Trancellular Labeling from Defined Taste Receptor Cells

描述（由申请人提供）：味蕾由许多伸长的修饰的上皮细胞组成，其中一些用作味觉的转导元件。不同的味觉细胞参与不同味觉品质的转导。R21提案中的实验旨在产生和测试转基因小鼠，其中在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芽凝集素（WGA）。WGA应该穿过突触进入味觉神经纤维，从而标记孤束核内的神经节细胞和可能的终末。如果有足够的WGA被运输，示踪剂也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编码区的侧翼是Iox位点，随后是WGA编码序列。当被cre重组酶作用时，BDNF编码区被切除，而产生WGA。我们将使用各种现有的转基因株系在颅神经节和脑干以及味蕾中的相关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仅在他莫昔芬的影响下在角蛋白14表达细胞中起作用，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，他莫昔芬处理将激活cre重组酶，从而导致由产生BDNF的味觉细胞产生WGA。正常表达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地标记参与“酸”转导的味觉细胞和神经节细胞。这些小鼠还将提供关于蛋白质处理、胞吐和味蕾内各种细胞类型摄取的动力学的信息。

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.