Exploring function of mu opioid receptor splice variants in rat by gene targeting
通过基因打靶探索大鼠μ阿片受体剪接变体的功能
基本信息
- 批准号:9181025
- 负责人:
- 金额:$ 25.71万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2016
- 资助国家:美国
- 起止时间:2016-07-15 至 2018-06-30
- 项目状态:已结题
- 来源:
- 关键词:Absence of pain sensationAdverse effectsAgonistAnalgesicsAnimal ModelBehavioral ModelC-terminalCellsClustered Regularly Interspaced Short Palindromic RepeatsCodeCommunitiesComplexConstipationEmbryoExonsFentanylFibroblastsGTP-Binding ProteinsGene TargetingGenerationsGenesGlucuronidesGrantHeroinHumanIntronsKnock-inKnock-in MouseKnock-outKnockout MiceLengthLiteratureLuciferasesMapsMediatingMessenger RNAMethadoneModelingMolecular CloningMorphineMorphine AbuseMusMutationOpioidOpioid AnalgesicsOrder ColeopteraPatientsPharmacologyProteinsPubMedRNA SplicingRattusReagentRegulationRenilla LuciferasesReporterResearchResourcesScientistSedation procedureTechniquesTechnologyTranscriptional RegulationTransmembrane DomainVariantaddictionbasedesigngenome editingin vivoinflammatory neuropathic painmRNA Precursormouse modelmu opioid receptorsmutantnovelnucleasepromoterreceptorresponsetool
项目摘要
Project Summary
Most opioid analgesics used clinically, including morphine and fentanyl, act primarily through the mu opioid
receptors. However, opioid analgesia and side-effects, such as constipation, itch, sedation and addiction, vary
immensely among patients and animal models. These observations suggest the existence of multiple mu
opioid receptors, a concept that was initially proposed based on pharmacological studies and has been
reinforced by molecular cloning of a vast array of mu opioid receptor variants, generated through extensive
alternative pre-mRNA splicing from the single-copy mu opioid receptor (OPRM1) gene. Increasing evidence
indicates that the OPRM1 splice variants are important in mediating the complex and variable actions of mu
opioids. Two distinct promoters, associated with either exon 1 (E1) or exon 11 (E11), control the expression of
E1-associated or E11-associated splice variants. E1-associated variants mainly encode full-length carboxyl (C-
) terminal, 7-transmembrane (7-TM) domain receptors, whereas the majority of E11-associated variants
encode truncated, 6-TM domain receptors. Gene targeted mouse models have revealed that E1-associated
full-length 7-TM C-terminal variants mediate the actions of morphine and methadone, whereas E11-associated
truncated 6-TM variants mediate a subset of mu opioids, including fentanyl, M6G and heroin, as well as IBNtxA
(3-iodobenzoyl-6β-naltrexamide), a novel analgesic that lacks many traditional opioid side-effects. While the
mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation.
Rats have been used extensively to study opioid actions for many decades, many of which have not been
conducted in mice. Rats are considered as better models than mice in a number of human conditions. Having
rat models would provide significant advantages and resources for the research community, and would expand
our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice. Splicing in
the rat OPRM1 gene is similar to that in the mouse and human OPRM1 genes, with an extensive array of both
E1-associated and E11-associated variants. Yet, the lack of appropriate rat gene targeting models limits further
exploration of their functions. We propose generating three rat OPRM1 knockout/knockin (KO/KI) models, E1-
KO/KI, E11-KO/KI and E1/E11-KO/KI, using recently developed CRISPR/Cas9 (Clustered Regularly
Interspaced Short Palindromic Repeats/Cas9 nuclease) technology. We will characterize E1-KO/KI and E11-
KO/KI models by examining the expression of OPRM1 splice variants, the analgesic actions of selected
opioids, as well as the endogenous E1 and E11 promoter activity. These mutant rat models will provide unique
and valuable tools for the research community to further explore the pharmacological functions and
transcriptional regulation of the rat OPRM1 gene. These studies will allow us to obtain a better understanding
of the complex actions of various opioids, and to design novel, potent opioid analgesics that lack traditional
opioid side-effects and abuse potential.
