Document Detail


Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference.
MedLine Citation:
PMID:  17714848     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Pascale Chomarat; Francis Cogé; Sophie P Guénin; François Mailliet; Fanny Vella; Catherine Mallet; Stéphanie Giraudet; Nadine Nagel; Stéphane Leonce; Gilles Ferry; Philippe Delagrange; Jean A Boutin
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Publication Detail:
Type:  Journal Article     Date:  2007-07-15
Journal Detail:
Title:  Biochimie     Volume:  89     ISSN:  0300-9084     ISO Abbreviation:  Biochimie     Publication Date:  2007 Oct 
Date Detail:
Created Date:  2007-10-08     Completed Date:  2008-02-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  1264604     Medline TA:  Biochimie     Country:  France    
Other Details:
Languages:  eng     Pagination:  1264-75     Citation Subset:  IM    
Affiliation:
Pharmacologie Moléculaire et Cellulaire, Institut de Recherches SERVIER, 125 chemin de Ronde, 78290 Croissy-sur-Seine, France.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Flow Cytometry
Green Fluorescent Proteins / genetics,  metabolism
Kinetics
Mice
Mice, Knockout
Microscopy, Fluorescence
NAD(P)H Dehydrogenase (Quinone) / genetics*,  metabolism
RNA Interference*
RNA, Small Interfering / genetics
Transfection
Chemical
Reg. No./Substance:
0/RNA, Small Interfering; 147336-22-9/Green Fluorescent Proteins; EC 1.6.5.2/NAD(P)H Dehydrogenase (Quinone)

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