Molecular Screening and Protein Expression Shared Resource

分子筛选和蛋白质表达共享资源

• 批准号: 10360641
• 负责人: Joseph M Salvino
• 金额: $ 20.74万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360641
• 关键词: Affinity; Affinity Chromatography; Agreement; Antibodies; Antineoplastic Agents; Automation; Bacteria; Baculoviruses; Basic Cancer Research; Biochemical; Biochemistry; Biological; Biological Assay; Budgets; CRISPR/Cas technology; Cancer Center; Cancer Research Project; Cells; Collaborations; Collection; Complementary DNA; Complex; Crystallization; Custom; Data; Data Set; Development; Dissection; Doctor of Philosophy; Engineering; Environment; Epigenetic Process; Equipment; Escherichia coli; Experimental Designs; Fostering; Funding; Gene Targeting; Genes; Genome; Glycerol; Grant; Image; Immunization; Industry; Insecta; Institutes; Interdisciplinary Study; Kinetics; Label; Laboratories; Lead; Lentivirus; Libraries; Liquid substance; Mammalian Cell; Metabolism; Miniaturization; Molecular; Molecular Biology; Mus; Nucleic Acids; Oncology; Oryctolagus cuniculus; PTPRC gene; Pharmaceutical Preparations; Philadelphia; Production; Proteins; Protocols documentation; Publications; Reader; Reagent; Recombinant DNA; Recombinant Proteins; Recombinants; Reporting; Research; Research Personnel; Resource Sharing; Retroviridae; Robotics; Services; Signal Pathway; Slide; Specificity; Structure-Activity Relationship; System; Technology; Therapeutic; Thermodynamics; Time; Training; Translations; United States National Institutes of Health; Validation; Work; assay development; base; clinical practice; computer infrastructure; data integration; density; design; drug development; drug discovery; early phase clinical trial; experience; high throughput screening; inhibitor; instrument; instrumentation; inter-institutional; interest; lead optimization; lead series; member; plasmid DNA; programs; protein expression; protein function; quality assurance; screening; screening services; small hairpin RNA; small molecule; small molecule libraries; therapeutic candidate; therapeutic target; tool; transcription activator-like effector nucleases; tumor; validation studies; vector

PROJECT SUMMARY – MOLECULAR SCREENING AND PROTEIN EXPRESSION The Molecular Screening and Protein Expression Shared Resource (MSPESR) provides Cancer Center (CC) members with state-of-the-art high-throughput screening capabilities of shRNA and small molecule libraries to identify genes and tool inhibitors of candidate therapeutic targets. Identifying drug-like, small molecules that regulate the activity of therapeutic targets holds promise in defining new treatment paradigms, especially for recalcitrant tumor types, where current clinical practice is suboptimal. In addition, expert technical assistance is provided in recombinant DNA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins, and production of high-titer retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells. CC investigators require high quality recombinant proteins for characterization of enzymatic activities, crystallization for structural analysis, characterization of structure- function relationships of protein-protein, protein-nucleic acid, and protein-small molecule interactions; and immunization of rabbits/mice to generate custom antibodies. Through an inter-institutional agreement with the Helen F. Graham Cancer Center (HFGCC), the MSPESR offers CC members access to CRISPR/Cas9 gene editing services. The MSPESR fosters collaboration by providing expertise in biochemical and cell-based assay development. Such assays enable researchers to identify small molecule compounds which can then be used as tools to further study the target protein functions and signaling pathways in cells, or alternatively they may represent hit or lead compounds and form the basis for 'hit-to-lead" optimization to identify a lead series for drug discovery initiatives. Currently, the MSPESR offers: 1) biochemical-, cell-, and high-content based assays amenable to high-throughput screening in 384-well microtiter plates; 2) libraries of small molecules and shRNA; 3) high-throughput screening of small molecule libraries; 4) analysis of biological and chemistry datasets; 5) characterization of potency and selectivity of newly identified compounds in secondary, orthogonal assays. These services are provided through a centralized laboratory equipped with robotics, libraries of drug- like molecules arrayed in high-density microplate formats, and computational infrastructure for efficient analysis, interpretation, and management of biological and chemistry datasets. The MSPESR is operated by an experienced Managing Director and dedicated laboratory staff, cross-trained in all services offered. This allows for timely project management, quality assurance, and dissemination/integration of data critical for translation of basic biological observations into potential therapeutic strategies. Through its activity over the last budget cycle, the MSPESR has enabled dissection of complex signaling pathways of tumor onset and progression, validation of anticancer agent(s), and proof of concept results that were ultimately incorporated into early phase clinical trials. As an engine for multidisciplinary research collaboration, the MSPESR has contributed to critical publications and grant funding across all three CC Programs.

项目摘要 - 分子筛选和蛋白质表达 分子筛选和蛋白质表达共享资源（MSPESR）提供癌症中心（CC） 具有最先进的shRNA和小分子库的最先进的高通量筛选功能的成员 确定候选治疗靶标的基因和工具抑制剂。识别类似药物的小分子 规范治疗靶标的活动有望定义新的治疗态度，尤其是针对 当前的临床实践次优。此外，专家技术帮助是 在重组DNA质粒工程，细菌中的蛋白质表达和杆状病毒感染中提供 昆虫细胞，重组蛋白的纯化以及用于高素质逆转录病毒（例如慢病毒）的产生 将shRNA和cDNA递送至哺乳动物细胞。 CC研究人员需要高质量的重组蛋白 为了表征酶活性，结晶的结晶，结构的表征 蛋白质蛋白质，蛋白质核酸和蛋白质 - 小分子相互作用的功能关系；和 兔子/小鼠的免疫以生成自定义抗体。通过与 Helen F. Graham癌症中心（HFGCC），MSPESR为CC成员提供CRISPR/CAS9基因的访问 编辑服务。 MSPESR通过提供生化和基于细胞的专业知识来促进合作 测定开发。这种测定使研究人员能够鉴定出小分子化合物，然后可以是 用作进一步研究细胞中靶蛋白功能和信号通路的工具，或者它们 可能代表命中率或铅化合物，并构成“命中至铅”优化以识别铅系列的基础 用于药物发现计划。当前，MSPESR提供：1）基于生化的，细胞和高含量 在384孔微量滴定板中适应高通量筛选的测定； 2）小分子和 shrna; 3）小分子库的高通量筛选； 4）生物学和化学分析 数据集; 5）在次级，正交中新鉴定的化合物的效力和选择性的表征 测定。这些服务是通过配备有机器人技术的集中实验室提供的，毒品图书馆 像以高密度微板格式成熟的分子，以及有效的计算基础架构 生物和化学数据集的分析，解释和管理。 MSPESR由 经验丰富的董事总经理和专门的实验室工作人员，在所有提供的服务中进行了交叉培训。这 允许及时的项目管理，质量保证以及数据的传播/集成至关重要 将基本生物学观测转换为潜在的治疗策略。通过它的活动 上一个预算周期，MSPESR已使肿瘤发作的复杂信号通路的解剖和 进展，抗癌剂的验证以及最终合并的概念证明结果证明 进入早期临床试验。作为多学科研究合作的引擎，MSPESR拥有 为关键出版物做出了贡献，并在所有三个CC计划中授予了资金。