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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325723 |
| Date | January 2006 |
| Contributors | Chan, Kai Man., 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, xvii, 119 leaves : ill. (some col.) ; 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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