Viral Vector Core

酒精滥用和依赖影响了大约8.5%的美国人口，并对 巨大的健康和社会成本。斯克里普斯大学酒精研究中心的首要目标 研究机构(TSRI-ARC)是为了了解易感性的分子和细胞机制 酒精依赖，重点是应激反应中兴奋性神经传递的失调 大脑区域，如杏仁基底外侧核(BLA)和终纹床核(BNST)。在……里面 此外，在戒断期间将对成人神经发生进行检查。病毒载体核心将发挥重要作用 在通过提供验证工具实现TSRI-ARC研究组件中提出的实验 暴饮暴食模型成年大鼠局部基因表达及新生神经元的标记 酗酒或酒精依赖。具体目标1是表征七种腺相关病毒的能力 (AAV)假型转导BLA和前额叶腹内侧皮质中的谷氨酸能细胞 (VmPFC)，向BLA和BNST发送兴奋性投射。这一目标的结果将用于 在特定目标2和3中选择最适合用于生产定制AAV载体的AAV伪型。 特异性目的2是为局部沉默BLA和BLA中单甘油脂肪酶的表达提供AAV载体 VmPFC，选择性下调血中谷氨酸能促肾上腺皮质激素释放因子受体1型 神经元。具体目的3是为功能敲除或谷氨酸能神经元特异性提供AAV载体 神经元五角蛋白2在白血球中的过度表达具体目的4是提供一种逆转录病毒载体 绿色荧光蛋白。该载体将标记成年大鼠大脑中的新生神经元，并使其能够表征其 形态和生理学。对于每个病毒载体，我们建议设计和克隆DNA结构，获得 从外部生产设施获得高滴度、纯化的病毒库存，并验证它们的沉默/过度表达 体内效率。总之，我们预计病毒载体提供的创新分子工具 CORE将有助于获得对过度饮酒的神经生物学机制的新见解。

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.