Generation of knockin mice expressing KOPR conjugated with a fluorescent protein

描述（由申请人提供）：kappa阿片受体（KOPR）的激活产生许多作用，包括镇痛，烦躁/厌恶，镇静，水利尿，止痒作用和体温过低。选择性KOPR激动剂纳氟萘芬用于治疗肾透析患者的尿毒症性瘙痒。Kappa激动剂可作为止痛剂和水利尿剂。KOPR拮抗剂可作为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，重复激活KOPR（通常是表位标记）会导致应答降低以及KOPR内化和下调。然而，很难检查KOPR是否在体内经历类似的调节，如果是，受体调节是否与行为反应的变化相关。同样具有挑战性的是，在行为范式（如压力和慢性药物滥用）导致KOPR介导的烦躁、抑郁和焦虑样反应的大脑区域中，KOPR的表达和/或体内贩运是否会发生改变，例如在压力暴露或长期滥用药物后。困难在于缺乏合适的KOPR抗体。来自不同实验室的KOPR抗体产生了不同的结果。在这里，我们提出产生一种表达KOPR与荧光蛋白融合的小鼠系，以绕过对特异性抗体的需要。Kieffer及其同事已经产生了一种表达与增强绿色荧光蛋白（DOPR- egfp）融合的δ阿片受体的敲入小鼠系，并被证明可用于将体内DOPR转运与DOPR介导的行为反应变化联系起来。我们构建了两个小鼠KOPR (mKOPR) cDNA构建物，mKOPR- egfp和mKOPR- tdtomato （mKOPR- tdt）。我们的初步结果表明，当KOPR在神经2a （N2A）小鼠神经母细胞瘤细胞中表达时，与eGFP或tdT结合并没有改变KOPR的特性。具体目的如下。(1)充分研究与eGFP或tdT融合是否影响KOPR的体外表达、配体结合、信号传导和激动剂诱导的调控。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。敲除基因小鼠的产生将与Brigitte Kieffer博士（法国伊尔基希的g<s:1> <s:1> <s:1>和生物研究所molsamuaire和Cellulaire）的团队合作完成。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，特别是产生DOPR-eGFP敲入小鼠。目标定位策略将与基弗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转运与KOPR介导的行为变化联系起来，而体内KOPR内化可能被用作KOPR激活的指标。这样的敲入小鼠系将为研究KOPR在药理学和病理生理条件下的转运提供宝贵的资源。

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.