2007 Papers - Robitschek


RNA Interference Knockdown of TGF-B2 in Human Fibroblasts

Jon Robitschek, Andrew Gilbert, Benjamin Cable Department of Otolaryngology, Tripler Army Medical Center


Introduction: Transforming Growth Factor Beta 2 (TGF-B2) is a principle gene of interest in the pathogenesis of subglottic stenosis and is a potential target for future therapies. We propose to introduce RNA Interference (RNAi) technology as a viable means of targeted gene knockdown in cultured human fibroblasts.

Objective: Demonstrate effective knockdown of TGF-B2 using RNAi technology in cultured human fibroblasts.

Methods: RNAi constructs targeted to the TGF-B2 transcript were encapsulated in microsomal lipids and applied to human fibroblasts in cell culture. Transfection efficiency and cell viability were measured by fluorescence microscopy. Messenger RNA (mRNA) for TGF-B2 was measured 48hrs post transfection using real time quantitative PCR.

Results: Transfection efficiency of over 80% was achieved with minimal induced cell death. Treated cells showed a selective knockdown of 80% of TGF-B2 mRNA, which was confirmed with negative controls.

Conclusions: RNA Interference technology is an effective means of localized and transient gene silencing in cultured human fibroblasts. Transfection can be achieved using microsome encapsulated RNAi with minimal induced cell death. This preliminary result shows great promise for future in vivo investigation utilizing topical RNAi therapy as an adjunct to surgical repair.