MODULATION OF OPIOID AND NMDA RECEPTOR MRNA BY RIBOZYMES

核酶对阿片类药物和 NMDA 受体 mRNA 的调节

• 批准号: 2414623
• 负责人: HUGH D ROBERTSON
• 金额: $ 13.27万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414623
• 关键词: NMDA receptors; animal genetic material tag; liposomes; messenger RNA; nucleic acid sequence; opioid receptor; pancreatic ribonuclease; receptor expression; ribozymes; tissue /cell culture; transfection; transfection /expression vector

We propose to conduct research designed to adapt the ribozyme approach to provide a transient or permanent "knockout" of a target protein (and its associated function) by cleaving the appropriate mRNA with high specificity. This approach is one which is already showing promise in the antiviral field, where a number of advantages for it have emerged. RNA- level approaches in general are succeeding in antiviral therapy for hepatitis B virus, for example; the ribozyme approach is most promising because one ribozyme can attack a number of RNAs because of its catalytic/enzymatic properties. The other major approach-antisense inhibition--works at a 1:1 level. We propose to adapt approaches which are showing promise in the antiviral field to key cellular RNAs, including mRNAs for mouse delta opioid receptors (DORs) and glutamate receptor channel subunits (NMDAs) in test tube experiments using cloned mRNA expression vectors; and in cultured mouse nerve cells, specifically the NGl08-l5 cell line. We plan to test the hypothesis that the ribozyme technique, using either delta hepatitis agent ribozymes retargeted to the relevant mRNAs; or external guide sequences (EGSs) which re-target the cellular tRNA processing enzyme RNase P to exogenous sequences, can provide a transient "knockout" of a targeted mRNA, and its associated functional protein. The cellular approach is the logical endpoint of the initial research effort, with work in animals to follow. The collaborative efforts of the Robertson Lab (RNA structure/function; ribozyme development) and the Inturrisi Lab (mRNA and cell characterization; RNA detection) have already led to several publications in the field of RNA quantitation. We will collaborate on testing the above mRNAs, comparing the ribozyme approaches mentioned to parallel efforts using antisense deoxyribonucleotides. In this way we should be able to introduce these approaches into the field of drug abuse research, where they should allow an increase in systematic studies of the effects of expression involving various receptor genes.

我们建议进行研究，旨在适应核酶的方法， 提供靶蛋白的瞬时或永久“敲除”（及其 相关功能）通过切割适当的mRNA， 的特异性这一方法已经显示出了希望， 在抗病毒领域，它已经出现了许多优势。核糖核酸 水平的方法一般是成功的抗病毒治疗， 例如，B型肝炎病毒;核酶方法是最有前途的 因为一个核酶可以攻击许多RNA， 催化/酶性质。另一种主要的方法-反义 抑制作用是1：1的我们建议采用以下方法 在抗病毒领域显示出对关键细胞RNA的承诺，包括 小鼠δ阿片受体（DORs）和谷氨酸受体的mRNA 使用克隆mRNA的试管实验中的通道亚基（NMDA） 表达载体;以及在培养的小鼠神经细胞中， NG 108 - 15细胞系。 我们计划测试的假设，核酶技术，使用 重新靶向相关mRNA的δ肝炎因子核酶;或 重新靶向细胞tRNA的外部指导序列（EGS） 加工酶RNase P向外源序列，可提供瞬时 靶向mRNA的“敲除”及其相关的功能蛋白。的 细胞方法是初始研究工作的逻辑终点， 在动物身上的工作也随之而来。罗伯逊夫妇的共同努力 实验室（RNA结构/功能;核酶开发）和Inturrisi实验室 (mRNA和细胞表征; RNA检测）已经导致 RNA定量领域的几篇出版物。我们将合作 在测试上述mRNA时，将提到的核酶方法与 使用反义脱氧核糖核苷酸的平行努力。这样我们 应该能够将这些方法引入药物滥用领域 研究，在那里他们应该允许增加系统的研究， 涉及各种受体基因的表达的影响。