Nucleic Acids Research Advance Access originally published online on April 10, 2007
Nucleic Acids Research 2007 35(9):e67; doi:10.1093/nar/gkm193
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2007, Vol. 35, No. 9 e67
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines
1Department of Pediatrics, University Hospital of North-Norway, 2Department of Pediatrics, Institute of Clinical Biology, University of Tromsø, 9037 Tromsø, Norway and 3Department of Human Genetics, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
*To whom correspondence should be addressed. Tel: +47 77 644735; Fax: +47 77 645350; Email: christer{at}fagmed.uit.no
Received January 5, 2007. Revised March 20, 2007. Accepted March 20, 2007.
Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox–doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications.