Zhuang Hanyi, Matsunami Hiroaki
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nat Protoc. 2008;3(9):1402-13. doi: 10.1038/nprot.2008.120.
A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.
嗅觉领域的一个基本问题是，给定的一种气味剂能激活哪些气味受体（OR）。在哺乳动物中，研究气味剂与OR关系的一个主要障碍是无法在适合筛选OR活性配体的异源细胞中表达OR。受体转运蛋白家族的发现促进了OR在异源细胞中的有效细胞表面表达。建立一个强大的哺乳动物OR异源表达系统，通过将这些受体与其同源配体匹配，有助于对它们进行高通量的“去孤儿化”。本方案详细介绍了用于评估瞬时表达的哺乳动物OR在HEK293T细胞中的细胞表面表达并测量其功能激活的方法。OR细胞表面表达的阶段包括细胞培养准备、细胞转移、转染、免疫细胞化学或流式细胞术、气味剂刺激和荧光素酶测定。从细胞转移到细胞表面表达检测和/或功能激活测量，本方案可在3天内完成。
