Viral Vector Core

病毒载体核心

• 批准号: 8788683
• 负责人: Candice Contet
• 金额: $ 12.84万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788683
• 关键词: Adult; Affect; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholism; Amygdaloid structure; Birth; Brain; Brain region; Cells; Cellular Neurobiology; Corticotropin-Releasing Hormone Receptors; Custom; DNA; Dependovirus; Development; Gene Expression; Genes; Glutamates; Goals; Health; Label; Modeling; Molecular; Monoacylglycerol Lipases; Morphology; Neurons; Physiology; Population; Prefrontal Cortex; Production; Rattus; Research; Research Institute; Retroviral Vector; Role; Stress; Structure of terminal stria nuclei of preoptic region; Viral; Viral Vector; Withdrawal; adeno-associated viral vector; adult neurogenesis; alcohol effect; alcohol research; alcoholism therapy; binge drinking; cost; design; in vivo; innovation; insight; interest; neurobiological mechanism; neuronal pentraxin; neurotransmission; newborn neuron; novel; overexpression; research study; tool; vector

Alcohol abuse and dependence affect an estimated 8.5% of the U.S. population and are responsible for substantial health and societal costs. The overarching goal of the Alcohol Research Center at The Scripps Research Institute (TSRI-ARC) is to understand the molecular and cellular mechanisms of vulnerability to alcohol dependence, with a focus on dysregulation of excitatory neurotransmission in stress-responsive brain regions, such as the basolateral amygdala (BLA) and the bed nucleus of the stria terminalis (BNST). In addition, adult neurogenesis will be examined during withdrawal. The Viral Vector Core will be instrumental in the realization of experiments proposed in TSRI-ARC Research Components by providing validated tools to manipulate gene expression locally and to label newborn neurons in adult rats exposed to models of binge drinking or alcohol dependence. Specific Aim 1 is to characterize the ability of seven adeno-associated virus (AAV) pseudotypes to transduce glutamatergic cells in the BLA and the ventromedial prefrontal cortex (vmPFC), which send excitatory projections to the BLA and BNST. Results from this Aim will be used to select the best-suited AAV pseudotype for the production of custom AAV vectors in Specific Aims 2 and 3. Specific Aim 2 is to provide AAV vectors for local silencing of monoacylglycerol lipase expression in BLA and vmPFC, and for knockdown of corticotropin-releasing factor receptor type 1 selectively in BLA glutamatergic neurons. Specific Aim 3 is to provide AAV vectors for functional knockdown or glutamatergic neuron-specific overexpression of neuronal pentraxin 2 in the BLA. Specific Aim 4 is to provide a retroviral vector expressing EGFP. This vector will label newborn neurons in the adult rat brain and enable characterization of their morphology and physiology. For each viral vector, we propose to design and clone DNA constructs, obtain high-titer, purified viral stocks from an outside production facility and validate their silencing/overexpression efficiency in vivo. Altogether, we anticipate that the innovative molecular tools provided by the Viral Vector Core will assist in gaining novel insights into the neurobiological mechanisms of excessive alcohol drinking.

酗酒和依赖影响估计占美国人口的8.5％，并负责 大量的健康和社会成本。斯克里普斯酒精研究中心的总体目标 研究所（TSRI-ARC）是了解脆弱性的分子和细胞机制 酒精依赖性，重点是压力反应性兴奋性神经传递的失调 大脑区域，例如基底外侧杏仁核（BLA）和质末端（BNST）的床核。在 此外，将在戒断期间检查成年神经发生。病毒矢量核心将具有工具性 在实现TSRI-ARC研究组件中提出的实验中，通过提供验证的工具 在局部操纵基因表达并在暴露于暴饮暴食模型的成年大鼠中标记新生神经元 饮酒或酒精依赖。特定目的1是表征七种腺相关病毒的能力 （AAV）假型将BLA和腹侧前额叶皮层转导谷氨酸能细胞 （VMPFC），将兴奋性预测发送到BLA和BNST。这个目标的结果将用于 选择最适合的AAV假型，用于在特定目标2和3中生产自定义AAV矢量。 具体目的2是提供AAV向量，以使BLA中的单酰甘油脂肪酶表达局部沉默 VMPFC，用于敲除抗皮质激素释放因子受体1型在BLA谷氨酸能 神经元。特定目标3是为功能敲低或谷氨酸神经元特异性的AAV矢量提供 BLA中神经元五肽2的过表达。特定目的4是提供表达逆转录病毒载体 EGFP。该载体将在成年大鼠大脑中标记新生神经元，并能够表征其 形态和生理学。对于每个病毒载体，我们建议设计和克隆DNA构建体，获得 来自外部生产设施的高敏机纯化病毒库存，并验证其沉默/过表达 体内效率。总之，我们预计病毒载体提供的创新分子工具 核心将有助于获得对过量饮酒的神经生物学机制的新见解。