CORE--SCIENTIFIC FACILITIES

核心——科学设施

• 批准号: 6111336
• 负责人: ANDREW J CZERNIK
• 金额: $ 30.36万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6111336
• 关键词: DARPP; animal colony; antibody; biomedical facility; gene deletion mutation; genetically modified animals; immunologic assay /test; laboratory mouse; protein purification; tissue /cell culture

A greater understanding of the intracellular signal transduction mechanisms which underlie dopaminergic neurotransmission in the basal ganglia can lead to novel therapeutic targets for the treatment of schizophrenia, and degenerative neurological disorders such as Parkinson's disease and Huntington's disease. A multi-disciplinary approach will be undertaken in the current proposal to investigate the phosphorylation-dependent functional regulation of key molecules in these signaling pathways. Studies will be performed at several levels of organizational complexity, ranging from in vitro biochemical studies with purified molecules to the characterization of phenotypes resulting from targeted deletion of key signal transduction molecules in whole animals. The overall goal of this project is to provide a Scientific Core facility for use by all the members of the Program Project. The responsibilities of the Core will include the production and supply of key reagents and the performance of routine, yet critical tasks that will be required to accomplish the studies described in the other sections of this Program Project. The centralization of routine tasks will facilitate an efficient, cost-effective means to ensure an adequate supply of essential materials and reagents to all the Projects. The Core will maintain a tissue culture facility to provide a continuous supply of cultured cells, including primary neuronal cultures, reaggregated cultures and immortalized cell lines. It will also maintain stocks of purified protein kinases, protein phosphatases, antibodies, and substrate proteins. Standard purification protocols will be used to obtain these enzymes from native sources, and recombinant technologies will also be employed to produce specific proteins in quantities sufficient to carry out detailed enzymological and structural studies.. The Core will be responsible for the expansion and maintenance of the colonies of "knockout" mice that are deficient in DARPP-32, inhibitor-1, protein phosphatase-1 alpha1 and gamma isoforms, and protein phosphatase- 2B, and transgenic animals harboring phosphorylation site mutations in DARPP-32.. Animal husbandry will include the setting up of appropriate matings and weaning of animals. The Core will also be responsible for the determination of the genotype of the offspring, using PCR and Southern blot strategies. The Core will be responsible for the production of phosphorylation state- specific antibodies to specific sites on DARPP-32, inhibitor-1, protein phosphatase-1, Na+,K+-ATPase alpha subunit isoforms and Na+ and Ca2+ channels. The ability to detect and quantitate changes in the state of phosphorylation of these key substrate proteins will be of great utility in the studies proposed to elucidate their functional significance.

对细胞内信号转导的进一步理解 基底膜多巴胺能神经传递的机制 神经节可以导致新的治疗靶点，用于治疗 精神分裂症和退行性神经系统疾病， 帕金森病和亨廷顿病。 一个多学科 在目前的建议中，将采取的办法是调查 磷酸化依赖的关键分子的功能调节， 信号通路研究将在以下几个水平进行： 组织的复杂性，从体外生物化学研究， 纯化的分子，以表征表型， 靶向删除整个动物中的关键信号转导分子。 该项目的总体目标是提供一个科学核心设施 供计划项目的所有成员使用。的责任 核心业务将包括生产和供应关键试剂， 执行例行但关键的任务， 完成本计划其他部分所述的研究 项目日常工作的集中化将有助于 有效、成本效益高的手段，以确保充分供应基本 所有项目的材料和试剂。 核心将维持一个组织培养设施， 供应培养细胞，包括原代神经元培养物， 重组培养物和永生化细胞系。它还将保持 纯化的蛋白激酶、蛋白磷酸酶、抗体和 底物蛋白标准纯化方案将用于 从天然来源和重组技术中获得这些酶 也将用于大量生产特定的蛋白质 足以进行详细的酶学和结构研究。 核心将负责扩大和维持 DARPP-32，抑制剂-1， 蛋白磷酸酶-1 α 1和γ亚型，以及蛋白磷酸酶- 2B中磷酸化位点突变的转基因动物， DARPP-32..畜牧业将包括建立适当的 动物的交配和断奶。核心还将负责 使用PCR和Southern杂交确定后代的基因型 印迹策略。 核心将负责磷酸化状态的产生 DARPP-32、抑制剂-1、蛋白质上特异性位点的特异性抗体 磷酸酶-1，Na+，K+-ATP酶α亚单位亚型和Na+和Ca 2 + 渠道 检测和定量的能力，在状态的变化， 这些关键底物蛋白的磷酸化将具有很大的用途 在研究中提出，以阐明其功能的意义。