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Studies on the Interaction Between Retinoschisin and the Retinal Na/K-Atpase

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Studies on the Interaction Between Retinoschisin and the Retinal Na/K-Atpase Studies on the Interaction between Retinoschisin and the Retinal Na/K-ATPase Towards Elucidating the Molecular Pathomechanism of X-linked Juvenile Retinoschisis Dissertation zur Erlangung des Doktorgrades der Biomedizinischen Wissenschaften (Dr. rer. physiol.) der Fakultät für Medizin der Universität Regensburg vorgelegt von Karolina Plößl aus Nabburg im Jahr 2017 Studies on the Interaction between Retinoschisin and the Retinal Na/K-ATPase Towards Elucidating the Molecular Pathomechanism of X-linked Juvenile Retinoschisis Dissertation zur Erlangung des Doktorgrades der Biomedizinischen Wissenschaften (Dr. rer. physiol.) der Fakultät für Medizin der Universität Regensburg vorgelegt von Karolina Plößl aus Nabburg im Jahr 2017 Dekan: Prof. Dr. Dr. Torsten E. Reichert Betreuer: Prof. Dr. Bernhard H.F. Weber Tag der mündlichen Prüfung: Parts of this work have already been published in peer-reviewed journals in an open access format Plössl, K., Weber, B. H. F., & Friedrich, U. (2017). The X-linked juvenile retinoschisis protein retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina. Journal of Cellular and Molecular Medicine, 21(4), 768–780. (a) Plössl, K., Royer, M., Bernklau, S., Tavraz, N. N., Friedrich, T., Wild, J., Weber, BHF, Friedrich, U. (2017). Retinoschisin is linked to retinal Na/K-ATPase signaling and localization. Molecular Biology of the Cell, mbc.E17-01-0064. (b) Table of Contents Zusammenfassung................................................................................................................... 1 Summary ................................................................................................................................... 3 1 Introduction ....................................................................................................................... 5 1.1 X-linked Juvenile Retinoschisis (XLRS) – Clinical Features ............................................... 5 1.2 Mutations in the RS1 Gene are Causative for XLRS ......................................................... 6 1.3 Rs1h Knock Out Mice – A Model System for XLRS ........................................................... 7 1.4 Prospect on Therapeutic Strategies for XLRS ................................................................... 7 1.5 The Retinoschisin Protein ............................................................................................... 8 1.6 Categorization of XLRS Associated Mutations .................................................................11 1.7 Towards Retinoschisin Function – Proposed Interaction Partners .....................................12 1.7.1 Phospholipids ........................................................................................................12 1.7.2 Galactose ..............................................................................................................13 1.7.3 Extracellular Matrix Proteins ....................................................................................13 1.7.4 L-Type Voltage Gated Calcium Channels.................................................................13 1.7.5 The Retinal Na/K-ATPase .......................................................................................14 1.8 The Na/K-ATPase .........................................................................................................15 1.8.1 Structure of the Na/K-ATPase .................................................................................15 1.8.2 The Na/K-ATPase as an Ionpump ...........................................................................17 1.8.3 Na/K-ATPase Mediated Signaling ...........................................................................18 1.8.4 Na/K-ATPases in Intracellular Adhesion...................................................................20 1.9 Aim of this Study ...........................................................................................................20 2 Material ............................................................................................................................. 21 2.1 Mouse Strains ...............................................................................................................21 2.2 Escherichia coli (E.coli) Strains .......................................................................................21 2.3 Eukaryotic Cell Lines .....................................................................................................21 2.4 Oligonucleotides for PCR and Sequencing Reactions ......................................................21 2.5 Oligonucleotides and Corresponding Probes Used for qRT-PCR ......................................24 2.6 Plasmids and Expression Constructs ..............................................................................25 2.7 Primary Antibodies ........................................................................................................27 2.8 Secondary Antibodies ....................................................................................................28 2.9 Molecular Weight Standards ..........................................................................................28 2.10 Enzymes.......................................................................................................................29 2.11 Kit Systems ...................................................................................................................29 2.12 Chemicals and Ready Made Solutions ............................................................................30 2.13 Buffers and Solutions .....................................................................................................32 2.14 Cell Culture Media and Supplements ..............................................................................34 2.15 Consumables ................................................................................................................35 2.16 Instruments ...................................................................................................................36 2.17 Software .......................................................................................................................37 3 Methods ............................................................................................................................ 38 3.1 Cloning of Expression Constructs ...................................................................................38 3.1.1 RNA Isolation.........................................................................................................38 3.1.2 cDNA Synthesis .....................................................................................................38 3.1.3 PCR Amplification of Coding Sequences .................................................................39 3.1.4 Agarose Gel Electrophoresis ...................................................................................39 3.1.5 Purification of PCR Products from Agarose Gels ......................................................40 3.1.6 Ligation into pGEM® -T ...........................................................................................40 3.1.7 Heat Shock Transformation of E. coli .......................................................................40 3.1.8 Plasmid DNA Miniprep............................................................................................40 3.1.9 Sanger Sequencing ................................................................................................41 3.1.10 Restriction Digestion...............................................................................................41 3.1.11 Ligation into Expression Vectors .............................................................................42 3.1.12 Colony PCR ...........................................................................................................42 3.1.13 Plasmid DNA "Midi" Preparation..............................................................................43 3.1.14 Preparation of Glycerolstocks for Long Term Storage ...............................................43 3.2 Generating Myc-tagged RS1 Variants .............................................................................43 3.3 Generating RS1-C59C Expression Constructs.................................................................43 3.4 Generating Expression Constructs for ATP1B2-ATP1B1 Chimeras ...................................44 3.5 Cell Culture ...................................................................................................................45 3.5.1 Cultivation of Hek293 Cells .....................................................................................45 3.5.2 Cultivation of Y-79 Cells..........................................................................................46 3.5.3 Transfection of Hek293 Cells – TransIT® -LTI Transfection Reagent .........................46 3.5.4 Transfection of Hek293 cells – Calcium-Phosphate Method ......................................46 3.6 Purification of Myc-tagged Retinoschisin Variants ............................................................47 3.7 Dialysis of Purified Retinoschisin Variants .......................................................................47 3.8 Sodiumdodecylsulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) ..........................47 3.8.1 Reducing SDS PAGE .............................................................................................47
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