Generation of knockin mice expressing KOPR conjugated with a fluorescent protein

描述(申请人提供)：激活kappa阿片受体(KOPR)产生许多作用，包括止痛、烦躁/厌恶、镇静、利水、止痒和降温。选择性KOPR激动剂那夫拉芬用于治疗肾透析患者尿毒症瘙痒炎。Kappa激动剂可能用作止痛剂和利水剂。KOPR拮抗剂可能用作抗抑郁和抗焦虑药物，并在缓解吸毒者的药物渴望方面有用。体外研究表明，重复激活KOPR(通常是表位标记的)会导致应答减少以及KOPR内化和下调。然而，很难研究KOPR在体内是否经历了类似的调节，如果是的话，受体调节是否与行为反应的变化相关。研究KOPR在体内的表达和/或转运是否在行为范式(如应激和慢性药物滥用)的脑区发生变化，从而导致KOPR介导的焦虑症、抑郁和焦虑样反应，如在压力暴露或长期使用药物滥用后，也是具有挑战性的。困难是由于缺乏适当的KOPR抗体。不同实验室的KOPR抗体产生了不同的结果。在这里，我们建议建立一个表达KOPR与荧光蛋白融合的小鼠系，以避开对特定抗体的需要。Kieffer和他的同事已经产生了一个表达增量阿片受体与增强的绿色荧光蛋白(DOPR-EGFP)的敲击小鼠系，并被证明有助于将体内DOPR贩运与DOPR介导的行为反应的变化联系起来。我们已经构建了两个小鼠KOPR(MKOPR)的cDNA体，mKOPR-EGFP和mKOPR-tdTomato(mKOPR-TdT)。我们的初步结果表明，当KOPR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)在体外研究融合EGFP或TDT是否影响KOPR的表达、配体结合、信号转导和激动剂诱导的调节。将使用FLAG-mKOPR进行比较。(2)根据我们的体外实验结果，建立表达KOPR-EGFP或KOPR-TDT的敲打小鼠系。敲门小鼠的产生将与Brigitte Kieffer博士(法国伊尔基奇的Génétique et de Biologie Moléculaire et Cellulaire)团队合作完成。我们选择与她合作，是因为她在培育突变小鼠方面的成功记录，特别是DOPR-EGFP敲门小鼠的一代。目标策略将类似于Kieffer和他的同事使用的策略。敲门小鼠的初步特征包括大脑和脊髓的分布以及大脑中KOPR融合蛋白的配体结合和信号传递。这些动物将非常有用地关联KOPR运输与KOPR介导的行为的变化，在体内KOPR的内化可能被用作KOPR激活的指标。对于在药理学和病理生理学条件下对KOPR贩运感兴趣的研究人员来说，这种敲击小鼠系将是一个有价值的资源。

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.