Specific and Potent RNA Interference in Terminally
Differentiated Myotubes*
Christopher E.
Yi
,
Janine M.
Bekker
,
Gaynor
Miller
,
Kent
L.
Hill§¶, and
Rachelle H.
Crosbie
§
From the
Department of Physiological Science,
University of California, Los Angeles, California 90025, § Molecular Biology Institute, University of California, Los
Angeles, California 90025, and ¶ Department of Microbiology,
Immunology, and Molecular Genetics, University of California, Los
Angeles, California 90025
Received for publication, June 14, 2002, and in revised form, October 18, 2002
 |
ABSTRACT |
Double-stranded RNA (dsRNA) interference
is a potent mechanism for sequence-specific silencing of gene
expression and represents an invaluable approach for investigating gene
function in normal and diseased states as well as for drug target
validation. Here, we report that skeletal muscle myoblasts and
terminally differentiated myotubes are susceptible to RNA interference.
We employed an approach in which dsRNA is generated by cellular
transcription from plasmids containing long (1 kilobase) inverted DNA
repeats of the target gene rather than using dsRNA synthesized in
vitro. We show that gene silencing by this method is effective
for endogenously expressed genes as well as for exogenous reporter
genes. An analysis of the expression of several endogenous genes and
exogenous reporters demonstrates that the silencing effect is specific
for the target gene containing sequences within the inverted repeat.
Our method eliminates the need to chemically synthesize dsRNA and is
not accompanied by global repression of gene expression. Furthermore, we show for the first time that sequence-specific dsRNA-mediated gene
silencing is possible in differentiated, multinucleated skeletal muscle
myotubes. These findings provide an important molecular tool for the
examination of protein function in terminally differentiated muscle
cells and provide alternative approaches for generating disease models.
 |
INTRODUCTION |
The availability of whole genome sequences for humans and model
organisms has greatly facilitated the identification of genes responsible for inherited human disease. In the post-genomics era,
efforts to understand the molecular basis of disease will be limited
primarily by the ability to determine the function of proteins encoded
by candidate disease genes and to evaluate novel drug targets. The
ability to silence expression of specific genes is a powerful mechanism
for analysis of gene function. Although targeted gene disruption via
homologous recombination is possible in mammals, currently available
strategies are costly and time-consuming. The generation of knock-out
animal models may also be confounded by unanticipated splice variants
that produce functional proteins, despite the removal of targeted exons
(1). Furthermore, when embryonic lethality results from gene deletion
(2), it may be impossible to evaluate the role of a target protein in
fully differentiated cells and adult tissues.
Sequence-specific gene silencing initiated by the presence of aberrant
RNA was first observed in plants containing inverted transgenes and
referred to as post-transcriptional gene silencing (3, 4). A similar
gene silencing phenomenon was observed in Caenorhabditis
elegans where insightful genetic studies demonstrate that
silencing was mediated by double-stranded RNA and occurred at the level
of mRNA abundance (5, 6). The term double-stranded RNA interference
(dsRNAi)1 was coined to
describe this RNA-mediated genetic interference. The use of RNAi to
target specific mRNAs for degradation provides an alternative
method for targeted gene silencing and has now been shown to function
in a variety of organisms including nematodes, planaria, trypanosomes,
hydra, zebrafish, and Drosophila (5-14). In these systems, the
presence of dsRNA composed of protein coding sequence induces
degradation of single-stranded mRNA that is complementary to the
dsRNA (5, 6, 15, 16). Genetic screens for RNAi-resistant mutants in
C. elegans has led to the identification of some components of the RNAi machinery including an RNA-dependent RNA
polymerase, eIF-2C, and RNase D (17-19). Although the precise
mechanism of RNAi is still unclear, the process has been heralded as
revolutionary because of its specificity, catalytic features, and
effectiveness in comparison with traditional sense or antisense
knock-out technology (20).
Despite the broad species specificity of RNAi (16), its utility
for examining gene function in mammalian cells has remained somewhat
dubious because of nonspecific repression of gene expression that is
induced by cellular responses to cytoplasmic duplex RNA in many
mammalian cell lines (21-25). A major component of this response is
presumed to be the dsRNA-dependent protein kinase PKR, which upon activation by dsRNA longer than 30 nucleotides, phosphorylates eIF-2
, leading to global
down-regulation of protein translation (21, 22). In an important
advance, Elbashir et al. (23) and Caplen et al.
(24) recently showed that by limiting the length of the synthetic dsRNA
to <30 bp, sequence-specific RNAi can be achieved in several commonly
used non-differentiated cultured mammalian cell lines. These studies
demonstrate that the machinery responsible for sequence-specific RNAi
is present in these cells, although the magnitude of the specific
decrease in target gene expression varies considerably depending upon
the cell type examined and choice of target gene (23, 24, 26). When the
length of the dsRNA duplex exceeds ~30 nucleotides, the specificity
of dsRNA-induced gene silencing is lost and genes unrelated to the
dsRNA duplex are also down-regulated (23, 24).
Because of our interest in gene products involved in the pathogenesis
of muscular dystrophy, we analyzed the efficacy of dsRNAi in cultured
skeletal muscle myoblasts and myotubes. To date, nearly all RNAi
approaches with mammalian cells have involved the introduction of
in vitro synthesized dsRNA into the cytoplasm of
undifferentiated cells (23, 26, 27). In this study, we use an alternate
approach of introducing plasmid DNA encoding inverted repeats of a
target sequence into cultured murine muscle cells. This approach has been dramatically successful in other non-mammalian systems (13) and
has recently been shown to be effective for RNAi in embryonic cell
lines (28). Using this method, we demonstrate that RNA interference is
effective and specific in cultured mammalian muscle cells.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Expression Constructs--
A human dyxin expression
construct was prepared by PCR amplification of the dyxin open reading
frame (GenBankTM accession number AF216709) (29) using
primers containing appropriate restriction sites for subcloning into
the mammalian expression vector pcDNA3 (Amersham Biosciences) A
human sarcospan expression construct was prepared by PCR amplification
of the sarcospan open reading frame from a human skeletal muscle
cDNA library (Clontech, Palo Alto, CA) (31).
Both the dyxin (pcDNA.dyxin) and the sarcospan (pcDNA.SSPN)
constructs were engineered to encode a Myc tag at the COOH
terminus followed by a stop codon. For dsRNA expression constructs,
standard cloning methods were used to place oppositely oriented copies
of the dyxin open reading frame at either end of the
GFPmut3b gene (GenBankTM accession number
AF110459.1) (32). This construct was inserted into the multicloning
site of pcDNA3 to generate pcDNA.dyxin.dsRNA with the 5' copy
of the dyxin gene oriented in the forward direction (Fig. 3). A similar
dsRNA expression plasmid was engineered using inverted repeats of the
GFPmut3b gene (nucleotides 1-714) (32). The sequences of
all constructs were verified by direct DNA sequence analysis performed
by the DNA Core Facility at the University of Iowa (Iowa City, IA).
Cell Culture Strains and Media--
C2C12 myoblasts, strain C3H
(American Type Cell Culture, Manassas, VA) (33, 34), were grown and
maintained in Dulbecco's modified essential medium (DMEM, Sigma) with
10% fetal bovine serum and 1% L-glutamine, and 1%
penicillin-streptomycin C2C12 myoblasts were differentiated into
myotubes by incubating in DMEM with 2% horse serum for 5-7 days as
described previously (34).
Transfection of C2C12 Myoblasts--
C2C12 cells were
trypsinized after reaching 90% confluency on a 150-mm plate and
resuspended in phosphate-buffered saline lacking CaCl2 and
MgCl2 (1.4 mM NaCl, 0.27 mM KCl, 1 mM Na2HPO4, and 0.18 mM
dibasic phosphate, pH 7.4). Myoblasts were transfected with 5 µg of
plasmid DNA by electroporation (340 V at 950 microfarads) in 1-cm
electroporation cuvettes and electroporated with a Bio-Rad Gene Pulser
II (Bio-Rad, Hercules, CA). Time constants ranged from 16 to 17 ms.
Immediately after electroporation, cells were resuspended in complete
medium and plated in 35-mm culture plates as described previously
(35).
Transfection of C2C12 myotubes--
Clonfectin
(Clontech, Palo Alto, CA) was diluted to 1 mg/ml in
10 mM HEPES-NaCl-buffered saline, and 5 µg was combined
with 5 µg of plasmid DNA in serum-free DMEM. The liposome plus DNA mixture was incubated for 30 min and then added to myotubes grown on a
35-mm culture dish with 2 ml of serum- free DMEM. Myotubes were treated
with the Clonfectin solution for 2 h at 37 °C, 5% CO2. After transfection, the medium was aspirated and the
cells were washed with phosphate-buffered saline and allowed to grow in
fresh complete DMEM. To quantitate transfection efficiency, myotubes
were transfected with pEGFP-C3 plasmid (Clontech,
Palo Alto, CA), which encodes green fluorescent protein (GFP). Myotubes were viewed under a Zeiss Axiovert 200 inverted fluorescence
microscope. Percent transfection efficiency was determined by counting
the number of GFP-positive cells relative to total cell number. Under these conditions, over 85% transfection efficiency was routinely achieved.
Western Blot Analysis--
For protein lysates, myoblasts, and
myotubes were washed with phosphate-buffered saline and lysed in lysis
buffer (300 µl of 50 mM HEPES, pH 7.8, 300 mM
NaCl, 1% Nonidet P-40, 1.2 mM EDTA, 5 mM
MgCl2, 0.1 mM phenylmethylsulfonyl fluoride,
0.75 mM benzamidine, 0.5 µg/ml aprotinin, and 0.5 µg/ml
leupeptin). Protein samples (30 µg/lane) were resolved on a 10%
SDS-polyacrylamide gel and transferred to polyvinyl difluoride membrane
(Immobilin-P, Millipore, Bedford, MA) as described previously (35). To
detect heterologous Myc-tagged proteins, immunoblots were probed with
mouse monoclonal antibodies to the Myc tag (9E10, 1:1000 dilution,
Sigma). For dyxin immunoblotting, membranes were probed with
affinity-purified polyclonal antibody (rabbit 2, 1:500 dilution)
generated against a COOH-terminal peptide-(KGQLLCPTCSKSKRS). The
monoclonal antibody E7 directed against
-tubulin was developed
previously (30) and obtained from the Developmental Studies Hybridoma
Bank maintained by the Department of Biological Sciences, University of
Iowa (Iowa City, IA). E7 supernatant was used at a 1:5000 dilution to
detect
-tubulin on immunoblots. Antibodies to vinculin
(1:250, Sigma) and eIF-2
(1:100, Santa Cruz Biotechnology Inc.) were
used for immunoblotting as indicated by the manufacturer. Following
incubation with the primary antibodies, blots were probed with either
anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary
antibodies (1:5000 dilution, Amersham Biosciences) and developed using
enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL).
Northern Blotting--
Total RNA was isolated from transfected
C2C12 cells using TRIzol (Invitrogen) according to the manufacturer's
instructions. Total RNA (2 µg) was fractionated by formaldehyde gel
and subjected to Northern blot analysis using probes, representing the
full-length dyxin,
-actin, and glyceraldehyde-3-phosphate dehydrogenase.
 |
RESULTS |
Inherited muscle disease provides an excellent example of a field
where the identification of candidate disease genes is outpacing efforts to understand function of the corresponding gene products. We
investigated whether RNAi could be used for sequence-specific gene
silencing in muscle cells as a method for analysis of gene function. As
a target gene, we chose a novel "LIM and cysteine-rich domain" gene
(LMCD1) (29) (GenBankTM accession number NM014583), also
known as dyxin (GenBankTM AF216709). Dyxin has two tandem
LIM domains in its COOH-terminal region and is highly expressed in
skeletal muscle (29). Although the precise function of dyxin is
unknown, it is postulated to be involved in DNA-protein interactions
during skeletal muscle development (29).
As a model system, we employed C2C12 cells, a well established murine
skeletal muscle cell line (33). C2C12 cells can be maintained in
culture as undifferentiated myoblasts or can be induced to fuse and
assemble into terminally differentiated, multinucleated myotubes in low
mitogen medium (33). Immunoblots of whole cell lysates from C2C12
myoblasts and myotubes were probed with affinity-purified anti-dyxin
polyclonal antibodies (Fig. 1). As shown
in Fig. 1, dyxin is expressed in mature differentiated myotubes but not
in myoblasts. Thus, by employing both myoblasts and myotubes, we are
able to assess the utility of RNAi for silencing the expression of
exogenous reporter genes (recombinant dyxin in myoblasts) as well as
endogenously expressed genes (dyxin in myotubes).

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Fig. 1.
Dyxin expression is developmentally regulated
and is only expressed upon differentiation of myoblasts into mature
myotubes. Protein extracts (30 µg) from murine C2C12 myoblasts
and differentiated myotubes were electrophoretically separated in 10%
SDS-polyacrylamide gels and transferred to polyvinyl difluoride
membrane. Blots were stained with affinity-purified rabbit polyclonal
antibodies against dyxin, a 48-kDa LIM domain protein (29, 30)
(GenBankTM NM014583). Molecular size standards are
indicated on the left (×103 Da).
|
|
To demonstrate that exogenous dyxin expression is possible in
myoblasts, a Myc-tagged human dyxin cDNA plasmid (pcDNA.dyxin) was introduced into C2C12 myoblasts by electroporation. Immunoblots of
protein lysates from transfected C2C12 cells were stained with either
anti-dyxin antibodies (Fig. 2) or
monoclonal antibodies to the Myc tag (data not shown). Dyxin protein
was not detected in mock-transfected cells (Fig. 2, lane 1)
but is expressed at high levels 30 h post-transfection with
pcDNA.dyxin (Fig. 2, lane 2).

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Fig. 2.
Expression of dyxin dsRNA leads to specific
loss of dyxin expression in cultured myoblasts. C2C12 myoblasts
were mock-transfected (lane 1, Mock) or
transfected with plasmid pcDNA.dyxin encoding dyxin
(lane 2, DYX) or plasmid pcDNA.dyxin.dsRNA,
encoding dyxin-inverted repeats
(lane 3, DIR). Parallel samples were
co-transfected with pcDNA.dyxin and pcDNA.dyxin.dsRNA (lane
4, DYX + DIR). As a control for specificity, cells were
co-transfected with pcDNA.dyxin and pcDNA.GFP.dsRNA containing
GFP-inverted repeats (lane
5, DYX + GIR). Dyxin expression is ablated by
expression of dyxin dsRNA but not by GFP dsRNA. Cell lysates were
harvested 24 h post-transfection and analyzed for dyxin levels by
immunoblotting with affinity-purified rabbit polyclonal antibodies
against dyxin (top panel). Identical blots were probed with
monoclonal antibodies to -tubulin, vinculin, and eIF-2 as
illustrated. Expression of dyxin dsRNA did not perturb protein levels
of these endogenous proteins. Molecular size markers are indicated
(×103 Da).
|
|
Previous efforts to use RNAi for sequence-specific gene silencing in
cultured mammalian cells have primarily employed dsRNA molecules that
were produced and annealed in vitro and then introduced into
the cytoplasm of cultured cells by injection or liposome-mediated transfection (23, 24, 26). When the length of the synthetic dsRNA
exceeds 30 bp, the target cells are found to exhibit a generalized suppression of gene expression (23, 24). This nonspecific inhibition of
gene expression is presumed to result from interferon-response pathways
that are activated by the presence of dsRNA injected into the
cytoplasm, a process that may mimic the response of the cell to viral
dsRNA (23, 24, 36). In this study, we employed an alternative strategy
in which the cell's own transcription machinery transcribes inverted
DNA repeats, producing dsRNA within the cell. For these experiments, we
generated a dyxin dsRNA expression vector (pcDNA.dyxin.dsRNA)
containing inverted copies of a 1-kilobase portion of the dyxin open
reading frame separated by 700 bp of nonspecific "spacer" DNA (Fig.
