Mechanisms of B-cell activation and lymphomagenesis [Elektronische Ressource] / vorgelegt von Caroline Hojer

Caroline Hojer

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Mechanisms of B cell activation and
lymphomagenesis




Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München









vorgelegt von
Caroline Hojer






02. Juni 2009 Versicherung
Ich, Caroline Hojer, geboren in München, versichere hiermit ehrenwörtlich, dass ich die
vorliegende Dissertation selbständig und ohne unerlaubte Beihilfe angefertigt habe.

München, den


Caroline Hojer



Erstgutachter: Prof. Dr. Dirk Eick

Zweitgutachterin: Prof. Dr. Elisabeth Weiß

Tag der mündlichen Prüfung: 06.10.2009 I
Contents

List of figures ........................................................................................................................... V
List of tables............................................................................................................................VI
List of abbreviations...............................................................................................................VI

VERSICHERUNG .................................................................................................................. II
1 INTRODUCTION..................................................................................................... 1
1.1 The immune system................................................................................................... 1
1.2 Humoral immunity and B lymphocytes .................................................................. 1
1.3 The CD40 receptor.................................................................................................... 4
1.4 The B cell receptor 7
1.5 The Notch receptor.................................................................................................. 10
1.6 Signaling pathways in lymphomagenesis .............................................................. 12
1.7 The LMP1/CD40 transgenic mouse strain............................................................ 13
2 AIM OF THE PROJECT....................................................................................... 15
3 RESULTS................................................................................................................. 17
3.1 Constitutive CD40 signaling in B cells in vivo....................................................... 17
3.1.1 Constitutive CD40 signaling in vivo selectively activates the noncanonical NF- B
pathway in B cells ..................................................................................................... 17
3.1.2 Constitutive CD40 signaling in vivo activates the MAPK Jnk and Erk.................... 20
3.1.3 Constitutive activation of Erk is essential for the improved survival of LMP1/CD40-
expressing B cells...................................................................................................... 22
3.1.4 Malignant B cells exhibit a diverse pattern of activated signaling pathways ........... 23 II
3.2 The role of B cell receptor signaling in LMP1/CD40-activated B cells .............. 24
3.2.1 The role of Igß in LMP1/CD40-expressing B cells .................................................. 25
3.2.2 The role of CD19 in LMP1/CD40-expressing B cells .............................................. 32
3.2.3 LMP1/CD40 signaling and function depend on CD19 ............................................. 40
3.3 Generation of a tetracycline regulatable Notch2-IC transgenic mouse strain .. 51
3.3.1 The Tet system .......................................................................................................... 51
3.3.2 Strategy...................................................................................................................... 52
3.3.3 ES cell targeting and screening ................................................................................. 55
3.3.4 Injection into blastocysts........................................................................................... 58
4 DISCUSSION 59
4.1 Constitutive CD40 signaling selectively activates the noncanonical NF-B
pathway and the MAPK Erk and Jnk................................................................... 59
4.2 Constitutive CD40 signaling renders B cells less dependent on BCR signals via
Ig , but requires expression of CD19 .................................................................... 61
4.2.1 The influence of Ig  on LMP1/CD40-expressing B cells in vivo............................. 62
4.2.2 The influence of CD19 on LMP1/CD40-expressing B cells in vivo......................... 64
4.2.3 A new role for CD19 in B cell lymphomagenesis .................................................... 66
4.2.4 CD19 deficiency impairs Erk activation in LMP1/CD40-expressing B cells........... 67
4.3 The generation of a Tet inducible Notch2-IC transgenic mouse strain ............. 71
4.4 Mechanisms of B cell activation and lymphomagenesis ...................................... 72
5 SUMMARY.............................................................................................................. 75
6 ZUSAMMENFASSUNG ........................................................................................ 77
7 MATERIAL............................................................................................................. 79
7.1 Plasmids.................................................................................................................... 79
7.2 Bacteria 80
7.3 Cell lines ................................................................................................................... 80 III
7.4 Mouse strains........................................................................................................... 81
7.5 Primer....................................................................................................................... 82
7.6 DNA probes.............................................................................................................. 83
7.7 Antibodies ................................................................................................................ 83
7.8 Small chemical inhibitors ....................................................................................... 84
7.9 Enzymes.................................................................................................................... 84
7.10 Software 84
8 METHODS .............................................................................................................. 85
8.1 Mice .......................................................................................................................... 85
8.1.1 Mouse breedings ....................................................................................................... 85
8.1.2 Isolation of primary lymphocytes ............................................................................. 85
8.1.3 Flow cytometry ......................................................................................................... 86
8.1.4 In vitro cultures of primary lymphocytes.................................................................. 86
8.1.5 In vivo 5-Bromo-2’-deoxyuridine (BrdU) assay ....................................................... 87
8.2 ES cell culture.......................................................................................................... 87
8.2.1 General cell culture techniques ................................................................................. 87
8.2.2 Thawing and freezing of cells ................................................................................... 87
8.2.3 Culturing of embryonic fibroblasts ........................................................................... 88
8.2.4 embryonic stem cells ............................................................................ 88
8.2.5 Transfection of ES cells ............................................................................................ 89
8.2.6 Selection and expansion of stably transfected ES cell clones................................... 89
8.2.7 Induction of transgene expression in transiently transfected ES cell clones............. 90
8.2.8 Culturing of stable ES cell clones for injection into blastocysts............................... 91
8.3 Standard methods of molecular biology................................................................ 91
8.3.1 DNA techniques ........................................................................................................ 91
8.3.2 Restriction digest of plasmid DNA ........................................................................... 93
8.3.3 Restriction digest of genomic DNA.......................................................................... 93
8.3.4 Ligation of plasmid DNA fragments......................................................................... 93 IV
8.3.5 Southern Blot analysis (Southern et al., 1997).......................................................... 95
8.3.6 Protein detection........................................................................................................ 96
8.3.7 Protein isolation.................................................................................................