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. 2004 Apr 6;101(14):4912-7.
doi: 10.1073/pnas.0401285101. Epub 2004 Mar 29.

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

Sebastian Schuck  1 , Aki Manninen, Masanori Honsho, Joachim Füllekrug, Kai Simons
Affiliations

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

Sebastian Schuck et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
Fig. 1.
RNAi-mediated reduction of target gene protein levels. Equal amounts of protein from MDCK cells, MDCK cells transduced with empty RVH1-puro (MDCK-RVH1), and knockdown (KD) cells were analyzed by immunoblotting. gp135 and transferrin receptor (TfR) were used as controls. Note that the annexin 13-2 antibody recognizes annexin 13a, annexin 13b, and annexin 2 (lowermost blot), and that both annexin 13a and b are depleted in annexin 13 knockdown cells.
Fig. 2.
Fig. 2.
Improvement of knockdowns by increasing virus titers. Five viruses expressing different shRNAs (VIP17/382, rab11a, anx2, TI-VAMP/230, and VIP17/230; anx 2, annexin 2) were pelleted by centrifugation and resuspended in 1, 0.2, or 0.05 times the original volume, resulting in 1, 5, or 20 times the original virus concentration. Percent mRNA reductions relative to the levels in control cells transduced with empty RVH1-puro were measured as in Table 1.
Fig. 3.
Fig. 3.
Internal energy profiles of functional versus nonfunctional siRNAs. Pentamer hybridization energies were calculated along the length of the siRNA duplexes starting from the 5′ end of the antisense strands. The average pentamer hybridization energies (ΔG) for the first 15 positions of functional siRNAs (>70% mRNA reduction) and nonfunctional siRNAs (<70% mRNA reduction) are shown.
Fig. 4.
Fig. 4.
Distribution of residual protein in a knockdown cell population. Caveolin-1 and the plasma membrane protein gp114 were visualized by immunofluorescence in caveolin-1 knockdown cells and control cells transduced with empty RVH1-puro (MDCK-RVH1).
Fig. 5.
Fig. 5.
Stability of target gene mRNA reductions in knockdown cell populations. MDCK cells were transduced with different viruses (VIP17/230, caveolin-1, rab11a, VIP36/453, and VIP36/626). Target gene mRNA levels were monitored between days 6 and 23 postinfection. Percent mRNA reductions relative to the levels in control cells transduced with empty RVH1-puro were measured as in Table 1. Note that the rab11a virus was concentrated 20-fold before infection.
Fig. 6.
Fig. 6.
RNAi-mediated reduction of target gene protein levels in single and double knockdown cells. Equal amounts of protein from MDCK cells transduced with empty RVH1-puro and empty RVH1-hygro (control) and single and double knockdown (KD) cells were analyzed by immunoblotting. Annexin 2 was used as control for the VIP17/annexin 13 knockdown (A), and annexin 2 and gp135 were used as controls for the rab11a/rab11b knockdown (B). Note that rab11a appears to be the major rab11 isoform in MDCK cells.
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