RECEPTOR CDNA EXPRESSION CLONING USING LIGAND AUTORADIOGRAPHIC SCREENING

使用配体放射自显影筛选进行受体 CDNA 表达克隆

• 批准号: 3853713
• 负责人: G R UHL
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3853713
• 关键词: PCP receptor; autoradiography; cocaine; complementary DNA; drug abuse; drug metabolism; genetic library; molecular cloning; opioid receptor; pharmacogenetics; plasmids; polymerase chain reaction

Understanding the receptor molecules that recognize abused drugs and the neurotransmitters impacted by drugs is an important step in determining the molecular mechanisms underlying drug abuse. Little is known about many of these molecules, because they have proven very difficult to purify through conventional approaches. The laboratory has continued to work to establish a method for directly cloning these molecules based on their ligand binding properties, and to exploit this approach. Over the past year, these workers have made substantial progress toward this end. Previously, a cloned beta adrenergic receptor cDNA was used to document and optimize expression of the beta adrenergic receptor binding site in COS cells. In studies ompleted during this year, improved DNA recovery techniques and electroporation have resulted in recoveries of the plasmid present in very small number of cells with a reasonable frequency. This results in documented enrichments of up to 300-fold in a single step. The workers have progressed in adapting the method for use with the cocaine and PCP receptors. Goals for the current year include using this technique to attempt receptor cloning based on both cDNA recovery and subfractionating libraries and adaptation of the polymerase chain reaction to aid in recovery of the very small numbers of plasmids present. This approach continues to provide promise for allowing direct cloning of genes key to the action of several classes of abused substances.

了解识别滥用药物的受体分子以及 受药物影响的神经递质是确定的重要一步 药物滥用的分子机制。鲜为人知的是 其中许多分子，因为事实证明它们很难 通过常规方法纯化。实验室继续 致力于建立一种直接克隆这些分子的方法 他们的配体结合特性，并利用这种方法。 一年来，这些工作人员在工作上取得了长足进步。 这结束。 此前，克隆的 β 肾上腺素受体 cDNA 用于 记录并优化 β 肾上腺素受体结合的表达 COS 细胞中的位点。 在今年完成的研究中，改进了 DNA 回收技术和电穿孔已导致回收 质粒存在于极少数细胞中，具有合理的 频率。 这导致记录中的富集度高达 300 倍 一步。 工人们在调整方法方面取得了进展 与可卡因和五氯苯酚受体一起使用。今年的目标 包括使用该技术尝试基于两者的受体克隆 cDNA 回收和亚分级文库以及适应性 聚合酶链反应有助于回收极少量的 存在质粒。 这种方法继续为 允许直接克隆对几类药物的作用至关重要的基因 滥用物质。