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The Gabaa Receptor 8 Subunit Gene Promoter Fragments to Direct Long - Term Neuron - Specific Expression

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The Gabaa Receptor 8 Subunit Gene Promoter Fragments to Direct Long - Term Neuron - Specific Expression THE GABAa RECEPTOR 8 SUBUNIT GENE PROMOTER : CHARACTERISATION AND USE Alexa Brett Roberts Division of Molecular Genetics Institute of Biomedical and Life Sciences University of Glasgow Glasgow A dissertation submitted for the degree of Doctor of Philosophy of the University of Glasgow February 1998 ProQuest Number: 13818612 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13818612 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 GIASGOW UNIVERSITY LIBRARY 11110 (coh O f GLASGOW 1 IUNIVERSITT I [LQgAW I For my husband Stuart and for my children Ross and Ailie with love Also to JR (Grandad) in loving memory Preface The work presented in this thesis was performed entirely by the author, except where acknowledged. I declare that my thesis contains unique work and will not be submitted for any other degree, diploma or qualification at any other University. A. Brett Roberts December 1997 Acknowledgements I would like to thank Professor R.W. Davies for giving me the opportunity to study for this degree and for his help throughout the last three years. I would also like to thank my colleagues for their help and advice. Particular thanks go to the technical staff - - Lynn Loughlin (Supertech) for her help and friendship, Irene Houghton and Gareth Westrop, and to Team Scotland : Nicola Craig (for keeping me sane and making me laugh), Vicki Laird and Thora Glencorse. I am especially indebted to Dr Thora Glencorse for her help, advice and encouragement. I would also like to thank Prof. B. Clements and Dr A. MacLean (Dept, of Virology, Glasgow) for help with the virus work. Thanks also to Dr Jackie Campbell and Neil Bennett (Dept, of Anatomy, Glasgow University) for performing the stereotactic injections. I acknowledge the Medical Research Council for supporting this work, and Glasgow University. Special thanks go to my husband Stuart for all his support and encouragement throughout the last 3 years and my children for letting me be an absent Mum for so long. Contents Page Preface i Acknowledgements ii Contents iii List of Figures x List of Tables xiv Abbreviations xv Summary xvii Chapter 1 General Introduction 1 1.1 GAB A RECEPTORS 2 1.1.1 The GABA a receptor 3 1.1.2 Pharmacology of the GABA a receptor 4 1.1.2.1 The GAB A binding site 5 1.1.2.2 The benzodiazepine site 5 1.1.2.3 The barbiturate site 7 1.1.2.4 The steroid site 7 1.1.2.5 Picrotoxin/TBPS site 7 1.1.2.6 Other sites 8 1.1.3 Structure of the GABA a receptor 8 1.1.4 Molecular biology of the GABA a receptor 11 1.1.4.1 Receptor heterogeneity 11 1.1.4.2 Receptor subunit families 12 1.1.4.2.1 a-subunits 12 1.1.4.2.2 p - subunits 14 1.1.4.2.3 y-subunits 14a 1.1.4.2.4 5 - subunit 15 1.1.4.2.5 e-subunit 15 1.1.4.2.6 7i-subunit 15 1.1.5 GABA a receptor expression patterns 16 1.1.5.1 Cell - specific expression 16 1.1.5.2 Developmental expression 17 1.2 NEURON - SPECIFIC GENE EXPRESSION 18 1.3 GABA a RECEPTOR 8 SUBUNIT GENE 28 1.3.1 Gene structure 28 1.3.2 8 expression patterns 31 iii 1.3.3 Pharmacology 3 2 1.3.4 Chromosomal assignment of human GABA a receptor 8 subunit gene 3 3 1.3.5 Recent research developments 3 4 1.4 RESEARCH PROJECT 34 Chapter 2 Materials and Methods 36 2.1 MATERIALS 37 2.1.1 Bacterial growth 3 7 2.1.1.1 Bacterial strains 37 2.1.1.2 Bacterial media requirements 3 7 2.1.2 Restriction and modifying enzymes 38 2.1.3 Biochemicals and chemicals 38 2.1.4 Molecular size standards 38 2.1.5 Radionucleotides 3 8 2.1.6 Large scale DNA purification 38 2.1.7 Vectors 39 2.1.8 Oligonucleotide primers 40 2.1.9 DNA sequencing 44 2.1.10 RNA isolation 45 2.1.10.1 Total RNA isolation 45 2.1.10.2 mRNA isolation 45 2.1.11 Cell culture 45 2.1.11.1 Cell lines 45 2.1.11.2 Medium for cell lines 46 2.1.11.2.1 BHK21/C13 46 2.1.11.2.2 NB4 1 A3 46 2.1.11.2.3 CGR8.8 46 2.1.11.3 Solutions for gene targeting 47 2.1.11.3.1 Trypsin solution 47 2.1.11.3.2 Freezing medium 47 2.1.11.4 Solutions for cell transfections 47 2.1.11.5 P - galactosidase assays 47 2.1.11.6 Luciferase assays 48 2.1.12 Cerebellar granule cell cultures 48 2.1.12.1 Medium for granule cells 48 2.1.12.2 Solutions for granule cell cultures 48 2.1.12.3 Solutions for granule cell transfections 49 2.1.12.4 Solutions for granule cells - histochemistry 49 2.1.13 Histochemistry of rat brains by X - gal staining 49 iv 2.2 METHODS 50 2.2.1 Basic molecular biology techniques 50 2.2.1.1 Bacterial growth 50 2.2.1.1.1 Bacterial transformation 50 2.2.1.1.2 Bacterial plasmid growth - large scale 51 2.2.1.2 Restriction enzyme digestion 51 2.2.1.3 DNA isolation 51 2.2.1.3.1 Small scale plasmid DNA isolation 51 2.2.1.3.2 Large scale plasmid DNA isolation 51 2.2.1.4 Ligation 52 2.2.1.5 Klenow reactions 52 2.2.1.6 Phenol extraction 52 2.2.1.7 Ethanol precipitation 52 2.2.1.8 Agarose gel electrophoresis 53 2.2.1.9 Radiolabelling of probes 5 3 2.2.1.9.1 Oligonucleotide probes 53 2.2.1.9.2 Random primed probes 53 2.2.1.10 Southern blotting 53 2.2.2 Subcloning of DNA from lambda clones 54 2.2.3 Sequencing of pBluescript double-stranded templates 54 2.2.3.1 Template DNA preparation 54 2.2.3.2 DNA sequencing of double-stranded templates 54 2.2.3.3 Analysis of DNA sequence data 55 2.2.4 The Polymerase Chain Reaction 55 2.2.5 RNA purification 55 2.2.5.1 Total RNA preparation 55 2.2.5.2 mRNA preparation 56 2.2.6 Oligo-capping 57 2.2.6.1 Removal of 5' - phosphate 57 2.2.6.2 Removal of 5' - cap 58 2.2.6.3 Ligation of r - oligonucleotide 58 2.2.6.4 First strand cDNA synthesis 58 2.2.6.5 Amplification of 5' - end of cDNA 58 2.2.7 Production of recombinant viruses 59 2.2.7.1 Insertion of promoter-reporter gene cassettesinto an HSV-1 shuttle vector 59 2.2.7.2 Recombination of shuttle vectors with an HSV-1 host 59 2.2.13 Isolation of single plaques from the recombinant virus stock 60 v 2.2.7.4 Screening for recombinant viruses 61 2.2.7.5 Large-scale viral DNA purification 61 2.2.7.6 Preparation of high titre viral stock 62 2.2.7.7 Titration of viral stocks 63 2.2.8 Gene targeting 64 2.2.8.1 