Core B: Molecular Tools Core

核心 B：分子工具核心

• 批准号: 9273576
• 负责人: Fredrick Jon Kull
• 金额: $ 16.3万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9273576
• 关键词: ART protein; Address; Adopted; Affinity; Alpha Cell; Bacteria; Binding; Biochemical; Biological; Biophysics; Biotechnology; Cell Line; Cells; Colon Carcinoma; Custom; Development; Ensure; Equipment; Escherichia coli; Faculty; Funding; Gene Expression; Genes; Glioblastoma; In Vitro; Individual; Institutes; Investigation; Laboratories; Leadership; Length; Mentors; MicroRNAs; Mission; Molecular; New Hampshire; Nightmare; Oligonucleotides; Pathway interactions; Peptidyl-Dipeptidase A; Performance; Physiological; Plasmid Cloning Vector; Plasmids; Probability; Procedures; Process; Production; Productivity; Protein Array; Proteins; Protocols documentation; RNA; RNA Interference; Reagent; Recombinant Proteins; Research; Research Infrastructure; Research Personnel; Resource Sharing; Resources; Respiratory Syncytial Virus Infections; Role; Services; Signal Pathway; Signal Transduction; Site; Source; Surveys; System; Techniques; Testing; Time; Training; Transfection; Validation; Viral; Viral Proteins; base; biophysical properties; career development; combinatorial; community college; cost; design and construction; enzyme activity; experience; inhibitor/antagonist; meetings; milligram; neutralizing antibody; novel; overexpression; pre-clinical; programs; promoter; protein complex; protein expression; small molecule inhibitor; structural genomics; success; targeted agent; tool; vector

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.

调节分子相互作用的能力是目标发展的所有阶段的重要先决条件， 从识别到验证再到临床前抑制。经过初级教师的迭代讨论 项目开发和审查会议期间的项目负责人，导师和ITARGET领导力是核心 董事能够系统地确定ITARGET调查人员的共同基础设施要求 蛋白质和细胞内靶向试剂。对共享资源的全面调查确认 在达特茅斯（Dartmouth）或我们目前提供或协调这样的区域构思合作伙伴中没有设施 服务。为了满足这些关键的未满足需求，我们建议建立一个分子工具核心（MTC；核心 b）。建立在结构基因组学和其他高通量表达计划中开发的技术的基础 我们已经建立了一条高效的管道，该管道使用并行结构设计，亚克隆和表达 测试策略快速识别细菌中重组蛋白的最佳条件 （特定目标1）或哺乳动物（特定目标2）细胞。一旦建立了表达条件，核心 将提供一套全面的色谱技术和设备，以允许有效测试， 优化和升级净化的性能。如果需要较大的培养量（例如，由于低 收益率），我们已经通过新罕布什尔州的生物技术计划建立了互惠安排。 INBRE（NH-INBRE）合作伙伴Great Bay社区学院，可提供流程规模的设施。到 促进细胞内靶向，我们将提供建议，并将简化对细胞内靶向的访问 策略，包括过表达和RNA干扰，适用于生理相关的细胞系 （特定目标3）。这些策略将包括商业和自定义质粒以及病毒式传递 系统和其他细胞内靶向剂。如果寡核苷酸（siRNA或Antagomir）或小分子 需要抑制剂，核心将资助或协调访问商业资源或合作伙伴设施。这 MTC还将充当希望发现，验证和的调查人员的资源和培训机构 抑制特定目标或途径。 MTC提供的专业知识和服务将允许 快速研究候选蛋白和蛋白质复合物，无论是在体外还是在细胞中，释放项目 研究人员专注于其目标系统的功能作用和生物学重要性。否 目前在达特茅斯（Dartmouth）或我们的区域思想合作伙伴中存在可比共享资源，MTC将有一个 对研究基础设施的重大积极影响广泛，尤其是研究生产率 在所有四个初级教师项目领导人，其他Itarget教师和我们的合作伙伴中。