The ability to modulate molecular interactions is an essential prerequisite for all stages of target development, from identification to validation to preclinical inhibition. Following iterative discussions among the junior faculty Project Leaders, Mentors, and iTarget leadership during project development and review meetings, the Core Director was able to systematically identify common infrastructure requirements of iTarget investigators for proteins and intracellular targeting reagents. A comprehensive survey of shared resources confirmed that there is no facility at Dartmouth or among our regional IDeA partners that currently provides or coordinates such services. To address these critical unmet needs, we propose to establish a Molecular Tools Core (MTC; Core B). Building on techniques developed in structural genomics and other high-throughput expression initiatives, we have established a highly efficient pipeline that uses parallel construct design, subcloning, and expression testing strategies to rapidly identify optimal conditions for production of recombinant proteins in bacterial (Specific Aim 1) or mammalian (Specific Aim 2) cells. Once expression conditions are established, the Core will provide a comprehensive set of chromatographic techniques and equipment to permit efficient testing, optimization, and performance of liter-scale purification. If larger culture volumes are required (e.g., due to low yield), we have established a reciprocal arrangement with the Biotechnology Program of our New Hampshire- INBRE (NH-INBRE) partner Great Bay Community College to provide access to process-scale facilities. To facilitate intracellular targeting, we will provide advice and will streamline access to intracellular targeting strategies, including overexpression and RNA interference, suitable for use in physiologically relevant cell lines (Specific Aim 3). These strategies will include commercial and custom plasmids, as well as viral delivery systems and other intracellular targeting agents. If oligonucleotide (siRNA or antagomir) or small-molecule inhibitors are required, the core will fund or coordinate access commercial sources or partner facilities. The MTC will also function as a resource and training facility for investigators who wish to discover, validate, and inhibit specific targets or pathways. The expertise and services provided by the MTC will thus allow for the rapid investigation of candidate protein and protein complexes, both in vitro and in cells, freeing project investigators to concentrate on the functional role and biological importance of their targeted systems. As no comparable shared resource currently exists at Dartmouth or our regional IDeA partners, the MTC will have a significant positive impact on the research infrastructure broadly, and in particular on the research productivity of all four junior faculty Project Leaders, other iTarget faculty, and our partners.
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