We propose to develop a novel MRI reporter gene system that uses MRI to monitor gene expression and the status of cell grafts in vivo. Current MRI technology is capable of high spatial resolution at 10-?m range and has found wide applications in research as well as in the clinical diagnosis of various diseases 1-7. In vivo monitoring of cell grafts is a critical function for the future development of cell replacement based therapy. Although applications of MRI in cell tracking and monitoring have shown great promise, technical challenges have also been recognized. As cell grafts are not expected to differ in water content from surrounding tissues, they alone do not generate signals detected by MRI. Several approaches have been explored to overcome the limitation of this sensitivity including the use of exogenous metal-chelate contrast agents 2, 3, 6, 8, 9. One of the most novel ideas for using contrast agents in MRI is to utilize transgene expression of metal based contrast materials endogenously. This approach has the potential for non-invasive, long-term in vivo monitoring of cell grafts, especially during cell division, and has recently received considerable attention 2, 10. An example of a reporter gene is the one coding for ferritin, an iron chelating protein for iron storage in living systems. Initial studies with ferritin have generated promising results that suggest the potential of MRI reporter genes 2. Another possible candidate is MagA, which regulates the transport of iron and the formation of magnetite (Fe3O4) crystal in certain types of bacteria 11. Magnetite is a supermagnetic particle that can induce substantial changes in water relaxation times, and is therefore considered an excellent MRI contrast agent 8, 9. Previously MagA hasn't received as much attention as ferritin since its expression has been limited to bacteria. Recently, our lab has successfully expressed MagA in mammalian cell lines and, for the first time, confirmed the formation of magnetosomes in mammalian cells, which can be readily detected in MRI 12. We hypothesize that MagA could be expressed in mouse embryonic stem cells (mESCs) without an adverse effect on the stem cell properties that allow an mESC graft to be monitored noninvasively by MRI. We proposed to expand this research and critically evaluate the potential of MagA as an MRI reporter gene. We are also interested in exploring its applications in cell graft monitoring in vivo, one of the major barriers in advancing cell replacement research. Our three specific aims are: (1) Determine the effects of expressing MagA genes, characterize magnetosomes in mammalian cells and investigate the sensitivity of MagA MRI reporter in vitro, (2) Determine whether MagA could be used as an MRI reporter in vivo using an animal model, and (3) Characterize the imaging properties of magnetosome produced from MagA and develop imaging methods for in vivo imaging and tracking of transplanted stem cell grafts expressing MagA.
The proposed project seeks to evaluate the novel transgenic MRI reporter and its application as a marker for noninvasive monitoring of embryonic stem (ES) cell grafts. Thus, the development of ES cells can be monitored and traced.
