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M. Duong, A. K. Ball; Stable Knockdown of Optineurin Expression in RGC-5 Cells Using Short Hairpin RNA Sequences. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5191. doi: https://doi.org/.
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RNA interference (RNAi) has provided investigators with a powerful tool to effectively study genes and their protein products by examining the effect of protein expression knockdown on cell function. Despite the power of this technology, limitations still exist in the delivery of siRNAs to target cells and obtain consistent knockdown results. The purpose of this study was to evaluate a novel method to deliver siRNAs to mediate the knockdown of the optineurin protein in the RGC-5 cell line.
BLOCK-iTTM miR RNAi Select hairpin sequences (Invitrogen), targeting the OPTN gene were cloned into BLOCK-iTTM Pol II miR RNAi expression vectors as per manufacturer’s instructions (Invitrogen). This technology allows short hairpin RNA (shRNA) sequences to be transcribed by the cell and subsequently processed into siRNAs used for protein knockdown. The expression vector includes a sequence encoding green fluorescent protein (GFP), to permit confirmation of positive transfection and expression. Lipofectamine 2000 was used to transfect RGC-5 cells with the constructed expression vectors. Cells were differentiated for 24 hours with 1 µM staurosporine to express neuronal characteristics of the RGC-5 cells. Transfection efficiency was determined based on the intensity of GFP fluorescence expressed by the vectors. Knockdown of optineurin was evaluated using immunohistochemistry, Western immunoblot analysis and RT-PCR at 4 hours, 8 hours, 24 hours, 2 days and 4 days post-transfection.
RGC-5 cells can be readily transfected with BLOCK-iTTM Pol II miR RNAi expression vectors using Lipofectamine 2000 in an efficient and consistent manner. shRNA sequences targeting the OPTN gene were effectively processed by RGC-5 cells, resulting in the generation of several OPTN-specific siRNAs, resulting in significant knockdown of the optineurin protein. Although the protein reduction levels were comparable to previously reported knockdown levels using separately generated siRNAs, the consistency and efficiency of protein knockdown was greatly increased.
We have shown that RNAi, mediated by the expression of shRNAs in RGC-5 cells can result in consistent knockdown of the target protein optineurin. This important finding addresses the limitations of traditional siRNA-mediated knockdown experiments including inconsistencies and poor efficiency of transfection. Furthermore, the expression vectors generated in these experiments can be combined with viral delivery vectors in the future to aid in the delivery of shRNAs in vivo, a limitation that has to date limited RNAi to in vitro studies.
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