MRI is non-invasive and allows longitudinal studies over the course of days and weeks to view into optically opaque subjects, and provides contrast among soft tissues at relatively high spatial resolution. In vivo imaging of gene expression using MRI has become a popular non-invasive imaging technology for biologists through the use of cellular metalloproteins, ex. ferritin, as contrast agents to monitor transferred gene expression under the control of interested promoter, such as DNA methyltransferase 1 (DNMT1). Metalloproteins load iron and become paramagnetic, thus enhancing the magnetic contrast in gene-transferred cells. This approach enables us to use high-resolution MRI to localize and image gene expression in a tissue or whole organism in three-dimensional (3D) format. Another novel approach proposed in this application is using fluorine-19 magnetic resonance spectroscopy/imaging (19F MRS/ MRI) as an in vivo """"""""confocal microscope"""""""" to measure DNMT1 gene activity based on the principle of fluorine chemical shift imaging. A yeast cytosine deaminase (CD) will be used as a reporter to convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Both 5-FC and 5-FU will be measured by 19F MRS/ MRI in vivo as a 5-FC/5-FU ratiometric index to indicate DNMT1 gene activity in a living organism and then superimposed to 3D anatomic MR images to illustrate the spatial distribution of DNMT1 gene activity. We will use both ferritin and CD/fluorine as MRI reporters along with optical markers (GFP/RFP) to visualize and quantify DNMT1 gene expression in mouse models. The outcomes of this application would produce an anatomic 3D image of DNMT1 gene activity in a living mouse throughout its lifetime from embryo to adult. The mouse model established by the synthetic genetic circuit proposed in this application is flexible and can be adapted to the study of other epigenetic activity. This mouse model, e.g. DNMT1 in this application, can serve as a living test tube for the treatments of different nutrients, diet, chemicals and environmental challenges to study the genome-environment interaction at cellular level or whole organism. The epigenetic effects resulted from these treatments can be quantified and visualized in vivo by MRI. The measurement of biological events by 19F MRS is precise and sensitive. Using both ferritin and CD/19F MRI/MRS can digitize the measured promoter activity in different tissues and organs in the same organism before and after experimental treatments;thereby, a database of the 3D anatomic MR images with gene activity digitized and pseudo-colored for computing and visualization can provide invaluable insights into the temporal and spatial nature of epigenetic change.
We will use MRI reporters along with optical markers to visualize and quantify DNMT1 gene activity in mouse models. The outcomes of this application would produce an anatomic 3D image of DNMT1 gene activity in a living mouse throughout its lifetime from embryo to adult to reveal the temporal and spatial nature of epigenetic change.