Chan Sin-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 154-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Contents --- p.viii / Abbreviations --- p.xiii / List of Figures and Tables --- p.xvi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Ontogeny of hematopoiesis --- p.1 / Chapter 1.1.2 --- Hiercharay of hematopoiesis --- p.2 / Chapter 1.2 --- Regulation of hematopoiesis --- p.5 / Chapter 1.2.1 --- Bone marrow stromal cell --- p.5 / Chapter 1.2.2 --- Hematopoietic growth factor --- p.6 / Chapter 1.2.3 --- Hematopoietic growth factor receptors and signal transduction --- p.10 / Chapter 1.2.4 --- Transcriptional regulation of myeloid cell development --- p.11 / Chapter 1.3 --- Deregulated hematopoiesis - Leukemia --- p.20 / Chapter 1.3.1 --- Classification of leukemia --- p.20 / Chapter 1.3.2 --- Molecular basis of leukemia --- p.20 / Chapter 1.4 --- Prostaglandin E2 induced WEHI-3B JCS and JCS-14 cell differentiation --- p.22 / Chapter 1.4.1 --- Induced leukemia cell differentiation --- p.22 / Chapter 1.4.2 --- Inducer of cell differentiation - Prostaglandin E2 --- p.22 / Chapter 1.4.3 --- WEHI-3B JCS and subline JCS-14 cells --- p.24 / Chapter 1.5 --- The aims of study --- p.26 / Chapter Chapter Two --- Identification of differentially expressed genes during PGE2-induced WEHI-3B JCS-14 cell differentiation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Strategy for studying PGE2-induced JCS-14 cell differentiation --- p.28 / Chapter 2.1.2 --- Method for studying differential gene expression: Microarry Technology --- p.29 / Chapter 2.2 --- Materials --- p.32 / Chapter 2.2.1 --- Cell line --- p.32 / Chapter 2.2.2 --- AtlasT M Mouse cDNA Expression Array --- p.32 / Chapter 2.2.3 --- Chemicals --- p.32 / Chapter 2.2.4 --- Solutions and buffers --- p.33 / Chapter 2.2.5 --- Reagents --- p.34 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Morphological study of PGE2-induced JCS-14 cell differentiation --- p.35 / Chapter 2.3.2 --- Preparation of total RNA from PGE2-induced JCS-14 cells --- p.35 / Chapter 2.3.2.1 --- Preparation of cell lysates --- p.35 / Chapter 2.3.2.2 --- Isolation of total RNA --- p.35 / Chapter 2.3.3 --- Preparation of cDNA probes --- p.36 / Chapter 2.3.3.1 --- Probe synthesis from total RNA --- p.36 / Chapter 2.3.3.2 --- Purification of the labeled cDNA probes --- p.37 / Chapter 2.3.4 --- Hybridization cDNA probes to the Atlas Array and stringency wash --- p.37 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Morphological changes in PGE2-treated JCS-14 cells --- p.39 / Chapter 2.4.2 --- Analysis of total RNA from PGE2-induced JCS-14 cells --- p.43 / Chapter 2.4.3 --- Hybridization of cDNA probes to AtlasT M cDNA Expression Array --- p.45 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Morphological study of JCS-14 cell differentiation --- p.73 / Chapter 2.5.2 --- Differentiation commitment of JCS-14 cell under PGE2 induction --- p.73 / Chapter 2.5.3 --- Gene expression profile by microarray --- p.74 / Chapter 2.5.4 --- Gene expression profile of 5 hours PGE2-induced JCS-14 cells --- p.74 / Chapter 2.5.5 --- Further analysis of regulatory genes in PGE2-induced JCS-14 cell differentiation --- p.77 / Chapter Chapter Three --- Expression profile of identified genes in WEHI-3B JCS-14 and JCS cell differentiation / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.1.1 --- Quantitation of mRNA by Real time RT-PCR --- p.80 / Chapter 3.1.2 --- Relative quantitation of gene expression --- p.83 / Chapter 3.2 --- Materials --- p.85 / Chapter 3.2.1 --- Cell lines --- p.85 / Chapter 3.2.2 --- SYBR® Green PCR core kit --- p.85 / Chapter 3.2.3 --- Chemicals --- p.85 / Chapter 3.2.4 --- Solutions and buffers --- p.86 / Chapter 3.2.5 --- Enzymes and nucleic acids --- p.86 / Chapter 3.3 --- Methods --- p.88 / Chapter 3.3.1 --- Preparation of total RNA from PGE2-induced JCS-14 and JCS cells --- p.88 / Chapter 3.3.1.1 --- Preparation of cell lysates --- p.88 / Chapter 3.3.1.2 --- Isolation of total RNA --- p.88 / Chapter 3.3.2 --- Reverse transcription (RT) --- p.88 / Chapter 3.3.3 --- Design of real-time PCR primers --- p.88 / Chapter 3.3.4 --- Determination of relative efficiency of target and reference amplification by validation experiment --- p.89 / Chapter 3.3.5 --- Confirmation of expression profile of identified genes in JCS-14 and JCS cells by comparative CT method in real-time PCR --- p.90 / Chapter 3.4 --- Results --- p.91 / Chapter 3.4.1 --- Analysis of total RNA from PGE2-induced JCS-14 and JCS cells --- p.91 / Chapter 3.4.2 --- Validation experiment of real-time PCR primers --- p.93 / Chapter 3.4.3 --- Expression profile of specific genes in JCS-14 and JCS cells by comparative CT method --- p.101 / Chapter 3.5 --- Discussion --- p.114 / Chapter 3.5.1 --- Study of gene expression profiles in JCS-14 and JCS cell differentiation --- p.114 / Chapter 3.5.2 --- Transcription analysis by real-time PCR --- p.114 / Chapter 3.5.3 --- Gene expression profiles during PGE2-induced JCS-14 and JCS cell differentiation --- p.115 / Chapter Chapter Four --- Inhibition of specific gene expression in WEHI-3B JCS-14 and JCS cells using antisense blocking technique / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.1.1 --- Antisense technique --- p.122 / Chapter 4.1.2 --- Design of antisense oligonucleotides --- p.125 / Chapter 4.1.3 --- Transfer of oligonucleotides to cells --- p.128 / Chapter 4.2 --- Materials --- p.129 / Chapter 4.2.1 --- Cell lines --- p.129 / Chapter 4.2.2 --- Chemicals --- p.129 / Chapter 4.2.3 --- Reagents --- p.129 / Chapter 4.2.4 --- Solutions --- p.129 / Chapter 4.3 --- Methods --- p.131 / Chapter 4.3.1 --- Design of antisense oligonucleotides --- p.131 / Chapter 4.3.2 --- Transfection of oligonucleotides into cells --- p.134 / Chapter 4.3.3 --- Morphological study of PGE2-induced JCS-14 and JCS cells --- p.134 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- Effect of antisense oligonucleotides on JCS-14 cell differentiation --- p.135 / Chapter 4.4.2 --- Effect of antisense oligonucleotides on JCS cell differentiation --- p.136 / Chapter 4.5 --- Discussion --- p.146 / Chapter 4.5.1 --- Effects of antisense B-myb on JCS-14 and JCS cell differentiation --- p.146 / Chapter 4.5.2 --- Effects of antisense thyroid hormone receptor (c-erbA) and transcription terminator factor (TTF-1) on JCS-14 and JCS cell differentiation --- p.147 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Introduction --- p.148 / Chapter 5.2 --- Differentiation program triggered by Prostaglandin E2 --- p.148 / Chapter 5.2.1 --- Lineage preference during differentiation --- p.148 / Chapter 5.2.2 --- Differentially expressed genes during PGE2-induced JCS-14 cell differentiation --- p.149 / Chapter 5.2.3 --- Expression patterns of the three differentially expressed genes in PGE2-induced JCS-14 and JCS cells --- p.149 / Chapter 5.2.4 --- Antisense blocking during differentiation --- p.151 / Chapter 5.3 --- Further studies --- p.152 / References --- p.154

