Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference.
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.
Chomarat
Pascale
P
Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, 125 chemin de Ronde, 78290 Croissy-sur-Seine, France.
Cogé
Francis
F
Guénin
Sophie P
SP
Mailliet
François
F
Vella
Fanny
F
Mallet
Catherine
C
Giraudet
Stéphanie
S
Nagel
Nadine
N
Leonce
Stéphane
S
Ferry
Gilles
G
Delagrange
Philippe
P
Boutin
Jean A
JA
D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D016660:NAD(P)H Dehydrogenase (Quinone)