In the translation medicine, a multimodality imaging approach together with using reporter genes plays an important role for detecting endogenous signal transduction activity or transgene expression for molecular therapy. Combining two different technologies (e.g., PET with optical) through a fusion gene would have the advantage of speed and ease of validating approaches in cells or small animals that in turn can be translated to humans. Furthermore, this approach can also overcome the shortcomings or limitations of each imaging modality. For such purpose, the HSV1-tk/GFP (TKGFP) dual reporter gene was created. This dual reporter gene combined with optical imaging and nuclear imaging modalities has been utilized for monitoring endogenous biological processes or transgene expression. Despite the TKGFP is widely used reporter in the molecular-genetic imaging, there are several unaddressed issues and potential problems associated with its applications in the translation medicine: (1) TKGFP fusion protein seems to be highly stable in mammalian cells and therefore, may has low temporal resolution for monitoring temporal dynamics of endogenous signal transduction activity or transgene expression , (2) The cytototoxicity and low total cellular TK enzymatic activity trigger by nuclear tropism of HSV1-TK limited in its applications in translation medicine. Therefore, it needs further studies to give an insight into these issues and overcome these potential problems of TKGFP as a reporter for use in the translation medicine. In this project, we are aim at evaluating the application of TKGFP dual reporter system in dynamic studies of transcriptional gene regulation and creating novel mutant forms of TKGFP reporter genes with high temporal resolution and low cytototoxicity for real-time monitoring the temporal dynamics and spatial heterogeneity of endogenous signal transduction activity or transgene expression. Three specific aims are involved in this project to achieve these study goals. In the specific aim 1, we propose to evaluate the cytototoxicity, protein degradation and temporal resolution of TKGFP as a reporter in vitro and in vivo. We hope to address some issues regarding TKGFP as a reporter in the molecular-genetic imaging. Although the detail information about the cytotoxicity and temporal resolution of TKGFP as reporter is not fully clear, the earlier studies and our preliminary results indicate TKGFP as a reporter may be high cytotoxicity and low temporal resolution for monitoring dynamic gene expression. In the specific aim 2, we attempt to create new mutant forms of TKGFP fusion gene with low cytototoxicity and high temporal resolution for real-time monitoring temporal dynamics of gene expression evens or signal transduction activities. By such studies, we attempt to develop an optimal TKGFP fusion gene as a reporter for the applications in the translation medicine. In the specific aim 3, we propose to apply our developed TKGFP reporters in monitoring temporal dynamics of endogenous signal transduction activity. Our design experiments will focus on in vitro and in vivo investigating whether our developed TKGFP fusion genes as reporters have enough temporal resolution for real-time monitoring the temporal dynamics and spatial heterogeneity of HIF-1 signal transduction activity in tumor hypoxia and reoxygenation. We expect that the information derived from our research could address some issues regarding TKGFP as a reporter in the molecular-genetic imaging and develop an optimal TKGFP fusion reporter for use in the translation medicine within the period of three years research.
