doi: 10.1073/pnas.082117599.
A DNA vector-based RNAi technology to suppress gene expression in mammalian cells
Affiliations
- PMID: 11960009
- PMCID: PMC122801
- DOI: 10.1073/pnas.082117599
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A DNA vector-based RNAi technology to suppress gene expression in mammalian cells
Proc Natl Acad Sci U S A.
.
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.
Figures
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.
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.)
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.
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