Stable suppression of gene expression by RNAi in mammalian cells

Abstract

In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, approximately 22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (approximately 500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of approximately 22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable "knock-down" cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.

Figure 1

RNAi in P19 embryonal carcinoma cells. Ten-centimeter plates of P19 cells were transfected by using 5 μg of GFP plasmid and 40 μg of the indicated dsRNA (or no RNA). Cells were photographed by fluorescent and phase-contrast microscopy at 72 h after transfection; silencing was also clearly evident at 48 h posttransfection.

Figure 2

RNAi of firefly and Renilla luciferase in P19 cells. (A) P19 cells were transfected with plasmids that direct the expression of firefly and Renilla luciferase and dsRNA 500 mers (25 or 250 ng, as indicated), that were either homologous to the firefly luciferase mRNA (dsFF) or nonhomologous (dsGFP). Luciferase activities were assayed at various times after transfection, as indicated. Ratios of firefly to Renilla activity are normalized to dsGFP controls. (B and C) P19 cells in 12-well culture dishes (2 ml of media) were transfected with 0.25 μg of a 9:1 mix of pGL3-Control and pRL-SV40 as well as 2 μg of the indicated RNA. Extracts were prepared 9 h after transfection. (B) Ratio of firefly to Renilla luciferase is shown. (C) Ratio of Renilla to firefly luciferase is shown. Values are normalized to dsGFP. The average of three independent experiments is shown; error bars indicate standard deviation.

Figure 3

Specific silencing of luciferase expression by dsRNA in murine embryonic stem cells. Mouse embryonic stem cells in 12-well culture dishes (1 ml of media) were transfected with 1.5 μg of dsRNA along with 0.25 μg of a 10:1 mixture of the reporter plasmids pGL3-Control and pRL-SV40. Extracts were prepared and assayed 20 h after transfection. The ratio of firefly to Renilla luciferase expression is shown for FF ds500; the ratio of Renilla to firefly is shown for Ren ds500. Both are normalized to ratios from the dsGFP transfection. The average of three independent experiments is shown; error bars indicate standard deviation.

Figure 4

RNAi in C2C12 murine myoblast cells. (A) Mouse C2C12 cells in 12-well culture dishes (1 ml of media) were transfected with 1 μg of the indicated dsRNA along with 0.250 μg of the reporter plasmids pGL3-Control and pRL-SV40. Extracts were prepared and assayed 24 h after transfection. The ratio of firefly to Renilla luciferase expression is shown; values are normalized to ratios from the no dsRNA control. The average of three independent experiments is shown; error bars indicate standard deviation. (B) C2C12 cells cotransfected with 1 μg of either plasmid alone or a plasmid containing a hyperactive mutant of vaccinia virus K3L (26). The absolute counts of Renilla and firefly luciferase activity are shown. (C) The ratios of firefly/Renilla activity from B, normalized to no dsRNA controls.

Figure 5

Expression of a hairpin RNA produces P19 EC cell lines that stably silence GFP. (A) A cartoon of the FLIP cassette used to construct the GFP hairpin. GFP represents the first 500 coding base pairs of EGFP. Zeo, zeocin resistance gene; L, Lox; P, the cytomegalovirus promoter in the expression plasmid pcDNA3. Homologous GFP fragments are first cloned as direct repeats into the FLIP cassette. To create inverted repeats for hairpin production, the second repeat is flipped by using Cre recombinase. When transcribed, the inverted repeat forms a GFP dsRNA with a hairpin loop. (B) P19 cell lines stably expressing the GFP hairpin plasmid, GFPhp.1 (clone 10) and GFPhp.2 (clone 12), along with wt P19 were transfected with 0.25 μg each of GFP and RFP reporter genes. Fluorescence micrographs were taken by using filters appropriate for GFP and RFP. Magnification is 200×. (C) P19 GFPhp.1 cells were transfected with pEGFP and 0, 0.5, or 1 μg of Dicer or firefly dsRNA. Fluorescence micrographs were taken at 48 h posttransfection and are superimposed with bright field images to reveal non-GFP expressing cells. Magnification is 100×. (D) In vitro and in vivo processing of dsRNA in P19 cells. In vitro Dicer assays were performed on S2 cells and three independently prepared P19 extracts by using ³²P-labeled dsRNA (30°C for 30 min). A Northern blot of RNA extracted from control and GFPhp.1 P19 cells shows the production of ≈22mer RNA species in hairpin-expressing cells but not in control cells. Blots were probed with a ³²P-labeled “sense” GFP transcript.

Figure 6

dsRNA induces silencing at the posttranscriptional level. P19 cell extracts were used for in vitro translation of firefly and Renilla luciferase mRNA (100 ng each). Translation reactions were programmed with various amounts of dsRNA 500mers, either homologous to firefly luciferase mRNA (dsLUC) or nonhomologous (dsGFP). Luciferase assays were carried out after a 1-h incubation at 30°C. Ratios of firefly to Renilla activity are normalized to no dsRNA controls. Standard deviations from the mean are shown.