Generation of knockin mice expressing KOPR conjugated with a fluorescent protein

表达与荧光蛋白缀合的 KOPR 的敲入小鼠的产生

基本信息

批准号：
8836513
负责人：
LEE-YUAN LIU-CHEN
金额：
$ 7.67万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-15 至 2016-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8836513
关键词：
Abbreviations Absence of pain sensation Address Adenylate Cyclase Affect Affinity Agonist Agreement Analgesics Animal Model Animals Anti-Anxiety Agents Antibodies Antidepressive Agents Antipruritic Effect Antipruritics Anxiety Area Behavior Binding Brain Brain region Cells Charge Chimeric Proteins Chronic Chronic stress Clinical Collaborations Complementary DNA Dialysis patients Diuresis Diuretics Down-Regulation Drug Addiction Drug abuse Drug usage Dynorphins Epitopes Family Forskolin France Generations Guanosine Triphosphate Health In Vitro Institutes Japan Ligand Binding Ligands MAP Kinase Gene MAPK14 gene Mediating Mental Depression Mus Mutant Strains Mice Neuroblastoma Opioid Opioid Receptor Peptides Pharmacology Phosphorylation Property Proteins Pruritus Receptor Activation Receptor Signaling Regulation Renal dialysis Research Personnel Resources Rhodopsin Scientist Sedation procedure Signal Transduction Source Specificity Spinal Cord Stress Water acute stress behavioral response behavioral study cost delta opioid receptor desensitization drug craving drug of abuse dysphoria enhanced green fluorescent protein experience in vivo interest natural hypothermia neuroblastoma cell painful neuropathy receptor receptor expression receptor internalization response success trafficking

项目摘要

DESCRIPTION (provided by applicant): Activation of the kappa opioid receptor (KOPR) produces many effects including analgesia, dysphoria / aversion, sedation, water diuresis, antipruritic effects and hypothermia. The selective KOPR agonist nalfurafine is used for treatment of uremic pruritis in kidney dialysis patients. Kappa agonists may be useful as analgesics and water diuretics. KOPR antagonists may be useful as antidepressants and anti-anxiety drugs and in alleviating drug craving in addicts. In vitro studies showed that repeated activation of the KOPR (usually epitope-tagged) resulted in reduced responses as well as KOPR internalization and downregulation. However, it has been difficult to examine if the KOPR undergoes similar regulation in vivo and, if so, whether receptor regulation correlates with changes in behavioral responses. It is also challenging to investigate whether KOPR expression and/or trafficking in vivo are altered in brain regions in behavior paradigms (such as stress and chronic drug abuse) that result in KOPR-mediated dysphoria-, depression- and anxiety-like responses, such as after stress exposure or chronic use of drugs of abuse. The difficulty is due to lack of appropriate KOPR antibodies. KOPR antibodies from different labs yielded different results. Here we propose to generate a mouse line expressing the KOPR fused with a fluorescent protein to circumvent the need for specific antibodies. A knockin mouse line expressing the delta opioid receptor fused with the enhanced green fluorescent protein (DOPR-eGFP) has been generated by Kieffer and colleagues and proven to be useful for relating in vivo DOPR trafficking with changes in DOPR-mediated behavior responses. We have generated two mouse KOPR (mKOPR) cDNA constructs, mKOPR-eGFP and mKOPR-tdTomato (mKOPR-tdT). Our preliminary results showed that conjugation with eGFP or tdT did not change the properties of the KOPR when expressed in Neuro2A (N2A) mouse neuroblastoma cells. The specific aims are as follows. (1) Investigate fully if fusion with eGFP or tdT affects expression, ligand binding, signaling and agonist-induced regulation of KOPR in vitro. FLAG-mKOPR will be used for comparison. (2) Generate a knockin mouse line expressing KOPR-eGFP or KOPR-tdT, depending on our in vitro results. Generation of knockin mice will be done in collaboration with the team of Dr. Brigitte Kieffer (Institut de G¿n¿tique et de Biologie Mol¿culaire et Cellulaire, Illkirch, France). We choose to collaborate with her because of her track record of success in generating mutant mice, in particular the generation of DOPR-eGFP knockin mice. The targeting strategy will be similar to that used by Kieffer and colleagues. Initial characterization of the knockin mice includes distribution in the brain and spinal cord and ligand binding and signaling of KOPR fusion protein in the brain. The animals will be very useful for correlating KOPR trafficking with changes in KOPR-mediated behaviors and in vivo KOPR internalization may be used as an indicator of KOPR activation. Such a knockin mouse line will be a valuable resource for researchers interested in KOPR trafficking under pharmacological and pathophysiological conditions.

描述（由申请人提供）：κ阿片受体（KOPR）的激活产生多种作用，包括镇痛、烦躁/厌恶、镇静、利尿、止痒作用和体温降低选择性KOPR激动剂纳芙拉芬用于治疗肾脏尿毒症瘙痒症。 Kappa 激动剂可用作镇痛药和利尿剂。拮抗剂可用作抗抑郁药和抗焦虑药，并可缓解成瘾者的药物渴望。体外研究表明，KOPR（通常带有表位标记）的重复激活会导致反应减少以及 KOPR 内化和下调。很难检查 KOPR 是否在体内行为上经历类似的调节，如果是的话，受体调节是否与反应的变化相关。研究 KOPR 体内表达和/或运输是否与反应变化相关也具有挑战性。行为范式（例如压力和长期药物滥用）的大脑区域发生改变，导致 KOPR 介导的烦躁、抑郁和焦虑样反应，例如在压力暴露或长期使用滥用药物后。由于缺乏合适的 KOPR 抗体，来自不同实验室的 KOPR 抗体产生了不同的结果，在此我们建议生成表达与荧光蛋白融合的 KOPR 的小鼠品系，以避免对表达 δ 阿片受体融合的敲入小鼠品系的需要。 Kieffer 及其同事生成了增强型绿色荧光蛋白 (DOPR-eGFP)，并证明可用于将体内 DOPR 运输与 DOPR 介导的行为反应的变化联系起来。我们生成了两种小鼠 KOPR (mKOPR) cDNA 构建体， mKOPR-eGFP 和 mKOPR-tdTomato (mKOPR-tdT) 我们的初步结果表明，在 KOPR 中表达时，与 eGFP 或 tdT 缀合不会改变 KOPR 的特性。 Neuro2A (N2A) 小鼠神经母细胞瘤细胞的具体目的如下： (1) 充分研究与 eGFP 或 tdT 的融合是否会影响 KOPR 的体外表达、配体结合、信号传导和激动剂诱导的调节。 (2) 根据我们的体外结果，生成表达 KOPR-eGFP 或 KOPR-tdT 的敲入小鼠系。 Brigitte Kieffer 博士团队（Institut de G¿不分子生物学与分子生物学culaire et Cellulaire，Illkirch，法国），因为她在生成突变小鼠方面取得了成功，特别是 DOPR-eGFP 敲入小鼠的生成，因此我们选择与她合作。敲入小鼠的初步特征包括大脑和脊髓中的分布以及大脑中 KOPR 融合蛋白的配体结合和信号传导，这些动物对于将 KOPR 运输与变化联系起来非常有用。 KOPR 介导的行为和体内 KOPR 内化可用作 KOPR 激活的指标。对于对药理学和病理生理条件下 KOPR 运输感兴趣的研究人员来说，这种敲入小鼠系将是宝贵的资源。