A NEW STRATEGY TO ASSESS GENE FUNCTION IN TOOTH FORMATION

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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.