The overall goal of this proposal is to develop widely applicable methods for non-invasive imaging of key molecular-biological and cellular events that could be useful in the assessment and management of patients with cancer and other diseases. We propose to image the expression of different endogenous genes and the activity of different signal transduction pathways in tissues and transgenic animals in this project. Imaging the regulation of expression of different endogenous genes will be performed at the levels of transcription and translation. Imaging the activity of different signal transduction pathways will be attempted at the levels of transactivation ( or repression) of pathway-specific target genes as well as specific protein-protein interactions. To achieve this goal, we combine an established method for non-invasive imaging of HSVl-tk reporter gene expression with existing molecular-biological methods that utilize in situ reporter genes ( e.g., CA T, beta-galactosidase, luciferase, GFP, etc.) for assessment of endogenous genes and signal transduction pathways. We propose to demonstrate that substitution of the HSVl-tk gene for the in situ reporter genes used in the existing in situ reporter systems would allow non-invasive imaging of the same molecular-biological and cellular events in transgenic tissues and animals. In addition, we will test the applicability and sensitivity of HSVl-tk reporter gene imaging to monitor changes in the expression of different endogenous genes and activity of different signal transduction pathways in response to specific exogenous modulatory stimuli. Initially, the HSVl-tk/GFP fusion gene-based reporter systems for will be evaluated in vitro. After optimization of the reporter systems in transgenic cell cultures, they will be tested in animals bearing the corresponding transgenic tissue. Then, we will generate transgenic animals with selected reporter systems and assess the activity of particular molecular-biological processes in response to different stimuli by non-invasive imaging. Once validated, these imaging techniques could be applied to naive (non-transgenic) tissues and animals, and possibly to human subjects. In the future, a dual-reporter gene vector system will be developed in which one reporter gene will be expressed constitutively to assess the magnitude of in vivo transfection, and another reporter gene will be expressed to image a specific molecular-biological process.
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