / Chapter 2.2.1.6 --- Construction of expression vectors for MuIFN αl and MuIFN a4 gene --- p.28 / Chapter 2.2.2 --- Preparation ofplasmid DNA --- p.29 / Chapter 2.2.3 --- Preparation of cell culture medium --- p.30 / Chapter 2.2.4 --- Production of recombinant MuIFN α (rMuIFN α) --- p.30 / Chapter 2.2.5 --- Production of native MuIFN α by polyI：polyC induction --- p.31 / Chapter 2.2.6 --- Influenza A virus strain A/NWS/33 preparation and titration --- p.31 / Chapter 2.2.7 --- Virus infection in Influenza A virus challenge assay --- p.32 / Chapter 2.2.8 --- Cell culture techniques --- p.32 / Chapter 2.2.9 --- "MTT cell proliferation assay of JCS cell line, for measuring MuIFN α anti-proliferation activity" --- p.33 / Chapter 2.2.10 --- Quantitative analysis of MuIFN α --- p.34 / Chapter 2.2.11 --- Flow cytofluorometric analysis of cell cycle of MuIFN α treated JCS cells by propidium iodide staining --- p.34 / Chapter 2.2.12 --- FACS study on the effect of MuIFN α on MHC-I up-regulation in JCS cells --- p.35 / Chapter 2.2.13 --- FACS study on the effect of MuIFN α on MHC-I up-regulation on primary macrophages from Balb/c mice --- p.35 / Chapter 2.2.14 --- Anti-viral activity by transfection of MuIFN α gene --- p.36 / Chapter 2.2.15 --- Sequencing of MuIFN al2 coding region from genomic DNA of L929 and JCS cell lines --- p.37 / Chapter 2.2.16 --- "RNA extraction from L929 cell lines, with or without Influenza A virus infection or polyI:polyC induction" --- p.37 / Chapter 2.2.17 --- RNA extraction from tissues of Balb/c mouse --- p.38 / Chapter 2.2.18 --- Reverse transcription --- p.39 / Chapter 2.2.19 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Overview --- p.40 / Chapter 3.2 --- "Subcloning of MuIFN α 12, MuIFN αl and MuIFN α4 coding sequences into the pEGFP-Nl vector" --- p.40 / Chapter 3.3 --- The growth inhibitory effect of different MuIFN α subtypes on murine myeloid leukemia cell line JCS --- p.41 / Chapter 3.4 --- Quantitation of MuIFN α subtype samples --- p.50 / Chapter 3.5 --- Cell cycle analysis of MuIFN α treated JCS cells --- p.50 / Chapter 3.6 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in JCS cell line --- p.57 / Chapter 3.7 --- FACS analysis of the effect of different MuIFN α subtypes on MHC-I expression in primary macrophages in Balb/c mice --- p.65 / Chapter 3.8 --- Effect of MuIFN α subtype transgenes on L929 cells challenged with Influenza A virus --- p.72 / Chapter 3.9 --- Sequencing of MuIFN αl2 coding region from genomic DNA of L929 and JCS cell line --- p.78 / Chapter 3.10 --- "MuIFN αl2 expression in untreated, Influenza A virus infected or polyl:polyC induced L929 cells" --- p.78 / Chapter 3.11 --- Detection of MuIFN α12 transcripts in tissues of the 8-10 week untreated Balb/c mice --- p.85 / Chapter Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- Overview --- p.89 / Chapter 4.2 --- rMuIFN α 12 has anti-proliferative and apoptotic effects on JCS cell line --- p.89 / Chapter 4.3 --- "Up-regulation of MHC-I expression in JCS cells and primary macrophages by rMuIFN αl2, rMuIFN αl, rMuIFN α4 and mixed type I IFN" --- p.91 / Chapter 4.4. --- Transfection of MuIFN α12 gene could induce anti-viral state in L929 cell line --- p.91 / Chapter 4.5 --- Gene regulation of MuIFN al2 in L929 cells infected with Influenza A virus or induced by polyI:polyC --- p.92 / Chapter 4.6 --- Gene expression of MuIFN αl2 in different tissues of Balb/c mice --- p.94 / Conclusion --- p.95 / Reference List --- p.96 / List of figures: / Fig. 1.1 The 3D structure of recombinant human interferon alpha (HuIFN α) subtype 2B --- p.11 / Fig. 1.2 Current model of lFN induction --- p.12 / Fig. 1.3 Activation of RNase L --- p.13 / Fig. 2.1 Graphical map of plasmid vector pEGFP-Nl --- p.17