Construction of vector 64 2.2.8.2 Growth of cells 64 2.2.8.3 Transfection of ES cells 64 2.2.8.4 Screening for targeted cell lines 65 2.2.8.4.1 Selection of resistant colonies 65 2.2.8.4.2 Freezing of selected colonies 66 2.2.8.4.3 Preparation of genomic DNA 66 2.2.8.4.4 Screening of genomic DNA 66 2.2.9 In vitro analysis of promoter-reporter constructs 67 2.2.9.1 Neuronal cell cultures 67 2.2.9.1.1 Preparation of cells 67 2.2.9.1.2 Transfection of cells 67 2.2.9.1.3 Harvesting of transfected cells 67 2.2.9.1.4 p - galactosidase assays of cell extracts 68 2.2.9.1.5 Luciferase assays of cell extracts 68 2.2.9.2 Cerebellar granule cell cultures 68 2.2.9.2.1 Preparation of cells 68 2.2.9.2.2 Transfection of cells 69 2.2.9.2.3 X - gal staining of granule cell cultures 70 2.2.10 Analysis of HSV-1 promoter constructs 70 2.2.10.1 In vitro analysis of cerebellar granule cells 70 2.2.10.1.1 Infection of granule cells 70 2.2.10.1.2 X - gal staining of infected cells 70 2.2.10.2 In vivo analysis by stereotactic injection into rats 70 2.2.10.2.1 Intracerebellar inoculation of animals 70 2.2.10.2.2 Histochemistry of extracted brains 71 Chapter 3 Characterisation of the GABAa Receptor 5 Subunit Gene Promoter 72 3.1 INTRODUCTION 73 3.2 RESULTS 74 3.2.1 Subcloning of the promoter region for sequence analysis 74 3.2.2 Identification of transcription start sites 75 3.2.3 Sequencing of the 5' - flanking region 81 vi 3.2.4 Analysis of the 5' - flanking region sequence 88 3.2.5 Comparison of murine and rat 5' - promoter regions 92 3.3 DISCUSSION 97 Chapter 4 Generation of GABAa Receptor 5 Subunit Gene Promoter Constructs 99 4.1 INTRODUCTION 100 4.2 RESULTS 100 4.2.1 Design and generation of reporter-gene vectors for promoter-deletion constructs 100 4.2.1.1 Luciferase vector construction 103 4.2.1.2 Luciferase control vector construction 103 4.2.1.3 lacZ vector construction 103 4.2.1.4 lacZ control vector construction 104 4.2.2 Construction of promoter-deletion vectors 110 4.3 DISCUSSION 113 Chapter 5 Analysis of Promoter Constructs in vitro 116 5.1 INTRODUCTION 117 5.2 RESULTS 117 5.2.1 Expression of promoter - reporter constructs in cell culture 117 5.2.2 Expression of promoter - reporter constructs in primary cell cultures 121 5.3 DISCUSSION 122 Chapter 6 Herpes Simplex Virus as a Delivery System 124 6.1 INTRODUCTION 125 6.1.1 Herpes simplex virus type 1 125 6.1.2 Molecular biology of HSV-1 126 6.1.3 HSV-1 latency 127 6.1.4 Selection of viral vector 128 6.1.5 Choice of HSV-1 mutant 129 6.1.6 Choice of insertion site for GABA a 6 promoter - reporter gene expression constructs 130 6.2 RESULTS 134 6.2.1 Generation of promoter construct - shuttle vectors 134 6.2.1.1 p35 and pBL promoter construct - shuttle vectors 134 6.2.1.2 pZ43 and pZLAT promoter construct - shuttle vectors 135 6.2.2 Generation of recombinant HSV-1 viruses 136 6.2.2.1 1764 viral variants 136 vii 6.22.2 1716 viral variants 142 6.2.2.2.1 pZ43 - 1.6 - lacZ 142 6.2.22.2 pZ43 - 10.5 - lacZ 147 6.2.2.2.3 Other pZ43 and pZLAT recombinant viruses 149 6.3 DISCUSSION 150 Chapter 7 Infection of
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