Die Rolle von HIF bei zellulären Transformationsprozessen [Elektronische Ressource] : I. HIF-2α im renalen Tubulus - ein transgenes Mausmodel ; II. Identifizierung und Charakterisierung von Lysyloxidasen als tumorfördernde HIF-Zielgene / vorgelegt von Ruth Elisabeth Schietke

Karl Xaver Knaup

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Die Rolle von HIF bei zellulären Transformationsprozessen:
I. HIF-2 α im renalen Tubulus – ein transgenes Mausmodel
II. Identifizierung und Charakterisierung von Lysyloxidasen
als tumorfördernde HIF-Zielgene






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades










vorgelegt von
Ruth Elisabeth Schietke
aus Tettnang




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






















Tag der mündlichen Prüfung: 22. Januar 2009
Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Thomas Winkler
Zweitberichterstatter: PD Dr. Michael Wiesener




für
Mama und Papa








The role of HIF for cellular transformation processes:
I. HIF-2 α in the renal tubulus – a transgenic mouse model
II. Identification and characterization of lysyl oxidases as
tumour promoting HIF target genes

















Table of Contents I
I. Table of Contents I

II. Table of figures VII

III. Abbreviations IX

1 INTRODUCTION..............................................................................................................1
1.1 Gene expression and regulation...............................................................................2
1.2 The hypoxia inducible factor (HIF)............................................................................3
1.3 Oxygen-dependent regulation of HIF .......................................................................6
1.4 Cellular transformation, cancer development and tumour progression.............10
1.5 Hypoxia in tumour biology ......................................................................................11
1.6 HIF in tumour biology ..............................................................................................13
1.7 Lysyl oxidases..........................................................................................................16
1.8 Aim of this study ......................................................................................................19
2 MATERIAL.....................................................................................................................21
2.1 Chemicals and disposable plastic materials .........................................................21
2.2 Solutions, buffer and media ....................................................................................21
2.2.1 Universal solutions .................................................................................................21
2.2.2 Solutions and media for cell culture........................................................................22
2.2.3 d media for bacterial cultivation ..........................................................23
2.2.4 Solutions for preparative plasmid-DNA isolation ....................................................23
2.2.5 nucleic acid analysis..........................................................................24
2.2.6 Agarose gel electrophoresis ...................................................................................25
2.2.7 Buffer for RNase Protection Assay (RPA) ..............................................................25
2.2.8 Solutions and buffer for Electromobility Shift Assay (EMSA)..................................26
2.2.9 Buffer for protein extraction ....................................................................................26
2.2.10 Solutions and buffers for protein gels .................................................................26 Table of Contents II
2.2.11 Solutions and buffer for protein transfer on PVDF-membrane („Western-Blot“)
and subsequent immunodetection .....................................................................................27
2.2.12 Staining solutions................................................................................................28
2.2.13 Solution for immunofluorescence analysis .........................................................28
2.2.14 Carrier material...................................................................................................29
2.3 Antibodies .................................................................................................................30
2.3.1 Primary antibodies..................................................................................................30
2.3.2 Secondary antibodies .............................................................................................31
2.4 Cell lines....................................................................................................................32
2.5 Bacterial strains........................................................................................................33
2.6 Commercial reagent kits..........................................................................................33
2.7 Enzymes34
2.8 Nucleic acids.............................................................................................................34
2.8.1 Cloning vectors.......................................................................................................34
2.8.2 Oligonucleotides .....................................................................................................35
2.8.2.1 Primers for polymerase chain reaction (PCR) ....................................................35
2.8.2.2 Primers for real-time RT-PCR ............................................................................36
2.8.2.3 Oligonucleotides for Electromobility Shift Assay (EMSA)...................................37
2.9 Size and molecular weight marker..........................................................................38
2.9.1 DNA size marker ....................................................................................................38
2.9.2 Protein weight marker.............................................................................................38
2.10 Equipment and instruments ....................................................................................39
2.11 Computer software...................................................................................................40
3 METHODS......................................................................................................................41
3.1 Cultivation of eukaryotic cells.................................................................................41
3.1.1 Passaging of eukaryotic cells42
3.1.2 Freezing and thawing of eukaryotic cells................................................................42
3.1.3 Transient transfection of eukaryotic cells43
3.1.3.1 Transient transfection with jetPEI™ (Polyplus) ..................................................43
®3.1.3.2 Transient Transfection with Fugene HD (Roche)...............................................44 Table of Contents III
3.2 Cultivation of bacteria..............................................................................................45
3.2.1 Cultivation of Escherichia coli.................................................................................45
3.2.2 Transformation of competent bacteria with circular plasmid DNA..........................45
3.3 Analysis of nucleic acids.........................................................................................46
3.3.1 Isolation of DNA......................................................................................................46
3.3.1.1 Analytical isolation of plasmid DNA from bacteria (mini preparation).................46
3.3.1.2 Isolation of plasmid DNA from bacteria (midi preparation).................................46
3.3.1.3 Isolation of genomic DNA from cultured eukaryotic cells ...................................47
3.3.1.4 Isolam mouse tail biopsies for PCR .............................47
3.3.1.5 Phenol/chloroform extraction of nucleic acids ....................................................48
3.3.1.6 Purification of DNA fragments from ethidium bromide agarose gels..................48
3.3.2 Enzymatic modification of DNA ..............................................................................49
3.3.2.1 DNA cleavage by restriction endonucleases......................................................49
3.3.2.2 Ligation of DNA fragments .................................................................................49
3.3.3 RNA extraction from cultivated cells...........................................