cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.
cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.
复制标题
通过对注射 mRNA 的非洲爪蟾卵母细胞进行电生理分析，克隆 5-羟色胺 5-HT1C 受体的 cDNA。
DOI：
10.1073/pnas.84.12.4332
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发表时间：
1987
影响因子：
11.1
通讯作者：
Davidson,N
中科院分区：
文献类型：
--
作者：
Lübbert,H;Hoffman,BJ;Snutch,TP;vanDyke,T;Levine,AJ;Hartig,PR;Lester,HA;Davidson,N
我们描述了一种克隆神经递质受体和离子通道cdna的策略，该策略基于对注射mrna的爪蟾卵母细胞的电生理分析。这种方法绕过了这些膜蛋白的纯化，因为它们的低丰度和疏水性阻碍了这些膜蛋白的纯化。它包括通过高分辨率凝胶电泳分离RNA的方法，在单链载体上定向克隆cDNA，以及通过电压钳测量mrna注射卵母细胞中血清素诱导的电流来筛选cDNA文库。从小鼠脉络膜丛乳头状瘤中分离血清素受体cDNA克隆证明了我们方法的适用性。通过杂交耗尽和杂交选择程序鉴定该克隆。该受体在卵母细胞中被混合选择RNA所表达，功能完全正常，这表明它是由一个5千碱基RNA编码的单个亚基组成的。杂交选择受体的药理学证实我们已经成功克隆了5-羟色胺5-HT1C受体cDNA。
We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.