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Superior Gene Silencing Using Adenoviral Vectors
Mediating RNA interference (RNAi) with recombinant
adenovirus expressing siRNAs has only recently been described
in the literature (1). Recombinant adenoviruses have several
advantages over other methods of nucleic acid delivery. Namely,
these viruses can infect dividing and non-dividing cells, and
can be used in animal model systems. They can infect many difficult-to-transfect
primary and transformed cell lines via their receptor mediated
delivery mechanism. The sequence of a chemically synthesized
siRNA capable of gene specific silencing can also be functionally
expressed from recombinant adenovirus, often at a lower cost.
Once made, the virus can be expanded, making it possible to
generate "stocks" for future experiments.
How Does pSilencer™ Adeno 1.0-CMV
Work?
An oligonucleotide insert encoding the
desired siRNA is cloned into the pre-linearized shuttle vector
(provided), which includes a modified CMV promoter for efficient
expression of the siRNA sequence. The shuttle vector, containing
the desired sequence, is then linearized and introduced into
HEK 293 packaging cells (not included) along with the linearized
adenovirus backbone (Figure 1). The HEK 293 packaging cells
contain the EIA and EIB genes which are not present in the
introduced vectors and are required to form infectious particles.
Inside the HEK 293 cells, the shuttle vector and the adenoviral
backbone plasmid recombine to produce an adenoviral genome
including the siRNA expression cassette. The siRNA expression
cassette is packaged into infectious virus particles that can
be harvested for experiments. Harvested adenoviral particles
can then be used to infect cells or animals, or stored for
future experiments.
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Figure 1. Schematic
of pSilencer Adeno 1.0 CMV
System Recombination and Packaging. The
shuttle vector and backbone plasmid are linearized
and then transfected into HEK 293 cells. The HEK 293
packaging cells contain the EIA and EIB genes which
are not present in the introduced vectors and are required
to form infectious particles. Inside the cell, the
plasmids recombine forming a functional genome that
is replicated and packaged into adenoviral particles.
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siRNA from Adenoviral Vectors Reduce Expression
from Transgenic Alleles in vivo
To demonstrate that adenoviruses are
useful for decreasing gene expression via the RNAi pathway
in animals, Dr. Beverly Davidson and colleagues, our collaborators
at the University of Iowa, injected recombinant adenoviruses
expressing siRNAs targeting green fluorescent protein (GFP)
or ß-glucuronidase (a negative control) into the brains
of transgenic GFP-expressing mice (1). Five days after injection,
brain sections were analyzed by fluorescence microscopy and
Western blot (Figure 2). GFP expression was reduced in the
injected brain hemisphere, indicating the utility of the method
for reducing gene expression in animals.
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Figure 2. Silencing
of Transgenic GFP in Mouse Brain with an Adenoviral
Vector (1). Adenovirus
engineered to express an siRNA targeting GFP (A, top
panels) or a negative control siRNA targeting human ß-glucuronidase
(A, bottom panels) were injected into the brain striatal
region of transgenic mice expressing GFP. The adenovirus
included a dsRed expression cassette to visualize the
localization of the virus. (A) Brain sections
were analyzed by fluorescence microscopy 5 days after
injection. Green: GFP. Red: dsRed. Left panels: GFP
detection. Middle panels: dsRed detection. Right panels:
merged images. (B) Brain sections were divided
into ipsilateral (il) and contralateral (cl) portions
and analyzed by Western blot using antibodies to GFP
and ß-actin (control).
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Allele-specific Silencing of Dominant Disease
Genes
Due to its inherent specificity, a potential
application of siRNAs is the treatment of dominantly inherited
diseases. One such neurodegenerative disease is Machado-Joseph
disease/spino-cerebellar ataxia type 3. In 70% of diseased
carriers, alleles contain a G to C mutation. Dr. Davidson's
laboratory constructed plasmids fusing GFP to normal (Atx-Q28-GFP)
or mutant polyglutamine (polyQ) alleles (Atx-Q166-GFP) containing
a CAG expansion in Ataxin-3 (2). Adenoviral vectors that expressed
hairpin siRNAs targeting either the wild type or the mutant
allele were designed generated from these plasmids and used
to infect Cos-7 cells that had previously been transiently
co-transfected with Atx-Q28-GFP (wt) and Atx-Q166 (mutant).
(Both plasmids were co-transfected into the Cos-7 cells to
mimic the heterozygous adenovirus.) The siRNA expressed from
AdG10i had perfect complementarity to the wild type allele,
and preferentially suppressed expression of the fusion construct
containing that allele (Atx-Q28-GFP, Figure 3A). Conversely,
AdC10i preferentially suppressed expression of the mutant allele
construct (Atx-Q166, Figure 3A). Finally, these two adenoviral
vectors were used to infect PC12 cells that had been created
to express normal (Q28) or (Q166) mutant ataxin-3. Again, the
viruses were able to specifically target the mutant allele
(Figure 3B). These results indicate the utility of viruses
for delivering siRNAs that target allele-specific disease genes.
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Figure 3. Allele-specific
Silencing with Adenoviral Vectors Expressing an siRNA
(2). Cos-7 cells
were co-transfected with the Atx-3-Q28-GFP (wt) and
Atx-Q166 (mutant) constructs and then infected (MOI
= 50) with adenovirus expressing siRNAs that had been
designed such that a single nucleotide polymorphism
(SNP) was in the middle of the target sequence. The
siRNA produced by Ad-G10i was perfectly complementary
to the wild type construct, whereas that of Ad-C10i
was complementary to the mutant construct. (A) Green:
GFP. Red: immunofluorescence of the polyQ construct
detected with 1C2 antibody. Ad-G10i reduced wt ataxin-3
levels 95% but did not affect mutant ataxin-3 levels.
Conversely, Ad-C10i reduced mutant ataxin-3 levels
91% but had no effect on WT ataxin-3 levels. (B) PC12
cells expressing WT (left) or mutant ataxin-3 (right)
were infected (MOI = 100) with the Ad-G10i or Ad-C10i
virus. Western analysis was performed with antibodies
to Ataxin-3 and GAPDH (as a control), and the siRNAs
produced by the viruses were able to specifically silence
the allele to which they were designed.
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The Complete Adenoviral Production System
The pSilencer adeno 1.0-CMV System
includes everything needed to produce five preparations of
recombinant adenovirus, except the siRNA template oligonucleotide
and the HEK 293 packaging cells (HEK 293 cells are available
from several sources, including ATCC). The kit includes linearized
Shuttle Vector 1.0-CMV (20 rxns), Negative Control Shuttle
Vector that encodes a scrambled siRNA sequence, a Positive
Control Oligonucleotide Insert that encodes an siRNA targeting
GAPDH, and the Adenoviral Backbone that includes a LacZ
sequence for monitoring transfection efficiency. Also included
are reagents for transfecting the HEK 293 cells with the Shuttle
and Backbone Vectors, forward and reverse sequencing primers
to verify clones, and 5X Annealing Buffer for preparing the
siRNA-encoding oligonucleotides for ligation.
The use of these materials is permitted for
research purposes only. Any other use requires a license from
the University of Iowa Research Foundation, 214 Technology
Innovation Center, Iowa City, IA 52242.
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