Post-transcriptional regulation of IgE [Elektronische Ressource] = Posttranskriptionelle Regulation von IgE / vorgelegt von Alexander Karnowski

Alex James

Sujets

• Biologie

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 65
• Langue: Deutsch

Extrait

Post-transcriptional regulation of IgE
Posttranskriptionelle Regulation von IgE
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Fakultät der Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau
vorgelegt von
Alexander Karnowski
aus Hamburg
Freiburg im Breisgau, April 2002
Dekan der Fakultät: Prof. Dr. Hans Kleinig


Promotionsvorsitzener: Prof. Dr. Samuel Rossel


Betreuer der Arbeit: Dr. Marinus Lamers


Referent der Arbeit: Prof. Dr. Gunther Neuhaus


Koreferent: Dr. Hans U. Weltzien


3. Prüfer: Prof. Dr. Christoph F. Beck


Tag der Verkündigung des Prüfungsergebnisses: 08.07.2002





















Die vorliegende Arbeit wurde in der Zeit von November 1998 bis April 2002 in der
Abteilung Entwicklung des Immunsystems von Prof. Dr. Thomas Boehm im Labor von
Dr. Marinus Lamers am Max-Planck Institut für Immunbiologie in Freiburg im
Breisgau durchgeführt Dedicated to Florienne LoderTable of contents
Abbreviations
1 Introduction..............................................................................................................1
1.1 General Introduction...........................................................................................1
1.2 Short introduction to the immune system ............................................................2
1.2.1 Innate Immunity..........................................................................................2
1.2.2 Adaptive Immunity......................................................................................3
1.2.2.1 Immunoglobulins and B cells...............................................................3
1.2.2.2 Fc receptors ....................................................................................... 10
1.2.2.3 T cell receptor and T cells .................................................................. 11
1.3 The IgE Network .............................................................................................. 12
1.4 Project and aims................................................................................................ 15
2 Material and Methods............................................................................................ 17
2.1 Material ............................................................................................................ 17
2.1.1 Chemicals ................................................................................................. 17
2.1.2 Nucleic acid modifying enzymes............................................................... 17
2.1.3 Plasmids.................................................................................................... 17
2.1.4 Bacteria..................................................................................................... 17
2.1.5 Antibodies 17
2.1.6 Cell lines ................................................................................................... 18
2.1.7 Mouse Strains............................................................................................ 18
2.1.8 Standard Buffers and Media ...................................................................... 18
2.2 Methods 20
2.2.1 Working with DNA................................................................................... 20
2.2.1.1 Analytical purification of plasmid DNA (Alkaline Lysis Miniprep).. 20
2.2.1.2 Preparative purification of plasmid DNA (Alkaline Lysis Midiprep
and Maxiprep) ............................................................................................... 21
2.2.1.3 Purification of eukaryotic genomic DNA ........................................... 21
2.2.1.4 Measuring DNA concentration in solutions........................................ 22
2.2.1.5 Restriction digest of DNA.................................................................. 22
2.2.1.6 Gel electrophoresis of DNA using agarose gels.................................. 22
2.2.1.7 Dephosphorylation of DNA with calf intestine phosphatase (CIP)...... 23
2.2.1.8 Repairing 3' or 5' overhanging DNA ends to generate blunt ends........ 23
2.2.1.9 Ligation of DNA fragments ............................................................... 24
2.2.1.10 Polymerase chain reaction (PCR)..................................................... 25
2.2.1.11 Amplification of cloning inserts by polymerase chain reaction
(PCR) ........................................................................................................... 25
2.2.1.12 Phosphorylation of PCR fragments by T4 polynucleotide kinase...... 26
2.2.1.13 Isolation and purification of DNA fragments from agarose gels with
DEAE membranes......................................................................................... 27
2.2.1.14 Isolation and purification of large DNA fragments from agarose
gels................................................................................................................ 27
2.2.1.15 Making of chemical competent Escherichia coli (E. coli) ................. 28
2.2.1.16 Transformation of chemical competent E.coli .................................. 292.2.1.17 Rapid amplification of cDNA ends (RACE)..................................... 29
2.2.1.18 Southern Blot................................................................................... 31
2.2.1.18.1 Southern blotting onto a nylon membrane with an alkaline
buffer........................................................................................................ 31
2.2.1.18.2 Radioactive labelling of DNA probes....................................... 32
2.2.1.18.3 Hybridisation of DNA probes to a Southern Blot ...................... 33
2.2.1.19 Calculating molecular weight and copy numbers of plasmid DNA ... 34
2.2.1.20 Quantitative real-time PCR .............................................................. 34
2.2.2 Working with RNA ................................................................................... 39
2.2.2.1 Purification of total RNA from cell culture and tissue ........................ 39
2.2.2.2 Measuring RNA concentration in solutions ........................................ 40
2.2.2.3 Removal of contaminating DNA by DNase I digests.......................... 40
2.2.2.4 First-Strand synthesis of cDNA.......................................................... 41
2.2.2.5 Northern Blot..................................................................................... 42
2.2.2.5.1 Electrophoresis of RNA denatured by glyoxal/DMSO treatment 42
2.2.2.5.2 Transfer of RNA from gel to membrane ..................................... 43
2.2.2.5.3 Non-formamide hybridisation of DNA probes to a
Northern Blot............................................................................................ 43
2.2.2.5.4 Removal of probes from Northern Blots 44
2.2.2.6 Improvement of the human mood by the synthesis and ingestion of
Apple Crumble .............................................................................................. 45
2.2.3 Cellular work 46
2.2.3.1 Stable transfection of eukaryotic cells through electroporation ........... 46
2.2.3.2 Isolating cells from mice.................................................................... 47
2.2.3.3 Lysis of erythrocytes.......................................................................... 47
2.2.3.4 Flowcytometric analysis of cells from cell culture or spleen............... 48
2.2.3.5 Dissociation of bound sIgE from FcεRII (CD23)................................ 49
2.2.3.6 Isolation of human peripheral blood mononuclear cells (PBMCs) ...... 49
2.2.3.7 Depletion of CD3+ cells by magnetic cell sorting (MACS) ................ 50
2.2.3.8 Cell culture ........................................................................................ 50
3 Results..................................................................................................................... 51
3.1 Expression of the murine ε HC locus ................................................................ 51
3.1.1 Expression analysis of ex vivo stimulated spleen cells by quantitative real-
time PCR ........................................................................................................... 51
3.1.2 Sequence and RACE analysis of the murine ε HC locus ............................ 55
3.1.2.1 Sequence analysis of the heavy chain isotypes ................................... 55
3.1.2.2 Determination of ex vivo poly(a) site usage by 3' RACE..................... 56
3.1.3 Regulation of alternative ε HC RNA processing ........................................ 59
3.1.3.1 Influence of the S-M intron regions on the mRNA expression pattern
of the ε HC gene............................................................................................ 59
3.1.3.1.1 The impact of regions in the S-M intron on the expression of m α
and m ε ...................................................................................................... 59
3.1.3.1.2 Construction of ε HC expression vectors .................................... 60
3.1.3.1.3 Quantitative real-time PCR analysis of ε HC intron deletion
expression vectors transfected cells...........................................