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Evaluation of RNA-knockdown strategies for modulation of cytochrome P450 reductase activity in mouse hepatocytes


Research Report

J RNAi Gene Silencing (2010), 6(2), 416-421

doi: jrgsxx

Published online: 26 November 2010

Full Text: (html | pdf ~406kb | refs)

Evaluation of RNA-knockdown strategies for modulation of cytochrome P450 reductase activity in mouse hepatocytes

Neil CW Mackenzie †, Simon G Lillico †, Ken Brown ‡, Charles R Wolf ‡¥ and Christopher BA Whitelaw †*

† Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, UK

‡ CXR Biosciences Limited, J ames Lindsay Place, Dundee Technopole, Dundee, DD1 5JJ, UK

¥ University of Dundee Biomedical Research Institute, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK

*Correspondence toNeil Mackenzie, Email: neil.mackenzie@roslin.ed.ac.uk, Tel: +44 (0)131 5274200, Fax: +44 (0)131 4400434

Received: 05 August 2010, Revised: 02 November 2010, Accepted: 18 November 2010

© Copyright The Authors

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ABSTRACT

Transgenic technologies can provide important animal models for studying drug-metabolizing enzymes. Our overall aim was to generate versatile cell and animal systems that exhibited varying levels of cytochrome P450 oxidoreductase (POR) activity, more accurately modelling the human population for pharmacological and toxicology studies. Towards this goal we evaluated RNA-interference constructs designed for use in vitro and in vivo for reducing POR activity in hepatocytes. This study clearly demonstrates that both POR protein level and reductase activity can be significantly knocked down in Hepa-1 cells in vitro, while highlighting the difficulty in predicting knockdown efficiency in transgenic animals. The high levels of embryonic lethality observed, and inability to produce multi-copy transgenic animals indicates that high levels of shRNA expression may be detrimental to embryonic development.

KEYWORDS: RNA-interference, drug metabolism, lentivirus vectors, liver metabolism, transgenic mice

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