by Chan Yick Bun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 145-165). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / List of Figures --- p.XVI / List of Tables --- p.XVII / Chapter Chapter One --- General introduction / Chapter 1.1 --- Leukemia: an overview --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Classification of leukemia --- p.1 / Chapter 1.1.3 --- Origin of leukemia --- p.3 / Chapter 1.1.4 --- Treatment of leukemia --- p.5 / Chapter 1.2 --- Introduction of leukemia cell re-differentiation --- p.8 / Chapter 1.2.1 --- Introduction --- p.8 / Chapter 1.2.2 --- Inducers of cell differentiation --- p.8 / Chapter 1.2.3 --- Genes involved in myeloid leukemia cell differentiation --- p.11 / Chapter 1.2.3.1 --- Transcription factors --- p.11 / Chapter 1.2.3.2 --- Signal transduction cascades --- p.16 / Chapter 1.2.3.3 --- Receptors --- p.18 / Chapter 1.2.3.4 --- Cytokines --- p.19 / Chapter 1.3 --- Tumor necrosis factor alpha induced WEHI 3B JCS cell differentiation --- p.21 / Chapter 1.3.1 --- Introduction --- p.21 / Chapter 1.3.2 --- Tumor necrosis factor alpha --- p.21 / Chapter 1.3.3 --- WEHI 3B JCS cells --- p.23 / Chapter 1.4 --- Aims of study --- p.25 / Chapter Chapter Two --- Isolation of differentially expressed genes during TNF-α induced WEHI 3B JCS cell differentiation / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.1.1 --- Overview of differential genes screening methods --- p.26 / Chapter 2.1.2 --- Differential hybridization for analysis of gene expression profiles --- p.29 / Chapter 2.1.3 --- Factors affect differential hybridization --- p.33 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.2.1 --- Cell line --- p.35 / Chapter 2.2.2 --- Mouse brain cDNA library --- p.35 / Chapter 2.2.3 --- E.coli strains --- p.35 / Chapter 2.2.3 --- Kits --- p.35 / Chapter 2.2.5 --- Chemicals --- p.35 / Chapter 2.2.6 --- Solutions and buffers --- p.36 / Chapter 2.2.7 --- Enzymes and reagents --- p.37 / Chapter 2.3 --- Methods --- p.38 / Chapter 2.3.1 --- Preparation of total RNA from TNF-a induced WEHI 3B JCS cells --- p.38 / Chapter 2.3.1.1 --- Preparation of cell lysates --- p.38 / Chapter 2.3.1.2 --- Extraction of total RNA --- p.38 / Chapter 2.3.2 --- Preparation of cDNA clones from cDNA library --- p.39 / Chapter 2.3.2.1 --- Rescue of phagemids from cDNA library --- p.39 / Chapter 2.3.2.2 --- Preparation of plasmids --- p.39 / Chapter 2.3.3 --- Primary differential hybridization --- p.40 / Chapter 2.3.3.1 --- Preparation of cDNA blots --- p.40 / Chapter 2.3.3.2 --- Preparation of cDNA probes --- p.40 / Chapter 2.3.3.3 --- Primary differential hybridization --- p.41 / Chapter 2.3.4 --- Subcloning of putative differential cDNA clones --- p.42 / Chapter 2.3.4.1 --- Preparation of DH5a competent cells --- p.42 / Chapter 2.3.4.2 --- Transformation of cDNA clones --- p.42 / Chapter 2.3.5 --- Secondary differential hybridization --- p.42 / Chapter 2.3.5.1 --- Preparation ofcDNA blots --- p.42 / Chapter 2.3.5.2 --- Secondary differential hybridization --- p.43 / Chapter 2.4 --- Results --- p.44 / Chapter 2.4.1 --- Analysis of total RNA prepared from TNF-α induced WEHI 3B JCS cells --- p.44 / Chapter 2.4.2 --- Spectrophotometric analysis of plasmid DNA --- p.46 / Chapter 2.4.3 --- Primary differential hybridization --- p.48 / Chapter 2.4.4 --- Secondary differential hybridization --- p.58 / Chapter 2.4.5 --- Comparison of two rounds of differential hybridization --- p.61 / Chapter 2.5 --- Discussions --- p.63 / Chapter 2.5.1 --- Study of gene