Neuroscience 126 (2004) 1–7
COMMENTARY
LOBOTOMY OF GENES: USE OF RNA INTERFERENCE IN
NEUROSCIENCE
T. HOLENa* AND C. V. MOBBSb
Although only discovered within the last decade, RNAi has
been used to reduce mRNA levels in hundreds of studies
in different species. In this commentary we highlight the
great potential, but also the possible pitfalls, for the use of
RNAi in neuroscience research. The rapid exploration of
RNAi the last 5 years has left many issues unaddressed,
and those who decide to use this technique may encounter
difficulties of a technical nature. Thus this commentary
focuses on experimental design using RNAi, but also aims
to give a starting point for further studies of RNAi and the
RNAi literature when the inevitable technical difficulties
arise with this potent, but yet still developing tool.
a
The National Hospital and University of Oslo, Center for Molecular
Biology and Neuroscience, P.b. 1105 Blindern, 0317 Oslo, Norway
b
Box 1639, Mt. Sinai School of Medicine, One Gustave Levy Pl., New
York, NY 10029, USA
Abstract—Galen of Pergamon studied nerve function by
shearing nerves in various species including monkeys, dogs,
bulls and even elephants (humans being off limits to researchers; Sarton, 1954). An analogous strategy to determine
gene function by ablating gene expression has recently been
developed. RNA interference (RNAi) is a cellular response to
double-stranded RNA (dsRNA) apparently as a defense
against viral or transposon activity (Denli and Hannon, 2003;
Dykxhoorn et al., 2003; Plasterk, 2002; Zamore, 2002). By
activating this ancient defense mechanism through the introduction of artificial dsRNA, it is now possible to inhibit expression of almost any gene in almost any cell type, among
them neuronal cells. In mammalian cells the active RNAi
species must be short, approximately 21 nucleotide RNAs;
these 21-bp species are called short interfering RNA (siRNA;
Fig. 1). © 2004 IBRO. Published by Elsevier Ltd. All rights
reserved.
RNAi reduces gene expression in neurons:
importance of delivery
In early studies in C. elegans there was evidence that
RNAi, while reducing gene expression in neurons, was not
as effective in neuronal tissue as in other tissues (Timmons et al., 2001; Timmons and Fire, 1998). However,
with the development of C. elegans strains that were more
sensitive to RNAi (Simmer et al., 2002), it became clear
that RNAi could indeed be used to reduce gene expression
in neurons (Bianchi et al., 2003). RNAi has now been used
to reduce gene expression and to study gene function in
neuronal tissues of several invertebrates, including Drosophila (Dearborn Jr. et al., 2002; Dzitoyeva et al., 2003;
Eaton et al., 2002; Kalidas and Smith, 2002; Schindelholz
et al., 2001; Yang et al., 2003) planaria (Cebria et al., 2002;
Gehring, 2002), and Aplysia (Korneev et al., 2002).
A major technical obstacle initially prevented effective
use of RNAi in mammals: in contrast to invertebrates, in
mammals long (greater than 30 bp) dsRNA produces a
PKR/interferon/response causing non-specific effects including general reduction in protein synthesis and even
cell death (Caplen et al., 2001; Williams, 1999). However,
dsRNA if shorter than 30 bp, can activate the RNAi response and reduce gene expression (Brummelkamp et al.,
2002; Castanotto et al., 2002; Jacque et al., 2002; Lee et
al., 2002; Miyagishi and Taira, 2002; Paddison et al., 2002;
Paul et al., 2002; Sui et al., 2002; Yang et al., 2002; Yu et
al., 2002). Therefore in mammals a common way to activate RNAi is to simply infuse synthetic siRNA (21–23 bp)
dsRNA species. An alternative approach is to infuse a
plasmid whose transcript produces a short dsRNA species. For a plasmid to produce short RNA species, transcription is driven by promoters that use RNA polymerase
III, which terminates transcription of a transcript after 5
Key words: siRNA, RNA interference, RNAi, neuroscience.
Contents
RNAi reduces gene expression in neurons: importance of
delivery
RNAi versus antisense oligonucleotides
A strategy for developing effective RNAi constructs
Plasmid-produced shRNAs
SiRNA
Designing RNAi constructs
Testing efficacy and specificity of RNAi
Verification
References
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*Corresponding author. Tel: ⫹47-2285-1018; fax: ⫹47-952-777-36.
E-mail address: torgeir.holen@basalmed.uio.no (T. Holen).
Abbreviations: AGRP, agouti-related protein; Dicer, a central protein in
RNAi: produces siRNA from dsRNA and shRNA, or microRNA from
longer transcripts from micro genes; dsRNA, double-stranded RNA,
mainly occurs due to viral infection and therefore produces an anti-viral
response, generally cleaved into short interfering RNA by Dicer; GFP,
green fluorescent protein; Nt, nucleotide(s); RNAi, RNA interference,
an immune system of the genome that recognizes and destroys target
sequences; shRNA, short hairpin RNA: a short RNA transcribed inside
cells, that can be processed to an active siRNA; siRNA, short interfering RNA: the active agent of RNAi, consists of approximately 21 nt
RNA strands, in duplex or single-stranded form.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2004.03.008
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T. Holen and C. V. Mobbs / Neuroscience 126 (2004) 1–7
Fig. 2. Putative pathway for shRNA. Transcription of the selfcomplimentary hairpin-transcript is performed by DNA polymerase III,
which release the shRNA-transcript after five uridines, cleaving after
the second uridine. The hairpin transcript is then processed into
siRNA, possibly by the nuclease Dicer, losing the loop and possibly
superfluous nucleotides at the 5⬘ and 3⬘ ends.
Fig. 1. A simplified schematic showing a cell with parts of the RNAi
and siRNA mechanism. When dsRNA appear, e.g. due to virus or
transposon activity, a cellular immune mechanism is activated and
siRNA are produced. These siRNA are incorporated the protein complex RISC (RNA induced silencing complex: an RNA:protein complex
that recognizes and cleaves the mRNA target) and RNAs containing
sequences corresponding to the siRNAs sequence, such as mRNA or
virus genomes, are depleted. This is exploited by introducing synthetic
siRNA to deplete a specific gene of interest, e.g. by transfection.
successive thymidines. The transcript is then cleaved after
the second uridine (Brummelkamp et al., 2002; Castanotto
et al., 2002; Jacque et al., 2002; Lee et al., 2002; Miyagishi
and Taira, 2002; Paddison et al., 2002; Paul et al., 2002;
Sui et al., 2002; Yang et al., 2002; Yu et al., 2002). To
make a short dsRNA species, the plasmid is designed with
complementary strands of the target sequence separated
by short non-target sequence (see below). The transcript
will therefore form a short hairpin loop, so the construct is
called shRNA (Lee et al., 2002; Miyagishi and Taira, 2002);
eventually the shRNA is cleaved to form a final siRNA
product (Fig. 2).
siRNA has now been used to reduce gene expression
in cell lines, including cells derived from neuroblastoma
(Fukumoto et al., 2002; Torocsik et al., 2002; Zhang et al.,
2003) and glioblastoma cells (Asai et al., 2003), that are
similar to those found in the nervous system. Some studies
have also used siRNA to reduce gene expression and
study gene function in primary cultures of mammalian
neurons (Gaudilliere et al., 2002; Krichevsky and Kosik,
2002) and glial cells (Nicchia et al., 2003).
Whereas RNAi in some invertebrates is so robust that
even systemic application, for example by feeding, reduces gene expression throughout the organism, this
widespread reduction does not occur in mammals. Therefore the use of RNAi in the whole organism, as opposed to
in vitro, requires direct delivery of the RNAi reagent to the
area of interest. The technical feasibility of using RNAi to
reduce expression of transfected genes in neuronal tissue
was first demonstrated in mouse embryos using electroporation of siRNA (Calegari et al., 2002), and using
adenoviral-mediated transfer to produce shRNA (Xia et al.,
2002), though in these studies it was not demonstrated if
the effect was observed in neurons in vivo.
However, RNAi has also now been shown to be effective in reducing expression of neuronal genes in vivo in
adult mammals (Makimura et al., 2002; Hommel et al.,
2003; Matsuda and Cepko, 2004). In the first such demonstration, liposome-mediated delivery to the hypothalamus of plasmid-based shRNA, complimentary to the
agouti-related protein (AGRP) gene, reduced hypothalamic AGRP mRNA and protein by about 50%, and led to
the expected reduction in body weight and increase in
metabolic rate (though interestingly not the expected decrease in food intake; Makimura et al., 2002). Since AGRP
is only expressed in neurons, this study demonstrated that
RNAi can be used to study the physiological function of a
gene expressed in neurons in adult mammals. The same
study also indicated the siRNA could be similarly effective
in reducing neuronal gene expression (Makimura et al.,
2002). In a subsequent study an shRNA construct directed
against tyrosine hydroxylase (expressed in neurons) was
delivered to substantia nigra by an adeno-associated viral
vector (Hommel et al., 2003). As with the AGRP study, the
shRNA construct directed against tyrosine hydroxylase reduced expression of the targeted gene and produced the
expected phenotype (Hommel et al., 2003). Finally, shRNA
was delivered to rodent photoreceptor cells by electroporation (Matsuda and Cepko, 2004), and again expression
of the targeted genes, neuronal transcription factors, was
reduced. On the other hand, in a recent report, siRNA
constructs that were effective in vitro were not effective
when infused into rat brain without transfection reagents
(Isacson et al., 2003). Since a previous report suggested
that even without a transfection reagent siRNA could be
effective (Makimura et al., 2002), the efficacy of siRNA in
mammalian brain probably depends on the specific construct, which vary widely in efficacy and even if effective in
vitro may not be effective at the lower cellular concentration obtained in vivo. Precise method of delivery and location in the brain may also influence the efficacy of siRNA.
The importance of delivery has been examined in more
detail in the liver. A single (high pressure) tail vein injection
of a plasmid which produces a dsRNA complimentary to
green fluorescent protein (GFP) reduces GFP in liver of
GFP-transgenic mice by about 50% (Makimura et al., unpublished observations). Detailed analysis demonstrated
T. Holen and C. V. Mobbs / Neuroscience 126 (2004) 1–7
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that before tail vein injection, nearly 100% of hepatocytes
of these GFP-transgenic mice express GFP. After tail vein
injection only about 50% of the hepatocytes express GFP,
but of those that do express GFP, the signal intensity is
about the same as in mice injected with a control plasmid.
These data suggest that the limiting factor in inhibition of at
least hepatic gene expression by RNAi is delivery: in cells
in which the plasmid is actually taken up, the target RNA
appears to be almost completely eliminated. Similar results
have been reported, in which a similar protocol for RNAi
delivery by tail vein injection was used to reduce transgenic luciferase gene expression in vivo in liver of adult
mice (McCaffrey et al., 2002). As a practical matter, because non-viral methods of transduction in vivo are not as
efficient as viral methods, reduction of gene expression by
much greater than 50% may be unrealistic for most experiments, unless viral transfection methods are used.
cantly reduce gene expression is greater than for antisense oligonucleotides. However, it remains to be demonstrated that this is true in mammalian systems, since the
21-base-pair constructs used for mammalian systems are
less robust than the full-length constructs used in nonmammalian systems.
However, a clear advantage of RNAi is that it is possible to use plasmid-based systems to produce long-term,
even permanent, expression of the active construct,
whereas anti-sense constructs must be delivered as oligonucleotides whose activity is relatively transient (on the
order of hours). Therefore RNAi can be implemented in a
plasmid-based system to study long-term phenotypes after
gene transfer of the RNAi construct. This strategy has now
been successfully used in mammalian brain (Hommel et
al., 2003).
RNAi versus antisense oligonucleotides
Based on these considerations, the following strategy can
be recommended for using RNAi to study gene function in
the mammalian nervous system. Although in principle the
same issues apply to using RNAi in invertebrate systems,
the relative lack of robustness of RNAi in vertebrate systems necessitates more stringent controls. First, the investigator must decide if synthetic double-stranded siRNA or
plasmid-based shRNA will be used. While siRNA may be
somewhat more robust than anti-sense oligonucleotides,
this has yet to be convincingly demonstrated in neurons in
adult mammalian brain (Isacson et al., 2003). A comparison of the two technologies performed by Vickers et al.
(2003), working at ISIS Pharmaceuticals, a company developing anti-sense technology, indicated that the most
developed forms of antisense technology might be as robust as siRNA in reducing mammalian gene expression
in vitro. Furthermore, like anti-sense oligonucleotides
dsRNAs must be produced and delivered anew for every
experiment, and the effects are quite transient, thus for
longer term studies may require chronic infusion, a possibly prohibitively expensive proposition.
Thus for initial phases of a study (e.g. screening for
sequences that produce effective down-regulation, see below) siRNA might save time, especially for those with little
experience in cloning, but RNAi in mammalian neurons,
especially in vivo, is probably most fully exploited using a
plasmid-based shRNA. shRNA may require somewhat
more effort in the first phase, to produce the plasmid, but
thereafter more construct can be made very easily and
cheaply, and the plasmid can be designed to produce
much more sustained down-regulation. Furthermore, since
delivery of the construct may ultimately constitute the limiting factor in the robustness of the down-regulation (Isacson et al., 2003). Ultimately plasmid delivery can be enhanced by a number of protocols, including packaging into
viral vectors.
Most investigators are interested in RNAi mainly as a
method to specifically reduce expression of a gene of
interest in order to assess the physiological function of that
gene, similar to the use of targeted gene ablation. For
many years, a similar strategy, using anti-sense oligonucleotides, has been used to study gene function in neurons
and other tissues (Abraham et al., 1997; Agrawal, 1999;
Lewis et al., 2000; McCarthy et al., 2000; Schobitz et al.,
1997; Van Oekelen et al., 2003). Such ablation techniques
are essentially pharmacological manipulations, the value
of which depends on efficacy and specificity. For example,
as with any pharmacological intervention, anti-sense oligonucleotides can produce effects that are independent of
reduction in expression of the target gene (Abraham et al.,
1997; Schobitz et al., 1997). Therefore the use of antisense oligonucleotides requires the use of controls that
address the issue of specificity, usually involving the use of
oligonucleotides with similar base composition but altered
sequence (McCarthy et al., 2000). Another key element of
studies using anti-sense oligonucleotides is the demonstration that expression of the targeted gene (especially
the protein encoded by the gene) is actually reduced, and
that the effect is specific to the targeted gene. Similar
issues must be addressed in studies using RNAi (see
below).
While the use of RNAi and the use anti-sense oligonucleotides entail strategically similar considerations, the two
approaches entail different advantages. A key advantage
of using anti-sense oligonucleotides is that the technology
is more mature than the technology of RNAi, and therefore
the essential reagents are currently less expensive and
time-consuming to produce. A potential advantage of using
RNAi rather than anti-sense oligonucleotides is that RNAi
may provide more robust inhibition than anti-sense oligonucleotides, in the sense that RNAi may produce inhibition
at lower concentrations and may therefore result in fewer
non-specific effects at effective doses. In invertebrates
RNAi is also probably more robust in the sense that when
plasmid-based delivery systems covering the entire gene
are used, the likelihood that a given construct will signifi-
A strategy for developing effective RNAi constructs
Plasmid-produced shRNAs
While all shRNAs are roughly similar, entailing two complementary strands separated by a non-coding loop, they
differ in some details, including the number of bases in the
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T. Holen and C. V. Mobbs / Neuroscience 126 (2004) 1–7
linking loop (Fig. 2). Although Brummelkamp et al. (2002)
found that a nine nucleotide (nt) linking loop between the
two siRNA strands is more effective than a five nt or seven
nt loop, corroborated by Castanotto et al. (2002), a wide
range of loops connecting the two strands can also be
effective: Paul et al. (2002) and Paddison et al. used four
loops (UUCG and UUAA, respectively), while Yu et al.
(2002) and Yang et al. (2002) used three loops (AUG and
UCU, respectively). Sui et al. (2002) report that a six-loop
can be effective, while Jacque et al. (2002) found no
difference between three, five and seven loops. The
shRNA seems to be processed by a nuclease to siRNA,
whether longer mimics of let-7 hairpins, or simple, 29 bp
hairpins (Paddison et al., 2002). At the moment for standard applications a nine nucleotide linking loop can be
recommended.
An additional variation of shRNA has been the development of plasmids that allow expression to be regulated.
For example, Ohkawa and Taira (2000) constructed a RNA
polymerase III promoter that is regulatable by fusing this
promoter with a tetracycline response element. Others
versions has been developed by Agami and coworkers
(van de Wetering et al., 2003) and Matsukura et al. (2003).
This system will obviously find many applications for tissue
and time-point specific induction of RNAi silencing. A variation of shRNA may be the production of transgenic mice
with RNAi constructs (Carmell et al., 2003). Similarly, an
improved delivery system for shRNA, developed for in vivo
targeting of viral sequences (Lee et al., 2002), involves
delivery by lentivirus, a virus suitable for neuronal cells (An
et al., 2003; Stewart et al., 2003).
SiRNA
Since plasmid-based shRNA production requires transcription to produce dsRNA species, siRNA may be preferable if an immediate reduction of mRNA is necessary.
On the other hand, even with siRNA, there is a time-lag for
establishing silencing (Holen et al., 2002, 2003; McManus
et al., 2002). As indicated above, delivery of siRNA in brain
in vivo may also be problematic (Isacson et al., 2003);
efficient delivery of siRNA will possibly require extensive
chemical modification. Fortunately, despite siRNAs having
to interact with the effector proteins in RISC (Fig. 1), they
show surprisingly high tolerance for chemical modifications. For example, the 3⬘ ends of siRNA can be modified
to DNA without losing significant activity (Elbashir et al.,
2001a,b). A systematic study to find the limit of the tolerance to chemical modifications in various parts of the two
21-nt strands found a surprisingly high tolerance of modifications (Amarzguioui et al., 2003). Most of the chemically
modified versions tested had initial activity near the activity
of the unmodified form, while the modification enhanced
long-term activity of certain siRNA species. When more
extensive 2⬘-O-methylation was introduced, a gradual decline of activity was observed and ultimately destroying
activity of even the most robust candidate siRNA. Other
studies have also found tolerance for chemical modifications (Chiu and Rana, 2003; Czauderna et al., 2003; Harborth et al., 2003). Tolerance to modification has important
implications for siRNA design, in particular in development
of siRNA as a therapeutic agent, where modifications of
siRNA might be necessary for stabilization of siRNA in
tissues, for avoidance of side effects and for delivery to the
tissues of interest, among them neuronal tissues.
Designing RNAi constructs
A key consideration in the use of RNAi is that siRNAs vary
extremely widely in their abilities to activate the RNAi
mechanism, and therefore in their ability to inhibit RNA
expression. Therefore regardless of whether siRNA or
shRNA is used, RNAi constructs must be designed that
either are or will produce 21-base-pair dsRNAs corresponding to the targeted gene, and will efficiently activate
the RNAi mechanism. Several companies now synthesize,
and even design, candidate dsRNAs. Constructs with
base-pair mismatches, preferably at least three, constitute
crucial controls. As with anti-sense, however, a “standard”
control, such as an RNAi constructs directed toward GFP,
is also a valuable addition to RNAi experimental designs.
Although several investigators and companies have
developed rules that are claimed to optimize the efficacy of
RNAi constructs, these rules have not been clearly validated in the published literature and in our experience do
not substantially increase the frequency of successful
down-regulation. Initially, it was predicted that it would be
possible to cleave a target RNA at almost any position, and
that RNAi differed from other types of antisense molecules
by not having to search for the optimal target sites (Elbashir et al., 2001b; Stein, 2001). However, clear RNAi
target position effects were found when siRNA constructs
were systematically tested against multiple targets in tissue factor mRNA, from the start to the stop codon (Holen
et al., 2002). Several siRNA constructs in this screen were
found to be inactive, and the effect verified in four different
assays and four different cell-lines, showing that identification of accessible target sites is critical. This natural
variation in siRNA activity has also subsequently been
demonstrated by systematic screens by other groups (Kawasaki et al., 2003; Vickers et al., 2003). Mechanisms
mediating this remarkable variability are speculative, but
may involve internal stability profiles of the siRNA (Khvorova et al., 2003; Schwarz et al., 2003).
The degree of specificity of RNAi remains unclear. A
recent review of RNAi claims that one of the unique features of RNAi is its exquisite sequence specificity (Shi,
2003), and a recent editorial in Nature Cell Biology, on a
Horizon RNAi symposium, proposes that a single nucleotide mismatch between the target RNA and the central
region of the RNAi construct can be used as an inactive
control (Editorial, 2003). On the other hand, this might not
be valid for all RNAi sequences, in particular for highly
active RNAi constructs. For example, a mismatch in the
central portion of the RNAi construct does not necessarily
completely ablate RNAi activity (Boutla et al., 2001), a
toleration effect also found by our group (Holen et al.,
2002). A systematic study introducing a series of G:C
mutations from one end to the other of the siRNA, found a
general tolerance to mutations, but with less tolerance for
T. Holen and C. V. Mobbs / Neuroscience 126 (2004) 1–7
mutations at the 3⬘ end of the siRNA (Amarzguioui et al.,
2003). This last point possibly facilitates rational design
siRNAs sensitive to single-nucleotide polymorphisms. A
similar mutation study was recently performed on an
shRNA-construct against the cell-cycle regulator p53
(Pusch et al., 2003). Reports on tolerance of single mutations are now available from other groups (Jacque et al.,
2002; Saxena et al., 2003; Vickers et al., 2003; Yu et al.,
2002; Zeng and Cullen, 2003).
Recent microarray investigations examining RNAi
specificity come to different conclusions about the specificity of RNAi, with two papers finding a very high degree of
specificity of siRNA (Chi et al., 2003; Semizarov et al.,
2003), while two other studies found surprisingly abundant
non-specific effects (Jackson et al., 2003; Persengiev et
al., 2004). Furthermore, another DNA microarray study
concluded that shRNA entailed relatively specific downregulation of the targeted gene (van de Wetering et al.,
2003), whereas Nicchia et al. (2003)reported that constructs targeted to the Aquaporin-4 gene in astrocytes also
produced significant alterations in expression of five genes
also differentially expressed in ischemia-induced brain
edema.
Since the specificity of RNAi constructs is a subject of
continuing debate, a key step in developing candidate
RNAi constructs is to ensure by computer search of databases that the candidate sequence appears only in the
target gene. A review of over 300 published and commonly
used siRNAs shows that 75% of these siRNAs exhibit
more than 18 of 21 positions in common with mRNAs other
than the intended target mRNA, and 25% of the siRNAs
have two or less mismatches (Snøve and Holen, unpublished observations). On the other hand, regardless of the
rules used, between 10% and 50% of RNAi constructs
tested can be expected to reduce expression of the targeted gene by more than 50%, assuming greater than 50%
transfection rate. Therefore, as with anti-sense oligonucleotides, control constructs should also be chosen with care.
Testing efficacy and specificity of RNAi
In general, it is best to first test each construct in vitro to
assess if it reduces expression of the target gene because
fewer than half of all 21-base-pair RNAi constructs produce significant decreases in gene expression in mammalian cells, regardless of the efficiency of the gene transfer.
Since transfection efficiency in most cell types of interest in
neuroscience research using non-viral methods is relatively low, it is best to assess the efficacy of the construct
in vitro under conditions of relatively reliable transfection
efficiency (which applies especially to dsRNA constructs,
whose transfection efficiency and reliability in vivo in mammals remains unclear (Isacson et al., 2003). A generally
reliable method to test construct efficacy is to transfect the
target gene into a cell type that is easily transfected, such
as 293-T cells, then assess if the RNAi construct reliably
reduces the expression of the transfected gene. In contrast
to anti-sense oligonucleotides, whose mechanism of action may entail attenuation of translation, RNAi works exclusively by reducing mRNA levels; therefore efficacy can
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be assessed by examining RNA levels, either by Northern
blot, RNAse protection assay, or real-time PCR. Protein
levels can of course also be measured, but generally
mRNA levels are easier to quantify and are a more accurate an indicator of efficacy of the construct (since, for
example, an mRNA can be down-regulated without an
immediate effect on protein levels, depending on the stability and abundance of the protein). Of course eventually
the goal is to reduce protein levels, but this question can be
addressed more thoroughly once the optimum RNAi construct has been obtained.
As with any study involving pharmacological intervention, a failure to observe an effect with RNAi will not
strongly support that the gene does not control the
phenotype under study: observing an effect is much
more informative. Therefore interpretation of the result
will always necessitate demonstration of significant
down-regulation of the targeted gene. Should downregulation in the tissue under observation be insufficient
to produce a phenotype, packaging the plasmid-based
construct into a viral, especially an adenoviral vector
(Xia et al., 2002) would provide a more rigorous assessment of the importance of the targeted gene in mediating
the target phenotype. As with production of the plasmidbased system, although the initial effort to produce such
a vehicle is considerable, once an effective RNAi vehicle
has been produced, a viral-based RNAi construct can be
reproduced quite easily. This may be a useful strategy if
extensive studies of the specific gene are planned, but
may not be practical if the experimental design is to
screen the effect of numerous genes.
Verification
Even should the RNAi construct reduce expression of the
targeted gene (compared with expression observed in the
presence of the control construct) and produce the expected phenotype, it cannot be definitively concluded that
the effect of the RNAi construct is due to down-regulation
of the targeted gene; it is still conceivable that the RNAi
construct has reduced expression of genes not targeted.
Such a concern, of course, applies to any study involving
pharmacological inhibition, including in particular those using anti-sense oligonucleotides. One way to assess nonspecific effects is to measure expression of non-target
genes. Nevertheless, demonstrating that the expression of
any given non-target gene is not reduced does not demonstrate that the observed phenotype is due to the downregulation of the target gene. On the other hand, RNAi
offers a unique opportunity to address this problem definitively. Since RNAi is less effective with increasing mismatches, the targeted gene, engineered with neutral mutations that cause RNAi mismatches but no alteration in
the coded protein, can be transfected along with the RNAi
construct. If the RNAi construct reduces expression of the
targeted gene and produces the expected phenotype,
whereas transfection of the mismatched construct reverses the effect, such a result would constitute extremely
strong evidence that the observed phenotype is in fact due
to down-regulation of the targeted gene.
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(Accepted 8 March 2004)