Application of in vivo siRNA knockdown mice in tooth studies

Objective:In searching for molecules critical for tooth development we identified a previously unknown growth factor, granulin/epithelin precursor (GEP) in odontoblast, ameloblast and chondrocytes of condyle. Instead of knockout of this gene, our goal is to use an siRNA approach to knockdown GEP in vivo. Methods: To do this, we first created GEP knockdown transgenic mice where siGEP, siRNA sequences specifically against GEP, was driven by the U6 promoter, a ubiquitous promoter that is disturbed by a loxP-flanked neomycin cassette (Note that the siGEP RNA expression will be activated only when crossed to Cre transgenic mice where Cre removes the neomycin). Next, U6-ploxPneo-GEP transgenic mice were crossed to Sox2-Cre transgenic mice, where Cre enzyme is activated in early progenitor cells, for tissue-specific knockdown of GEP in tooth and condyle. Results: Our results showed that knockdown of GEP in condyle led to reductions of condyle size as measured by both x-ray and histological assays, suggesting that GEP, the novel growth factor, is likely critical for condyle formation. Second, we showed that knockdown of GEP in ameloblasts led to reduction of enamel thickness. Third, we showed that knockdown of GEP in odontoblasts led to reduction of dentin thickness. Fourth, our in vitro data showed that overexpression of GEP led to up-regulations of markers critical for odontogenesis and amelogenesis, which is in consistent with the in vivo data. Conclusion: in vivo siRNA knockdown approach is quick and efficient, although there is a problem and the pros and cons will be discussed. (This work is supported by NIH grants: DE018486)