Analysis of S/MAR vectors for gene therapy in muscle

3 ACKNOWLEDGEMENTS.5 GLOSSARY OF ABBREVIATIONS.6 TABLE OF CONTENTS.7 LIST OF FIGURES. 13 CHAPTER 1: INTRODUCTION . 17 1.1 MUSCULAR DYSTROPHY . 17 1.2 SKELETAL MUSCLE FUNCTION . 19 1.2.1 General Muscle Structure . 19 1.2.2 The Role of Dystrophin Protein In Skeletal Muscle Function.21 1.2.3 The Role of Dystrophin-Associated Protein Complex (DAPC) In Skeletal Muscle Function.25 1.2.4 Utrophin Protein in Skeletal Muscle.29 1.2.5 Therapeutic Approaches to Muscular Dystrophy.29 1.3 MUSCLE STEM CELLS . 30 1.3.1 Satellite Cells . 30 1.3.2 Satellite Cell Identifiable Markers and Transcription Factors During Quiescence, Activation, and Differentiation.32 1.3.3 The C2C12 Cell Line As A Model For Quiescent Satellite Cells, Proliferating Myoblasts, And Myofibres . 35 1.4 MUSCULAR DYSTROPHY TREATMENT USING CELL THERAPY . 36 1.5 GENE THERAPY. 37 1.6 MUSCULAR DYSTROPHY TREATMENT . 40 1.6. 1 Viral Approaches for Gene Delivery . 40 1.6. 2 Muscular Dystrophy Treatment Using Non-Viral Gene Transfer . 41 1.7 EPIGENETICS . 45 1.7. 1 Histones.45 1.7. 2 Plasmid DNA And Epigenetics.48 1.7. 3 Methylation.49 1.8 IMPROVING PLASMID TRANSGENE EXPRESSION. 51 1.9 SCAFFOLD/MATRIX ATTACHMENT REGION (S/MAR) ELEMENTS, DNA ORGANISATION, AND THE NUCLEAR MATRIX . 54 1.9. 1 DNA Replication Initiation Complex And The Binding of Associated Proteins To S/MAR Elements.57 1.9. 2 The Role of S/MARs In Transcription And Its Related Factors . 58 1.10 S/MAR STUDIES. 63 1.11 S/MAR DATABASE AND ALTERNATIVE S/MARS. 67 1.11.1 The c-MYC Proto-oncogene S/MAR Element . 68 1.12 AIMS AND OBJECTIVES . 69 CHAPTER 2: MATERIALS AND METHODS. 73 8 2.1. MATERIALS . 73 2.1.1. General Buffers and Chemical Reagents . 73 2.1.2. Cell Culture . 73 2.1.2.1. Cell Types . 73 2.1.2.2. General Cell Culture Materials and Reagents.74 2.1.3. Bacterial Culture.74 2.1.3.1. General Bacterial Culture Materials and Reagents . 74 2.1.3.2. Plasmid Vectors Recieved/Purchased . 75 2.1.3.3. Plasmid Vectors Constructed within RHUL.79 2.1.4. Immunostaining . 83 2.1.4.1. Antibodies . 83 2.1.4.2. General Immunostaining Materials and Reagents . 83 2.1.5. DNA Extraction Reagent . 83 2.1.6. Fluorescent In-Situ Hybridisation (FISH) Reagents:. 83 2.1.7. Polymerase Chain Reaction (PCR) Reagents and Primers: . 84 2.2. METHODS. 85 2.2.1. Cell Culture Methods . 85 2.2.1.1. Culture and maintenance of cells.85 2.2.1.2. Frozen Cell Storage.85 2.2.1.3. Thawing Frozen Cell Stocks . 85 2.2.1.4. Induction of Myogenic Differentiation In C2C12 Cultures.86 2.2.1.5. Lipofection: Liposome-Mediated DNA Transfection . 86 2.2.1.6. G-418 Sensitivity Curves.87 2.2.1.7. C2C12 Cell Arrest In G0 Phase of Cell Cycle.87 2.2.1.8. Reserve Cell Isolation . 87 2.2.2 Bacterial Culture and Molecular Cloning.88 2.2.2.1. Preparation of selective agar . 88 2.2.2.2. Bacterial streaks . 88 2.2.2.3. T4 DNA Polymerase mediated conversion of 5’ overhangs to blunt ended termini . 88 2.2.2.4. Ligation of a DNA insert into a plasmid vector backbone . 89 2.2.2.5. Plasmid DNA transformation of bacteria by electroporation.89 2.2.2.6. Screening of transformants.90 2.2.2.7. Analysis of DNA By Restriction Endonuclease Digestion and Electrophoresis . 90 2.2.2.8. Amplification and purification of plasmid DNA . 91 2.2.2.9. Glyc