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

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Identification and Characterizationof Genes with Specific Expressionin Dendritic CellsDissertation zur Erlangung desDoktorgrades der Naturwissenschaften (Dr. rer. Nat.)der Naturwissenschaften Fakultät IV– Chemie und Pharmazie –der Universität Regensburgvorgelegt vonSven Heinzaus Kassel2002The work presented in this thesis was carried out in the Department of Hematology and Oncology atthe University Hospital Regensburg from October 1997 to December 2000 and Mai 2001 throughDecember 2001.Parts of this work have been published in:Heinz, S., Krause, S.W., Gabrielli, F., Wagner, H.M., Andreesen, R., Rehli, M. (2002). GenomicOrganization 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 2002Die Arbeit wurde angeleitet von: PD Dr. med. S.W. KrausePrüfungsausschuß:Vorsitzender: Prof. Dr. Pfitzner1. Gutachter: Prof. Dr. Buschauer2. Gutachter: PD Dr. med. Krause3. Prüfer: Prof. Dr. SteinemTag der mündlichen Prüfung: 18. Juli 2002ToHeathermy wifeand mybest friend"Forty-two."Deep Thought, on the question of life,the universe and everythingin"Hitchhiker's Guide to the Galaxy"by Douglas AdamsTable of Contents1 Introduction ...................................................................................................... 11.1 The Immune System........................................................................................

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Published 01 January 2003
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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
3.7.3 Electrophoretic Mobility Shift Assay ............................................................... 52
3.7.4 Immunoprecipitation....................................................................................... 55
3.7.5 Discontinuous SDS-PAGE ............................................................................. 55
3.7.6 Western Blot .................................................................................................. 57
3.7.7 Immunostaining of Blotted Proteins 57
3.7.8 Flow Cytometry .............................................................................................. 58
4 Results........................................................................................................... 59
4.1 Identification of genes with DC-specific expression by Representational
Difference Analysis ........................................................................................ 59
4.2 Expression analyses by Northern blot hybridization....................................... 62
4.3 MCP-4............................................................................................................ 64
4.3.1 Analysis of MCP-4 expression during DC differentiation................................ 64
4.3.2 Blood DC produce MCP-4 mRNA .................................................................. 66
iiTable of Contents
4.3.3 DCs utilize the same MCP-4 promoter as dermal fibroblasts......................... 67
4.3.4 Different regulatory pathways govern MCP-4 expression in DCs and dermal
fibroblasts....................................................................................................... 67
4.3.5 CpG methylation analysis of the proximal MCP-4 promoter........................... 68
4.3.6 Dendritic cell-specific demethylation of the MCP-4 promoter allows binding of a
nuclear factor ................................................................................................. 71
4.3.7 Nuclear factor binding at the -80 bp CpG is necessary for maximal MCP-4
promoter activity in reporter assays in transiently transfected THP-1 cells .... 74
4.3.8 CpG demethylation of the MCP-4 promoter during monocyte to dendritic cell
differentiation is differentiation stage-dependent............................................ 75
4.4 Hep27 ............................................................................................................ 77
4.4.1 Predominant expression of Hep27 in monocyte-derived dendritic cells ......... 77
4.4.2 Tissue expression of Hep27 mRNA ............................................................... 78
