Transcellular Labeling from Defined Taste Receptor Cells

来自特定味觉感受器细胞的跨细胞标记

• 批准号: 6811227
• 负责人: Thomas E Finger
• 金额: $ 15.16万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6811227
• 关键词: agglutinins; biological models; biological signal transduction; biotechnology; brain derived neurotrophic factor; cell cell interaction; chemoreceptors; ganglion cell; gene expression; gene targeting; genetically modified animals; laboratory mouse; model design /development; neural transmission; neuronal transport; neurons; recombinase; tamoxifen; taste; taste buds

DESCRIPTION (provided by applicant): Taste buds consist of numerous elongate modified epithelial cells, some of which serve as the transducing elements for taste. Different taste cells are involved in transduction of different taste qualities. Experiments in this R21 proposal are designed to generate and test a transgenic mouse in which under the influence of cre-recombinase, synaptically-connected taste cells will produce the transneuronal tracer, Wheatgerm Agglutinin (WGA). The WGA should cross the synapse into the gustatory nerve fibers thereby labeling ganglion cells and perhaps terminals within the nucleus of the solitary tract. If sufficient WGA is transported, the tracer may also label postsynaptic cells in the nucleus of the solitary tract. The construct to be employed for generation of the mouse is a knock-in whereby the BDNF coding region is flanked by Iox sites and followed by the WGA coding sequence. When acted upon by cre-recombinase, the BDNF coding region is excised and WGA is produced instead. We will use various existing transgenic lines to express crerecombinase in relevant BDNF-expressing populations in cranial ganglia and the brainstem as well as in taste buds. For selective recombination in taste buds, we will utilize an existing K14tamcre line where crerecombinase can function only in keratin 14-expressing cells under the influence of tamoxifen, thus providing us with good spatial and temporal control. Since basal cells of taste buds express keratin 14, tamoxifen treatment will activate cre-recombinase thereby resulting in production of WGA by taste cells that would have produced BDNF. The population of taste cells that normally express BDNF are synaptically-connected cells that include those specifically implicated in "sour" (H'+)transduction. Thus the WGA-expressing transgenic mice that we propose will specifically mark taste cells and ganglion cells involved in "sour" transduction. These mice also will be informative about the kinetics of protein handling, exocytosis and uptake by various cell types within the taste bud.

描述（由申请人提供）：味蕾由许多细长的修饰上皮细胞组成，其中一些充当味觉转导元件。不同的味觉细胞参与不同味觉品质的转导。 R21 提案中的实验旨在生成并测试转基因小鼠，在 cre 重组酶的影响下，突触连接的味觉细胞将产生跨神经元示踪剂小麦胚凝集素 (WGA)。 WGA 应该穿过突触进入味觉神经纤维，从而标记神经节细胞，并且可能标记孤束核内的末端。如果运输了足够的 WGA，示踪剂还可以标记孤束核中的突触后细胞。用于产生小鼠的构建体是敲入，其中BDNF编码区侧翼是Iox位点，后面是WGA编码序列。当受到 cre 重组酶作用时，BDNF 编码区被切除并产生 WGA。我们将使用各种现有的转基因系在颅神经节和脑干以及味蕾的相关 BDNF 表达群体中表达 crerecombinase。对于味蕾中的选择性重组，我们将利用现有的 K14tamcre 系，其中 crerecombinase 只能在他莫昔芬的影响下在表达角蛋白 14 的细胞中发挥作用，从而为我们提供良好的空间和时间控制。由于味蕾的基底细胞表达角蛋白 14，他莫昔芬治疗将激活 cre 重组酶，从而导致本来会产生 BDNF 的味觉细胞产生 WGA。通常表达 BDNF 的味觉细胞群是突触连接的细胞，其中包括那些特别参与“酸味”(H'+) 转导的细胞。因此，我们提出的表达 WGA 的转基因小鼠将特异性标记参与“酸味”转导的味觉细胞和神经节细胞。这些小鼠还将获得有关味蕾内各种细胞类型的蛋白质处理、胞吐作用和摄取动力学的信息。