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An investigation into gene regulation involved in human gamma-globin gene reactivation induced by a lead compound.

Chan Kai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Title --- p.i / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Abstract (English) --- p.vii / Abstract (Chinese) --- p.ix / Table of contents --- p.xi / List of Figures --- p.xvi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Human Hemoglobin --- p.1 / Chapter 1.2 --- Hemoglobinopathies --- p.4 / Chapter 1.3 --- Hereditary Persistence of Fetal Hemoglobin (HPFH) and β - Thalassemias --- p.6 / Chapter 1.4 --- Globin Genes Switching --- p.7 / Chapter 1.5 --- Pharmaceutical Agents for γ-Globin Gene Reactivation --- p.9 / Chapter 1.6 --- Discovery of LC978: A Novel Fetal Hemoglobin Inducing Agent --- p.10 / Chapter 1.7 --- Aim of Study --- p.11 / Chapter Chapter 2: --- Specific Induction of Gamma Globin Gene Transcription in K562 Leukemia Cell Line by Lead Compound LC978 --- p.12 / Chapter 2.1 --- K562 Cell Line as a Model for Gamma Globin Gene Induction Studies --- p.12 / Chapter 2.2 --- LC978-Induced Fetal Hemoglobin Expression in K562 Cell Line --- p.12 / Chapter 2.3 --- Materials --- p.14 / Chapter 2.3.1 --- "Chemicals, Kits and Reagents" --- p.14 / Chapter 2.3.2 --- Buffers and Solutions --- p.15 / Chapter 2.3.3 --- Cell Line --- p.16 / Chapter 2.3.4 --- Instruments and Equipments --- p.16 / Chapter 2.3.5 --- Enzymes --- p.16 / Chapter 2.3.6 --- Nucleic Acids --- p.17 / Chapter 2.3.7 --- Oligo Primers --- p.17 / Chapter 2.4 --- Methods --- p.17 / Chapter 2.4.1 --- In vitro Bioassay for Total Hemoglobin Production --- p.17 / Chapter (a) --- Preparation of Treatment Cell Culture Plates --- p.17 / Chapter (b) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (c) --- Signal Development --- p.18 / Chapter 2.4.2 --- Detection of Fetal Hemoglobin Production by HbF Sandwich ELISA --- p.18 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.18 / Chapter (b) --- Preparation of Capture Antibody-Coated and BSA-Blocked ELISA Plate --- p.19 / Chapter (c) --- Preparation of K562 Cell Lysates --- p.19 / Chapter (d) --- Antigen Capture and Signal Development --- p.19 / Chapter 2.4.3 --- Detection of Gamma Globin mRNA Level by Real-time RT-PCR --- p.20 / Chapter (a) --- Treatment of K562 Cells by LC978 --- p.20 / Chapter (b) --- Preparation of K562 Cell Lysate in Guanidium Thiocyanate (GT) Solution --- p.20 / Chapter (c) --- Isolation of Total RNA from LC978-treated K562 Cells --- p.21 / Chapter (d) --- cDNA Synthesis from mRNA by Reverse Transcriptase (RT) --- p.22 / Chapter (e) --- Real-Time Quantitative Polymerase Chain Reaction (PCR) --- p.23 / Chapter 2.5 --- Results --- p.24 / Chapter (a) --- In vitro Bioassay for Total Hemoglobin Production --- p.24 / Chapter (b) --- Fetal Hemoglobin Sandwich ELISA --- p.24 / Chapter (c) --- LC978-Induced Gamma Globin mRNA Accumulation --- p.25 / Chapter 2.6 --- Discussion --- p.31 / Chapter Chapter 3: --- Construction of Promoter-Reporter Plasmid Constructs --- p.33 / Chapter 3.1 --- The Human Gamma Globin Gene Promoter --- p.33 / Chapter 3.2 --- SEAP as a Reporter Gene for Promoter Deletion Study --- p.34 / Chapter 3.3 --- Materials --- p.35 / Chapter 3.3.1 --- "Chemicals, Kits and Reagents" --- p.35 / Chapter 3.3.2 --- Buffers and Solutions --- p.35 / Chapter 3.3.3 --- Bacterial Strain --- p.36 / Chapter 3.3.4 --- Cell Line --- p.36 / Chapter 3.3.5 --- Enzymes --- p.37 / Chapter 3.3.6 --- Nucleic Acids --- p.37 / Chapter 3.3.7 --- Oligo Primers --- p.37 / Chapter 3.4 --- Methods --- p.38 / Chapter 3.4.1 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pBlueScript II SK (-) --- p.38 / Chapter (a) --- Design and Synthesis of Oligo Primers --- p.38 / Chapter (b) --- Isolation of Genomic DNA from K562 Cells --- p.39 / Chapter (c) --- PCR Amplification of Gamma Globin Gene Promoter and 3' Enhancer --- p.40 / Chapter (d) --- Ligation of PCR Fragments into EcoR V-cut pBlueScript II SK (-) --- p.41 / Chapter (e) --- Preparation of E coli DH5a Competent Cells --- p.43 / Chapter (f) --- Heat-Shock Transformation of E. coli DH5a Competent Cells --- p.44 / Chapter (g) --- PCR Screening and Plasmid Purification of Putative pBlu2SKM-γAP and pBlu2SKM-γAE --- p.44 / Chapter (h) --- Isolation of Putative pBlu2SKM-γAP and pBlu2SKM-γAE Plasmid DNA --- p.45 / Chapter (j) --- Nucleotide Sequencing of Putative pBlu2SKM-yAP and pBlu2SKM-γAE --- p.47 / Chapter (j) --- Graphical Summary of Section 3.6.1 Sub-cloning Procedures --- p.49 / Chapter 3.4.2 --- Molecular Cloning of A-Gamma Globin Gene Promoter and 3' Enhancer into pSEAP2-Enhancer --- p.51 / Chapter (a) --- Sub-cloning of Promoter Fragment into pSEAP2-Enhancer --- p.51 / Chapter (b) --- Sub-cloning of 3' Enhancer Fragment into p 1224γAP-SEAP2 --- p.52 / Chapter (c) --- Graphical Summary of Section 3.6.2 Sub-cloning Procedures --- p.54 / Chapter 3.4.3 --- Construction of p 1224γAP-SEAP2-γAE Promoter Deletions Constructs --- p.56 / Chapter (a) --- Restriction Digestion at 5' End of A-Gamma Promoter Deletions --- p.56 / Chapter (b) --- Restriction Digestion at 3' Ends of A-Gamma Promoter Deletions --- p.56 / Chapter (c) --- Blunting 5'-overhangs and Self-Ligation of Linearized Plasmid Constructs --- p.57 / Chapter (d) --- Graphical Summary of Section 3.6.3 5,Deletions Procedures --- p.58 / Chapter 3.5 --- Results --- p.59 / Chapter (a) --- Nucleotide Sequence Confirmed by Cycle Sequencing --- p.60 / Chapter (b) --- "Resulting Plasmid Constructs p 1224γAP-SEAP2-yAE, p754yAP-SEAP2-yAE and p205yAP-SEAP2-γAE" --- p.64 / Chapter 3.6 --- Discussion --- p.67 / Chapter Chapter 4: --- Mapping of LC978-Responsive Elements on Human A-Gamma Globin Gene Promoter --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.69 / Chapter 4.3 --- pSV-β-Galactosidase as a Transfection Normalization Standard --- p.70 / Chapter 4.4 --- Materials --- p.72 / Chapter 4.4.1 --- "Chemicals, Kits and Reagents" --- p.72 / Chapter 4.4.2 --- Buffers and Solutions --- p.73 / Chapter 4.4.3 --- Cell Line --- p.74 / Chapter 4.4.4 --- Nucleic Acids --- p.74 / Chapter 4.4.5 --- Instruments and Equipments --- p.74 / Chapter 4.5 --- Methods --- p.74 / Chapter 4.5.1 --- Determination of Optimal Dose of Transfection Reagent for --- p.74 / Chapter (a) --- Sterilization of Plasmid DNA for Transfection --- p.74 / Chapter (b) --- Transient Transfection of K562 Cells by pEGFP-N 1 --- p.75 / Chapter (c) --- Examination of EGFP Expression Level --- p.76 / Chapter 4.5.