Alternate Modes of Gene Silencing
by RNA Interference
Authors: Nitin Puri, Richard A. Jarvis, Vince
Pallotta, Mike Byrom, Zhenhai Lu, Lance P. Ford and David Brown
Ambion, Inc., 2130 Woodward Street, Austin,
TX 78744-1832 • www.ambion.com
INTRODUCTION
Small Interfering RNAs (siRNAs) are short double-stranded
RNA molecules that can target and degrade complementary mRNAs via
a cellular process termed RNA interference (RNAi). Alternate methods
for generating siRNA are: (i) vector based in vivo expression,
(ii) chemical synthesis, (iii) in vitro transcription (IVT),
(iv) RNAse III mediated hydrolysis and (v) PCR based siRNA expression
cassettes. We have evaluated these diverse methodologies and found
them to offer varying advantages over one another.
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| Figure 1. Use
of Chemically Synthesized and in Vitro Transcribed siRNAs
to Induce Gene Silencing. siRNAs
targeting ß-Actin were prepared by chemical synthesis
(Ambion) or by in vitro transcription using Ambion's Silencer™ siRNA
Construction Kit. HeLa cells were plated at 30,000 cells
per well in a 24 well tissue culture plate containing glass
slides. The cells were transfected 24 hours after plating,
using 2 µl siPORT™ Lipid (Ambion)
according to the manufacturer's protocol, at a final siRNA
concentration of 75 nM. Immunofluorescence analysis was
performed 96 hr post transfection using mouse anti-Human ß-Actin
primary antibody and a FITC conjugated anti-mouse IgG secondary
antibody. Photographs were taken using the appropriate
fluorescent filters and quantified using MetaMorph software.
Note that both siRNA preparation methods resulted in >_
95% reduction in ß-actin protein levels. |
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| Figure 2. Schematic
of a Typical siRNA Expression Vector. A. Vector
Diagram. B. siRNA encoding insert. C. Hairpin
siRNA. |
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| Figure
3. Long Term Silencing
of GFP with pSilencer™ 2.1-U6 hygro. HeLa
cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6
hygro containing an insert encoding an siRNA targeting
cycle 3 GFP or pSilencer 2.1-U6 hygro without an
siRNA-encoding insert. Following transfection, the cells
were selected with hygromycin. Three weeks following selection,
the cells were analyzed for GFP expression by fluorescence
microscopy. Green: GFP. Blue: DAPI stained
nuclei. GFP levels were remarkably reduced (94%) in cells
transfected with the GFP siRNA-encoding pSilencer 2.1-U6
hygro siRNA Expression Vector as compared to those transfected
with an "empty" siRNA expression vector. |
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| Figure 4. siRNA
Expression Plasmids Under Transient Selection to Induce
Gene Silencing. HeLa
cells were plated at 60,000 cells per well and then co-transfected,
using 1.2 µl FuGENE 6 (Roche) according to the manufacturer's
protocol, with 400 ng GFP-expressing plasmid and 400 ng
of each of 6 different siRNA expressing plasmids containing
a selectable marker (Ambion) and encoding an siRNA targeting
GFP. The cells were placed under selection at 24 hours
post transfection using 300 µg/ml Hygromycin, 2.5 µg/ml
Puromycin, or 2000 µg/ml G418. At 48 hours post transfection,
the antibiotic containing media was removed and replaced
with normal growth media. At 72 hours post transfection,
the cells were harvested and analyzed. Blue: DAPI
stained nuclei. Green: GFP. |
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| Figure 5. Silencing
of Gene Expression by PCR-based siRNA Expression Cassettes. A. Schematic
representation of the PCR strategy used to yield the PCR-based
siRNA expression cassettes [Castanotto et al., (2002) RNA 8:1454]. B. Inhibition
of exogeneous EGFP expression using siRNA-encoding PCR
cassettes. HeLa cells were co-transfected with PCR cassettes
encoding an EGFP or scrambled control siRNA and a target
EGFP expressing plasmid and analyzed by fluorescence microscopy.
High levels of EGFP expression can be detected in control
cells, but not in cells transfected with EGFP siRNA-encoding
PCR products. C. Inhibition of endogeneous human
GAPDH expression using siRNA-encoding PCR cassettes. PCR
cassettes encoding a human GAPDH and a scrambled control
siRNA were transfected into HeLa, DU145, and HEK 293 cells
and analyzed by Northern Blot. GAPDH expression levels
were reduced by >80% in cells treated with GAPDH siRNA-encoding
PCR cassettes over control cells. |
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| Figure 6. Comparison
of siRNAs Prepared by Chemical Synthesis vs RNase III Digestion
of Long dsRNA. A population
of siRNAs targeting GAPDH was prepared by synthesizing
and purifiying a 200 bp dsRNA, corresponding to the first
200 bases downstream of the AUG start site of GAPDH mRNA,
with the MEGAscript RNAi Kit (Ambion) and then cleaving
15 µg of the transcript with 15 U RNase III (Ambion;
Panel A). HeLa cells were plated at 30,000 cells per well
in a 24 well tissue culture plate on glass cover slips.
The cells were transfected 24 hours later with 100 nM,
50 nM, 25 nM, or 12.5 nM final concentration of the siRNA
population or a chemically synthesized siRNA known to efficiently
silence GAPDH. The cells were harvested after 48 hours
and the reduction in protein levels was examined by immunofluorescence
microscopy. The level of GAPDH in the cells was reduced
efficiently by siRNAs prepared by both chemical synthesis
and RNase III digestion. |
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