High throughput in vivo functional analysis of human odorant receptors.

人类气味受体的高通量体内功能分析。

• 批准号: 7977232
• 负责人: PAUL FEINSTEIN
• 金额: $ 19万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7977232
• 关键词: Alleles; Axon; Binding; Biological Assay; Brain; Cells; Cloning; Communities; Complement; Detection; Dorsal; Environment; Epithelium; Exons; Failure; Feedback; GTP-Binding Proteins; Gene Targeting; Gene Transfer Techniques; Genes; Genome; Goals; Golf; Health; Human; In Vitro; Individual; Knowledge; Length; Libraries; Maintenance; Methods; Mus; Mutation; Nasal Epithelium; Neurons; Odorant Receptors; Odors; Olfactory Epithelium; Open Reading Frames; Perception; Population; Property; Protein Isoforms; Pseudogenes; Rattus; Receptor Gene; Robotics; Scheme; Side; Smell Perception; Social Welfare; Stem cells; Structure; Subgroup; System; Technology; Testing; Transgenes; Transgenic Organisms; Variant; axon guidance; embryonic stem cell; food consumption; genetic manipulation; improved; in vivo; neural circuit; novel; olfactory bulb; public health relevance; rat genome; receptor; research study; response; tool; vector

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.

描述（由申请人提供）：人们认为人类嗅觉系统可检测数千种气味，从而可以适当的食物消耗和厌恶。人类鉴定气味的能力取决于鼻上皮细胞的嗅觉神经元内的气味受体。气味受体（OR）在成熟的嗅觉神经元（一个或一个神经元的一个或等位基因）中表达。因此，研究可识别的个体神经元中的气味反应等同于研究个体OR。人类嗅觉基因组由〜900或基因组成；这些基因属于七跨膜受体超家族。但是，只有〜40％编码全长开放阅读框（ORF）。因此，〜355或基因可用于主要嗅觉系统中的气味鉴定。 ORS的该亚组分为三个或类：I类：〜50基因； II类：〜300个基因；和taars：〜5基因。随着SNP分析的出现，具有全长ORF的变体的人数继续增长。 I级和II类或基因的整个集已经闻名了十年，但是它们在体内发挥作用的能力仍然难以捉摸。缺乏三件事是为了理解所有〜355或基因的功能以及它们的变体的功能：第一个）在人类嗅觉神经元中表达了哪些ORS的知识，证明ORF可以将ORF与嗅觉G-蛋白（Golf）相结合的证据（在体内和3RD环境中）可以允许ORFS允许GLOMERIMDERIBLES ANNOMERIMTARITIAN vIVS ANTOMILTATION。某些人或ORF无法驱动肾小球形成或夫妇去高尔夫球的失败会将这些基因归类为“假基因”。因此，拥有全长ORF的OR并不构成使用体外技术研究其气味结合特性的足够理由。需要有一种方法来快速识别人类或ORF可以在体内发挥作用，并同时利用该体内系统来识别气味或活性相关。在AIM 1 A节中，将制定高通量基因靶向策略，以便可以快速地“交换”人或ORF代替小鼠或ORF，并在体内进行功能测试。初始实验将确定在两只小鼠或基因座上这种方法的功效。该子目标的目的是生成一个包含独特人或ORF的胚胎干细胞库。最初，一个人或将要测试的几种同工型。在AIM 1 B中，将开发出高通量转基因方法，从而在小鼠嗅觉上皮中所有嗅觉神经元（数百万）中有70％将在克隆中表达给定的人或。同样，一个人的几种同工型或将被测试。该项目旨在补充基因靶向策略，并为有气味鉴定的机器人方法提供大量神经元。简而言之，该提议的唯一目的是促进我们对人类受体的理解，可用于人类对气味的感知。 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重要方面。嗅觉关键取决于通常在嗅觉神经元中表达的数百种不同的人类气味受体。但是，很少有气味/气味受体匹配。缺乏知识是无力表达和表征非富特性细胞中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显着帮助弥合这种鸿沟。