Identification and characterization of genes with specific expression in dendritic cells [Elektronische Ressource] / vorgelegt von Sven Heinz

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• Biologie

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2003
• Nombre de lectures: 3
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Identification and Characterization
of Genes with Specific Expression
in Dendritic Cells
Dissertation zur Erlangung des
Doktorgrades der Naturwissenschaften (Dr. rer. Nat.)
der Naturwissenschaften Fakultät IV
– Chemie und Pharmazie –
der Universität Regensburg
vorgelegt von
Sven Heinz
aus Kassel
2002The work presented in this thesis was carried out in the Department of Hematology and Oncology at
the University Hospital Regensburg from October 1997 to December 2000 and Mai 2001 through
December 2001.
Parts of this work have been published in:
Heinz, S., Krause, S.W., Gabrielli, F., Wagner, H.M., Andreesen, R., Rehli, M. (2002). Genomic
Organization of the Human Gene HEP27: Alternative Promoter Usage in HepG2 Cells and Monocyte-
Derived Dendritic Cells. Genomics 79, 608-615.
Promotionsgesuch eingereicht am: 19. Juni 2002
Die Arbeit wurde angeleitet von: PD Dr. med. S.W. Krause
Prüfungsausschuß:
Vorsitzender: Prof. Dr. Pfitzner
1. Gutachter: Prof. Dr. Buschauer
2. Gutachter: PD Dr. med. Krause
3. Prüfer: Prof. Dr. Steinem
Tag der mündlichen Prüfung: 18. Juli 2002To
Heather
my wife
and my
best friend
"Forty-two."
Deep Thought, on the question of life,
the universe and everything
in
"Hitchhiker's Guide to the Galaxy"
by Douglas AdamsTable of Contents
1 Introduction ...................................................................................................... 1
1.1 The Immune System........................................................................................ 1
1.2 Dendritic Cells: Antigen Presenting Cells Bridging Innate and Adaptive
Immunity .......................................................................................................... 2
1.2.1 Dendritic Cell Ontogeny ................................................................................... 2
1.2.2 DC Model Systems .......................................................................................... 5
1.3 Dendritic Cell Function..................................................................................... 5
1.3.1 Antigen Uptake ................................................................................................ 6
1.3.2 Antigen Processing and Presentation .............................................................. 7
1.3.3 Costimulation ................................................................................................... 8
1.3.4 Helper T cell polarization.................................................................................. 9
1.3.5 Tolerance Induction........................................................................................ 10
1.3.6 Interactions with other Cells of the Immune System ...................................... 10
1.4 DC Morphology and Function at the Different Stages of Differentiation......... 11
1.4.1 Characteristic Molecules................................................................................ 13
2 Research Objectives...................................................................................... 15
3 Materials and Methods................................................................................... 17
3.1 Equipment and Materials ............................................................................... 17
3.1.1 Equipment...................................................................................................... 17
3.1.2 Materials ........................................................................................................ 17
3.1.3 Chemicals 18
3.1.4 DNA Oligonucleotides.................................................................................... 18
3.1.5 Antibodies 19
3.1.6 Enzymes, Inhibitors and Kits.......................................................................... 19
3.1.7 Molecular Weight Standards 20
3.1.8 Primary Cells and Cell Lines 20
3.1.9 Bacterial E.Coli Strains .................................................................................. 20
3.1.10 Plasmid Vectors ............................................................................................. 21
3.2 Cell Isolation .................................................................................................. 21
3.2.1 Monocytes...................................................................................................... 21
3.2.2 Blood Dendritic Cell Isolation by MACS ......................................................... 22
3.2.3 Isolation of Granulocytes from Buffy Coats.................................................... 22
3.3 Cell Culture .................................................................................................... 23
3.3.1 Cell Culture Conditions and Passaging.......................................................... 23
3.3.2 Assessing Cell Vitality by Trypan Blue Exclusion........................................... 23
3.3.3 Freezing and Thawing Cells........................................................................... 24
3.3.4 Primary Cells 24
3.3.4.1 Dendritic Cells..................................................................................... 24
3.3.4.2 Macrophages ...................................................................................... 24
3.3.5 Cell Lines ....................................................................................................... 25
3.3.6 Mycoplasma Assay ........................................................................................ 25
3.3.7 Mixed Leukocyte Culture................................................................................ 25
3.4 DNA ............................................................................................................... 27
3.4.1 Transient transfection of THP-1 cells with DEAE-Dextran ............................. 27
iTable of Contents
3.4.2 Agarose Gel Electrophoresis ......................................................................... 28
3.4.3 Denaturing Alkaline Agarose Gels for Analysis of Single-Stranded DNA....... 29
3.4.4 Purification of DNA Fragments by Gel Extraction .......................................... 30
3.4.5 Representation Difference Analysis ............................................................... 30
3.4.5.1 cDNA-Synthesis.................................................................................. 31
3.4.5.2 Generation of Representations ........................................................... 31
3.4.5.3 Difference Analysis ............................................................................. 33
3.4.6 Reverse Dot Blot............................................................................................ 35
3.4.7 PCR ............................................................................................................... 36
3.4.8 RT-PCR ......................................................................................................... 37
3.4.8.1 SMART-RACE .................................................................................... 38
3.4.9 Precipitation of DNA using PEG..................................................................... 38
3.4.10 PCR-based Site-Specific Mutagenesis .......................................................... 39
3.4.11 Genome Walking ........................................................................................... 40
3.4.12 Preparation of Genomic DNA and Bisulfite Sequencing ................................ 41
3.4.13 DNA Sequencing and Sequence Analysis ..................................................... 42
3.5 RNA 42
3.5.1 RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform
Extraction....................................................................................................... 42
3.5.2 CsCl purification of RNA ................................................................................ 44
3.5.3 Poly-A mRNA Isolation................................................................................... 45
3.5.4 Electrophoresis of RNA in Denaturing Formaldehyde Agarose Gels ............. 45
3.5.5 Northern Blot – RNA Transfer........................................................................ 45
3.5.6 Radioactive Labeling of DNA ......................................................................... 46
3.5.7 NHybridization ............................................................................ 47
3.6 Molecular Cloning .......................................................................................... 48
3.6.1 Bacterial Culture ............................................................................................ 48
3.6.2 Preparation of Chemically Competent E.Coli ................................................. 49
3.6.3 Transformation of Chemically Competent E.Coli ........................................... 49
3.6.4 Cloning........................................................................................................... 50
3.6.5 Plasmid DNA Preparation .............................................................................. 51
3.7 Protein Methods............................................................................................. 51
3.7.1 Nuclear Extraction Procedure ........................................................................ 51
3.7.2 BCA Protein Assay......................................................................................... 52