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Your Data: Silencing Hepatitis C Virus Replication
With In Vitro Transcribed siRNAs
The Silencer™ siRNA
Construction Kit uses an in vitro transcription reaction and
column purification to prepare transfection-ready siRNAs. With
this kit, multiple siRNAs can be prepared in ~24 hours. Below
is one example of how siRNAs prepared by the Silencer siRNA
Construction Kit were used in gene silencing studies.
Kapadia S, Brideau-Anderson A, and
Chisari FV (2003) Interference of hepatitis C virus RNA replication
by short interfering RNAs. Proc Natl Acad Sci
USA 100: 2014-2018.
Dr. Sharookh B. Kapadia and colleagues at
the Scripps Research Institute used siRNAs prepared with
the Silencer siRNA Construction Kit to demonstrate that
RNAi can be used to inhibit Hepatitis C Virus (HCV) replication.
The researchers generated multiple siRNAs targeting various regions
of the HCV subgenomic replicon sequence. These siRNAs were transfected
into an Huh-7 cell line that stably expressed the HCV RNA replicon
and screened for their gene silencing activities
by real-time RT-PCR. Two of the siRNAs, NS3-1948 and NS5B-6133,
showed the greatest specific inhibition of HCV RNA replication
and were tested further for their ability to suppress HCV RNA
and protein expression. As shown in Figure 1, HCV protein expression
levels were significantly reduced on Day 4 (Figure 1B) and Day
6 (Figure 1C) post-transfection. Similar results are seen at
the mRNA level (Figure 2). Viral replication was also inhibited.
These results suggest that RNAi might represent a new approach
for the treatment of HCV infection.
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Figure 1. The
Effect of RNAi on HCV Protein Expression in S11791 Cells. Western
blot analysis was performed on total cell lysates harvested from
mock- or siRNA-transfected S11791 cells at various days post-transfection
by using NS3- and NS5B-specific antibodies. Molecular mass markers
(in kilodaltons) are shown to the right of each gel. Shown are representative
data of three independent experiments. |
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2. Effect
of HCV-specific siRNAs on HCV and GAPDH RNA Transcript
Levels. Total RNA
was isolated from S11791 cells that were transfected
with siRNA specific for NS3, NS5B, GAPDH, or an irrelevant
scrambled (scr) siRNA 2, 4, and 6 days previously. Equal
amounts of RNA were separated on a denaturing agarose
gel, transferred to membrane and hybridized with probes
to HCV and GAPDH. GAPDH signal was used for normalization
of the HCV signal. |
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