Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference

Sebastian Schuck, Aki Manninen, Masanori Honsho, Joachim Füllekrug, Kai Simons

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)4912-4917
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number14
DOIs
Publication statusPublished - Apr 6 2004

All Science Journal Classification (ASJC) codes

  • General

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