Currently very little is known regarding the tissue-specific gene regulation of tooth extracellular matrix proteins during odontogenesis. The purpose of this proposal is to study the regulatory mechanisms that control the process of dentine Sialophosphoprotein (DSPP) gene expression during tooth formation, while gaining information to its biological function. DSPP is a large processor protein that is specifically cleaved giving rise to two dentine proteins dentine sialoprotein (DSP) and dentine phosphoprotein also known as phosphophoryn (DPP). In situ hybridization studies show the restricted pattern of DSPP. The application plan to elucidate the molecular mechanism that control the expression of DSPP in the odontoblasts (enhancers) and down-regulate expression (silencers) in ameloblasts. The hypothesis is that tissue and developmental regulation of DSPP is regulated both up and down through discrete interactions of transcription factors with their cognate cis-elements in specific region of the promoter and that this protein has an important role in tooth mineralization. To test this hypothesis, four specific aims are proposed: 1) to determine the DNA sequences of the 5' upstream promoter region of previous identified mouse and human genomic clones; 2) to determine the region of the DSPP promoter that confers the potential tissue specific expression of DSPP gene using novel immortalized dental cell lines and in vivo using a transgenic approach; 3) to generate transgenic Knock-in DSPP LacZ mouse model to investigate the in vivo endogenous patterns of expression of native DSPP promoter, while allowing the functional role to be explored in homozygous null DSPP animals; and 4) to determine the effects of DSPP over-expression using trasgenic mouse model. These experiments will provide needed preliminary data and background for the future potential use of the DSPP promoter in human gene therapy strategy for the numerous dentine and enamel genetic diseases. Furthermore, once fully characterized, the DSPP promoter could be used in other therapeutic applications to derive tooth specific expression of beneficial target genes related to pulp capping modalities.
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