Cellular knock-down of quinone reductase 2: A laborious road to successful inhibition by RNA interference
| Autor: | Fanny Vella, Catherine Mallet, Sophie P. Guénin, Nadine Nagel, Francis Cogé, Pascale Chomarat, Jean A. Boutin, François Mailliet, Stephanie Giraudet, Gilles Ferry, Philippe Delagrange, Stéphane Léonce |
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| Rok vydání: | 2007 |
| Předmět: |
Mice
Knockout Small interfering RNA Green Fluorescent Proteins Trans-acting siRNA General Medicine Transfection Biology Flow Cytometry Biochemistry Cell Line Small hairpin RNA Kinetics Mice RNA silencing Microscopy Fluorescence RNA interference NAD(P)H Dehydrogenase (Quinone) Animals Gene silencing RNA Interference RNA Small Interfering Cellular model |
| Zdroj: | Biochimie. 89:1264-1275 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/j.biochi.2007.07.004 |
| Popis: | NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48 h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71–80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42–48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application. |
| Databáze: | OpenAIRE |
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