Characterization of Maga Expression and Iron Uptake in P19 Cells: Implications for Use as a Gene-Based Contrast Agent

.............................................................................................................................. II Acknowledgments ............................................................................................................. III List of Tables ................................................................................................................. VIII List of Figures .................................................................................................................. IX List of Abbreviations ......................................................................................................... X Chapter 1 ............................................................................................................................. 1 1.1 Molecular and Cellular Imaging ............................................................................. 1 1.1.1 MRI Relaxometry ....................................................................................... 3 1.1.2 Contrast Agents for Cell Tracking with MRI Relaxometry ........................ 5 1.2 Role of MagA Expression in MRI .......................................................................... 6 1.2.1 Magnetotactic Bacteria ............................................................................... 6 1.2.2 MagA in Magnetotactic Bacteria ................................................................ 7 1.2.3 Using MagA for MRI-based Cell Tracking ................................................ 7 1.3 Overview of This Thesis ......................................................................................... 9 1.3.1 Hypothesis................................................................................................... 9 1.3.2 Motivation ................................................................................................... 9 1.3.3 Choice of Cell Line ................................................................................... 10 1.3.4 Thesis Objectives ...................................................................................... 10 1.4 References ............................................................................................................. 11 Chapter 2 ........................................................................................................................... 15 2.1. Introduction ........................................................................................................... 15 2.2. Methods................................................................................................................. 15 2.2.1 Generation of a MagA-HA Expression Construct .................................... 15 2.2.2 P19 Cell Culture, Transfection and Iron-loading ...................................... 16 VI 2.2.2.1 Cell of Choice ........................................................................................... 16 2.2.2.2 Transfection Information .......................................................................... 16 2.2.2.3 Iron Loading .............................................................................................. 17 2.2.3 Protein Expression .................................................................................... 19 2.2.3.1 Western Blot ............................................................................................. 19 2.2.3.2 Immunocytochemistry (ICC) .................................................................... 19 2.2.4 Trace Element (Iron) Analysis .................................................................. 20 2.2.5 MRI of MagA-HA-expressing Cells ......................................................... 21 2.2.5.1 Phantom Preparation ................................................................................. 21 2.2.5.2 Scanner and Pulse Sequences ................................................................... 21 2.2.5.3 Region of Interest (ROI) ........................................................................... 22 2.2.5.4 Calculation of R1, R2, R2* and R2' .......................................................... 23 2.2.6 Data Analysis ............................................................................................ 24 2.2.6.1 Sample Groups .......................................................................................... 24 2.2.6.2 Statistical analysis ..................................................................................... 24 2.3. Results ................................................................................................................... 25 2.3.1 Generation of a Stably-expressing Cell Line ............................................ 25 2.3.2 Localization of MagA-HA in P19 Cells ................................................... 26 2.3.3 Analysis of Cellular Iron in MagA-HA-expressing P19 Cells ................. 28 2.3.4 MRI of MagA-HA-expressing Cells ......................................................... 31 2.4. Discussion ............................................................................................................. 41 2.5. Conclusion ............................................................................................................ 45 2.6. References ............................................................................................................. 46 Chapter 3 ........................................................................................................................... 49 3.1 Summary ............................................................................................................... 49