High throughput in vivo functional analysis of human odorant receptors.

描述（由申请人提供）：人类嗅觉系统被认为可以检测到数千种气味，从而允许适当的食物消费和厌恶。人类识别气味的能力依赖于排列在鼻上皮上的嗅觉神经元内的气味受体。嗅觉受体（ORs）在成熟的嗅觉神经元中无性表达（每个神经元有一个OR等位基因）。因此，在可识别的单个神经元中研究气味反应相当于研究单个ORs。人类嗅觉基因组由约900个OR基因组成；这些基因属于七个跨膜受体超家族。然而，只有约40%的编码是全长开放阅读帧（ORF）。因此，在主嗅觉系统中，大约有355个OR基因可用于气味识别。这个OR亚群分为三个OR类：第一类：约50个基因；II类：约300个基因；TAARs：约5个基因。随着SNP分析的出现，具有全长orf的人类变异orf的数量不断增加。整个I类和II类OR基因已经被发现了十年，但它们在体内的功能仍然难以捉摸。为了了解所有约355个OR基因及其变体的功能，严重缺乏三件事：1)了解哪些OR在人类嗅觉神经元中表达；2)在体内环境下orf可以与嗅觉G-蛋白（Golf）偶联的证据；3)在体内环境下orf可以允许肾小球形成的证据。一些人类OR orf不能驱动肾小球形成或与Golf配对，将这些基因归类为“假基因”。因此，具有全长ORF的ORs并不构成使用体外技术研究其气味结合特性的充分有效理由。需要有一种方法来快速识别哪些人类OR orf可以在体内发挥作用，同时利用这种体内系统来识别气味-OR活性相关物。在目标1 A部分，将开发一种高通量基因靶向策略，使人类OR ORF可以快速“交换”代替小鼠OR ORF，并在体内测试其功能。初步实验将确定该方法在两个小鼠OR位点的有效性。这个子目标的目的是产生一个胚胎干细胞库，每个干细胞都包含一个独特的人类OR ORF。最初，将测试一种人类OR的几种同种异构体。在目标1 B部分中，将开发一种高通量转基因方法，使小鼠嗅上皮中70%的嗅觉神经元（数百万）克隆表达给定的人类OR。同样，一个人类OR的几个同种异构体将被测试。该项目旨在补充基因靶向策略，并为可能的机器人气味识别方法提供大量神经元。简而言之，这项提议的唯一目的是推进我们对哪些人类受体对人类气味感知起作用的理解。

DESCRIPTION (provided by applicant): The human olfactory system is thought to detect thousands of odorants, allowing for proper food consumption and aversions. The ability to identify odors by humans is dependent on odorant receptors within the olfactory neurons that line the nasal epithelium. Odorant receptors (ORs) are clonally expressed in mature olfactory neurons (one OR allele per neuron). Thus studying odorant responses in identifiable individual neurons is equivalent to studying individual ORs. The human olfactory genome consists of ~900 OR genes; these genes belong to the seven-transmembrane receptor superfamily. However, only ~40% encode a full-length open reading frame (ORF). Thus, ~355 OR genes are available for odor identification in the main olfactory system. This subgroup of ORs breaks down into three OR Classes: Class I: ~50 genes; Class II: ~300 genes; and the TAARs: ~5 genes. With the emergence of SNP analysis, the number of variant, human ORs with full-length ORFs continues to grow. The entire set of Class I and II OR genes have been known for a decade, yet their ability to function in vivo has remained elusive. Three things are severely lacking in order to understand the functionality of all ~355 OR genes plus their variants: 1st) knowledge of which ORs are expressed in human olfactory neurons 2nd) evidence that the ORFs can couple to the olfactory G- protein (Golf) in an in vivo setting and 3rd) evidence that the ORFs can allow for glomerular formation in an in vivo setting. Failure for some human OR ORFs to drive glomerular formation or couple to Golf would classify these genes as "pseudogenes". Thus ORs having a full-length ORF does not constitute a valid enough reason for studying their odorant binding properties using in vitro technologies. There needs to be a method for quickly identifying which human OR ORFs can function in vivo and simultaneously make use of this in vivo system for the identification of odorant-OR activity correlates. In Aim 1 section A, a high-throughput gene-targeting strategy will be developed such that human OR ORFs can be rapidly "swapped" in place of a mouse OR ORF and be tested for functionality in vivo. Initial experiments will determine the efficacy of this approach at two mouse OR loci. The intent of this sub aim would be to generate a library of Embryonic Stem cells that each contain a unique human OR ORF. Initially, several isoforms for one human OR will be tested. In Aim 1 section B, a high-throughput transgenic approach will be developed whereby 70% of all olfactory neurons (millions) in the mouse olfactory epithelium will clonally express a given human OR. Again, several isoforms for one human OR will be tested. This project is aimed at complementing the gene- targeting strategy and providing a large number of neurons for possible robotic approaches in odorant identification. In short, the sole aim of this proposal is to move forward our understanding of which human receptors are functional for human perception of odors. PUBLIC HEALTH RELEVANCE: The ability for humans to detect odors (odorants) remains an important aspect of human health and welfare. The sense of smell is critically dependent on hundreds of different human odorant receptors normally expressed in olfactory neurons. However, very few odorant/odorant receptor matches have been made. This lack of knowledge is a sole reflection of the inability to express and characterize human odorant receptor proteins in non-olfactory cells. Thus, the functional analysis of human odorant receptors expressed within olfactory neurons should significantly help bridge this divide.