Purchase this article with an account.
Cailing Liu, Diego Ogando, Joseph Bonanno; Effect of SOD2 Knockdown on ROS Production in Rabbit Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6456.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress is associated with age related corneal endothelial cell loss and dystrophy. Corneal endothelial cells are rich in mitochondria that could be a source of reactive oxygen species (ROS). In this study we examined whether the mitochondrial superoxide dismutase, SOD2 contributes to anti-oxidative activity.
An shRNA specific to rabbit SOD2 was designed and cloned into FIV-derived RNAi lentivector (pFIV-H1-puro) and packaged into lenti-pseudoviral pPACKF1 (System Biosciences). Primary rabbit corneal endothelial (RCE) cells were transduced with either shSOD2-puro or scrambled-sequence shRNA pseudoviral particles at an m.o.i. of 20 in presence of 7 mg/ml transduction reagent Polybrene (Sigma). Five days post transduction SOD2 gene knockdown efficiency was analyzed by Western blot. SOD2 enzyme activity was examined by Superoxide Dismutase Assay kit (Cell Technology). ROS production was measured with 5-(and-6) - carboxy-2’, 7 ’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) fluorescence by flow cytometry. Cells exposed under UV for two minutes were served as positive control for cellular ROS production.
Western blot results showed that SOD2 expression was reduced by ~90% in primary RCE cells at 5 days post transduction with shSOD2. SOD2 activity was reduced by ~65 % in shSOD2 virus treated cells relative to scrambled-sequence control. Flow cytometry showed that positive DCFDA fluorescence was present in 0.4 ± 0.1% of cells for untreated, polybrene only and scrambled-sequence virus treated cells. This increased to 9.0 ± 6.1% of cells for shSOD2 virus treated cells. With 2 minutes UV, DCFDA positive fluorescence increased to 12.7% for scrambled sequence treated cells and 28.6 ± 5.4% for shSOD2 treated cells.
shSOD2 lentiviral vectors can efficiently knockdown SOD2 expression and reduce enzyme activity in RCE cells, while increasing ROS production and sensitivity to oxidative stress (UV). SOD2 is a significant contributor to anti-oxidative activity in RCE cells.
This PDF is available to Subscribers Only