A NEW STRATEGY TO ASSESS GENE FUNCTION IN TOOTH FORMATION

评估牙齿形成中基因功能的新策略

• 批准号: 7039225
• 负责人: Yiping Chen
• 金额: $ 3.7万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039225
• 关键词: RNA interference; dental development; developmental genetics; epithelium; functional /structural genomics; gene induction /repression; genetic screening; genetically modified animals; histogenesis; laboratory mouse; organ culture; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): Mammalian tooth development relies largely upon sequential and reciprocal epithelial-mesenchymal interactions, and has long been used as a model system to study gene function and tissue interactions during organogenesis. A large number of genes have been found to be expressed in developing mouse teeth, but their functions remain largely unknown. Gene targeting and transgenic techniques have been widely used as powerful tools to study loss-of- and gain-of-function of a gene of interest in organogenesis and pathogenesis in mice. However, the standard transgenic/gene knockout technologies have proven to be laborious, expensive, and time consuming in practice. RNA interference (RNAi) has emerged recently as a new tool to silence gene expression n cells, but its application in assessing gene function in mammalian organ development remains highly limited. We have recently established a methodology to generate a well-differentiated tooth organ from a single-cell suspension of embryonic mouse tooth germ. In this study we propose to use recombinant viral vectors that express siRNAs intracellularly to examine the function of specific genes in this novel, highly amenable model of tooth organ development that has been reproducibly established in our lab. In Aim 1, we will establish a method to knock down target gene expression specifically in embryonic dental mesenchymal cells by lentivirus-mediated RNAi, and assay for tooth development in organ culture and subrenal culture. In Aim 2, an adenovirus-mediated RNAi method will be established to assay for gene function in the dental epithelium during odontogenesis. The proposed studies will establish a reliable, convenient, and fast assay that obviate the need for both conventional and conditional gene targeting strategies that require considerable investments of time, money, and labor, making it possible to conduct large scale screen of gene function in mammalian tooth development. Such large screening would provide invaluable insights for studying genetic tooth abnormalities in humans.

描述（由申请人提供）：哺乳动物牙齿发育很大程度上依赖于顺序和相互的上皮-间质相互作用，并且长期以来被用作研究器官发生过程中基因功能和组织相互作用的模型系统。 已发现大量基因在发育中的小鼠牙齿中表达，但它们的功能仍然很大程度上未知。 基因打靶和转基因技术已被广泛用作研究小鼠器官发生和发病机制中感兴趣的基因功能丧失和功能获得的有力工具。 然而，标准的转基因/基因敲除技术在实践中已被证明是费力、昂贵且耗时的。 RNA干扰（RNAi）最近作为一种沉默细胞基因表达的新工具出现，但其在评估哺乳动物器官发育中的基因功能方面的应用仍然非常有限。 我们最近建立了一种方法，可以从小鼠胚胎牙胚的单细胞悬浮液中产生分化良好的牙齿器官。 在这项研究中，我们建议使用在细胞内表达 siRNA 的重组病毒载体来检查这种新型、高度适合的牙齿器官发育模型中特定基因的功能，该模型已在我们的实验室中重复建立。 在目标1中，我们将建立一种通过慢病毒介导的RNAi特异性敲低胚胎牙齿间充质细胞中靶基因表达的方法，并在器官培养和肾下培养中检测牙齿发育。 在目标 2 中，将建立一种腺病毒介导的 RNAi 方法来测定牙发育过程中牙上皮的基因功能。 拟议的研究将建立一种可靠、方便、快速的检测方法，消除传统和条件基因靶向策略的需要，这些策略需要大量的时间、金钱和劳动力投入，从而使大规模筛选哺乳动物基因功能成为可能。牙齿发育。 如此大规模的筛查将为研究人类遗传性牙齿异常提供宝贵的见解。