| Autor: |
Deroose, Christophe |
| Přispěvatelé: |
Debyser, Zeger, Baekelandt, Veerle, Mortelmans, Luc |
| Jazyk: |
angličtina |
| Rok vydání: |
2007 |
| Popis: |
Table of Contents Dankwoord 1 Table of Contents 5 Abbreviations 9 Chapter 1: General Introduction 13 1. Imaging Modalities 14 1.1. Radionuclide Imaging Techniques 15 1.1.1. Single Photon Emission Computed Tomography (SPECT) 15 1.1.2. Positron Emission Tomography 16 1.2. Magnetic Resonance Imaging and Spectroscopy 16 1.2.1. Magnetic Resonance Imaging (MRI) 16 1.2.2. Magnetic Resonance Spectroscopy (MRS) 17 1.3. Optical Imaging Strategies 17 1.3.1. Fluorescence Imaging 18 1.3.2. Bioluminescence Imaging 18 2. Reporter Gene Concept and Existing Imaging Reporter Genes 19 2.1. Radionuclide Imaging Techniques 20 2.1.1. The Herpes Simplex Virus Thymidine Kinase Reporter System 20 2.1.2. Other Enzymatic Reporter Genes 22 2.1.3. Receptor Reporter Genes 22 2.1.4. Transporter Reporter Genes 23 2.2. Optical Imaging Techniques 25 2.2.1. Bioluminescence Imaging Reporter Genes 25 2.2.2. Fluorescence Imaging Reporter Genes 27 2.3. Magnetic Resonance Techniques 28 2.3.1. Magnetic Resonance Spectroscopy Reporter Genes 28 2.3.2. Magnetic Resonance Imaging Reporter Genes 30 3. Vectors for Gene Transfer 32 3.1. Oncoretroviral Vectors 34 3.2. Lentiviral Vectors 34 3.2.1. Molecular Biology of Lentiviral Vectors 35 3.2.2. Lentiviral Vector Biosafety 36 3.2.3. Improvements in Lentiviral Vector Design 37 3.2.4. Applications of Lentiviral Vectors 39 3.3. DNA Containing Viral Vectors 40 3.3.1. Adenoviral Vectors 40 3.3.2. Adeno-Associated Viral Vectors 40 3.3.3. Herpes Simplex Virus Type-1 Vectors 42 3.4. Non-Viral Vectors for Gene Delivery 42 3.4.1. Physical Methods 43 3.4.2. Chemical Methods 43 Chapter 2: Objectives 45 Chapter 3: Materials & Methods 47 1. Radiotracer Synthesis 47 2. Cell Culture and Lentiviral Vector Production 48 2.1. Cell Lines and Culture Conditions 48 2.2. Lentiviral Vector Transfer Plasmid Construction 49 2.3. Lentiviral Vector Production 50 2.4. Cell Transduction 51 3. Detection of Reporter Gene Expression 51 3.1. Western Blot 51 3.2. Flow Cytometry Analysis of eGFP Expression 52 3.3. Bioluminescence Measurement in Living Cells 52 3.4. In Vitro Firefly and Renilla Luciferase Activity Assay 52 3.5. Herpes Simplex Virus type 1 Thymidine Kinase Activity Assay 53 4. Cell Experiments 53 4.1. Radiotracer Uptake Experiments 53 4.2. High Pressure Liquid Chromatography (HPLC) 54 5. Creation of Animal Models 54 5.1. Inoculation of Lentiviral Vector in Mice and Rats 55 5.2. Inoculation of Tumor Cells 55 6. Small Animal Imaging Procedures 56 6.1. In Vivo Bioluminescence Imaging of Firefly Luciferase Activity and Image Analysis 56 6.2. In Vivo Bioluminescence Imaging of Renilla Luciferase Activity and Image Analysis 57 6.3. Combined Positron Emission Tomography – Computed Tomography Procedure 57 6.4. Image Fusion and Quantitative Analysis 58 6.5. Positron Emission Tomography Imaging 59 7. Ex vivo Confirmation Studies 59 7.1. Histology and Stereological counting 59 7.2. Ex-Vivo Brain Bioluminescence Imaging 60 7.3. Ex-Vivo Xenograft Analysis 60 8. Statistical Analysis 61 Chapter 4: Results 63 1. Noninvasive Monitoring of Long-Term Lentiviral Vector-Mediated Gene Expression in Rodent Brain with Bioluminescence Imaging 63 1.1. BLI Imaging Allows Quantification of Luciferase Expression in LV-Transduced Cells 66 1.3. Characterization of the BLI Signal After Stereotactic Injection of LV-Fluc into the Mouse Striatum 69 1.4. The Localisation of the BLI Signal Reflects the Anatomical Site of Injection 70 1.5. BLI Allows Quantification of Luciferase Activity In Vivo 73 1.6. Imaging of Long-Term Lentiviral Vector-Mediated Luciferase Expression in the Mouse Brain 73 1.7. The BLI Signal Reports Expression of a Transgene of Interest 76 1.8. BLI Detects Luciferase Expression in the Rat Substantia Nigra 78 1.9. BLI can Detect Migration of Endogenous Neural Stem Cells from the Subventricular zone to the Olfactory Bulb 78 2. Multimodality Imaging of Tumor Xenografts and Metastasis in Mice with Combined Micro Positron Emission Tomography, Small Animal Computed Tomography and Bioluminescence Imaging 83 2.1. Establishment of a Fused MicroPET-CT Imaging Strategy 84 2.2. [18F]-FDG MicroPET-CT is Capable of Monitoring Growth of Tumor Xenografts 85 2.3. [18F]-FDG MicroPET-CT Detects Pulmonary Melanoma Metastases 86 2.4. [18F]-FHBG MicroPET-CT and Bioluminescence Imaging of Systemic Melanoma Metastases 87 2.5. [18F]-FHBG MicroPET-CT is More Sensitive than [18F]-FDG MicroPET-CT for Detection of Systemic Metastases 90 3. Validation of Varicella Zoster Virus Thymidine Kinase as a Novel Reporter Gene for PET 97 3.1. Uptake Kinetics of Radiolabeled BCNAs in Stably Transduced 293T Cells 100 3.2. High Pressure Liquid Chromatography Shows Presence of Polar Metabolites in VZV-tk Expressing Cells 101 3.5. Thymidine Blocks Uptake of Radiolabeled BCNAs in Stably Transduced 293T Cells 102 3.6. Variable Intracellular Retention of Radiolabeled BCNAs 104 3.7. The Accumulation of Radiolabeled BCNA Reflects the LV Titer 104 3.8. Radiolabeled BCNAs Do Not Accumulate in Cells Expressing an HSV1-sr39TK-Containing Fusion Protein 106 3.9. MicroPET Imaging of Accumulation of 4-[18F]-FEP-BCNA in VZV-tk Expressing Prostate Cancer Xenografts 107 Chapter 5: General Discussion & Perspectives 115 Summary 123 Samenvatting 125 Curriculum Vitae 127 Publication List 129 References 133 status: published |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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