Study of human specific microglial receptor siglec-11 and generation of transgenic mice expressing human siglec 11 [Elektronische Ressource] / submitted by Yiner Wang

Yiner Wang

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• Gesundheit

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Study of Human-Specific Microglial Receptor Siglec-11 and
Generation of Transgenic Mice Expressing Human Siglec-11








PhD Thesis
In fulfillment of the requirements for the degree
“Doctor of Philosophy (PhD)/Dr. rer. nat.”
at the
Faculty of Mathematics and Natural Sciences
of the
Rheinischen Friedrich-Wilhelms University of Bonn





Submitted by
Yiner Wang
born in
Zhejiang, China

Bonn, March 2009 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Rheinischen Friedrich-Wilhelms Universität Bonn.

































1. Referent: Prof. Dr. Harald Neumann
2. Referent: Prof. Dr. Waldemar Kolanus


Tag der Promotion: 25.06.2009

1














Dedicated to my families























2 Contents
TABLE OF CONTENTS

ABBREVIATIONS..........................................................................................................5
1 INTRODUCTION ........................................................................................................8
1.1 Siglecs........................................................................................................................................ 8
1.1.1 Definition and nomenclature of Siglecs.......................................................................... 8
1.1.2 Subfamilies of Siglecs................................................................................................... 10
1.1.3 Expression pattern of human Siglecs ............................................................................ 11
1.1.4 Ligands of Siglecs......................................................................................................... 12
1.1.5 Siglecs and intracellular signaling ................................................................................ 18
1.1.6 Function of Siglecs in the immune system ................................................................... 18
1.1.7 Siglec-11 ....................................................................................................................... 20
1.2 Microglia.................................................................................................................................. 21
1.2.1 Microglia: parenchymal macrophage of the central nervous system (CNS)................. 21
1.2.2 Origin and turnover of microglia .................................................................................. 22
1.2.3 Role of microglia in the CNS........................................................................................ 23
1.2.4 Molecules and signaling pathways involved in microglial activation........................... 24
1.3 Lentiviral vector system........................................................................................................... 27
1.3.1 General concept of viral vectors ................................................................................... 27
1.3.2 Constitution of lentiviral vectors................................................................................... 28
1.4 Transgenic mice ....................................................................................................................... 31
1.4.1 Transgenic mouse as an invaluable model.................................................................... 31
1.4.2 Generation of transgenic mouse through pronuclear injection ..................................... 33
1.4.3 ES cell-mediated transgenic mice ................................................................................. 34
1.4.4 The Cre/loxP system ..................................................................................................... 35
1.5 Aim of the study....................................................................................................................... 37
2 MATERIALS AND METHODS ................................................................................. 38
2.1 Materials .................................................................................................................................. 38
2.1.1 Buffers and Solutions.................................................................................................... 38
2.1.2 Cell culture media and reagents .................................................................................... 41
2.1.3 Cells and animals .......................................................................................................... 43
2.1.4 Antibodies ..................................................................................................................... 43
2.1.5 Primer (purchased from MWG, Germany).................................................................... 44
2.1.6 Consumables................................................................................................................. 46
2.1.7 Equipment..................................................................................................................... 46
2.1.8 Software ........................................................................................................................ 47
2.1.9 Kits and additional reagents.......................................................................................... 48
2.1.10 Anesthethics ................................................................................................................ 48
2.2 RT-PCR analysis of Siglec-11 ................................................................................................. 48
2.3 Plasmids construction .............................................................................................................. 49
2.4 Viral particle production .......................................................................................................... 49
2.5 Lentivial transduction of cells.................................................................................................. 50
2.6 PCR analysis of the incorporation of the lentiviral vector to the transduced cells................... 51
3 Contents
2.7 Culture of primary microglia ................................................................................................... 51
2.8 Culture of 5637 cell line (human urinary bladder carcinoma)................................................. 51
2.9 Culture and differentiation of human monocytic cell line ....................................................... 52
2.10 Culture of ES cells ................................................................................................................. 52
2.11 Immunocytochemistry of cultured cells................................................................................. 52
2.12 Analysis of cytokine gene transcripts by real-time RT-PCR ................................................. 53
2.13 Western blot analysis of the protein expression..................................................................... 53
2.14 Removal of PSA by EndoN treatment ................................................................................... 54
2.15 Microglia-neuron co-culture .................................................................................................. 54
2.16 Neurite and neuroal cell body evaluation............................................................................... 55
2.17 Aβ phagocytosis assay ........................................................................................................... 55
2.18 Generation of transgenic mice from embryo-stem cell aggregation ...................................... 56
2.19 Generation of transgenic mice from pronuclear injection...................................................... 56
2.20 Genotyping of mice................................................................................................................ 57
2.21 Statistics ................................................................................................................................. 57
3 RESULTS ................................................................................................................... 58
3.1 Detection of splice variant 2 of Siglec-11 in human brain tissue............................................. 58
3.2 Induction of Siglec-11 in macrophages differentiated from human monocytes....................... 60
3.3 Lentiviral expression of Siglec-11 ........................................................................................... 61
3.3.1. Molecular cloning of the lentiviral vectors .................................................................. 61
3.3.2 Verification of Siglec-11 expression in 293 cells.......................................................... 63
3.3.3 Transduction of primary mouse microglia ..............