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