The function of Krüppel-like factor 2 in B cell development [Elektronische Ressource] = Die Funktion von Krüppel-like Faktor 2 in der B-Zellentwicklung / vorgelegt von Rebecca Winkelmann geb. Heidbüchel

Rwinkelm

Sujets

• Naturwissenschaften

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

The function of Krüppel-like factor 2 in B cell development

Die Funktion von Krüppel-like Faktor 2 in der B-Zellentwicklung










Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.











vorgelegt von
Rebecca Winkelmann geb. Heidbüchel

aus Kleve





Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

















Tag der mündlichen Prüfung: 17. 12. 2010

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Hans-Martin Jäck
Zweitberichterstatter: Prof. Dr. Falk Nimmerjahn
2 Table of contents
Table of contents
1 Zusammenfassung ............................................................................. 1
2 Summary ............................................................................................. 2
3 Introduction......................................................................................... 3
3.1 B cell development and homeostasis................................................................ 3
3.1.1 Central B cell development................................................................................3
3.1.2 Peripheral B cell development...........................................................................6
3.1.3 Plasma cell generation and migration................................................................9
3.2 Krüppel-like factor 2 (KLF2) ............................................................................ 11
3.2.1 Krüppel-like transcription factors .....................................................................11
3.2.2 KLF2 in T lymphocytes....................................................................................12
3.2.3 Regulation of KLF2..........................................................................................13
4 Specific Aims .................................................................................... 15
5 Results............................................................................................... 16
5.1 B cell speficic KLF2 deletion in vivo ................................................................ 16
5.1.1 KLF2 expression profile during B cell development .........................................16
5.1.2 Normal B cell development in the bone marrow of KLF2-deficient animals......18
5.1.3 Enlarged spleen size and increased numbers of splenic B cell populations
in KLF2-deficient animals ................................................................................20
5.1.4 Decreased frequencies of B cells in the peritoneal cavity, the blood and
peyers patches of KLF2-deficient mice............................................................25
5.1.5 Reduced numbers of plasma cells in the bone marrow after boost
immunization with TD antigen..........................................................................28
5.1.6 Distribution of peripheral B cell subsets is controlled by KLF2 regulating
L-selectin and α β integrin but not S1P1 ........................................................32 4 7
5.1.7 KLF2 determines Fo B cell identity ..................................................................35
5.2 Biochemical analyses of KLF2 ........................................................................ 37
5.2.1 KLF2 protein modifications in splenic B cells and non B cells..........................37
5.2.2 In silico phosphorylation analysis of KLF2 amino acid sequence.....................38
5.2.3 KLF2 is posttranslationally modified by phosphorylation..................................40
I Table of contents
6 Discussion......................................................................................... 42
6.1 KLF2-deficiency results in impaired B cells homeostasis and plasma cell
homing ............................................................................................................ 42
6.2 Biochemical analyses of KLF2 ........................................................................ 47
7 Material and Methods ....................................................................... 50
7.1 Material ........................................................................................................... 50
7.1.1 Chemicals .......................................................................................................50
7.1.2 Antibodies .......................................................................................................50
7.1.3 PCR primer .....................................................................................................53
7.1.4 Plasmids..........................................................................................................54
7.1.5 Cell lines..........................................................................................................55
7.1.6 Mouse strains..................................................................................................55
7.1.7 Bacteria...........................................................................................................56
7.1.8 Technical Equipment.......................................................................................56
7.1.9 Plastic material................................................................................................57
7.1.10 Consumable material.......................................................................................58
7.2 Methods .......................................................................................................... 59
7.2.1 Cell culture ......................................................................................................59
7.2.1.1 Cultivation and harvest of vertebrate cell lines.................................................59
7.2.1.2 Cryoconservation ............................................................................................59
7.2.1.3 Thawing of cryoconserved cells.......................................................................59
7.2.1.4 Cell counting ...................................................................................................60
7.2.2 Isolation of primary B and T lymphocytes from different organs.......................60
7.2.2.1 Erythrocyte depletion.......................................................................................60
- +
7.2.2.2 MACS-sorting of CD43 splenic and CD19 bone marrow B cells ....................60
7.2.3 Flow cytometry ................................................................................................61
7.2.3.1 Analyses of surface expression and intracellular proteins................................61
TM
7.2.3.2 MoFlo cell sorting .........................................................................................61
7.2.4 Histology .........................................................................................................62
7.2.5 Molecular biological methods ..........................................................................62
7.2.5.1 Plasmid DNA and RNA preparation.................................................................62
7.2.5.2 DNA restriction analyses .................................................................................62
7.2.5.3 DNA fragment agarose gel electrophoresis .....................................................63
II Table of contents
7.2.5.4 Isolation of genomic tail DNA...........................................................................63
7.2.5.5 DNA/RNA concentration..................................................................................63
7.2.5.6 Polymerase chain reaction (PCR)....................................................................63
7.2.5.7 cDNA synthesis...............................................................................................64
7.2.5.8 Qualitative RT-PCR.........................................................................................64
®
7.2.5.9 Quantitative TaqMan -RT-PCR.......................................................................65
7.2.5.10 Affymetrix microarray analysis.........................................................................65
7.2.6 Antibody isotype detection using ELISA ..........................................................65
7.2.7 IgG specific Elispot..........................................................................................66
7.2.8 Proteinbiochemistry.........................................................................................66
7.2.8.1 Cell lysates......................................................................................................66
7.2.8.2 Nuclear extracts ..............................................................................................66
7.2.8.3 BCA test..........................................................................................................67
7.2.