Vaccinia virus-mediated MR imaging of tumors in mice [Elektronische Ressource] : overexpression of iron-binding proteins in colonized xenografts / vorgelegt von Ulrike Geißinger

Ulrike Geissinger

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 107
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Bayerische Julius-Maximilians-Universität Würzburg

Fakultät für Biologie









Vaccinia
Virus-mediated MR Imaging of Tumors in Mice:
Overexpression of Iron-binding Proteins in Colonized
Xenografts


Dissertation


Zur Erlangung des naturwissenschaftlichen Doktorgrades der bayerischen
Julius-Maximilians-Universität Würzburg


vorgelegt von
Ulrike Geißinger
aus Aalen

Würzburg, 2010












































meiner Familie

























Eingereicht am:...................................................................................................................




Mitglieder der Promotionskommission:


Vorsitzender: Prof. Dr. T. Dandekar
Gutachter: Prof. Dr. A. A. Szalay
Gutachter: Prof. Dr. G. Krohne


Tag des Promotionskolloquiums:........................................................................................

Doktorurkunde ausgehändigt am:.......................................................................................





Erklärung gemäß § 4 Absatz 3 der Promotionsordnung der Fakultät für Biologie
der Bayerischen Julius-Maximilians-Universität Würzburg:

Hiermit erkläre ich, die vorgelegte Dissertation selbständig angefertigt zu haben und
keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu
haben.

Des Weiteren erkläre ich, dass die vorliegende Arbeit weder in gleicher noch in
ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat.

Zuvor habe ich neben dem akademischen Grad “Diplombiologin, Univ.” keine
akademischen Grade erworben oder zu erwerben versucht.






Würzburg, 2010






Ulrike Geißinger






Contents


Summary 1
Zusammenfassung 4


1 Introduction 7

1.1 Iron metabolism 7
1.1.1 Iron 7
1.1.2 Iron Absorption and Transport 8
1.1.3 Cellular Iron Uptake 10
1.1.4 Iron Storage and Release 12
1.1.5 Regulation of Iron Homeostasis 13
1.1.6 Iron-accumulating Proteins Used in this Study 17
1.1.6.1 Ferritin 17
1.1.6.2 Bacterioferritin 18
1.1.6.3 Transferrin Receptor 19
1.1.6.4 Divalent Metal Transporter 20
1.1.6.5 MagA from Magnetospirillum magnetotacticum 21
1.1.6.6 Synthetic Phytochelatin EC20 23

1.2 Vaccinia Virus 24
1.2.1 History 24
1.2.2 Taxonomy 24
1.2.3 Morphology 25
1.2.4 Vaccinia Virus Replication Cycle 26
1.2.5 Oncolytic Viruses 28
1.2.6 Vaccinia Viruses Used in this Study 30

1.3 Cancer 31
1.3.1 Classification and Morphology 33
1.3.2 Cancer Cell Lines Used in this Study 34
1.3.2.1 GI-101A Cell Line 34
1.3.2.2 A549 Cell Line 35

1.4 MRI Technology 35
1.4.1 History 35
1.4.2 Principle 36
1.4.2.1 Magnetization 36
1.4.2.2 T1 and T2 Relaxation 37
1.4.3 MRI Cancer Diagnosis with Iron-accumulating Proteins 39

1.5 Aim of this Study 40




2 Material 42

2.1 Chemicals and Enzymes 42

2.2 Cell Lines and Cell Culture Media 46

2.3 Kits 47

2.4 Synthetic Oligonucleotides 47

2.5 Antibodies 48

2.6 Recombinant Viral Constructs 48

2.7 Bacterial Strains 55

2.8 Laboratory Animals 55

2.9 Laboratory Equipment and other Materials 56


3 Methods 59

3.1 Generation of Recombinant Vaccinia Virus 59
3.1.1 Cloning of Plasmids for Homologous Recombination with Virus DNA 59
3.1.2 Co-Transfection of Plasmid DNA with Parental Virus GLV-1h68 Infection 60
3.1.3 Plaque Selection 60
3.1.4 Screening of Marker Gene Expression 62
3.1.5 gpt Screening 64
3.1.6 Isolation of DNA to Verify Sequence 65
3.1.7 Amplification and Purification of Recombinant Viruses 66
3.1.8 Determination of the Virus Titer by Plaque Assay 67

3.2 Virological Methods 68
3.2.1 Infection of Cells with Vaccinia Virus 68
3.2.2 Viral Replication 68
3.2.3 Plaque Assay 68