expression profile by differential hybridization --- p.63 / Chapter 2.5.1.1 --- cDNA library --- p.63 / Chapter 2.5.1.2 --- Blots --- p.64 / Chapter 2.5.2 --- Two rounds of differential hybridization --- p.66 / Chapter 2.5.3 --- Comparison of two rounds of differential hybridization --- p.68 / Chapter Chapter Three --- Sequence analysis of putative differentially expressed genes / Chapter 3.1 --- Introduction --- p.70 / Chapter 3.1.1 --- Basic structure of cDNA clones --- p.70 / Chapter 3.1.2 --- Strategies for DNA sequencing --- p.71 / Chapter 3.1.2.1 --- Primer walking --- p.71 / Chapter 3.1.2.2 --- Restriction digestion and subcloning --- p.71 / Chapter 3.1.2.3 --- Nested deletion sets --- p.72 / Chapter 3.1.2.4 --- Shotgun sequencing --- p.72 / Chapter 3.1.2.5 --- Other sequencing strategies --- p.73 / Chapter 3.1.3 --- Sequence alignment and database search --- p.74 / Chapter 3.1.3.1 --- Sequence database --- p.74 / Chapter 3.1.3.2 --- Sequence alignment --- p.74 / Chapter 3.1.3.3 --- BLAST algorithm --- p.75 / Chapter 3.2 --- Materials --- p.76 / Chapter 3.2.1 --- Kits --- p.76 / Chapter 3.2.2 --- Restriction enzymes --- p.76 / Chapter 3.2.3 --- Solutions and buffers --- p.76 / Chapter 3.2.4 --- Enzymes and reagents --- p.77 / Chapter 3.3 --- Methods --- p.78 / Chapter 3.3.1 --- Restriction digestion --- p.78 / Chapter 3.3.2 --- Subcloning --- p.79 / Chapter 3.3.2.1 --- Gel purification --- p.79 / Chapter 3.3.2.2 --- Ligation --- p.79 / Chapter 3.3.2.3 --- Transformation --- p.80 / Chapter 3.3.3 --- Shotgun sequencing --- p.80 / Chapter 3.3.4 --- Sequencing reaction --- p.81 / Chapter 3.3.4.1 --- Preparation of sequencing gel --- p.81 / Chapter 3.3.4.2 --- Sequencing reaction --- p.81 / Chapter 3.4 --- Results --- p.83 / Chapter 3.4.1 --- Restriction mapping of cDNA inserts --- p.83 / Chapter 3.4.2 --- Sequencing results --- p.85 / Chapter 3.4.3 --- Sequence analysis --- p.90 / Chapter 3.5 --- Discussions --- p.103 / Chapter 3.5.1 --- Sequencing strategies --- p.103 / Chapter 3.5.2 --- Sequence analysis --- p.104 / Chapter Chapter Four --- Characterization of the putative differentially expressed genes / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Midazolam induced WEHI 3B JCS cells differentiation --- p.107 / Chapter 4.1.2 --- Gene expression profiles in embryogenesis --- p.108 / Chapter 4.2 --- Materials --- p.110 / Chapter 4.2.1 --- Mouse embryo multiple tissue Northern (MTN´ёØ) blot --- p.110 / Chapter 4.2.2 --- Megaprime´ёØ DNA labelling system --- p.110 / Chapter 4.2.3 --- Chemicals --- p.110 / Chapter 4.2.3 --- Solutions and buffers --- p.111 / Chapter 4.3 --- Methods --- p.112 / Chapter 4.3.1 --- Preparation of Northern blots --- p.112 / Chapter 4.3.1.1 --- Preparation of total RNA from midazolam induced WEHI 3B JCS cells --- p.112 / Chapter 4.3.1.2 --- Preparation of Northern blots --- p.112 / Chapter 4.3.2 --- Preparation of DNA probes --- p.113 / Chapter 4.3.2.1 --- Preparation of DNA templates --- p.113 / Chapter 4.3.2.2 --- Preparation of 32P labelled probes --- p.114 / Chapter 4.3.3 --- Northern blot analysis --- p.115 / Chapter 4.3.3.1 --- Northern hybridization --- p.115 / Chapter 4.3.3.2 --- Stripping of Northern blot --- p.115 / Chapter 4.4 --- Results --- p.117 / Chapter 4.4.1 --- Analysis of midazolam induced JCS cells total RNA --- p.117 / Chapter 4.4.2 --- Preparation of DNA templates for probe syntheses --- p.119 / Chapter 4.4.3 --- Northern Hybridization --- p.121 / Chapter 4.4.4 --- Comparison of the results of differential hybridization and Northern hybridization --- p.126 / Chapter 4.5 --- Discussions --- p.127 / Chapter 4.5.1 --- Northern hybridization --- p.127 / Chapter 4.5.1.1 --- Gene expression patterns under TNF-α induction --- p.127 / Chapter 4.5.1.2 --- Normalization of Northern hybridization --- p.129 / Chapter 4.5.1.3 --- Gene expression patterns under midazolam induction --- p.130 / Chapter 4.5.1.4 --- Gene expression pattern during embryo development --- p.133 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Identification of differentially expressed genes in TNF-α induced WEHI 3B JCS diffentiation --- p.135 / Chapter 5.2 --- Differentially expressed genes and myeloid leukemia cell differentiation --- p.137 / Chapter 5.3 --- Differentially expressed genes and embryogenesis --- p.142 / Chapter 5.4 --- Further studies --- p.144 / References --- p.145

by Yip Mei Chu Pandora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 207-233). / Abstract also in Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER ONE ... --- GENERAL INTRODUCTION / Chapter 1.1 --- the blood cells formation - hematopoiesis --- p.1 / Chapter 1.1.1 --- Hierarchy of hematopoiesis --- p.2 / Chapter 1.1.2 --- Malfunction in the process of hematopoiesis - hematologic neoplasia - Leukemia --- p.6 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Differentiation therapy ´ؤ a new hope in the treatment of leukemia --- p.9 / Chapter 1.2 --- Understanding the pathogenesis of leukemia --- p.12 / Chapter 1.2.1 --- General regulation of hematopoiesis --- p.12 / Chapter 1.2.2 --- Regulation of the differentiation of myeloid lineage --- p.15 / Chapter 1.2.2.1 --- Regulation of myeloid cell differentiation by hematopoietic regulatory protein --- p.16 / Chapter 1.2.2.2 --- Signal transduction pathways in myeloid cell differentiation --- p.20 / Chapter 1.2.2.3 --- Gene regulation of myeloid cell differentiation --- p.22 / Chapter 1.2.2.3.1 --- Transcription factors --- p.23 / Chapter 1.2.2.3.2 --- Myeloid specific genes --- p.31 / Chapter 1.2.2.3.3 --- Protooncogenes and tumor suppressor genes --- p.37 / Chapter 1.2.2.3.4 --- Homeobox genes --- p.42 / Chapter 1.2.2.3.5 --- Cell cycle control in myeloid growth and differentiation --- p.47 / Chapter 1.3 --- Induction of differentiation in myeloid leukemia cell --- p.48 / Chapter 1.3.1 --- Induced myeloid leukemia cell differentiation --- p.48 / Chapter 1.3.2 --- Inducers of myeloid cell differentiation --- p.52 / Chapter 1.3.3 --- Chemical inducers ´ؤ Flavonoids --- p.57 / Chapter 1.3.4 --- Murine myeloid leukemia cell ´ؤ WEHI-3B JCS --- p.60 / Chapter 1.4 --- Aim of study --- p.53 / Chapter CHAPTER TWO ... --- ISOLATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED DURING BIOCHANIN A INDUCED WEHI-3B (JCS) MYELOID LEUKEMIA CELL DIFFERENTIATION / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.1.1 --- Strategy for searching differentially expressed genes - RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP- PCR) --- p.65 / Chapter 2.1.2 --- Reamplification of PCR products by Touchdown PCR --- p.67 / Chapter 2.1.3 --- Methods for eliminating false positives : Dot blot hybridization screening --- p.68 / Chapter 2.2 --- Materials --- p.70 / Chapter 2.2.1 --- "Cell line, Bacterial strain and Vector" --- p.70 / Chapter 2.2.2 --- Chemicals --- p.70 / Chapter 2.2.3 --- Reagents and nucleic acids --- p.71 / Chapter 2.2.4 --- Kits --- p.72 / Chapter 2.2.5 --- Solutions --- p.72 / Chapter 2.2.6 --- Equipments --- p.73 / Chapter 2.3 --- Methods --- p.74 / Chapter 2.3.1 --- Induction of murine myeloid leukemia cell line -WEHI-3B (JCS) cells by biochanin-A --- p.74 / Chapter 2.3.2 --- Isolation of total RNA by guanidium thiocyanate cesium chloride ultracentrifugation --- p.74 / Chapter 2.3.