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.76 / Chapter (a) --- Transient Transfection of K562 Cells by pSV-β-Gal --- p.76 / Chapter (b) --- Determination of β-Galactosidase Expression Level --- p.76 / Chapter 4.5.3 --- Mapping of LC978-Responsive Elements on Human Gamma Globin Gene Promoter --- p.77 / Chapter (a) --- Co-Transfection of K562 Cells by p1224/754/205γAP-SEAP2 -γAE and pSV-β-Gal --- p.77 / Chapter (b) --- Treatment of Co-Transfected K562 Cells by LC978 --- p.77 / Chapter (c) --- Determination of β-Galactosidase Expression Level --- p.78 / Chapter (d) --- Determination of Secreted Alkaline Phosphatase (SEAP) Expression Level --- p.78 / Chapter (e) --- Determination of Fetal Hemoglobin Expression Level --- p.79 / Chapter 4.5.4 --- Mapping of Hydroxyurea-Responsive Elements on Human Gammm Globin Gene Promoter --- p.80 / Chapter (a) --- Determination of Optimal Biological Dose (OBD) of Hydroxyurea --- p.80 / Chapter (b) --- Co-Transfection of K562 Cells and Subsequent Treatment by Hydroxyurea --- p.80 / Chapter (c) --- "Assay for β-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.81 / Chapter 4.5.5 --- Sodium Butyrate-Induced SEAP Expression --- p.81 / Chapter (a) --- Determination of Optimal Biological Dose (OB(d) of Sodium Butyrate --- p.81 / Chapter (b) --- Co-Transfection of K562 Cells and Treatment by Sodium Butyrate --- p.82 / Chapter (c) --- "Assay for p-Galactosidase (β-Gal), Secreted Alkaline Phosphatase (SEAP) and Fetal Hemoglobin (HbF) Expression Level" --- p.83 / Chapter 4.5.6 --- Data Analysis --- p.83 / Chapter (a) --- "Data Processing, Normalization and Graphing" --- p.83 / Chapter (b) --- Statistical Analysis --- p.83 / Chapter 4.6 --- Results --- p.84 / Chapter 4.6.1 --- Optimal Dose of Transfection Reagent for K562 --- p.84 / Chapter 4.6.2 --- β-Galactosidase as Normalization Standard for K562 Transfections --- p.84 / Chapter 4.6.3 --- LC978 Induction on Co-Transfected K562 Cells --- p.84 / Chapter 4.6.4 --- Hydroxyurea Induction on Co-Transfected K562 Cells --- p.85 / Chapter 4.6.5 --- Sodium Butyrate Induction on Co-Transfected K562 Cells --- p.86 / Chapter 4.7 --- Discussion --- p.98 / Chapter 4.7.1 --- Theme Question to be Answered --- p.98 / Chapter 4.7.2 --- Optimal Dose of DMRIE-C Transfection Reagent on K562 Cell Line --- p.98 / Chapter 4.7.3 --- pSV-β-gal as an Internal Normalization Control --- p.99 / Chapter 4.7.4 --- Responsive Element Mapping --- p.99 / Chapter (a) --- LC978-Induced Response --- p.100 / Chapter (b) --- Hydroxyurea-Induced Response --- p.100 / Chapter (c) --- Sodium Butyrate-Induced Response --- p.101 / Chapter 4.7.5 --- LCR-Dependent Gamma Globin Gene Reactivation --- p.101 / Chapter 4.7.6 --- Induction of Gamma Globin by Histone Deacetylase Inhibitor --- p.104 / Chapter 4.7.7 --- Basal SEAP Expression Levels of the Promoter-Reporter Constructs --- p.105 / Chapter 4.7.8 --- Summary --- p.105 / Chapter Chapter 5: --- General Discussion --- p.106 / References Cited --- p.109

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325723
Date January 2006
ContributorsChan, Kai Man., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xvii, 119 leaves : ill. (some col.) ; 30 cm.
RightsUse 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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