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：
8623020
负责人：
LEE-YUAN LIU-CHEN
金额：
$ 7.75万
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 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8623020
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 Behavioral 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 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 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 Génétique et de Biologie Moléculaire 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（由应用提供）：Kappa阿片受体（KOPR）的激活产生许多影响，包括镇痛，烦躁不安 /厌恶，镇静，水利尿，抗尿素效应和体温过低。选择性Kopr激动剂Nalfurafine用于治疗肾脏透析患者的尿毒性瘙痒性炎。卡帕激动剂可能可作为镇痛药和水利尿剂。 KOPR拮抗剂可能可作为抗抑郁药和抗焦虑药，并减轻吸毒者中的渴望。体外研究表明，重复激活KOPR（通常是表位标记）导致反应减少以及Kopr内在化和下调。但是，很难检查KOPR是否在体内受到类似的调节，如果是的，则受体调节是否与行为反应的变化相关。还要调查在行为范例（例如压力和慢性药物滥用）的大脑区域的KOPR表达和/或流量在体内是否改变，这会导致Kopr介导的烦躁不安，抑郁症和动画样反应，例如在压力暴露或慢性使用滥用药物后的压力暴露或慢性使用。困难是由于缺乏适当的KOPR抗体。来自不同实验室的KOPR抗体产生了不同的结果。在这里，我们建议生成一条与荧光蛋白融合的Kopr的小鼠系，以规避对特定抗体的需求。 Kieffer及其同事生成了与增强的绿色荧光蛋白（DOPR-EGFP）融合的Delta Ooid受体的敲击小鼠系，被证明可用于将体内DOPR运输与DOPR介导的行为反应的变化相关。我们已经生成了两个小鼠KOPR（MKOPR）cDNA构建体Mkopr-EGFP和Mkopr-Tdtomato（Mkopr-TDT）。我们的初步结果表明，在Neuro2a（N2A）小鼠神经母细胞瘤细胞中表达时，与EGFP或TDT的结合不会改变KOPR的性质。具体目标如下。（1）完全研究与EGFP或TDT融合是否会影响表达，配体结合，信号传导和激动剂诱导的KOPR体外调节。 Flag-Mkopr将用于比较。（2）根据我们的体外结果产生表达KOPR-EGFP或KOPR-TDT的敲击小鼠系。敲蛋白小鼠的产生将与Brigitte Kieffer博士（InstitutdeGénétiqueet de BiologieMoléculaireet Cellulaire，France，Illkirch，Illkirch）合作完成。我们之所以选择与她合作，是因为她在产生突变小鼠方面的成功记录，尤其是DOPR-EGFP敲蛋白小鼠的产生。定位策略将与Kieffer及其同事使用的策略相似。敲击蛋白小鼠的初始表征包括大脑中的分布，脊髓和大脑中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Kopr介导的行为的变化相关联和体内KOPR内在化将非常有用，可以用作KOPR激活的指标。对于在药物和病理生理条件下对Kopr贩运感兴趣的研究人员来说，这种敲击小鼠系将是一种宝贵的资源。