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

This project was the first study of the genetics and embryo-pathology of exencephaly in a partially inbred mouse stock, SELH/Bc. Exencephaly was found in 17% of SELH fetuses. Analysis of day 8-9 gestation embryos indicated that SELH embryos were collectively normal in general development, but delayed in neural tube closure relative to overall or general development compared to two normal strains of mice, ICR/Be and SWV/Bc. Exencephaly was observed to be caused by a failure of fusion of the cranial neural folds in the mesencephalon region in SELH. All SELH embryos appeared to be abnormal in their pattern of cranial neural tube closure. They fail to make initial contact at the prosencephalon/mesencephalon junction region of the cranial neural folds (the first fusion in the cranial neural folds in normal embryos). SELH embryos, fused their anterior neural folds via an alternate (possibly passive) mechanism compared to normal strains of mice (SWV/Bc, and ICR/Be), by fusing the folds in a "zipper-like" fashion from the rostral base of the prosencephalon. This closure of the neural tube in genetically liable embryos by an abnormal sequence of events suggests a new model for anterior neural tube closure failure. Liability to exencephaly appeared to be fixed in the SELH stock. Of the 53 SELH males tested, all produced exencephaly. SELH animals were found to be heterogeneous in the frequency of exencephaly they produced, indicating that there are still genes segregating in the stock which affect the ability of embryos to complete anterior neural tube closure. Exencephaly in SELH does not appear to be caused by an autosomal dominant, sex-linked dominant or recessive, or simple autosomal recessive single gene, although F2, BCl, and BC2 exencephaly frequencies (after an outcross to ICR/Be) suggest that only a small number of genes are involved. A marked excess of female exencephalics was observed in SELH, F2, BCl, and BC2 fetuses. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

by Pui-kuen Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 168-182). / ACKNOWLEDGMENT --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.IV / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER1 --- INTRODUCTION AND BACKGROUND --- p.1 / Chapter 1.1 --- ROLE OF CYTOKINES IN MOUSE EMBRYONIC DEVELOPMENT --- p.1 / Chapter 1.1.1 --- Why mouse model --- p.1 / Chapter 1.1.2 --- Embryonic development of mouse --- p.1 / Chapter 1.1.3 --- An overview of cytokines --- p.4 / Chapter a. --- Classes of cytokines --- p.5 / Chapter i) --- Growth factors --- p.5 / Chapter ii) --- Interleukins --- p.7 / Chapter iii) --- Colony-stimulating factors --- p.9 / Chapter iv) --- Interferons --- p.10 / Chapter v) --- Tumor necrosis factor --- p.11 / Chapter b. --- Cytokine networks --- p.12 / Chapter c. --- Role of cytokines in the whole organism --- p.13 / Chapter 1.1.4 --- Cytokine and receptor gene expression in mouse embryonic development --- p.15 / Chapter a. --- Murine embryonic stem cell model --- p.15 / Chapter b. --- Leukemia Inhibitory Factor (LIF) in mouse embryos --- p.16 / Chapter c. --- IL-6 in mouse embryo --- p.19 / Chapter d. --- Ciliary Neurotrophic Factor (CNTF) in mouse embryo --- p.19 / Chapter e. --- TNF-a and TNF-β in mouse embryos --- p.20 / Chapter f. --- TGF-a in mouse embryos --- p.20 / Chapter g. --- TGF-P in mouse embryos --- p.20 / Chapter h. --- Stem cell factor / c-kit --- p.21 / Chapter i. --- Other cytokines in mouse embryos --- p.22 / Chapter j. --- Cytokine receptors --- p.24 / Chapter 1.2 --- NEUROPOIETIC CYTOKINES --- p.28 / Chapter 1.2.1 --- Family members --- p.28 / Chapter 1.2.2 --- Shared signal transducer gpl30 --- p.29 / Chapter 1.2.3 --- "LIF, CNTF and OSM inhibit differentiation of embryonic stem cells" --- p.31 / Chapter 1.3 --- BIOLOGY OF ONCOSTATIN M (OSM) --- p.33 / Chapter 1.3.1 --- Physical properties of OSM --- p.33 / Chapter 1.3.2 --- Biological activities of OSM --- p.34 / Chapter 1.3.3 --- Molecular aspect of OSM --- p.35 / Chapter 1.4 --- AIMS OF THE STUDY --- p.38 / Chapter CHAPTER2 --- CYTOKINE GFNE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT --- p.40 / Chapter 2.1 --- INTRODUCTION --- p.40 / Chapter 2.1.1 --- Rationale --- p.40 / Chapter 2.1.2 --- Design of primers --- p.43 / Chapter 2.2 --- MATERIALS --- p.44 / Chapter 2.2.1 --- Chemicals and Reagents --- p.44 / Chapter 2.2.2 --- Enzymes --- p.45 / Chapter 2.2.3 --- Buffers --- p.45 / Chapter 2.2.4 --- Solutions --- p.47 / Chapter 2.2.5 --- Probe labeling and detection kits --- p.48 / Chapter 2.2.6 --- Primers and internal probes --- p.49 / Chapter 2.3 --- METHODS --- p.52 / Chapter 2.3.1 --- Preparation of total RNA from mouse embryos at different stages --- p.52 / Chapter a. --- Mice dissection for embryo --- p.52 / Chapter b. --- Guanidinium thiocyanate cell lysate --- p.52 / Chapter c. --- Isolation of RNA by centrifugation through CsCl gradient --- p.53 / Chapter d. --- Spectrophotometric determination of RNA amount --- p.54 / Chapter 2.3.2 --- Preparation of embryo sections --- p.54 / Chapter 2.3.3 --- Primers and internal probes --- p.55 / Chapter 2.3.4 --- Cytokine mRNA Phenotyping by Reverse transcription-Polymerase chain reaction --- p.56 / Chapter a. --- Reverse transcription (First strand cDNA synthesis) --- p.56 / Chapter b. --- Polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.5 --- Analysis of PCR products with agarose gel electrophoresis --- p.57 / Chapter 2.3.6 --- Analysis of PCR products with Southern blotting --- p.58 / Chapter a. --- DNA transfer from gel to nylon membrane --- p.58 / Chapter b. --- Probe labeling --- p.61 / Chapter c. --- Prehybridization --- p.61 / Chapter d. --- Hybridization --- p.62 / Chapter e. --- Detection of DIG-labeled probe --- p.62 / Chapter 2.3.7 --- Cycle titration of PCR and dot blotting of regulatory cytokine mRNA --- p.63 / Chapter a. --- Cycle titration of PCR --- p.63 / Chapter b. --- Dot blotting --- p.63 / Chapter 2.4 --- RESULTS --- p.65 / Chapter 2.4.1 --- Sagittal sections of mouse embryos --- p.65 / Chapter 2.4.2 --- Preparation of total RNA --- p.69 / Chapter 2.4.3 --- Cytokine mRNA phenotyping --- p.71 / Chapter a. --- Southern hybridization for 'no expression' cytokines --- p.74 / Chapter b. --- Consistent' and 'regulatory ´ة cytokines in embryo and placenta --- p.79 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter 2.5.1 --- Isolation of embryo RNA by guanidinium thiocyanate/ cesium chloride centrifugation --- p.95 / Chapter 2.5.2 --- mRNA Quantitation --- p.96 / Semi-quantitative PCR --- p.98 / Chapter 2.5.3 --- Cytokine mRNA phenotyping by RT-PCR --- p.99 / Chapter a. --- Reverse Transcription --- p.99 / Chapter b. --- GAPDH as a control for normalization --- p.100 / Chapter c. --- PCR for cytokine transcripts --- p.101 / Chapter 2.5.4 --- Cytokines and receptors in embryonic development --- p.103 / Chapter 2.5.4.1 --- Cytokines in hematopoietic development of mouse fetus --- p.104 / Chapter 2.5.4.2 --- Other cytokines --- p.113 / Chapter 2.5.5 --- Expression Pattern in placenta: maternal and fetal communication --- p.116 / Chapter CHAPTER3 --- MOLECULAR ANALYSTS OF MOUSE ONCOSTATIN M --- p.117 / Chapter 3.1 --- INTRODUCTION --- p.117 / Chapter 3.2 --- MATERIALS --- p.121 / Chapter 3.2.1 --- Chemicals and Reagents --- p.121 / Chapter 3.2.2 --- Enzymes --- p.121 / Chapter 3.2.3 --- Buffers --- p.122 / Chapter 3.2.4 --- Solutions --- p.122 / Chapter 3.2.5 --- Culture media --- p.124 / Chapter 3.2.6 --- Competent cell --- p.125 / Chapter 3.2.7 --- DNA materials --- p.125 / Chapter 3.2.8 --- Primers --- p.126 / Chapter 3.3 --- METHODS --- p.127 / Chapter 3.3.1 --- Primers and internal probes --- p.127 / Chapter 3.3.2 --- Cloning of human Oncostatin M exon 2 and exon 3 by PCR --- p.127 / Chapter 3.3.3 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.128 / Chapter a. --- Preparation of human OSM exons and plasmid --- p.128 / Chapter i) --- Purification of PCR products --- p.128 / Chapter ii) --- T4 DNA polymerase ´بblunt-end´ة reaction for PCR products --- p.129 / Chapter iii) --- Sma I digestion of pUC18 --- p.129 / Chapter b. --- Ligation --- p.129 / Chapter c. --- Preparation of competent cell --- p.130 / Chapter d. --- Transformation --- p.131 / Chapter e. --- Screening of recombinants by PCR --- p.131 / Chapter f. --- Screening of recombinants by restriction enzyme digestion --- p.132 / Chapter i) --- Preparation of plasmids --- p.132 / Chapter ii) --- Double restriction enzymes digestion of pUC18 --- p.133 / Chapter 3.3.4 --- Verification of the clones of human OSM exons 2 and 3 by cycle sequencing --- p.135 / Chapter 3.3.5 --- Purification of human OSM exons from plasmid for making probe --- p.136 / Chapter 3.3.6 --- Southern blotting --- p.136 / Chapter a. --- Probe making and labeling --- p.136 / Chapter b. --- Preparation of mouse genomic DNAs --- p.137 / Chapter c. --- DNA transfer --- p.138 / Chapter i) --- Digestion of genomic DNA with restriction endonucleases --- p.138 / Chapter ii) --- Gel electrophoresis and DNA blotting --- p.139 / Chapter d. --- Hybridization --- p.139 / Chapter 3.4 --- RESULTS --- p.142 / Chapter 3.4.1 --- Cloning of human OSM exon 2 and exon 3 by PCR --- p.142 / Chapter 3.4.2 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.142 / Chapter a. --- Screening of recombinants by PCR --- p.142 / Chapter b. --- Screening of recombinants by restriction enzymes digestion --- p.143 / Chapter 3.4.3 --- Sequence of subcloned exons 2 and3 --- p.147 / Chapter 3.4.4 --- Southern hybridization --- p.149 / Chapter a. --- Genomic DNA preparation --- p.149 / Chapter b. --- Digestion of genomic DNAs --- p.151 / Chapter c. --- Hybridization signal --- p.154 / Chapter 3.5 --- DISCUSSION --- p.158 / Chapter 3.5.1 --- Cross-species hybridization --- p.158 / Chapter 3.5.2 --- Hybridization of human OSM exon fragments against mouse genome --- p.158 / Chapter a. --- hOSM exon 2 as probe --- p.158 / Chapter b. --- hOSM exon 3 as probe --- p.160 / Chapter c. --- Feasibility of using hOSM as probe for fishing out the mOSM gene --- p.160 / Chapter d. --- The cloning of mouse OSM by Yoshimura's group --- p.161 / Chapter CHAPTER4 --- CONCLUSION --- p.162 / Chapter 4.1 --- SUMMARY OF CYTOKINE AND CYTOKINE RECEPTOR GENES EXPRESSION DURING EMBRYONIC DEVELOPMENT --- p.162 / Chapter 4.2 --- FURTHER STUDIES OF THE CYTOKINE ACTIONS ON EMBRYOGENESIS --- p.165 / Chapter 4.3 --- MOLECULAR ANALYSIS OF MOUSE OSM GENE --- p.167 / REFERENCES --- p.168

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

Thesis (Ph. D.)--University of Glasgow, 2003. / Ph. D. thesis submitted to the Faculty of Veterinary Medicine, University of Glasgow, 2003. Includes bibliographical references. Electronic version also available via Glasgow University e-Theses service.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences