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Retroviral-mediated Delivery of siRNA
Enhanced
siRNA Delivery and Long-term Gene Silencing
The pSilencer™ 5.1 Retro System
enables researchers to study the long-term effects of reducing
the expression of specific genes in cell culture models. These
retroviral vectors make use of H1 or U6 Polymerase III promoters
to stably express siRNAs, even in difficult-to-transfect cell
types. This technology expands RNA interference research to address
long-term alteration of the expression of genes that cause disease
phenotypes.
Introduction: Getting siRNAs into Cells with Viruses
The use of siRNAs to induce gene silencing has enormous potential for identifying
and understanding gene function and may eventually be used as a therapeutic
strategy. Approaches to produce siRNA molecules include chemical synthesis,
enzymatic synthesis, and DNA-based expression vectors. In general, production
of siRNA in vitro by chemical synthesis is preferred for most transient experiments.
However, DNA-based approaches have distinct advantages when performing long-term
RNAi studies or when conducting studies in cells or organisms into which
siRNAs are particularly difficult to deliver.
The DNA-mediated siRNA expression techniques
can be divided into viral and nonviral methods. Both involve
the expression of a short hairpin RNA transcript that is processed
endogenously to an siRNA. Although the nonviral mediated approach
is effective for transient RNA interference experiments, low
transfection efficiency is a commonly encountered obstacle for
production of stable siRNA expressing
cell lines, and it presents severe limitations for performing
animal studies. (In general, chemically synthesized siRNAs are
a better choice for transient experiments in most immortalized
cell lines.) In contrast, viral approaches facilitate effective
siRNA delivery into many difficult-to-transfect cell types, including
primary cells, because infection is a much more efficient delivery
mechanism than transfection. In addition, since retroviruses
typically integrate into the host’s genome, long-term,
stable reduction of gene expression is relatively easy to achieve
with these constructs.
Several groups, including Ambion, offer adenoviral,
retroviral, and other viral vectors for siRNA expression. For
example, Ambion's pSilencer adeno
1.0-CMV System, Cat# 5790, offers an adenoviral-mediated
approach to deliver siRNA into cells and animals. pSilencer adeno
vectors are ideal for transient siRNA expression in hard-to-transfect
cells and in animal tissue.
Now Ambion introduces a retroviral system (pSilencer 5.1
Retro System) that facilitates stable siRNA expression for studies
in mammalian systems (see sidebar, Production of Retroviruses
as a Research Tool).
The pSilencer retroviral
vectors effectively induce gene silencing through efficient siRNA
delivery into a wide variety of cells, including hard-to-transfect
primary cells. This system is also ideal for long-term, stable
knockdown of gene expression, which is critical for understanding
and studying how reducing expression of disease-relevant genes
cause disease phenotypes over extended periods of time.
This article describes how Ambion’s retroviral
system was used to elicit RNAi in both transformed and primary
cell types. Strong gene silencing was observed even in an extremely
difficult-to-transfect primary cell type, NHDF-neo cells. The
pSilencer 5.1 Retro System was also used for long-term
stable reduction of gene expression in an experiment continued
for over eight months. Surprisingly, some changes in cellular
morphology and differences in growth characteristics became evident
only after very prolonged gene silencing.
Choice of Promoter Allows Optimal Gene Knockdown
Ambion provides two pSilencer 5.1 Retro
vectors—one that features the
polymerase III U6 promoter, and one that includes the polymerase III H1 promoter.
siRNAs are expressed differentially from these promoters; thus the choice of
promoter can affect the level of gene silencing. To demonstrate this, inserts
encoding siRNA hairpins targeting several genes including cyclophillin A, GAPDH,
ATM, BRCA-1, and CDK2 were cloned into both U6 and H1 promoter-containing pSilencer
retroviral vectors. HeLa cells infected with the resulting viruses were placed
under selection with puromycin. Surviving cells were analyzed for reduction
in mRNA levels. Figure 1A demonstrates that mRNA expression reduction differences
were dependent on which promoter was driving siRNA expression. For example,
mRNA levels for ATM and cyclophilin were more strongly reduced when their respective
siRNA inserts were expressed from the H1 promoter compared to the U6 promoter.
