Bioanalytic Core

生物分析核心

• 批准号: 10090657
• 负责人: MAR M SANCHEZ
• 金额: $ 27.79万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10090657
• 关键词: Acetylcholinesterase; Anatomy; Autoradiography; Biological Assay; Brain; Cells; Choline O-Acetyltransferase; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Data; Detection; Documentation; Dopamine D1 Receptor; Dopamine D2 Receptor; Ensure; Faculty; Future; Genes; Genetic Polymorphism; Genotype; Goals; Green Fluorescent Proteins; Histologic; Histological Techniques; Histology; Human Resources; Immediate-Early Genes; Immunochemistry; Immunohistochemistry; In Situ Hybridization; Injections; Learning; Macaca; Mediating; Melanocortin 4 Receptor; Memory; Messenger RNA; Microscopy; Microtome - medical device; Microtus; Mission; Molecular; Neuroanatomy; Neuromodulator; Neurons; Neuropeptide Receptor; Oxytocin; Oxytocin Receptor; Population; Postdoctoral Fellow; Primates; Productivity; Proteins; Rattus; Research; Research Activity; Research Personnel; Research Support; Rodent; Services; Site; Slice; Stains; Standardization; Steroid Receptors; Students; System; Technical Expertise; Techniques; Time; Training; Validation; Viral Vector; brain tissue; career development; cell type; cryostat; experience; histological stains; improved; innovation; member; molecular phenotype; novel; optogenetics; prairie vole; programs; receptor binding; reconstruction; relating to nervous system; social cognition; transgene expression

PROJECT SUMMARY (Bioanalytic Core, Sanchez) The Bioanalytic Core is a multifunctional core that will support the research efforts of each of the Projects by providing histological services, developing new innovative techniques and assays, and by training members of the Center to improve their own technical skills. The Aims of the Bioanalytic Core are: (1) To Provide basic neurohistology services for verification of probe placement or injection sites for Projects 1-4, including brain sectioning, histological staining, and immunohistochemistry for viral vector detection, and microscopy for reconstruction of recording and injection sites. (2) To provide assay services and to develop new techniques for molecular characterization of specific cell types, and for validating novel CRISPR viral vectors for Projects 1-4. These services include OXTR autoradiography, and the highly sensitive RNAScope mRNA in situ hybridization (ISH) technique to co-localize mRNA in cells for the purpose of molecular phenotyping. This technique will allow for the co-localization of mRNA for OXTR with other neuromodulator systems such as dopamine D1 and D2 receptors or with choline acetyltransferase (ChAT). We will also develop PCR/sequencing or Western assays to validate viral vector mediated CRISPR editing of OXTR or OT gene for Projects 1 and 3. Finally, the core will perform genotyping for OXTR polymorphisms in prairie voles in support of Projects 1 and 2. (3) To provide training in the techniques used in the Core to make sure that students, postdoctoral fellows, staff and trainees of the Center gain experience in basic histology and staining, autoradiography, ISH, IHC, neuroanatomy and fluorescent microscopy, which will be useful for their future career development. The services of the Bioanalytic Core will facilitate progress in every project and will serve as the center memory for techniques as trainees come and go. We will also develop new assays as needed and will perform basic neurohistology with standardization across projects while freeing up time for trainees to make progress in their own projects. The Bioanalytic Core is supported by the strong neuroanatomical expertise of the PI (Sanchez) in mapping neuropeptide and steroid receptors in the primate brain, studies performed in collaboration with the Center PI (Young), who has additional expertise with OT receptor systems both in rodent and primate brain. Co-investigator Kiyoshi Inoue brings critical extensive expertise in situ hybridization using RNAScope, OXTR autoradiography, general histology for probe anatomical verification, immunohistochemistry, and genotyping the prairie vole OXTR gene. These services will facilitate the integrated research programs of the Conte Center by providing unified and standardized services for each of the Projects, and allow investigators for the first time to identify the molecular phenotype of neurons activated by the OT system, and facilitate validation of new techniques such as optogenetic transgene expression and CRISPR editing of the OT system. These services will streamline the research activity of the Projects to maximize their productivity, while also providing valuable training on each of the techniques used to all Conte personnel who wish to learn these techniques.

项目总结（生物分析中心，Sanchez） 生物分析核心是一个多功能核心，将支持每个项目的研究工作， 提供组织学服务，开发新的创新技术和分析，并通过培训 中心，以提高自己的技术能力。生物分析核心的目的是：（1）提供基本的 神经组织学服务，用于验证项目1-4的探针放置或注射部位，包括大脑 切片、组织学染色和免疫组织化学用于病毒载体检测，显微镜检查用于 记录和注射部位的重建。(2)提供化验服务及发展新技术， 用于特定细胞类型的分子表征，以及用于验证项目1-4的新型CRISPR病毒载体。 这些服务包括OXTR放射自显影和高度敏感的RNAScope mRNA原位杂交。 (ISH)在细胞中共定位mRNA以进行分子表型分析的技术。这项技术将允许 用于OXTR的mRNA与其他神经调节剂系统如多巴胺D1和D2的共定位 受体或胆碱乙酰转移酶（ChAT）。我们还将开发PCR/测序或Western分析， 验证项目1和3的病毒载体介导的OXTR或OT基因的CRISPR编辑。最后，核心将 在草原田鼠中进行OXTR多态性的基因分型，以支持项目1和2。(3)提供 在核心技术的培训，以确保学生，博士后研究员，工作人员和学员的核心 中心在基础组织学和染色、放射自显影、ISH、IHC、神经解剖学 荧光显微镜，这将是他们未来的职业发展有用。生物分析服务 核心将促进每一个项目的进展，并将作为中心记忆的技术作为学员来 然后离开我们还将根据需要开发新的检测方法，并进行标准化的基本神经组织学检查 同时腾出时间让学员在自己的项目中取得进展。生物分析核心 由PI（Sanchez）在绘制神经肽和类固醇方面的强大神经解剖学专业知识支持 受体在灵长类动物的大脑中，研究与中心PI（年轻），谁有额外的合作进行 在啮齿动物和灵长类动物大脑中的OT受体系统的专业知识。共同研究员井上清带来了关键的 在使用RNAScope进行原位杂交、OXTR放射自显影、探针的一般组织学方面具有广泛的专业知识 解剖学验证，免疫组织化学，和草原田鼠OXTR基因分型。这些服务将 通过提供统一和标准化的服务，促进康特中心的综合研究计划 为每个项目，并允许研究人员首次确定神经元的分子表型 通过OT系统激活，并促进新技术的验证，如光遗传转基因 OT系统的表达和CRISPR编辑。这些服务将使联合国的研究活动更加合理化。 项目，以最大限度地提高他们的生产力，同时也提供了宝贵的培训，每一个技术， 所有希望学习这些技术的Conte人员。