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