High throughput in vivo functional analysis of human odorant receptors.

描述(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。人类辨别气味的能力依赖于鼻腔上皮内嗅觉神经元内的气味感受器。气味受体(ORs)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，研究可识别的单个神经元的气味反应等同于研究单个OR。人类嗅觉基因组由900个OR基因组成，这些基因属于七跨膜受体超家族。然而，只有约40%的人编码全长开放阅读框架(ORF)。因此，~355个OR基因可用于主嗅觉系统的气味识别。ORs的这个亚组分为三个OR类：I类：约50个基因；II类：约300个基因；以及TAARs：~5个基因。随着SNP分析的出现，具有全长ORF的变异型人类ORs的数量继续增长。一整套I类和II类OR基因已经知道了十年，但它们在体内发挥作用的能力仍然难以捉摸。要了解所有~355 OR基因及其变体的功能，严重缺乏三点：1)了解哪些ORs在人类嗅神经元中表达；2)有证据表明ORF可以在活体环境中与嗅觉G蛋白(Golf)偶联；3)有证据表明ORF可以在体内环境中允许肾小球形成。如果一些人类或ORF不能驱动肾小球的形成或与高尔夫偶联，则会将这些基因归类为“假基因”。因此，具有全长ORF的ORS不构成使用体外技术研究其气味结合特性的足够有效的理由。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，同时利用这个体内系统来识别气味OR活性相关。在目标1的A部分中，将开发一种高通量的基因靶向策略，使人的OR ORF可以快速地“交换”，以取代老鼠的OR ORF，并在体内进行功能测试。初步实验将确定这种方法在两个小鼠或基因座上的效果。这个子目标的目的是建立一个胚胎干细胞库，每个干细胞库都包含一个独特的人类或ORF。最初，将测试一个人OR的几种异构体。在目标1的B部分中，将开发一种高通量转基因方法，通过该方法，小鼠嗅觉上皮中70%(数百万)的嗅觉神经元将克隆地表达给定的人类OR。再一次，将测试一个人OR的几种异构体。该项目旨在补充基因打靶策略，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，这项提议的唯一目的是推动我们对哪些人类受体对人类气味感知起作用的理解。 公共卫生相关性：人类检测气味(气味)的能力仍然是人类健康和福利的一个重要方面。嗅觉在很大程度上依赖于数百种不同的人类气味受体，这些受体通常在嗅觉神经元中表达。然而，很少有气味/气味受体匹配。这种知识的缺乏是无法在非嗅觉细胞中表达和表征人类气味受体蛋白的唯一反映。因此，对嗅觉神经元中表达的人类气味受体的功能分析应该会大大有助于弥合这一分歧。

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.