Generation of Single and Double Knockdowns in Polarized Epithelial Cells by Retrovirus-Mediated RNA Interference

RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Her...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 101; no. 14; pp. 4912 - 4917
Main Authors Schuck, Sebastian, Manninen, Aki, Honsho, Masanori, Füllekrug, Joachim, Simons, Kai
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 06.04.2004
National Acad Sciences
Subjects
Annexins
Antibodies
Biological Sciences
Cell lines
Cell Polarity
Cells
Cloning, Molecular
Epithelial Cells - virology
Eukaryotes
Gene expression
Gene Expression Regulation, Viral
Genetics
Infections
Kidney cells
Kidneys
Messenger RNA
Plasmids
Retroviridae
Retroviridae - physiology
Retrovirus
Reverse Transcriptase Polymerase Chain Reaction
Ribonucleic acid
RNA
RNA Interference
RNA, Small Interfering - metabolism
Small interfering RNA
Viruses
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Summary:RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell.
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To whom correspondence should be addressed. E-mail: simons@mpi-cbg.de.
Present address: Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Present address: Internal Medicine IV, University of Heidelberg, 69120 Heidelberg, Germany.
Abbreviations: MDCK, Madin-Darby canine kidney; miRNA, micro-RNA; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA.
S.S. and A.M. contributed equally to this work.
Contributed by Kai Simons, February 24, 2004
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0401285101