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: 8418728
• 负责人: Sue C. Kinnamon
• 金额: $ 21.05万
• 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 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8418728
• 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.

描述(申请人提供)：味蕾是味觉的传递元素。因此，它们是检测营养物质(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)区分开来的第一步。了解这一过程中的步骤可能会导致可用于调节食物摄入量的治疗干预措施，这是控制肥胖的关键因素。每个味蕾由三种类型的细长味觉细胞和一群基细胞组成。了解每种细胞类型的功能作用是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基础。在三种类型的梭形细胞中，表达苦味、甜味和鲜味信号转导的受体和信号效应器的II型细胞是最了解的。它们释放三磷酸腺苷来激活传入神经纤维上的嘌呤能受体。III型，或“突触前”细胞，探测酸刺激并释放5-羟色胺和去甲肾上腺素。I型，或“神经胶质样”细胞是味蕾中最丰富的细胞，但也是最不被了解的细胞。与神经系统中的神经胶质细胞类似，它们的膜紧密包裹着其他味觉细胞，并表达NTPDase2，这是一种降解从II型细胞释放的ATP的胞外ATPase。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型细胞的调制。与II型和III型细胞不同的是， 目前尚无鉴定I型味觉细胞的荧光记者，这给鉴定和功能鉴定带来了困难。拟议的研究将利用现有的试剂和技术来开发一种基因靶向的小鼠，它将表达来自NTPDase2启动子的核靶向荧光报告(GFP)和Cre重组酶。绿色荧光蛋白的核定位将确保I型细胞能够与其他类型的细胞区分开来，无论是在芽中还是在分离后。此外，Cre重组酶将允许选择性地删除I型味觉细胞的序列，当这些小鼠与携带适当的“FLOXED”等位基因的小鼠杂交时。在第一个目的中，我们将通过将mce与商业上可用的Cre报告系rosa26-tdTomato杂交来验证目标等位基因的表达，该报告系将在Cre介导的剪除报告基因上游loxP位点的序列后表达红色荧光报告基因。在第二个目标中，我们将利用小鼠来测试I型细胞是阿米洛利敏感盐味所必需的假设。全细胞记录将测试I型细胞是否表达功能性的阿米洛利敏感的Na+电流。此外，我们将开发使用白喉毒素受体靶向特异性消融I型味觉细胞的方法。 到类型I品尝细胞。总而言之，这些实验将使我们能够定义I型细胞群体，并提供一个新的工具来剖析这种最常见的味觉细胞类型的功能。