3.3 Bacteriological Methods 69
3.3.1 Growth Curve 69
3.3.2 Preparation of Bacterial Cultures for Heavy Metal ICP-MS Measurements 69

3.4 Protein Analytical Methods 71
3.4.1 Preparation of Protein Lysates from Mammalian Cells 71
3.4.2 Protein Assay 72
3.4.3 SDS-Polyacrylamid Gel Electrophoresis 72
3.4.4 Coomassie Staining of Protein Gels 72
3.4.5 Protein Transfer to PVDF Membrane (Western Blot) 73
3.4.6 Native Blot 74
3.4.7 Colorimetric Immunodetection 75
3.4.8 Stripping of Membranes 75
3.4.9 ELISA 76

3.5 Detection of Gene Expression Mediated by Recombinant Vaccinia Virus 76
3.5.1 Analysis of Gene Transcription by RT-PCR 76
3.5.1.1 Isolation of RNA from Adherent Mammalian Cells 77
3.5.1.2 Synthesis of cDNA 77
3.5.1.3 Polymerase Chain Reaction (PCR) 77
3.5.1.4 Agarose Gel Electrophoresis 78

3.6 Determination of the Iron Content in Cell Culture 78
3.6.1 QuantiChrom™ Iron Assay 78
3.6.2 Ferrozine Assay 79
3.6.3 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 80
3.6.3.1 Sample Preparation for ICP-MS 81

3.7 In vivo Studies 81
3.7.1 Tumor Monitoring 81
3.7.2 Preparation of Virus for Mouse Injection 82
3.7.3 Production of Mouse Serum against Bacterioferritin 83
3.7.4 Tumor and Organ Preparation for Virus Titration 83
3.7.5 Tumor Preparation for Western Blot 84
3.7.6 Tumor Preparation for Ferrozine Assay 84
3.7.7 Histology 84
3.7.7.1 Dehydration 84
3.7.7.2 Embedding 85
3.7.7.3 Sectioning 85
3.7.7.4 Deparaffinization and Rehydration of Tissue Sections 85
3.7.7.5 Hematoxylin and Eosin (H&E) Staining 86
3.7.7.6 Prussian Blue Iron Staining with DAB Intensification 87
3.7.7.7 Immunohistochemical Staining of Vaccinia Virus 87
3.7.7.8 Immunohistochl Staining of Ferritin 88

3.8 MRI Measurements 88


4 Results 91

4.1 Characterization of Iron-collecting Virus Strains in Cell Culture 91
4.1.1 Virus-mediated Ferritin Expression
4.1.1.1 Analysis of Viral Replication in the Cancer Cell Lines GI-101A and A549 91
4.1.1.2 Analysis of Recombinant Protein Expression in Infected Cell Cultures 93
4.1.1.2.1 Coomassie Staining of Protein Gels 93
4.1.1.2.2 Western Blot 96
4.1.1.2.3 Native PAGE 98
4.1.1.2.4 ELISA 100
4.1.2 Virus-mediated Bacterioferritin Expression 102
4.1.2.1 Analysis of Viral Replication in the Cancer Cell Lines GI-101A and A549 103
4.1.2.2 Analysis of Recombinant Protein Expression in Infected Cell Cultures 105
4.1.2.2.1 Coomassie Staining of Protein Gels 105
4.1.2.2.2 Western Blot 107
4.1.3 Virus-mediated Transferrin Receptor Expression 109

4.1.3.1 Analysis of Viral Replication in GI-101A and A549 Cells 109
4.1.3.2 Analysis of Recombinant Protein Expression in Infected Cell Cultures 111
4.1.3.2.1 Coomassie Staining of Protein Gels 111
4.1.3.2.2 Western Blot 113
4.1.4 Virus-mediated Ferritin and Transferrin Receptor Expression 115
4.1.4.1 Analysis of Viral Replication in GI-101A and A549 Cells 115
4.1.4.2 Analysis of Recombinant Protein Expression in Infected Cell Cultures 117
4.1.4.2.1 Coomassie Staining of Protein Gels 117
4.1.5 Virus-mediated Ferritin Light Chain Expression 119
4.1.5.1 Analysis of Viral Replication in GI-101A and A549 Cells 119
4.1.5.2 Analysis of Recombinant Protein Expression in Infected Cell Cultures 121
4.1.5.2.1 Coomassie Staining of Protein Gels 121
4.1.6 Virus-mediated Divalent Metal Transporter Expression 122
4.1.6.1 Analysis of Viral Replication in GI-101A and A549 Cells 123
4.1.6.2 Analysis of Recombinant Protein Expression in