RECEPTOR CDNA EXPRESSION CLONING USING XENOPUS OOCYTE EXPRESSION

表达克隆是一种有吸引力的鉴定基因的方法， 编码滥用物质受体和 与药物滥用有关的神经递质（见上文）。 的能力 非洲爪蟾卵母细胞适当地翻译， 修改，并适当地插入几个受体到它的膜， 引导我们建立了这个系统作为这些cDNA的筛选工具， 受体。 该项目的进展包括：1）获得电生理 来自卵母细胞的神经降压素、胆囊收缩素和红藻氨酸的信号 注射这些合成的mRNA转录物，2）克隆单个 在卵母细胞中诱导CCK-A反应的cDNA， 药理学上适当的，但可能是由于上调 内源性CCK受体基因通过注射的转录本。 该cDNA “CCK-UP”在几个大脑区域中表达，并且可以提供重要的 对上调机制的深入了解对其中一个主要的 大脑中的多巴胺共递质，3）表征有趣的 在该系统中表达的GABA-Rho 1受体的特性，包括 不敏感性 因此，这种方法增强了以下能力： 通过“同胞选择”技术克隆感兴趣的DNA， 表征表达的cDNA的性质的能力。

Expression cloning is an attractive approach to identifying the genes and cDNAs encoding receptors for abused substances and for the neurotransmitters implicated in drug abuse (see above). The ability of the Xenopus oocyte to appropriately translate, post-translationally modify, and appropriately insert several receptors into its membrane has led us to establish this system as a screening tool for these cDNAs and receptors. Progress in this project has included: 1) Obtaining electrophysiologic signals for neurotensin, cholecystokinin, and kainic acid from oocytes injected with these synthetic mRNA transcripts, 2) Cloning of a single cDNA that induces CCK-A responses in oocytes that are pharmacologically-appropriate, but likely due to upregulation of the endogenous CCK receptor gene by the injected transcript. This cDNA "CCK-UP" is expressed in several brain regions, and may provide important insights into the upregulation mechanisms important for one of the chief dopamine cotransmitters in the brain, 3) Characterizing interesting properties of the GABA-Rho1 receptor expressed in this system, including bicucculing-insensitivity. This approach thus enhances abilities to clone interestingc DNAs through "sib selection" techniques, and improves abilities to characterize the properties of expressed cDNAs.