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C.J. Scott, L.A. Levin; Gene Silencing in a Differentiated Retinal Ganglion Cell Line With RNAi . Invest. Ophthalmol. Vis. Sci. 2006;47(13):890.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal ganglion cell (RGC)–like RGC–5 line differs from RGCs in morphology, ability to proliferate, and changes in ion channel behavior. Differentiated RCG–5 cells differentiated with staurosporine are more similar to RGCs (IOVS, in press). We studied gene silencing and transfection efficiency in these cells using short interfering RNA (siRNA) to knockdown various gene products.
RGC–5 cells were transfected with lamin a/c siRNA and incubated for 48 and 72 hours. 24 hours post–transfection, staurosporine at 316 nM was added to cultures and compared to untreated cultures. Silencing efficiency of protein expression was determined by Western blot with antibodies against lamin a/c. Transfection efficiency was measured by cytotoxic RNA assay and cell viability was measured 48 hours post–transfection with calcein assay. Transfection efficiency was also measured in lamin a/c siRNA 48 hours post–transfection with fluorescent labels expressing intracellularly.
After 48 hours, high transfection efficiency was observed in RGC–5 cells using a cytotoxic RNA assay showing decreased cell viability, and lamin a/c siRNA demonstrated robust transfection with positive intracellular fluorescence. Silencing efficiency in undifferentiated RGC–5 cells was minimal at 48 hours post–transfection, but was 78.8% of control at 72 hours post–transfection. In staurosporine treated cells, silencing efficiency was not observed at 48 hours post–transfection, but was 23.3% of control at 72 hours post–transfection.
Knockdown of lamin a/c is observed in differentiated RGC–5 cells treated with staurosporine. Transfection of interfering RNA into RGC–5 cells which are subsequently differentiated will allow studying the effect of modulating gene products in cells similar to primary RGCs.
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