Analysis of 'knockout, knockin' mice that express a functional Fas Ligand molecule lacking the intracellular domain [Elektronische Ressource] / von Katharina Maria Lückerath

Katharina Lückenrath

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2010
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Analysis of ‘knockout/knockin’ mice that
express a functional Fas Ligand molecule
lacking the intracellular domain




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich 15 (Biowissenschaften)
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main


von Katharina Maria Lückerath
aus Solingen


Frankfurt 2010
(D 30)



















vom Fachbereich 15 (Biowissenschaften) der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.



Dekan: Prof. Dr. A. Starzinski-Powitz

Gutachter : Prof. Dr. A. Starzinski-Powitz und PD Dr. M. Zörnig

Datum der Disputation :

Table of contents
Zusammenfassung ........................................................................................................1
Summary.........................................................................................................................6
1 Introduction ...............................................................................................................10
1.1 Apoptosis................................................................................................................10
1.1.1 The extrinsic pathway of apoptosis....................................................................11
1.1.2 The intrinsic pathway of apoptosis12
1.2 The FasL/Fas system.............................................................................................13
1.2.1 Molecular mechanism of FasL/Fas-induced apoptosis......................................13
1.2.2 Apoptotic Fas signaling in the immune system..................................................14
1.2.2.1 Lymphocyte death in the periphery: activation-induced cell death (AICD)
and activated cell autonomous cell death (ACAD)......................................15
1.2.2.2 FasL/Fas-induced apoptosis in B cell function............................................16
1.2.3 Apoptotic Fas signaling outside the immune system.........................................17
1.2.4 FasL/Fas signaling in the establishment of immune privilege and in tumor
biology................................................................................................................17
1.2.5 Non-apoptotic Fas signaling..............................................................................19
1.3 The Fas Ligand.......................................................................................................21
1.3.1 FasL structure ...................................................................................................21
1.3.2 Regulation of FasL expression and activity .......................................................22
1.3.2.1 Transcriptional control ................................................................................22
1.3.2.2 FasL sorting and storage ............................................................................22
1.3.2.3 Regulation of FasL activity by dynamic localization in lipid rafts.................23
1.3.2.4 FasL processing .........................................................................................24
1.3.2.5 Regulation of FasL by proteins that bind to its intracellular domain............25
1.4 Reverse ligand signaling.......................................................................................26
1.4.1 Reverse signal-transduction by TNF family ligands...........................................26
1.4.2 FasL reverse signaling ......................................................................................27
1.4.3 Functional implications of FasL reverse signaling .............................................28
1.5 Aims of the project.................................................................................................30
2 Materials and methods .............................................................................................31
2.1 Materials...............................................................................................................31
2.1.1 Equipment .....................................................................................................31
2.1.2 Consumables.................................................................................................31
2.1.3 Chemical reagents.........................................................................................32
2.1.4 Inhibitors ........................................................................................................33
2.1.5 Enzymes34
2.1.6 Size standards...............................................................................................34
2.1.7 Commercial kits .............................................................................................34
2.1.8 Buffer and solutions.......................................................................................34
2.1.9 Antibodies......................................................................................................36
2.1.10 Vectors37
2.1.11 Oligonucleotides ..........................................................................................38
2.1.12 Mouse lines .................................................................................................39
2.2 Methods ..................................................................................................................40
2.2.1 Animal models...................................................................................................40
2.2.2 Genotyping ........................................................................................................40
2.2.3 Cell culture methods..........................................................................................41
2.2.3.1 Isolation of naïve murine lymphocytes........................................................41
2.2.3.2 Generation of T cell blasts ..........................................................................41
2.2.3.3 Lymphocyte maintenance and stimulation..................................................42
2.2.3.4 Erythrocyte lysis42
2.2.3.5 Determination of cell number and viability42
2.2.3.6 Cultivation of cell lines ................................................................................43
2.2.3.7 Freezing and thawing of cells .....................................................................43
2.2.3.8 Transfection of cells....................................................................................43
2.2.4 Flow cytometry ..................................................................................................44
2.2.4.1 Analysis of cell surface marker expression.................................................44
2.2.4.2 Intracellular stainings ..................................................................................44
2.2.4.3 Staining of unfixed cells with Annexin V and propidium iodide ...................45
2.2.4.4 Propidium iodide staining of ethanol fixed cells – Nicoletti test45
2.2.4.5 Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay for
lymphocyte proliferation..............................................................................45
2.2.4.6 Co-culture of primary lymphocytes with Fas-sensitive A20 target cells ......46
2.2.4.7 β-Galactosidase activity assay for the analysis of Wnt signaling in vivo.....46
2.2.5 Enzyme-linked immunoabsorbant assay (ELISA) .............................................47
2.2.5.1 Cell-based ELISA .......................................................................................47
2.2.5.2 Standard solid phase sandwich ELISA.......................................................49
2.2.6 Molecular biological methods ............................................................................50
2.2.6.1 Isolation of total RNA..................................................................................50
2.2.6.2 Quantification of nucleic acid concentration and purity...............................50
2.2.6.3 Complementary DNA (cDNA) synthesis .....................................................50
2.2.6.4 Polymerase chain reaction (PCR)...............................................................51
2.2.6.5 Electrophoresis of PCR products................................................................51
2.2.6.6 Quantitative real time polymerase chain reaction (qRT-PCR) ....................51
2.2.6.7 mRNA expression profiling of Wnt signaling-related genes ........................53
2.2.7 Protein-biochemical methods ............................................................................54
2.2.7.1 Protein-extraction from mammalian cells....................................................54
2.2.7.2 Determination of protein concentration using the Bradford method............54
2.2.7.3 SDS polyacrylamide gel electrophoresis (PAGE) .......................................54
2.2.7.4 Immunoblotting ...........................................................................................55
2.2.8 In vivo studies...............................................................................................