4.4.3 DC and HepG2 cells utilize different Hep27 promoters.................................. 80
4.4.4 Genomic organization of Hep27..................................................................... 81
4.4.5 Exclusive utilization of the upstream Hep27 promoter by monocyte-derived
dendritic cells and alternative splicing............................................................ 82
4.4.6 Butyrate treatment activates the downstream Hep27 promoter ..................... 84
5 Discussion...................................................................................................... 87
5.1 Identification of genes with DC-specific expression ....................................... 87
5.2 MCP-4............................................................................................................ 87
5.3 Hep27 97
5.4 Complement C1q C-chain.............................................................................. 99
5.5 15-Lipoxygenase.......................................................................................... 101
5.6 Folate Receptor β ........................................................................................ 103
6 Summary 105
7 References................................................................................................... 107
iiiABBREVIATIONS
aa amino acid MBN Mung Bean Nuclease
AA Acrylamide MCP Monocyte Chemotactic Protein
AP Ammonium Peroxodisulfate MeOH Methanol
ATCC American Tissue Type Collection MIP Macrophage Inflammatory Protein
BCA Bicinchoninic Acid MLC Mixed Leukocyte Culture
BLAST Basic Local Alignment Search Tool MLR Mixed Leukocyte Reaction
BLOTTO Bovine Lacto Transfer Technique MMLV-RT Moloney Murine Leukemia Virus
Optimizer Reverse Transcriptase
BSA Bovine Serum Albumine MNC Mononuclear Cells
cDNA Complementary DNA MOPS 3-(N-Morpholino) Propanesulfonic
CNS Central nervous system Acid
CpG Cytidine-Phosphate-Guanosine mRNA Messenger RNA
cpm Counts per Minute NaOAc Sodium Acetate
dATP deoxyadenosine triphosphate NCBI National Center for Biotechnology
DC dendritic cell Information
dCTP deoxycytidine triphosphate NH OAc Ammonium Acetate4
DEAE Diethylaminoethyl NHDFC Normal Human Dermal Fibroblast
DEPC Diethyl Pyrocarbonate Cells
dGTP deoxyguanosine triphosphate NP-40 Nonidet P-40
DMEM Dulbecco's Modified Eagle Medium OD Optical Density (Absorbance)
DMSO Dimethyl Sulfoxide PAA Polyacrylamide
DNA Deoxyribonucleic Acid PAGE Polyacrylamide Gel Electrophoresis
dNTP Deoxyribonucleotide Triphosphates PCR Polymerase Chain Reaction
DP Difference Product PE Phycoerythrin
ds double-stranded PEG Polyethyleneglycol
DSM Deutsche Sammlung für PBS Phosphate Buffered saline
Mikroorganismen PLB Passive Lysis Buffer
DTT Dithiothreitol PMSF Phenylmethylsulfonic acid
dTTP deoxythymidine triphosphate PVDF Polyvinylidene Difluoride
ECL Enhanced Chemiluminescence RACE Rapid Amplification of cDNA Ends
EDTA Ethylenediaminetetraacetic Acid RDA Representational Difference
ELC Epstein-Barr virus-induced molecule Analysis
1 (EBI-1) Ligand Chemokine RNA Ribonucleic Acid
EPPS 4-(2-Hydroxyethyl)piperazine-1- rpm Revolutions per Minute
propanesulfonic acid rRNA Ribosomal RNA
ERV Endogenous Retroviral Sequence RT Room Temperature
EtOH Ethanol RT-PCR Reverse Transcription Polymerase
FACS Fluorescence-Activated Cell Sorting Chain Reaction
FCS Fetal Calf Serum SAGE Serial Analysis of Gene Expression
FITC Fluorescein Isothiocyanate SDS Sodium Dodecylsulfate
GM-CSF Granulocyte-Macrophage Colony SMART Switching Mechanism At 5' end of
Stimulating Factor RNA Transcript
GTC Guanidine Thiocyanate SSC Saline-Sodium Citrate
HBSS Hank's Balanced Salt Solution STBS Suspension TBS
HEPES 4-(2-hydroxyethyl)-1- STTBS Slimfast-TTBS
Piperazineethane Sulfonic Acid TAE Tris-Acetate/EDTA Electrophoresis
HOAc Acetic Acid Buffer
HUGO Human Genome Organization TBS Tris-Buffered Saline
HRP Horseradish Peroxidase TCA Trichloracetic A
IAA Isoamyl Alcohol TE Tris-EDTA
IL Interleukin TEMED N,N,N',N'-Tetramethylenediamine
LAMP lysosome-associated membrane TGE Tris-Glycine/EDTA
glycoprotein TNF Tumor Necrosis Factor
LARC Liver and Activation-regulated Tris Tris(hydroxymethyl)aminomethane
Chemokine tRNA transfer RNA
LB Luria Bertani TTBS 0.1% Tween-20/TBS
LPS Lipopolysaccharide UV Ultraviolet
MACS Magnetic Cell Sorting