Signaling events at the {β-selection [beta-selection] checkpoint during thymocyte development [Elektronische Ressource] / von Marei Dose

Marei Dose

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2007
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

Signaling events at the β-selection checkpoint
during thymocyte development
Von der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades
Doktorin der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Dipl.-Biochem. Marei Dose
geboren am 15.02.1979, in Preetz, Holst
2007Referent: Prof. Dr. Walter Müller
Korreferentin: Fotini Gounari, PhD, H.D.R.
Tag der Promotion: 23. Mai 2007Table of Contents 3
Table of contents
1 ZUSAMMENFASSUNG .................................................................................... 7
2 ABSTRACT .................................................................................................... 10
3 INTRODUCTION............................................................................................. 13
3.1 The thymus.................................................................................................................................................... 14
3.2 Getting there- extrathymic precursors in bone marrow and blood ...................................................... 16
3.3 Inside the thymus- stages of T cell development...................................................................................... 20
3.4 Migration of developing thymocytes ......................................................................................................... 23
3.5 The preTCR.................................................................................................................................................. 24
3.5.1 The pTα chain........................................................................................................................................ 24
3.5.2 preTCR signaling mediators.................................................................................................................. 26
3.6 Notch signaling in thymocyte development .............................................................................................. 27
3.6.1 Notch signaling in γδ T cells ................................................................................................................. 30
3.7 Wnt signaling in thymocyte development................................................................................................. 31
3.8 c-Myc in thymocyte development .............................................................................................................. 34
3.9 Aims ............................................................................................................................................................... 37
4 MATERIALS AND METHODS........................................................................ 39
4.1 In vivo experiments ...................................................................................................................................... 40
4.1.1 Mice........................................................................................................................................................ 40
4.1.2.1 TetOβ.............................................................................................................................................. 40
fl/fl
4.1.2.2 LckCre Myc ................................................................................................................................ 41
ex34.1.2.3 LckCre Ctnnb ............................................................................................................................. 41
4.1.2.4 Lck-MyrAkt HA ............................................................................................................................ 42
4.1.2 Anti-CD3ε injection............................................................................................................................... 42Table of Contents 4
4.1.3 Tetracycline treatment of animals ......................................................................................................... 42
4.1.4 Preparation of embryonal thymi............................................................................................................ 43
4.2 Molecular Biology ........................................................................................................................................ 43
4.2.1 Bacterial strains...................................................................................................................................... 43
4.2.2 Transformation and culture of E. coli cells........................................................................................... 43
4.2.3. Plasmid purification.............................................................................................................................. 44
4.2.4 Detection of DNA on agarose gels........................................................................................................ 44
4.2.5 Restriction digest.................................................................................................................................... 45
4.2.5.1 Cloning of pIRES_TetOβ_Neo..................................................................................................... 45
4.2.6 Genomic DNA extraction from tail biopsies ........................................................................................ 45
4.2.7 Genomic DNA extraction from single cell suspensions....................................................................... 46
4.2.8 PCR based genotyping........................................................................................................................... 46
4.2.9 Semi-quantitative PCR .......................................................................................................................... 47
4.2.10 Quantitative real-time PCR ................................................................................................................. 48
4.2.11 Microarray analyses............................................................................................................................. 49
4.3 Cell Biology ................................................................................................................................................... 50
4.3.1 Mammalian cell lines............................................................................................................................. 50
4.3.2 Transfection............................................................................................................................................ 50
4.3.2.1 Calcium phosphate transfection for retrovirus production........................................................... 50
4.3.2.2 Transfection of SciET27F cells..................................................................................................... 51
4.3.3 Retroviral spin infection of TetOβ positive clones............................................................................... 52
4.3.4 OP9-DL1 coculture................................................................................................................................ 53
4.3.4.1 Primary cells................................................................................................................................... 53
4.3.4.2 SciET27F and SCB.29 coculture on OP9 stromal cells ............................................................... 53
4.3.5 CFSE labeling of primary thymocytes.................................................................................................. 54
4.3.6 Flow Cytometry and Antibodies ........................................................................................................... 54
4.3.6.1 Surface stainings ............................................................................................................................ 54
4.3.6.2 Intracellular FACS staining........................................................................................................... 54
4.3.6.3 Detection of apoptotic cells and cell cycle analysis ..................................................................... 55
4.3.6.4 Enrichment of DN cells by bead depletion................................................................................... 55
4.3.6.5 Cell sorting..................................................................................................................................... 56
4.3.7 Western Blot........................................................................................................................................... 56
5 RESULTS ....................................................................................................... 58
5.1 The role of c-Myc mediated proliferation at the preTCR checkpoint .................................................. 59Table of Contents 5
5.1.1 PreTCR signaling induces c-Myc expression....................................................................................... 59
-/-5.1.1.1 preTCR induction in TetOβ-LTH Rag1 mice ............................................................................ 59
-/-5.1.1.2 c-Myc induction in TetOβ-LTHCre Rag1 thymocytes.............................................................. 61
-/-5.1.1.3 c-Myc induction upon preTCR signals in Rag mice.................................................................. 62
5.1.2 Abnormal thymocyte development upon conditional c-Myc ablation in mice.................................... 64
fl/fl5.1.2.1 Generation of LckCre Myc mice............................................