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鼻上皮细胞内嗅觉神经元的气味受体。气味受体（OR）在成熟的嗅觉神经元中克隆表达（每个神经元一个OR等位基因）。因此，研究可识别的个体神经元中的气味反应相当于研究个体OR。人类嗅觉基因组由约900个OR基因组成;这些基因属于七跨膜受体超家族。然而，只有~40%编码全长开放阅读框架（ORF）。因此，约355个OR基因可用于主要嗅觉系统中的气味识别。该OR亚组分为三个OR类别：I类：~50个基因; II类：~300个基因;以及TAAR：~5个基因。随着SNP分析的出现，具有全长ORF的变体、人类OR的数量持续增长。整个I类和II类OR基因的集合已经知道了十年，但它们在体内发挥功能的能力仍然难以捉摸。为了理解所有~355个OR基因及其变体的功能，严重缺乏三件事：1）哪些OR在人类嗅觉神经元中表达的知识; 2）ORF可以在体内环境中与嗅觉G蛋白（Golf）偶联的证据;以及3）ORF可以在体内环境中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高尔夫偶联，将这些基因归类为“假基因”。因此，具有全长ORF的OR不构成使用体外技术研究其气味剂结合性质的有效的足够理由。需要一种方法来快速鉴定哪些人OR ORF可以在体内起作用，同时利用该体内系统来鉴定气味剂-OR活性相关物。在目标1部分A中，将开发高通量基因靶向策略，使得人OR ORF可以快速“交换”代替小鼠OR ORF，并在体内测试功能性。最初的实验将确定这种方法在两个小鼠OR基因座的功效。这个子目标的目的是产生一个胚胎干细胞库，每个细胞都含有一个独特的人类OR ORF。最初，将检测一个人OR的几种亚型。在目标1部分B中，将开发高通量转基因方法，由此小鼠嗅上皮中所有嗅觉神经元（数百万）的70%将克隆表达给定的人OR。同样，将检测一个人OR的几种亚型。该项目旨在补充基因靶向策略，并为气味识别中可能的机器人方法提供大量神经元。简而言之，这项提案的唯一目的是推进我们对人类感受器对人类感知气味的功能的理解。 公共卫生相关性：人类检测气味（气味剂）的能力仍然是人类健康和福利的重要方面。嗅觉严重依赖于嗅觉神经元中通常表达的数百种不同的人类气味受体。然而，很少有气味剂/气味剂受体匹配。这种知识的缺乏是在非嗅觉细胞中无法表达和表征人类气味受体蛋白的唯一反映。因此，嗅觉神经元内表达的人类气味受体的功能分析应显着帮助弥合这一鸿沟。

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.