A DNA vector-based RNAi technology to suppress gene expression in mammalian cells

Abstract

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.

Figure 1

A siRNA synthesized from a DNA template in vivo inhibited expression of a transfected gene. (A) Strategy for generating siRNA from DNA template in vivo. An inverted repeat is inserted at the +1 position of the U6 promoter (−315 to + 1). The individual motif is 21 nt long and corresponds to the coding region of the gene of interest. The two motifs that form the inverted repeat are separated by a spacer of 6 nt. The transcriptional termination signal of five Ts is added at the 3′ end of the inverted repeat. The resulting siRNA is predicted to fold back to form a hairpin dsRNA as shown (drawing not to scale). The selection of the nucleotide sequence to be included in the siRNA vector is empirical and it is unknown whether certain regions of a given mRNA would be more or less susceptible to RNAi. (B) Analysis of gfp siRNA on GFP expression in HeLa cells. Vector BS/U6 or the plasmid carrying DNA templates that direct synthesis of gfp siRNA (BS/U6/gfp) were cotransfected with CMV-GFP and CMV-HA-ERK-5 into HeLa cells with a ratio of 20:1 (effector versus target plasmids) to ensure that cells that received GFP and HA-ERK-5 also received the RNAi plasmid. (a–c) Cells transfected with BS/U6 vector together with CMV-GFP and CMV-HA-ERK-5. (a) GFP-positive cells; (b) same field of cells stained with the anti-HA antibody to detect HA-ERK-5 expression; (c) same cells stained with 4′,6-diamidino-2-phenylindole (DAPI) to indicate all cells in the field. (d–f) Cells transfected with BS/U6/gfp and the GFP and HA-ERK-5 plasmids. Solid arrows indicate cells that are positive for HA-ERK-5 but display nearly undetectable GFP. All corresponding images were taken at the same exposure. (Magnification: ×60.) (C) Western blot analysis of GFP expression in cells cotransfected with either BS/U6 or BS/U6/gfp. (Upper) GFP proteins. (Lower) HA-ERK-5 detected with the anti-HA monoclonal antibody. Lane 1: BS/U6 vector control. Lanes 2 and 3: Cells transfected with 1.5 and 3.0 μg of the BS/U6/gfp plasmid. In all transfections, 100 ng of CMV-GFP and 0.5 μg of HA-ERK-5 plasmids were used.

Figure 2

Human lamin A/C siRNA derived from DNA template in vivo specifically inhibited expression of endogenous lamin A/C. HeLa cells were transfected with either BS/U6 vector or the RNAi plasmid BS/U6/lamin A/C together with CMV-GFP to mark transfected cells. (a and d) Cells stained with the anti-lamin A/C antibody. (g) BS/U6/lamin A/C-transfected cells stained with the secondary antibody alone to indicate background signals. b, e, and h, corresponding to a, d, and g, respectively show the same sets of cells positive for GFP, as a marker of transfected cells. c, f, and i are images of the same fields (as in a, d, and g, respectively) of cells stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify all cells. Cells transfected with either the vector BS/U6 or the RNAi plasmid BS/U6/lamin A/C were also stained with antibodies that recognized the related lamin B protein (j and m). Corresponding images were taken at the same exposure. (Magnifications: ×60.)

Figure 3

Inhibition of expression of a cell cycle control gene and a DNA methyltransferase. (A) Inhibition of CDK-2 expression. HeLa cells were transfected with either BS/U6 vector or BS/U6/cdk-2 and CMV-GFP to mark transfected cells as above. Solid arrows indicate two of the GFP-positive cells (transfected cells) in which CDK-2 expression was below the level of detection. Open arrows indicate two GFP-negative cells in which CDK-2 expression is also undetectable. (B) Inhibition of a DNA methyltransferase (DNMT-1) expression. HeLa cells were transfected with either BS/U6 vector or BS/U6/dnmt-1 and CMV-GFP to mark transfected cells as above. Solid arrows indicate two GFP-positive cells (transfected cells) in which DNMT-1 expression was barely detectable. Open arrows indicate two GFP-negative cells in which DNMT-1 expression is also undetectable. The latter are likely to have received the RNAi plasmid only, because of the excess ratio of the RNAi vector versus GFP-encoding plasmid used in transfection.