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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322768 |
| Date | January 1999 |
| Contributors | Chan, Yick Bun., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvi, 165 leaves : ill. ; 30 cm. |
| Rights | Use 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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