Conversely, BRCA-1 mRNA levels were more strongly reduced when the U6 promoter
was used to express BRCA-1 siRNA compared to the H1 promoter.
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Figure 1. Gene
Silencing After Retroviral Delivery of siRNA. Retroviral
vectors expressing the indicated siRNA inserts from
either the H1 or U6 promoter were transfected into
a retrovirus packaging cell line in 6 well plates.
Three days following infection, virus was collected
and used to infect HeLa cells in triplicate. (A) Infected
HeLa cells were placed under puromycin selection (4
days). After two months of continued growth, RNA was
purified from the cells and analyzed by real-time PCR
for target gene expression levels. Data were expressed
relative to cells expressing negative control siRNA.
Standard deviations were all below 5%. (B) The expression
level of CDK2 siRNA regulated by the H1 promoter (H1CDK2)
was monitored using the mirVana™ miRNA
Isolation and Detection Kits (Ambion). |
In order to measure siRNA expression
levels in infected cells, small RNAs were purified using the mirVana™ miRNA
Isolation Kit (Ambion), and the specific siRNA was identified
with the mirVana
miRNA Detection Kit (Ambion), a solution-based hybridization assay. As
observed in Figure 1B, CDK2 siRNA was robustly expressed. (Note: The correlation
between siRNA expression level and subsequent target gene knockdown will
differ across siRNAs studied. This is because some siRNAs are more efficient
at eliciting effective silencing than others.)
To achieve the greatest amount of siRNA expression and consequent target gene
silencing, it may be advantageous to analyze both promoter systems for each
gene being studied. Ambion’s new pSilencer 5.1 Retro System provides
vectors with each of the polymerase U6 and H1 promoters for this purpose. Because
both vectors use the same restriction sites, siRNA-encoding inserts can be
readily cloned from one to the other.
Knockdown of Gene Expression in Primary Cell Lines
In addition to transformed cells, target
gene silencing was also observed in a primary cell line that
is ordinarily very difficult to transfect—NHDF-neo
cells. Retroviruses expressing either a negative control siRNA or an siRNA
targeting cyclophilin were generated and used to infect HeLa cells and a
primary NHDF-neo cell type (NHDF-neo cells were plated in 24 well plates
in the presence of 2 µg/ml of Sequa-brene™ (Sigma-Aldrich) to
enhance viral uptake). Infected cells were placed under selection with puromycin
at 1, 1.5, and 2 µg/ml, respectively. A control infection was also
performed with packaging cell supernatant that was not transfected with viral
DNA.
Once control cells were killed by puromycin,
the experimental cells were removed from antibiotic selection
(~4 days). A fraction of the cells was harvested to analyze cyclophilin
expression in cells that received the cyclophilin siRNA relative
to cells that received the negative control siRNA. The remaining
cells were propagated in long-term culture. pSilencer retroviral
vectors expressing cyclophilin siRNA effectively reduced cyclophilin
mRNA levels in both cell types, indicative of effective siRNA
delivery and expression (Figure 2). In fact, gene silencing of
cyclophilin was more effective in the primary NHDF-neo cells
than in the HeLa cells, suggesting that certain inserts may be
more effective in one cell line compared to another.
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Figure 2. Retroviral
Delivery Used to Elicit RNAi in Primary Cells. Retrovirus
expressing cyclophilin A siRNA or a negative control
siRNA (nc) from the H1 promoter were transfected into
a packaging cell line in 6 well plates in triplicate.
Three days post-transfection, virus was collected and
used to infect either HeLa cells or NHDF-neo cells.
