| Autor: |
Chomarat P; Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, 125 chemin de Ronde, 78290 Croissy-sur-Seine, France., Cogé F, Guénin SP, Mailliet F, Vella F, Mallet C, Giraudet S, Nagel N, Leonce S, Ferry G, Delagrange P, Boutin JA |
| Jazyk: |
angličtina |
| Zdroj: |
Biochimie [Biochimie] 2007 Oct; Vol. 89 (10), pp. 1264-75. Date of Electronic Publication: 2007 Jul 15. |
| DOI: |
10.1016/j.biochi.2007.07.004 |
| Abstrakt: |
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 48h 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: |
MEDLINE |
| Externí odkaz: |
|
Full Text from ScienceDirect