3).

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Fig. 3.
Schematic diagram of the dyxin dsRNA
construct used to block dyxin expression by dsRNA-interference.
The complete dyxin cDNA (nucleotides 1-1091) was cloned into a
pcDNA3 mammalian expression vector in the forward and reverse
orientations as illustrated. This construct, pcDNA.dyxin.dsRNA, is
designed to generate a double-stranded, step-loop RNA upon
transcription. The cytomegalovirus promoter (CMV) is located
upstream of the double-inverted dyxin repeats. A similar dsRNAi
expression plasmid was engineered using inverted repeats of GFP,
pcDNA.GFP.dsRNA.
|
|
To test the efficacy of RNAi on exogenous dyxin expression, C2C12
myoblasts were co-transfected with pcDNA.dyxin together with
pcDNA.dyxin.dsRNA. Whole cell lysates were assayed for dyxin expression by immunoblotting (Fig. 2). These experiments demonstrate that exogenous dyxin expression is blocked by expression of the dyxin
dsRNA construct. The specificity of this gene silencing was tested
using three separate approaches. First, the expression of several
endogenous genes was examined in cells transfected with
pcDNA.dyxin.dsRNA. As shown in Fig. 2, dyxin dsRNA has no effect on
the expression of
-tubulin, vinculin, or eIF-2
. Therefore, gene
silencing is specific for the target gene. We next asked whether dyxin
expression was affected by a non-homologous dsRNA construct. For this
purpose, we constructed a plasmid (pcDNA.GFP.dsRNA) containing inverted
repeats of the GFP open reading frame. Dyxin remains abundantly
expressed in myoblasts co-transfected with pcDNA.dyxin and
pcDNA.GFP.dsRNA (Fig. 2, lane 5), demonstrating that GFP
dsRNA has no effect on exogenous dyxin expression. As shown earlier for
dyxin dsRNA, GFP dsRNA also has no effect on endogenous expression of
-tubulin, vinculin, or eIF-2
(Fig. 2, lane 5). As an
additional test of the specificity of RNAi in myoblasts, we examined
the effect of dyxin dsRNA on the expression of another exogenous
reporter gene. As a reporter gene, we used sarcospan (SSPN), a
component of the dystrophin-glycoprotein complex (31, 35, 37).
Myoblasts do not normally express SSPN (data not shown), but cells
transfected with a sarcospan cDNA express high levels of sarcospan
protein (Fig. 4, lane 1).
Expression of sarcospan was not affected by co-expression of
dyxin.dsRNA (Fig. 4, lane 2).

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Fig. 4.
Specificity of double-stranded RNA
interference. Myoblasts were transfected with an expression vector
encoding Myc-tagged human sarcospan, pcDNA.SSPN (lanes 1 and 3, SSPN), or co-transfected with
pcDNA.SSPN and pcDNA.dyxin.dsRNA (lanes 2 and
4, SSPN + DIR). Immunoblots of protein lysates
were separately stained with 9E10 anti-Myc antibodies to detect SSPN
(lanes 1 and 2) or monoclonal antibody E7 (42) to
detect -tubulin (lanes 3 and 4).
Expression of dyxin dsRNA does not affect expression of the
heterologous reporter SSPN or endogenous -tubulin. Molecular mass
markers are indicated (×103 Da).
|
|
A key feature of RNA interference is that it exerts its effect at the
post-transcriptional level by destruction of targeted mRNA (for
review see Ref. 16). To test whether the loss of dyxin protein occurred
at the level of mRNA abundance, we analyzed dyxin mRNA levels
by Northern blotting. Total RNA was prepared from the samples analyzed
previously for dyxin protein expression. Northern blots were probed
with a full-length dyxin cDNA probe. As shown in Fig.