项目概要
临床上使用的大多数阿片类镇痛药,包括吗啡和芬太尼,主要通过 mu 阿片类药物发挥作用
受体。然而,阿片类药物的镇痛效果和副作用(如便秘、瘙痒、镇静和成瘾)各不相同
在患者和动物模型中影响巨大。这些观察表明存在多个 mu
阿片受体这一概念最初是根据药理学研究提出的,并已被
通过大量 mu 阿片受体变体的分子克隆得到加强,这些受体变体是通过广泛的研究产生的
来自单拷贝 mu 阿片受体 (OPRM1) 基因的选择性前 mRNA 剪接。越来越多的证据
表明 OPRM1 剪接变体对于介导 mu 的复杂且可变的作用非常重要
阿片类药物。两个不同的启动子,与外显子 1 (E1) 或外显子 11 (E11) 相关,控制
E1 相关或 E11 相关剪接变体。 E1相关变体主要编码全长羧基(C-
) 末端 7 跨膜 (7-TM) 结构域受体,而大多数 E11 相关变体
编码截短的 6-TM 结构域受体。基因靶向小鼠模型显示,E1 相关
全长 7-TM C 端变体介导吗啡和美沙酮的作用,而 E11 相关
截短的 6-TM 变体介导 mu 阿片类药物的子集,包括芬太尼、M6G 和海洛因,以及 IBNtxA
(3-碘苯甲酰-6β-纳曲酰胺),一种新型镇痛药,没有许多传统阿片类药物的副作用。虽然
小鼠模型很有价值,大鼠在行为建模和体内操作方面都有很多优势。
几十年来,老鼠被广泛用于研究阿片类药物的作用,其中许多尚未被研究过。
在小鼠中进行。在许多人类条件下,大鼠被认为是比小鼠更好的模型。拥有
大鼠模型将为研究界提供显着的优势和资源,并将扩大
我们有能力用在小鼠中不可行的方法和技术探索μ阿片类药物的药理学。拼接进去
大鼠 OPRM1 基因与小鼠和人类 OPRM1 基因相似,具有广泛的
E1 相关和 E11 相关变体。然而,缺乏适当的大鼠基因靶向模型进一步限制了
探索它们的功能。我们建议生成三种大鼠 OPRM1 敲除/敲入 (KO/KI) 模型,E1-
KO/KI、E11-KO/KI 和 E1/E11-KO/KI,使用最近开发的 CRISPR/Cas9(定期聚类)
间隔短回文重复序列/Cas9 核酸酶)技术。我们将表征 E1-KO/KI 和 E11-
KO/KI模型通过检查OPRM1剪接变体的表达,选择的镇痛作用
阿片类药物,以及内源性 E1 和 E11 启动子活性。这些突变大鼠模型将提供独特的
为研究界进一步探索药理功能和
大鼠 OPRM1 基因的转录调控。这些研究将使我们对
各种阿片类药物的复杂作用,并设计出传统的、有效的阿片类镇痛药
阿片类药物的副作用和滥用的可能性。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('YING-XIAN PAN', 18)}}的其他基金
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Alternative pre-mRNA splicing of mu opioid receptor gene and mu opioid actions
mu阿片受体基因的选择性前mRNA剪接和mu阿片作用
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10166814 - 财政年份:2020
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Alternative pre-mRNA splicing of mu opioid receptor gene and mu opioid actions
mu阿片受体基因的选择性前mRNA剪接和mu阿片作用
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Mapping mu agonist-induced receptor-protein interactions for OPRM1 7TM variants
绘制 OPRM1 7TM 变体 mu 激动剂诱导的受体-蛋白质相互作用图谱
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$ 25.71万 - 项目类别:
Alternative pre-mRNA splicing of mu opioid receptor gene and mu opioid actions
mu阿片受体基因的选择性前mRNA剪接和mu阿片作用
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Alternative pre-mRNA splicing of mu opioid receptor gene and mu opioid actions
mu阿片受体基因的选择性前mRNA剪接和mu阿片作用
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