Optimisation of siRNA-mediated RhoA silencing in neuronal cultures

Research output: Contribution to journalArticle

Colleges, School and Institutes

Abstract

In investigating the consequences of gene silencing in axon growth disinhibition strategies in cultured retinal ganglion cells (RGC), we conducted experiments designed to silence RhoA signalling in PC12 and primary adult rat retinal cell cultures (containing RGC) by siRNA-mediated RhoA mRNA knockdown. We demonstrate wide differences in the levels of RhoA mRNA knockdown, dose-dependent cell toxicity, and induction of endogenous inflammatory cytokine and interferon responses to siRNA therapy. Toxicity effects observed with RhoA-siRNA was significantly reduced with "Stealth" chemical modification of the sequence, promoting approximately 50% and 70% knockdown of RhoA mRNA and protein in retinal cells, respectively, while promoting significant disinhibited RGC neurite outgrowth in the presence of inhibitory CNS myelin. Our results highlight differential responsiveness of cell lines compared to primary cultured cells, and demonstrate the efficacy of the "Stealth" modification to reduce siRNA-induced interferon responses, thereby increasing target cell viability and reducing off-target effects of the delivered nucleic acids.

Details

Original languageEnglish
Pages (from-to)451-462
Number of pages12
JournalMolecular and Cellular Neuroscience
Volume40
Early online date30 Jan 2009
Publication statusPublished - 30 Jan 2009