Characterization of the Popeye domain containing gene family in zebrafish [Elektronische Ressource] / Bettina Carmen Kirchmaier. Betreuer: Thomas Brand

Bettina Mrosowski

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Characterization of the Popeye domain containing gene family
in zebrafish






Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg










vorgelegt von
Bettina Carmen Kirchmaier
aus Frankfurt am Main


Würzburg, 2010






























Eingereicht am:………………………………

Mitglieder der Promotionskommission:

Vorsitzender: Prof. Dr. Thomas Dandekar
1. Gutachter: Prof. Dr. Thomas Brand
2. Gutachter: Prof. Dr. Christoph Winkler

Tag des Promotionskolloquiums:…………………………….

Doktorurkunde ausgehändigt am:……………………………








Für meine Familie, in Liebe

Index 4

CONTENTS
Contents .................................................................................................................................................................. 4
Figures .... 9
Tables .................................................................................................................................................................... 10
1 Summary ......... 11
2 Zusammenfassung.......................................................................................................................... 12
3 Introduction ..................................... 13
3.1 The vertebrate heart........................................................................................................................ 13
3.1.1 Formation of the heart field .............. 13
3.1.2 Heart tube assembly and development of polarity ........................................................................... 14
3.1.3 Chamber formation and cardiac conduction development ............................... 15
3.1.4 Heart remodeling ............................................................................................................................. 17
3.2 Electrical activity of the adult heart .................................. 17
3.2.1 The nodes and the fast conduction Purkinje fiber networks ............................................................ 19
3.2.2 Gap junctions ................................................................................................... 19
3.2.3 Cardiac currents involved in generation of myocardial action potentials ......... 20
3.3 Danio rerio ...................................................................................................................................... 23
3.3.1 The zebrafish as model system for cardiovascular research ........................... 23
3.3.2 Zebrafish heart development ........................................................................................................... 24
3.3.3 Development of the cardiac conduction system in zebrafish ........................................................... 25
3.4 The Popeye domain containing (Popdc) gene family ...................................... 27
3.4.1 Expression pattern ........................................................................................................................... 27
3.4.2 Protein structure and distribution ..................................... 28
3.4.3 Interaction partners .......................................................................................................................... 29
3.4.4 Function ........................................................................................................................................... 29
3.5 Aim of the study .............................................................................................................................. 31



Index 5

4 Material ........................................................................................................................................... 32
4.1 Biological material ........................................................................................................................... 32
4.1.1 Bacterial strains ............................... 32
4.1.2 Vectors ............................................................................................................................................ 32
4.1.3 Fish lines ......... 33
4.1.4 Primary and secondary antibodies .................................................................................................. 33
4.2 Molecularbiological material ............ 33
4.2.1 DNA and protein ladders ................................................................................................................. 33
4.2.2 Kits ................................................... 34
4.3 Software .......................................................................................................... 34
4.4 Equipment ....................................... 35
4.5 Reagents ......................................................................................................... 36
4.6 Enzymes ......................................... 37
4.7 Primers ............................................................................................................ 38
4.8 Morpholinos .................................... 38
5 Methods .......................................................................................................... 39
5.1 Fish care ......................................... 39
5.1.1 Setting up pair crosses .................................................................................................................... 39
5.1.2 PTU treatment to prevent melanization of embryos ......... 40
5.2 Genexpression manipulation in Danio rerio .................................................................................... 40
5.2.1 Agarose plates for holding embryos ................................ 40
5.2.2 Microinjections ................................................................................................. 40
5.2.3 Morpholinos ..................................... 41
5.2.4 Chemical treatment with cyclopamine ............................................................................................. 41
5.3 Physiological methods/Imaging methods ........................ 42
5.3.1 Recording of cardiac activity ............................................................................................................ 42
5.3.2 Isolated calcium transient recordings by selective plane illumination microscopy (SPIM) ............... 42
5.4 Microbiological methods .................................................................................................................. 43
5.4.1 Ligation ............................................ 43
5.4.2 Transformation................................................................................................. 43 Index 6

5.4.3 Cloning into plasmid vectors ............................................................................................................ 45
5.4.4 Screening bacterial colonies using X-Gal and IPTG ........ 45
5.4.5 Glycerol stocks ................................................................................................................................ 45
5.5 Molecularbiological methods ........... 46
5.5.1 Preabsorption of antibody for whole mount in situ hybridization ...................................................... 46
5.5.2 Whole mount RNA in situ probe generation ..................................................... 46
5.5.3 Whole mount in situ hybridization using a single digoxygenin-labeled probe .................................. 47
5.5.4 RNA isolation using Trizol ................................................................................................................ 50
5.5.5 In vitro transcription ......................... 51
5.5.6 In vitro synthesis of capped RNA ..................................................................................................... 51
5.5.7 Preparation of plasmid DNA by alkaline lysis with SDS (Minipreparations 1-2 ml) .......................... 51
5.5.8 Restriction analysis ................................................................................................ 53
5.5.9 Primer design................................... 53
TM5.5.10 Endpoint PCR using Phusion DNA polymerase ........................................... 53
5.5.11 Endpoint PCR using Taq polymerase .............................................................................................. 54
5.5.12 DEPC-treated water ......................................................... 54
5.6 Immunological methods .................................................................................. 55
5.6.1 Phalloidin staining ............................................................ 55
5.6.2 Whole mount antibody staining with DAB ........................................................................................ 55
5.6.3 Whole mount immunocytochemistry (double staining MF20/S46) ................... 56
5.7 Histological methods ..............................................................................................................