Characterization and modification of genetically encoded indicators to monitor neural activity in Drosophila melanogaster [Elektronische Ressource] / Alexandra Ihring

Alexandra Ihring

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 23 Mo

Extrait

CHARACTERIZATION AND MODIFICATION OF
GENETICALLY ENCODED INDICATORS TO
MONITOR NEURAL ACTIVITY IN DROSOPHILA
MELANOGASTER





Dissertation


zur Erlangung des Doktorgrades der Naturwissenschaften


an der Fakultät für Biologie
der Ludwig-Maximilians-Universität München



Alexandra Ihring


München, 2006




























Erstgutachter: Prof. Dr. Alexander Borst
Zweitgutachter: Prof. Dr. Rainer Uhl
Tag der mündlichen Prüfung: 18.Juli 2006



TABLE OF CONTENTS
Table of Contents

Table of Contents…………………………………………………..……….…I
Table of Figures……………………………………………….….……..…….V
Abbreviations……………………………………………………………...…IX

1 Abstract...............................................................................................1
2 Introduction........................................................................................3
2.1 General..........................................................................................................3
2.2 Green Fluorescencent Protein ....................................................................5
2.3 Fluorescence and FRET ..............................................................................7
2.4 Genetically Encoded Indicators................................................................10
2.4.1 Voltage Sensors...................................................................................10
2.4.2 Sensor for Vesicle Release: SynaptopHluorin.....................................11
2.4.3 Calcium Indicators...............................................................................12
2.5 Drosophila melanogaster............................................................................17
2.5.1 D. melanogaster Life Cycle.................................................................17
2.5.2 The Genome .............................................................18
2.6 GAL4 -UAS System18
2.7 Analysis of Genetically Encoded Indicators............................................19
2.7.1 The Drosophila Neuromuscular Junction19
3 Material and Methods .....................................................................23
3.1 Laboratory Animals and Preparations....................................................23
3.1.1 Fly Strains............................................................................................23
3.1.2 Driver Lines.........................................................................................28
3.1.3 Fly Food...............................................................................................28
3.1.3.1 Appleagar Dishes.............................................................................28
3.1.3.2 Drosophila Food...............................................................................28
3.1.4 Breeding28
3.1.5 Engineering of Transgenic Flies ..........................................................29
3.1.5.1 Spin Dialysis....................................................................................29
3.1.5.2 DNA-Precipitation and Injection Mix .............................................29
3.1.5.3 Injection of Fly Embryos with DNA ...............................................29
I TABLE OF CONTENTS
3.1.5.4 Generation of Transgenic Fly Stocks...............................................30
3.1.6 Crossing for GAL4-UAS flies .............................................................30
3.2 Material.......................................................................................................30
3.2.1 Chemicals.............................................................................................30
3.2.2 Instruments...........................................................................................33
3.2.2.1 Imaging Setup..................................................................................33
3.2.2.2 Confocal Microscopy.......................................................................34
3.2.3 Consumables........................................................................................34
3.2.4 Antibodies............................................................................................35
3.2.5 Plasmids...............................................................................................35
3.2.6 Enzymes36
3.2.7 Oligonucleotides..................................................................................36
3.2.8 Antibiotics............................................................................................40
3.3 Media, Buffer and Solutions .....................................................................40
3.4 Molecular Methods ....................................................................................43
3.4.1 Spectrometric Determination of DNA Concentration .........................43
3.4.2 DNA Amplification using Polymerase Chain Reaction ......................43
3.4.3 Restriction site cleavage of DNA ........................................................44
3.4.4 Ligation of DNA Fragments................................................................44
3.4.5 Transformation of Chemically Competent E. coli...............................45
3.4.6 Isolation of Nucleic Acids ...................................................................45
3.4.6.1 Small-scale Plasmid Isolation from E. coli (Miniprep) ...................45
3.4.6.2 Large-scale Plasmid Isolation from E. coli (Midiprep)45
3.4.7 Agarose Gel Electrophoresis................................................................45
3.4.8 Site-directed Mutagenesis by PCR ......................................................46
3.5 Cell culture .................................................................................................47
3.5.1 Maintenance of Drosophila Neuronal Cell Culture.............................47
3.5.1.1 Media...............................................................................................47
3.5.1.2 Splitting Cells...................................................................................47
3.5.2 Transfection of Drosophila Neuronal Cells.........................................47
3.6 Histology and Immunohistochemistry .....................................................48
3.6.1 Antibody Staining of Larvae................................................................48
3.6.1.1 Antibody Staining with Primary and Secondary Antibodies...........48
3.6.1.2 Antibody Staining with α-GFP Alexa Fluor488..............................48
II TABLE OF CONTENTS
3.6.2 Dissection of Drosophila Brains..........................................................49
3.7 Stimulation and Optical Imaging .............................................................49
3.7.1 Larval Preparation................................................................................49
3.7.2 Physiology and Imaging......................................................................50
3.7.3 Data Analysis.......................................................................................51
3.7.4 Signal-to-Noise-Ratio..........................................................................52
4 Results ...............................................................................................53
4.1 Analyzed Indicators and Transgenic Flies ..............................................53
4.1.1 Expression............................................................................................54
4.1.2 Experimental Procedure.......................................................................55
4.2 In Vivo Measurements of Genetic Calcium Indicators and SpH...........57
4.3 Superecliptic SpH.......................................................................................58
4.4 Calcium Indicators Based on One Chromophore...................................59
4.4.1 Inverse Pericam....................................................................................59
4.4.2 Camgaroo Variants..............................................................................60
4.4.2.1 Camgaroo-1......................................................................................60
4.4.2.2 Camgaroo-260
4.4.3 Flash Pericam.......................................................................................61
4.4.4 GCaMP Variants..................................................................................62
4.4.4.1 GCaMP 1.362
4.4.4.2 GCaMP 1.6......................................................................................64
4.5 Calcium Indicators Based on Two Chromophores.................................65
4.5.1 Yellow Cameleon 2.0...........................................................................65
4.5.2 Yellow Cameleon 2.366
4.5.3 Yellow Cameleon 3.366
4.5.4 TNL 15.................................................................................................68
4.6 Signals Over a Wide Range of Neural Activity.......................................69
4.7 Kinetics and SNR Depend on the Expression Level ...............................71 <