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

• 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
• 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

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（由申请方提供）：κ阿片受体（KOPR）的激活产生许多作用，包括镇痛、烦躁/厌恶、镇静、利尿、抗过敏作用和体温过低。选择性KOPR激动剂纳呋拉芬用于治疗肾透析患者的尿毒症性肾炎。κ激动剂可用作镇痛剂和利尿剂。KOPR拮抗剂可能用作抗抑郁药和抗焦虑药物，并可缓解成瘾者的药物渴望。体外研究表明，KOPR（通常是表位标记的）的重复激活导致反应降低以及KOPR内化和下调。然而，它一直难以检查，如果KOPR在体内经历类似的调节，如果是这样，受体调节是否与行为反应的变化相关。研究KOPR表达和/或体内运输是否在导致KOPR介导的烦躁、抑郁和焦虑样反应的行为范式（例如应激和慢性药物滥用）中的脑区域中改变也是具有挑战性的，例如在应激暴露或慢性使用滥用药物之后。困难是由于缺乏适当的KOPR抗体。来自不同实验室的KOPR抗体产生不同的结果。在这里，我们建议产生一个小鼠系表达KOPR融合荧光蛋白，以规避需要特异性抗体。Kieffer及其同事已经产生了表达与增强型绿色荧光蛋白融合的δ阿片受体（DOPR-eGFP）的敲入小鼠系，并证明其可用于将体内DOPR运输与DOPR介导的行为反应变化联系起来。我们已经产生了两种小鼠KOPR（mKOPR）cDNA构建体，mKOPR-eGFP和mKOPR-tdTomato（mKOPR-tdT）。 我们的初步结果表明，与eGFP或tdT的缀合在Neuro 2A（N2 A）小鼠神经母细胞瘤细胞中表达时不改变KOPR的性质。具体目标如下。(1)充分研究与eGFP或tdT的融合是否影响体外KOPR的表达、配体结合、信号传导和激动剂诱导的调节。 FLAG-mKOPR将用于比较。(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。将与Brigitte Kieffer博士（Institut de G <$n <$tique et de Biologie Mol <$culaire et Cellulaire，Illkirch，France）的团队合作生成敲入小鼠。我们选择与她合作，是因为她在产生突变小鼠方面的成功记录，特别是DOPR-eGFP敲入小鼠的产生。靶向策略将与Kieffer及其同事使用的策略相似。敲入小鼠的初步表征包括在脑和脊髓中的分布以及KOPR融合蛋白在脑中的配体结合和信号传导。这些动物将非常有助于将KOPR运输与KOPR介导的行为变化相关联，并且体内KOPR内化可用作KOPR活化的指标。这样的敲入小鼠系将是一个宝贵的资源，研究人员有兴趣在药理学和病理生理条件下KOPR贩运。