Transcellular Labeling from Defined Taste Receptor Cells

描述（由申请人提供）：味蕾由许多细长修饰的上皮细胞组成，其中一些细胞作为味觉的转导元件。不同的味觉细胞参与不同味觉品质的转导。本R21提案的实验旨在产生和测试一种转基因小鼠，在cree -recombinase的影响下，突触连接的味觉细胞会产生跨神经元示踪剂小麦胚芽凝集素（WGA）。WGA应穿过突触进入味觉神经纤维，从而标记神经节细胞，并可能标记孤束核内的终端。如果足够的WGA被运输，示踪剂也可以标记孤束核的突触后细胞。用于生成小鼠的结构是敲入，其中BDNF编码区两侧是Iox位点，随后是WGA编码序列。当cree -recombinase作用于BDNF编码区时，BDNF编码区被切除并产生WGA。我们将利用现有的各种转基因系，在颅神经节和脑干以及味蕾中相关的bdnf表达群体中表达crere组合酶。对于味蕾的选择性重组，我们将利用现有的K14tamcre系，其中creerecombinase在他莫昔芬的影响下只能在表达角蛋白14的细胞中发挥作用，从而为我们提供了良好的时空控制。由于味蕾基底细胞表达角蛋白14，他莫昔芬治疗将激活crer -recombinase，从而导致味觉细胞产生WGA，而味觉细胞本应产生BDNF。通常表达BDNF的味觉细胞群是突触连接的细胞，包括那些特别涉及“酸”（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”转导的味觉细胞和神经节细胞。这些小鼠还将提供有关味蕾内各种细胞类型的蛋白质处理、胞吐和摄取动力学的信息。

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.