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D. L. Harris, N. C. Joyce; p21WAF1/cip1 siRNA-Induced Proliferative Activity of Rat Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3938.
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To determine whether siRNA down-regulation of p21WAF1/cip1 will overcome G1-phase arrest and promote cell cycle progression in rat corneal endothelial cells (RCEC).
Confluent cultures of RCEC were trypsinzed and electroporated using a Bio-rad Gene Pulser system. Proper conditions were determined for voltage (150-450V) and pulse length (100-400µs) by viewing Cy3-labeled non-silencing siRNA electroporation efficiency with a fluorescent microscope. Optimal number of pulses (1-3) was determined using G3PDH siRNA and 3 different p21WAF1/cip1 siRNAs (Ambion, Austin, TX). Controls included cells not electroporated or electroporated with non-silencing siRNA. To determine the most efficient downregulation of the corresponding protein, cells were electroporated, seeded in dishes, maintained for 72 hrs in 10% FBS, followed by western blot analysis. To determine the effect of p21WAF1/cip1 siRNA treatment on proliferation, cells were electroporated and then incubated for 24, 72, or 120 hours in 10% FBS. Cell numbers were determined at each time-point using WST-8-based spectrophotometric analysis.
The following conditions were determined to be optimal for siRNA electroporation in RCEC: voltage of 300V, pulse length of 400 µs, and 2 or 3 pulses. Two of the three p21WAF1/cip1 siRNAs significantly reduced p21WAF1/cip1 protein expression. Treatment of RCEC with one of the p21WAF1/cip1 siRNAs resulted in significantly higher cell numbers by 120 hours compared with controls electroporated with non-silencing siRNA.
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