A NEW STRATEGY TO ASSESS GENE FUNCTION IN TOOTH FORMATION

描述（由申请人提供）：哺乳动物牙齿发育在很大程度上依赖于顺序和相互上皮-间充质相互作用，长期以来一直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。 已经发现大量基因在发育中的小鼠牙齿中表达，但它们的功能在很大程度上仍然未知。 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的功能丧失和获得的有力工具。 然而，标准的转基因/基因敲除技术在实践中被证明是费力、昂贵且耗时的。 RNA干扰（RNA interference，RNAi）是近年来出现的一种抑制细胞基因表达的新技术，但其在哺乳动物器官发育过程中基因功能研究的应用还很有限。 我们最近建立了一种方法，从胚胎小鼠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，我们建议使用重组病毒载体，表达siRNA细胞内检查特定基因的功能，在这个新的，高度顺从的牙齿器官发育模型，已在我们的实验室可重复地建立。 目的1：建立一种利用慢病毒介导的RNAi技术特异性敲减胚胎牙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。 目的二：建立一种腺病毒介导的RNAi方法，用于检测牙胚发育过程中牙上皮细胞中基因的功能。 这些研究将建立一种可靠、方便、快速的检测方法，避免了需要大量时间、金钱和人力投入的常规和条件性基因靶向策略，使大规模筛选哺乳动物牙齿发育中的基因功能成为可能。 这种大规模的筛查将为研究人类牙齿遗传异常提供宝贵的见解。

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.