Novel transgenic reporter/deleter allele for Type I taste cells

I 型味觉细胞的新型转基因报告基因/删除等位基因

• 批准号: 8302602
• 负责人: Sue C. Kinnamon
• 金额: $ 18.81万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-03 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302602
• 关键词: Ablation; Acids; Alkaloids; Alleles; Amiloride; Amino Acids; Apical; Basal Cell; Cell Aging; Cell membrane; Cell physiology; Cells; Contracts; Data; Development; Eating; Elements; Enzymes; Epithelium; Esthesia; Event; Excision; France; Fusiform Cell; G-Protein-Coupled Receptors; Gene Targeting; Hormonal; Immune Sera; Internal Ribosome Entry Site; Kinetics; Knockout Mice; Label; Lead; Mediating; Membrane; Methodology; Methods; Molecular; Mus; NTPDase2; Nerve; Nerve Fibers; Nervous system structure; Neuroglia; Norepinephrine; Nuclear; Nutritional; Obesity; Oral cavity; Pattern; Physiology; Pilot Projects; Population; Process; Proteins; Purinoceptor; Reagent; Receptor Cell; Receptor Signaling; Reporter; Rest; Role; Salts; Sequence Deletion; Serotonin; Site; Staining method; Stains; Stimulus; Sum; Synapses; System; Taste Buds; Taste Perception; Technology; Testing; Therapeutic Agents; Therapeutic Intervention; Tongue; Toxin; Transgenic Organisms; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; Type III Epithelial Receptor Cell; Whole-Cell Recordings; afferent nerve; basolateral membrane; cell age; cell type; chorda tympani; diphtheria toxin receptor; dosage; ectoATPase; genetic manipulation; immunocytochemistry; killings; novel; patch clamp; presynaptic; promoter; rat Gnat3 protein; receptor; recombinase; research study; response; salt sensitive; sugar; tool; uptake; vector; voltage

DESCRIPTION (provided by applicant): Taste buds are the transducing elements of gustatory sensation. As such, they represent the first step in the process by which nutritious substances, such as Na+ salts, sugars, and amino acids can be detected and distinguished from harmful substances, such as acids and toxic bitter compounds. Understanding the steps in this process may lead to therapeutic interventions that can be used to modulate food intake, a critical factor in controlling obesity. Each taste bud comprises three types of elongate taste cells and a population of basal cells. Understanding the functional role of each cell type is fundamental to understanding how each type of stimulus is detected and how this information is transmitted to the nervous system. Of the three types of fusiform cells, the Type II cells, which express the receptors and signaling effectors for bitter, sweet, and umami transduction, are the best understood. They release ATP to activate purinergic receptors on afferent nerve fibers. Type III, or "presynaptic" cells, detect sour stimuli and release serotonin and noradrenalin. Type I, or "glial-like" cells are the most abundant cells in the taste bud but also the least understood. Similar to glial cells in the nervous system, their membranes closely envelope other taste cells and they express NTPDase2, an ectoATPase that degrades ATP that is released from Type II cells. Recent data suggest that Type I cells may have additional functions, including transduction of Na+ salts and modulation of Type II cells. Unlike Type II and Type III cells, there are no fluorescent reporters for identification of Type I taste cells, making identification and functional characterization difficult. The proposed studies will utilize existing reagents and technology to develop a gene-targeted mouse that will express a nuclear-targeted fluorescent reporter (GFP) and Cre recombinase from the NTPDase2 promoter. Nuclear localization of GFP will insure that Type I cells can be distinguished from the other cell types, both within the bud and after isolation. Moreover, Cre recombinase will allow selective deletion of sequence from Type I taste cells, when these mice are crossed with mice carrying an appropriate "floxed" allele. In the first aim, we will validate the expression of the targeted allele, by crossing the mce to a commercially available Cre reporter line, Rosa26-tdTomato, which will express a red fluorescent reporter upon Cre-mediated excision of sequence flanked by loxp sites upstream of the reporter. In the second aim, we will utilize the mice to test the hypothesis that Type I cells are necessary for amiloride-sensitive salt taste. Whole cell recording will test whether Type I cells express functional amiloride- sensitive Na+ currents. Further, we will develop the methodology to specifically ablate type I taste cells, using the diphtheria toxin receptor targeted to Type I taste cells. In summary, these experiments will allow us define the Type I cell population and provide a new tool to dissect the functions of this most common taste cell type. PUBLIC HEALTH RELEVANCE: The function of the gustatory system is to detect and discriminate nutritious substances, such as salts, sugars, and amino acids from harmful substances, such as bitter alkaloids and acids. Understanding the molecular events involved in this process will lead to the development of therapeutic agents that can modulate food intake, a major factor in controlling obesity. The proposed studies will develop new tools to uncover the functional significance of the Type I taste cell, the most abundant but least understood cell type in the taste bud.

