A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing

Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi's, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies.