Electrophysiological reporter for monitoring gene manipulations

用于监测基因操作的电生理报告器

• 批准号: 8244231
• 负责人: Jeffrey C Petruska
• 金额: $ 22.38万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8244231
• 关键词: Address; Adult; Afferent Neurons; Aging; Animals; Biological Models; Candidate Disease Gene; Cell Line; Cells; Characteristics; Code; Complex; Data; Diabetes Mellitus; Diabetic Neuropathies; Disease; Electrodes; Electrophysiology (science); Functional disorder; Gene Delivery; Genes; Human; In Vitro; Individual; Information Systems; Injury; Ion Channel; Kinetics; Learning; Ligands; Light; Link; Locomotion; Maintenance; Mammalian Cell; Mediating; Memory; Mental disorders; Methods; Modeling; Molecular Genetics; Monitor; Motor; Motor Neurons; Nerve Degeneration; Nerve Growth Factor Receptors; Nervous System Trauma; Nervous system structure; Neurobiology; Neurodegenerative Disorders; Neurons; Neurosciences; Neurotrophic Tyrosine Kinase Receptor Type 3; Neurotrophin 3; Newborn Animals; Pathology; Physiologic pulse; Physiological; Physiology; Play; Preparation; Process; Production; Proline; Property; Publishing; Rattus; Receptor Signaling; Reporter; Reporter Genes; Reporting; Research; Research Personnel; Role; Signal Transduction; Spinal; Spinal Ganglia; Stimulus; Substance abuse problem; Synapses; System; Technology; Time; Viral Vector; Virus; Work; addiction; cell type; fighting; genetic manipulation; improved; in vivo; injured; innovation; interest; knock-down; mutant; nervous system disorder; neurophysiology; novel; receptor; relating to nervous system; research study; response; small hairpin RNA; therapeutic target; tool; voltage

DESCRIPTION (provided by applicant): This proposal is an innovative effort to develop a novel tool that could revolutionize the execution and application of in vivo sharp electrode intracellular electrophysiology, the fundamental approach for examining cellular and synaptic mechanisms of systems-level functions. This tool is a reporter gene that will be detectible by electrophysiological methods in real-time by sharp electrodes in vivo, without the need for secondary factors (i.e., light stimulus or exogenous ligands). This could provide a necessary link to enable a more thorough incorporation of classic-type systems physiology with the reduced preparations and lower species that make up the majority of current experimental approaches used to understand genetic manipulations. An ideal electrophysiological reporter is one that is genetically-encoded, is "visible" to electrophysiological methods, has a signal that is easily recognized and distinguished from native processes in real time, and does not interfere with normal physiological function. We have identified a lab-generated mutant ion channel that fits these criteria, and appears to be an excellent candidate. We will make use of new viral vector technology to deliver candidate gene to adult rats. Aim 1 will use both in vivo and in vitro single-cell electrophysiological approaches to determine if the signal from the reporter gene can be detected against the well characterized background of the model systems (dorsal root ganglion sensory neurons and spinal motoneurons). Aim 2 is hypothesis driven and will determine if constitutive signaling by trkC, the receptor for neurotrophin-3, plays a necessary role in maintenance of the cellular and synaptic properties of adult motoneurons. The issue of maintenance of cellular properties by target-derived factors is in a question of fundamental importance in terms of the principles of neuroscience/neurobiology, and also in terms of biomedical issues in the adult nervous system such as aging, neurodegeneration, traumatic injury, and learning and memory. PUBLIC HEALTH RELEVANCE: This project is aimed at developing a new tool for research - a gene that will report the status of genetic manipulations to electrophysiological probes. If successful, it will greatly facilitate the execution of, and interpretation of data from, systems-level in vivo neurophysiology, an experimental approach that is particularly relevant to functional studies of the role of specific genes/cell-types in systems-level processes such as aging, locomotion, sensori-motor integration, models of psychiatric disorders and addiction/substance abuse, and nervous system disease/injury/insult and the responses to treatments. This project also Aims to determine the effect of altering the expression of trkC (the receptor for neurotrophin-3) on the cellular and synaptic electrophysiological properties of a subpopulation of neurons, which is relevant to conditions or disease states that may include reduced neurotrophin-receptor signaling in the pathophysiology, such as aging, diabetic neuropathy, traumatic nervous system injury, and neurodegeneration.

描述（由申请人提供）：该提案是开发一种新颖工具的创新努力，该工具可以彻底改变体内锐电极细胞内电生理学的执行和应用，这是检查系统级功能的细胞和突触机制的基本方法。 该工具是一种报告基因，可以通过体内尖锐电极通过电生理学方法实时检测，而不需要二次因素（即光刺激或外源配体）。 这可以提供必要的联系，使经典型系统生理学与减少的制剂和低等物种更彻底地结合起来，这些制剂构成了当前用于理解基因操作的大多数实验方法。 理想的电生理报告基因是一种基因编码的报告基因，对电生理学方法“可见”，具有易于实时识别并与天然过程区分开的信号，并且不干扰正常的生理功能。 我们已经确定了一种符合这些标准的实验室生成的突变离子通道，并且似乎是一个很好的候选者。 我们将利用新的病毒载体技术将候选基因传递给成年大鼠。 目标 1 将使用体内和体外单细胞电生理学方法来确定是否可以在模型系统（背根神经节感觉神经元和脊髓运动神经元）的良好特征背景下检测到来自报告基因的信号。 目标 2 是假设驱动的，将确定 trkC（神经营养素 3 的受体）的组成性信号传导是否在维持成体运动神经元的细胞和突触特性中发挥必要的作用。 靶标衍生因子维持细胞特性的问题对于神经科学/神经生物学原理以及成人神经系统的生物医学问题（例如衰老、神经变性、创伤性损伤、以及学习和记忆。 公共健康相关性：该项目旨在开发一种新的研究工具——一种能够向电生理探针报告基因操作状态的基因。 如果成功，它将极大地促进系统级体内神经生理学数据的执行和解释，这是一种实验方法，与特定基因/细胞类型在系统级过程中的作用的功能研究特别相关，例如如衰老、运动、感觉运动整合、精神疾病模型和成瘾/药物滥用、神经系统疾病/损伤/侮辱以及对治疗的反应。 该项目还旨在确定改变 trkC（神经营养素 3 受体）的表达对神经元亚群的细胞和突触电生理特性的影响，这与可能包括神经营养素受体减少的状况或疾病状态有关病理生理学中的信号传导，例如衰老、糖尿病神经病变、创伤性神经系统损伤和神经变性。