描述（申请人提供）：味蕾是味觉的传导元件。因此，它们代表了该过程中的第一步，通过该步骤，可以检测营养物质，如Na+盐，糖和氨基酸，并将其与有害物质，如酸和有毒苦味化合物区分开来。了解这个过程中的步骤可能会导致治疗干预，可用于调节食物摄入量，这是控制肥胖的关键因素。每个味蕾包括三种类型的细长味细胞和一群基底细胞。了解每种细胞类型的功能作用对于了解每种类型的刺激是如何被检测到的以及这些信息是如何传递到神经系统的至关重要。在三种类型的梭形细胞中，II型细胞表达苦、甜和鲜味转导的受体和信号效应器，是最好理解的。它们释放ATP以激活传入神经纤维上的嘌呤能受体。第三型，或称“突触前”细胞，检测酸刺激并释放血清素和去甲肾上腺素。I型，或“胶质样”细胞是味蕾中最丰富的细胞，但也是最不了解的细胞。类似于神经系统中的神经胶质细胞，它们的膜紧密地包裹其他味觉细胞，并且它们表达NTPDase 2，这是一种降解从II型细胞释放的ATP的胞外ATP酶。最近的数据表明，I型细胞可能具有额外的功能，包括Na+盐的转导和II型细胞的调节。与II型和III型细胞不同， 没有用于识别I型味细胞的荧光报告基因，使得识别和功能表征变得困难。拟议的研究将利用现有的试剂和技术开发一种基因靶向小鼠，该小鼠将表达核靶向荧光报告基因（GFP）和来自NTPDase 2启动子的Cre重组酶。GFP的核定位将确保I型细胞可以在芽内和分离后与其他细胞类型区分开。此外，当这些小鼠与携带适当的“floxed”等位基因的小鼠杂交时，Cre重组酶将允许从I型味觉细胞中选择性缺失序列。在第一个目标中，我们将通过将mce与市售Cre报告基因系Rosa 26-tdTomato杂交来验证靶向等位基因的表达，所述Cre报告基因系Rosa 26-tdTomato将在Cre介导的切除报告基因上游的loxp位点侧翼序列后表达红色荧光报告基因。在第二个目标中，我们将利用小鼠来检验I型细胞对于阿米洛利敏感的盐味是必需的这一假设。全细胞记录将测试I型细胞是否表达功能性阿米洛利敏感性Na+电流。此外，我们将开发专门消融I型味觉细胞的方法，使用靶向白喉毒素受体， I型味觉细胞总之，这些实验将使我们能够定义I型细胞群，并提供一种新的工具来剖析这种最常见的味觉细胞类型的功能。 公共卫生相关性：味觉系统的功能是检测和区分营养物质，如盐、糖和氨基酸，以及有害物质，如苦味生物碱和酸。了解这个过程中涉及的分子事件将导致治疗剂的发展，可以调节食物摄入量，控制肥胖的一个主要因素。拟议的研究将开发新的工具来揭示I型味觉细胞的功能意义，I型味觉细胞是味蕾中最丰富但最不了解的细胞类型。