[Double knockdowns with retroviral delivery in mammalian cells]
>>Published online before print March 29, 2004
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0401285101
Genetics
Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference
Sebastian Schuck *, Aki Manninen *, Masanori Honsho , Joachim Füllekrug , and Kai Simons
Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
Contributed by Kai Simons, February 24, 2004
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.<<
These guys are from Tuschl's old employer, but are not collaborators.
>>Without transfection, would delivery issues be much less complex?<<
Might help a little. It definitely eliminates a step in gene expression studies, though. How the technique would work in vivo is not clear to me.
Cheers, Tuck |