Cells were placed under selection (4 days), and after
two months of culture, cyclophilin expression was analysed
by real-time PCR. Cyclophilin expression in these cells
was compared to expression levels in cells infected
with virus expressing the negative control siRNA. Standard
deviations were below 5%.
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In this experiment, NHDF-neo cells with reduced cyclophilin
mRNA levels became “sick” (showed increased cell
death), and after two months it was no longer possible to propagate
the cells; remaining cells were cultured until they eventually
died. In contrast, HeLa cells with reduced cyclophilin expression
did not show the same level of cell death and could still be
expanded, but the cells exhibited morphological changes (Figure
3C). The morphological differences between the primary NHDF-neo
cell cultures and transformed HeLa cell line could be due to
differences in cyclophilin reduction, since cyclophilin levels
were reduced more effectively in NHDF-neo cells compared to HeLa
cells. They could also be due to a biological difference between
the two cell types; e.g. their ability to overcome or adapt to
reduced cyclophilin expression. Additional experiments using
cloned populations of infected cells expressing different levels
of siRNA will be required to distinguish between these two scenarios.
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Figure 3. Long-term
Reduction in Target Gene Expression Results in Morphological
Changes. Retroviral DNA, expressing cyclophilin
A, BRCA 1, or a negative control siRNA from the H1
promoter, was transfected into a packaging cell line
in a 6 well dish in triplicate. Three days later virus
was collected and used to infect HeLa cells. The HeLa
cells were placed under puromycin selection (4 days).
(A) Cells were periodically analyzed over an 8 month
period for gene reduction. Data are depicted as the
percent of gene expression compared to cells expressing
the negative control siRNA (nc), and standard deviations
were below 5%. (B) Cells were also analyzed for expression
of siRNA inserts (8 month time point) using the mirVana™ miRNA
Detection Kit (Ambion). (C) Morphological changes were
observed for cells expressing cyclophilin and BRCA1
siRNAs after 8 months of culture post selection. |
Stable Knockdown of Gene Expression
Long-term stable reduction of gene expression
by siRNAs has the potential to mimic specific disease states
in which a gene is not expressed or is mutated. To further examine
the effects of long-term stable knockdown of gene expression,
pSilencer 5.1–H1 Retro vectors expressing siRNAs targeting several
genes, including cyclophilin and BRCA1, were prepared. After infecting and
selecting cells, qRT-PCR was used to confirm target mRNA reduction, and cells
were grown and passaged for eight months.
Several interesting observations about cell
morphology were noted. Morphological changes were not initially
evident even in cells where target gene expression had been reduced
for 1–2
months; however, cells in which expression of cyclophilin and
BRCA1 had been reduced long-term began to show significant phenotypic
differences compared to a stable cell line expressing the negative
control siRNA (Figure 3C). Although BRCA1 mRNA levels were 60%
of that observed in the cells expressing a negative control siRNA
(Figure 3A, B), pronounced morphological phenotypes arose in
the BRCA1 siRNA-expressing cells. Thus, relative reduction of
mRNA levels by 70% or more is not always necessary to achieve
a morphological response. Indeed, long-term reduction of gene
expression to more modest levels may mimic the effects seen with
heterogeneous disease causing alleles such as BRCA1, where only
one of the alleles needs to be mutated to see a disease phenotype,
and which often takes extended time to develop.
pSilencer Retro System
The data presented here highlight the utility of using retroviral-mediated
delivery of siRNA for long-term reduction in gene expression in transformed
and primary cell cultures. The pSilencer 5.1 Retro System comes linearized
and ready for cloning. For convenience, the new vectors have restriction
sites that are compatible with the original series of pSilencer plasmid vectors.
This system enables researchers to perform long-term gene silencing studies
to expose cellular phenotypes that are not detectable in transient or short-term
experiments.
Scientific Contributors
Lance P Ford*, Kevin Kelnar, and Lesslie
Beauchamp • Ambion, Inc.
*Corresponding Author: lford@ambion.com
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