5, a 1.35-kilobase dyxin transcript is
expressed in C2C12 myoblasts transfected with pcDNA.dyxin, but this
dyxin mRNA is dramatically reduced in myoblasts co-transfected with pcDNA.dyxin and pcDNA.dyxin.dsRNA (Fig. 5, lane
4). As a control for specificity, dyxin mRNA levels are not
affected by co-transfection with a non-homologous dsRNA construct
(pcDNA.GFP.dsRNA) (Fig. 5, lane 5). Northern blots
hybridized with probes for
-actin (Fig. 5, lower panel)
and glyceraldehyde-3-phosphate dehydrogenase (data not shown) further
demonstrate that silencing is specific for dyxin.

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Fig. 5.
Dyxin dsRNA-induced gene silencing occurs at
the level of mRNA abundance. Northern blots containing 2 µg
of total RNA from mock (lane 1, Mock),
pcDNA.dyxin (lane 2, DYX), or
pcDNA.dyxin.dsRNA (lane 3, DIR) transfected
myoblasts. Parallel samples were taken from cells co-transfected with
pcDNA.dyxin and pcDNA.dyxin.dsRNA (lane 4, DYX + DIR). As a control for specificity, RNA was analyzed from
myoblasts co-transfected with pcDNA.dyxin and pcDNA.GFP.dsRNA
(lane 5, DYX + GIR). Blots were hybridized with
dyxin (top panel) and -actin (bottom panel)
probes.
|
|
The ability to silence specific genes in mammalian cells holds great
promise for uncovering the function of proteins that are known to be
defective in certain inherited diseases. However, many disease genes
are expressed only in terminally differentiated cells and tissues. For
example, components of the dystrophin-glycoprotein complex are required
for normal muscle function and prevention of Duchenne muscular
dystrophy (38) but are expressed only upon differentiation of myoblasts
into myotubes (39). To determine whether RNAi can be used for
sequence-specific gene silencing in differentiated mammalian cells, we
induced the differentiation of C2C12 myoblasts into skeletal muscle
myotubes in culture. Myotubes possess fully functional sarcomeres,
exhibit contractile properties in culture, and are important models for
mammalian skeletal muscle physiology (33). We introduced the dyxin
dsRNA expression vector (pcDNA.dyxin.dsRNA) into myotubes by
liposome-mediated transfection. Whole myotube protein lysates were
analyzed at 0, 12, and 24 h post-transfection by Western blotting
with affinity-purified anti-dyxin antibodies (Fig.
6). Endogenous dyxin expression in
myotubes was completely abolished 12 and 24 h after transfection
with the dyxin dsRNA expression vector (Fig. 6). Dyxin levels were
constant for all mock-treated samples (Fig. 3) (data not shown). As is
the case for myoblasts, dyxin.dsRNA-mediated gene silencing is specific to dyxin because
-tubulin, vinculin, and eIF-2
expression
remained constant in all samples (Fig. 6) (data not shown).

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Fig. 6.
dsRNA-induced silencing of endogenous dyxin
in differentiated myotubes. C2C12 myoblasts were differentiated
into multinucleated myotubes in culture as described under
"Experimental Procedures." Myotubes were transfected by liposome
transfection with pcDNA.dyxin.dsRNA, and cellular lysates were
analyzed for dyxin expression 0, 12, and 24 h after transfection
as indicated. Immunoblots were probed with anti-dyxin polyclonal
antibody (left panel) or anti- -tubulin (E7) monoclonal
antibody (30) (right panel). Molecular mass markers are
indicated (×103 Da).
