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Identification of Novel Mouse Delta1 Target Genes: Combination of Transcriptomics and Proteomics

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Identification of Novel Mouse Delta1 Target Genes: Combination of Transcriptomics and Proteomics Institut für Experimentelle Genetik GSF – Forschungszentrum für Umwelt und Gesundheit Identification of novel mouse Delta1 target genes: Combination of transcriptomics and proteomics Christine Hutterer Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. A. Gierl Prüfer der Dissertation: 1. apl. Prof. Dr. J. Adamski 2. Univ.-Prof. Dr. W. Wurst Die Dissertation wurde am 31.01.2005 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 13.05.2005 angenommen. DANKSAGUNG Besonderer Dank gebührt Prof. Dr. Martin Hrabé de Angelis, Leiter des Instituts für experimentelle Genetik. Unter anderem durch seine Leidenschaft für die Wissenschaft wollte ich am IEG arbeiten. Ich möchte mich jedoch nicht nur für die wissenschaftlichen Ratschläge und Diskussionen bedanken, sondern auch für viele interessante nicht-wissenschaftliche Gespräche. Ebenfalls besonders bedanke ich mich bei meinem Doktorvater Dr. Jurek Adamski, dessen fachliche Meinung und positive Einstellung mich sehr motivierte. Großen Dank möchte ich der gesamten „Beckers-Arbeitsgruppe“ aussprechen. Allen voran Dr. Johannes Beckers, Leiter der Genregulation und Expression Profiling Arbeitsgruppe, für die Überlassung des Themas und die Betreuung meiner Doktorarbeit, die mir viel Entscheidungsfreiraum ließ, sowie für zahlreiche gute Ideen und lehrreiche Gespräche. Bei Tomek Mijalski möchte ich mich für seine stetige Hilfsbereitschaft und die entstandene Freundschaft besonders bedanken. Weiterhin bedanke ich mich ganz herzlich bei Dr. Sonja Becker für wertvolle Labortricks und Kniffe, die Durchsicht des Manuskripts und ihre herzliche Art. Vielen Dank an dieser Stelle auch an Kathrin Seidel für die gute technische Unterstützung im Labor und mit der Mausarbeit. All den anderen (Ex-) Mitgliedern der Arbeitsgruppe, besonders aber Alexei Drobyshev und Oliver Ehm, möchte ich herzlich für wertvolle Ratschläge und die gute Arbeitsatmosphäre danken. Mein Dank gilt ebenfalls vielen weiteren Mitgliedern des IEG, vor allem Dr. Gerhard Przemeck und Sabine Pfister für ihre Diskussions- und Hilfsbereitschaft. Herzlich bedanke ich mich bei Dr. Thomas Halder, Dr. Alois Harder und Michael Kersten von der Toplab GmbH und Dr. Monika Zubawa und Prof. Dr. Friedrich Lottspeich vom MPI für Biochemie für die Einführung in die Proteomtechnologie und die Durchführung der primären Proteomanalysen. Great thanks to Dr Aidan Parte for the linguistic revision of the manuscript. Ein liebes Dankeschön geht an Philipp Hutterer für Alles. Bei meinen Eltern möchte ich mich von ganzem Herzen dafür bedanken, dass sie immer an mich geglaubt und mich mit allen Kräften unterstützt haben. TABLE OF CONTENTS Abstract .................................................................................................................................. 1 Zusammenfassung................................................................................................................ 3 1 Introduction ................................................................................................... 5 1.1 The Delta/Notch signal transduction pathway .................................................. 6 1.1.1 Components of Delta/Notch signal transduction.............................................. 7 1.1.1.1 Receptors .................................................................................................. 8 1.1.1.2 Ligands...................................................................................................... 9 1.1.1.3 Effector genes ......................................................................................... 10 1.1.1.4 Target genes ........................................................................................... 11 1.1.2 Levels of Notch regulation.............................................................................. 11 1.1.2.1 Regulation of receptor-ligand interaction via glycosylation...................... 11 1.1.2.2 Regulated degradation of proteins .......................................................... 11 1.1.2.3 Further regulatory mechanisms............................................................... 13 1.1.3 Functions of Delta/Notch signalling................................................................ 13 1.1.3.1 Lateral inhibition / Lateral induction......................................................... 13 1.1.3.2 Boundary formation ................................................................................. 15 1.1.3.3 Left/right specification.............................................................................. 17 1.2 Technologies for transcriptome and proteome analysis................................. 18 1.2.1 DNA-microarray technology........................................................................... 18 1.2.2 Tools for proteome analysis........................................................................... 19 1.2.3 Correlation between protein and mRNA abundance...................................... 21 1.3 Aim of this work .............................................................................................. 22 2 Material & Methods ..................................................................................... 23 2.1 Material........................................................................................................... 23 2.1.1 Antibodies ...................................................................................................... 23 2.1.2 Buffers and Solutions..................................................................................... 24 2.1.3 Chemicals and Reagents............................................................................... 25 2.1.4 Clones............................................................................................................ 25 2.1.5 Competent cells ............................................................................................. 25 2.1.6 DNA-Chips ..................................................................................................... 25 2.1.7 Flurescent dyes.............................................................................................. 26 II Table of Contents 2.1.8 Enzymes.........................................................................................................26 2.1.9 Laboratory equipment ....................................................................................26 2.1.10 Mice................................................................................................................27 lacZ 2.1.10.1 Dll1 ......................................................................................................27 pu 2.1.10.2 Dll3 ........................................................................................................27 htu 2.1.10.3 Jag1 ......................................................................................................27 2.1.11 Molecular weight markers ..............................................................................28 2.1.12 Ready-to-use-systems ...................................................................................28 2.1.13 Software and databases.................................................................................28 2.2 Methods ..........................................................................................................29 2.2.1 Dissection of embryos ....................................................................................29 2.2.2 Genomic typing ..............................................................................................29 2.2.2.1 Isolation of DNA.......................................................................................29 2.2.2.2 PCR .........................................................................................................29 2.2.3 Isolation of RNA .............................................................................................31 2.2.4 Preparation of slides.......................................................................................31 2.2.5 Target labelling...............................................................................................32 2.2.6 Hybridisation and image processing...............................................................33 2.2.7 Washing with increasing stringency for fractionation experiments and image analysis .........................................................................................................33 2.2.8 Data analysis..................................................................................................34 2.2.9 Plasmid preparation .......................................................................................35 2.2.10 Whole mount in situ hybridisation...................................................................35 2.2.10.1 Riboprobe synthesis ................................................................................35 2.2.10.2 In situ hybridisation ..................................................................................35 2.2.11 Reverse Transcription-PCR (RT-PCR) and Real-time PCR........................... 37 2.2.11.1 Primers ....................................................................................................37
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