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.75 / Chapter 2.3.3.1 --- Synthesis of first strand cDNA --- p.75 / Chapter 2.3.3.2 --- Normalization of RNA samples --- p.75 / Chapter 2.3.3.3 --- RAP-PCR --- p.76 / Chapter 2.3.3.4 --- Reamplification of differentially amplified fragment --- p.77 / Chapter 2.3.4 --- First round dot blot hybridization screening --- p.78 / Chapter 2.3.4.1 --- Dot blot --- p.78 / Chapter 2.3.4.2 --- Preparation of cDNA probe --- p.79 / Chapter 2.3.4.3 --- 32P-labelling of cDNA probe --- p.79 / Chapter 2.3.4.4 --- Removal of unincorporated probe by NICK´ёØ column --- p.80 / Chapter 2.3.4.5 --- Estimation of 32P labelling efficiency by scintillation counting --- p.80 / Chapter 2.3.4.6 --- Prehybridization and hybridization --- p.81 / Chapter 2.3.4.7 --- Quantitation of hybridization signal by scanning densitometry --- p.81 / Chapter 2.3.5 --- Second round dot blot hybridization screening --- p.81 / Chapter 2.3.5.1 --- Subcloning of differentially amplified fragments --- p.82 / Chapter 2.3.5.1.1 --- Preparation of vector DNA --- p.82 / Chapter 2.3.5.1.2 --- Synthesis of blunt end PCR product --- p.84 / Chapter 2.3.5.1.3 --- Blunt end ligation --- p.34 / Chapter 2.3.5.1.4 --- Transformation --- p.85 / Chapter 2.3.5.1.5 --- Selection and confirmation by polymerase chain reaction --- p.85 / Chapter 2.3.5.2 --- Dot blot hybridization screening --- p.85 / Chapter 2.4 --- Results --- p.87 / Chapter 2.4.1 --- Spectrophotometric analysis of total RNA --- p.87 / Chapter 2.4.2 --- Normalization of RNA samples --- p.88 / Chapter 2.4.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.39 / Chapter 2.4.4 --- Reamplification of isolated RAP-PCR products --- p.91 / Chapter 2.4.5 --- First round of dot blot hybridization screening --- p.92 / Chapter 2.4.6 --- Subcloning of differentially amplified fragments --- p.100 / Chapter 2.4.7 --- Second round of dot blot hybridization screening --- p.102 / Chapter 2.4.8 --- Comparison of the first and second round of dot blot hybridization screening --- p.106 / Chapter 2.5 --- Discussion --- p.108 / Chapter 2.5.1 --- RNA fingerprinting by arbitrarily primed PCR --- p.108 / Chapter 2.5.2 --- Limitation of RAP-PCR --- p.110 / Chapter 2.5.3 --- Two rounds of dot blot hybridization screening --- p.111 / Chapter CHAPTER THREE... --- CHARACTERIZATION OF THE ISOLATED GENE FRAGMENTS / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.1.1 --- Automated DNA sequencing and analysis --- p.113 / Chapter 3.1.2 --- GenBank and the BLAST homology search --- p.115 / Chapter 3.2 --- Materials --- p.118 / Chapter 3.2.1 --- Selected recombinant plasmids --- p.118 / Chapter 3.2.2 --- Chemicals --- p.118 / Chapter 3.2.3 --- Reagents --- p.118 / Chapter 3.2.4 --- Kits --- p.119 / Chapter 3.2.5 --- Solutions --- p.119 / Chapter 3.2.6 --- Equipment --- p.119 / Chapter 3.3 --- Methods --- p.120 / Chapter 3.3.1 --- Preparation of selected recombinant plasmid DNA --- p.120 / Chapter 3.3.2 --- Restriction digestion of recombinant plasmid DNA --- p.120 / Chapter 3.3.3 --- Automated DNA sequencing --- p.120 / Chapter 3.3.3.1 --- Primer annealing to template --- p.120 / Chapter 3.3.3.2 --- Sequencing reactions --- p.121 / Chapter 3.3.3.3 --- Polyacrylamide gel electrophoresis --- p.121 / Chapter 3.3.3.4 --- Data analysis by ALF manager and DNAsis --- p.122 / Chapter 3.3.4 --- Sequence homology search with databases --- p.122 / Chapter 3.4 --- Results --- p.123 / Chapter 3.4.1 --- Spectrophotometric analysis of selected recombinant plasmid DNAs subcloned with differentially amplified fragments --- p.123 / Chapter 3.4.2 --- Restriction digestion of selected recombinant plasmid DNA --- p.124 / Chapter 3.4.3 --- Sequences of the subcloned differentially amplified fragments --- p.126 / Chapter 3.4.4 --- Sequence analysis of the subcloned differentially amplified fragments --- p.144 / Chapter 3.5 --- Discussion --- p.157 / Chapter 3.5.1 --- Sequence analysis of the isolated gene fragment --- p.157 / Chapter CHAPTER FOUR … --- "EXPRESSION PROFILE OF ISOLATED GENES FRAGMENTS IN MYELOID LEUKEMIA CELL, MOUSE EMBRYO, AND TISSUES" / Chapter 4.1 --- Introduction --- p.162 / Chapter 4.1.1 --- Quantitation of mRNA by Reverse transcription-polymerase chain reaction --- p.162 / Chapter 4.1.2 --- Internal primer design by OLIGO´ёØ ver 34 --- p.167 / Chapter 4.2 --- Materials --- p.168 / Chapter 4.2.1 --- Mice --- p.168 / Chapter 4.2.2 --- Cell lysate --- p.168 / Chapter 4.2.3 --- Total RNAs --- p.168 / Chapter 4.3 --- Methods --- p.169 / Chapter 4.3.1 --- Internal primer design by OLIGO´ёØ ver 34 --- p.169 / Chapter 4.3.2 --- "Isolation of total RNA from biochanin A induced JCS cells, mouse embryos and tissue" --- p.169 / Chapter 4.3.2.1 --- Preparation of cell lysate from mouse embryo and postnatal mouse brain --- p.169 / Chapter 4.3.2.2 --- Isolation of RNA by guanidium thiocyanate cesium chloride method --- p.170 / Chapter 4.3.3 --- Preparation of saggital section of mouse embryo --- p.170 / Chapter 4.3.4 --- Confirmation of differential expression of isolated genes fragments during biochanin A and midazolam induced WEHI 3B (JCS) differentiation and the expression profile in mouse tissues and during mouse embryo development by reverse transcription-polymerase chain reaction --- p.171 / Chapter 4.4 --- Results --- p.173 / Chapter 4.4.1 --- Internal primer design of the sequenced fragments --- p.173 / Chapter 4.4.2 --- Spectrophotometric analysis of total RNA --- p.175 / Chapter 4.4.3 --- Saggital section of mouse embryo --- p.176 / Chapter 4.4.4 --- Normalization of RNA samples --- p.180 / Chapter 4.4.5 --- Analysis of mRNA expression of differentially amplified fragmentsin biochanin A or midazolam induced JCS cells and mouse embryos by RT- PCR --- p.182 / Chapter 4.4.5.1 --- "Genes downregulated at 1 hour, 5 hours and 48 hours after biochanin A induction of JCS cells" --- p.183 / Chapter 4.4.5.2 --- Genes up-regulated at 48 hours after biochanin A induction --- p.183 / Chapter 4.4.5.3 --- Genes constitutively expressed during the course of biochanin A treatment --- p.184 / Chapter 4.4.5.4 --- Genes showing undetectable level of expression in biochanin A induced JCS cells --- p.184 / Chapter 4.4.6 --- Tissue expression of the biochanin A induced-differentially expressed fragments by RT-PCR --- p.188 / Chapter 4.5 --- Discussion --- p.191 / Chapter 4.5.1 --- Expression profiles of isolated differentially amplified fragments --- p.191 / Chapter 4.5.2 --- Comparison of the expression profiles of the isolated gene fragments analyzed by dot blot hybridization screening and RT-PCR --- p.197 / Chapter CHAPTER FIVE ... --- GENERAL DISCUSSION --- p.200 / REFERENCES --- p.207 / APPENDIX --- p.234

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137