|
|
 |
DISCUSSION |
Skeletal muscle fibers are specialized multinucleated cells with
well organized contractile filaments. Investigating the function of
muscle-specific gene products is complicated by the limitations of
traditional cell lines, which lack the contractile apparatus and
therefore lack many muscle-specific proteins. Although cultured myotubes provide an excellent model for studying skeletal muscle physiology, the lack of a method to easily eliminate gene expression in
these cells has restricted in vivo analysis of protein
function. RNAi offers a number of advantages over gene disruption,
because it is rapid, potent, and can be achieved with a fraction of the effort required for generating knock-out animal models. Recent ground-breaking experiments have demonstrated that sequence-specific RNAi is feasible in certain types of undifferentiated cultured mammalian cells using chemically synthesized or in vitro
generated duplex RNAs (23, 26, 27, 40). To extend these studies to
terminally differentiated myofibers, we tested a method whereby plasmid
DNA containing large inverted repeats of a target mRNA is
introduced into myoblasts and myotubes. In this way, dsRNA is generated
intracellularly by transcription from the expression vector. We
demonstrate that gene silencing induced by this method results in a
loss of target gene expression without perturbing overall gene
expression. Importantly, we show that the target mRNA can be an
endogenous transcript or an exogenous reporter gene and that the loss
of the target mRNA is correlated with concomitant loss of target
protein. Only the target gene is affected, and inverted repeats
containing non-relevant sequences have no effect on target gene
expression. In addition, we demonstrate that gene silencing by this
approach is effective in mature differentiated skeletal muscle myotubes.
Although the mechanism of dsRNA-mediated gene silencing
observed here in mammalian muscle cells remains to be determined, several lines of evidence suggest that this is occurring through a
phenomenon mechanistically related to RNA interference pathways that
have been characterized in C. elegans, Drosophila, and other systems (16). First, the loss of target gene expression is not accompanied by global down-regulation of protein synthesis. Second, target gene silencing is sequence-specific, because non-homologous dsRNA does not interfere with target gene expression. Third, silencing of the target gene occurs at the level of mRNA abundance.
Importantly, the ability to silence endogenously expressed genes
demonstrates the utility of this approach for examining the function of
proteins that are only expressed in differentiated muscle cells.
Recent work from several laboratories indicates that the effectiveness
of long dsRNA for inducing sequence-specific RNAi in cultured mammalian
cells varies from cell type to cell type (23, 26, 27, 40). During
preparation of this paper, Paddison and colleagues (28) report using a
500-nucleotide synthetic luciferase dsRNA to knockdown expression of an
exogenous reporter gene (firefly luciferase) in murine myoblasts. Our
results compliment and extend this work by demonstrating potent and
sequence-specific silencing of endogenous gene expression in both
myoblasts and myotubes. Together, these results suggest that murine
myoblasts and myotubes may be more susceptible to sequence-specific
RNAi than other mammalian cell types thus far tested as has been
previously been reported for embryonic cell lines (28).
We have shown that double-stranded RNA-induced gene silencing is
possible in terminally differentiated muscle cell lines. This
represents the first example of dsRNA-induced gene silencing in
terminally differentiated mammalian cells and demonstrates that the
target gene can be an endogenous transcript that is expressed only upon
cellular differentiation. These findings provide an alternative to traditional gene "knock-out" studies for generating models of inherited muscle disease. It has recently been reported that
RNAi induced by short synthetic-interfering RNA duplexes (siRNA) can be
used to knockdown expression of several endogenous genes in mammalian
cell lines (23, 24, 41). Our studies extend these findings by
demonstrating that sequence-specific dsRNA-mediated gene silencing can
be used for functional analysis of muscle gene products within the
context of a biologically relevant cell culture model for mammalian
muscle. With advances in gene-silencing techniques, diseases that are
caused by genetic mutations leading to loss of protein expression
(i.e. recessive muscular dystrophies) can be investigated
with greater ease.
 |
ACKNOWLEDGEMENTS |
We thank K. M. Lipman for
technical assistance.
 |
FOOTNOTES |
*
This work was supported by Grant 1RO1AR48179-01 from
the National Institutes of Health (to R. H. C.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
California, Life Sciences Bldg. 5804, 621 Charles E. Young Dr., S., Los
Angeles, CA 90025. Tel.: 310-794-2103; Fax: 310-206-3987; E-mail:
rcrosbie@physci.ucla.edu.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M205946200
 |
ABBREVIATIONS |
The abbreviations used are:
dsRNAi, double-stranded RNA interference;
dsRNA, double-stranded RNA;
eIF, eukaryotic initiation factor;
DMEM, Dulbecco's modified Eagle's
medium;
GFP, green fluorescent protein;
SSPN, sarcospan.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.