SPECIAL COMMUNICATION
HSV-1 amplicon vectors are a highly efficient gene delivery system
for skeletal muscle myoblasts and myotubes
Y.
Wang1,
C.
Fraefel2,
F.
Protasi1,
R. A.
Moore3,
J. D.
Fessenden3,
I. N.
Pessah3,
A.
DiFrancesco1,
X.
Breakefield4, and
P. D.
Allen1
1 Department of Anesthesia, Brigham and
Women's Hospital, Boston 02115; 4 Molecular
Neurogenetics Unit, Department of Neurology, Massachusetts General
Hospital, Boston, Massachusetts 02129;
2 University Institution for Virology, 8057 Zurich, Switzerland; and 3 Department of
Molecular Biosciences, University of California, Davis, School of
Veterinary Medicine, Davis, California 95616
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ABSTRACT |
Analysis of RyR1 structure function in
muscle cells is made difficult by the low (<5%) transfection
efficiencies of myoblasts or myotubes using calcium phosphate or
cationic lipid techniques. We inserted the full-length 15.3-kb RyR1
cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector,
pHSVPrPUC between the ori/IE 4/5 promoter sequence and the
HSV-1 DNA cleavage/packaging signal (pac). pHSVGN and
pHSVGRyR1, two amplicons that expressed green fluorescent protein, were
used for fluorescence-activated cell sorter analysis of transduction
efficiency. All amplicons were packaged into HSV-1 virus
particles using a helper virus-free packaging system and yielded
106 transducing vector units/ml. HSVRyR1, HSVGRyR1, and
HSVGN virions efficiently transduced mouse myoblasts and myotubes,
expressing the desired product in 70-90% of the cells at
multiplicity of infection 5. The transduced cells appeared healthy and
RyR1 produced by this method was targeted properly and restored
skeletal excitation-contraction coupling in dyspedic myotubes. The
myotubes produced sufficient protein to allow single-channel analyses
from as few as 10 100-mm dishes. In most cases this method could
preclude the need for permanent transfectants for the study of RyR1
structure function.
helper virus-free herpes simplex virus type 1 packaging; RyR1; excitation-contraction coupling; large complementary DNA
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INTRODUCTION |
ALTHOUGH EXCITATION-CONTRACTION (E-C) coupling in
skeletal muscle cells represents one of the best understood forms of
cellular signal transduction (13), the molecular basis for its
signaling mechanisms has not been elucidated and remains the principal
unresolved question regarding the mechanism of muscle contraction.
Currently it is believed that, in skeletal muscle, the dihydropyridine
receptor (DHPR or slow-voltage-sensitive Ca2+
channel) of the plasmalemma and t-tubule serves as the
voltage sensor. It translates the depolarization of the surface
membrane of the muscle into a signal that is transmitted to the
Ca2+-release channel (ryanodine receptor, RyR1), located in
the sarcoplasmic reticulum (SR). It is possible to study
structure-function relationships of these E-C coupling proteins in
heterologous cells such as HEK-293 (3, 4) or Chinese hamster ovary
cells (12, 21), which are easily transfected. They target normally
SR-targeted E-C coupling proteins, such as RyR1, to the endoplasmic
reticulum and sarcolemmal-targeted proteins, such as the
DHPR, to the surface membrane. Studies done in these heterologous cells
have yielded important data on channel function, but the fact remains
that they may not provide complete information on the function of
native channels. Further, it is only possible to study how ryanodine
receptor structure relates to functional reconstitution of E-C coupling
within the context of the skeletal muscle phenotype. This is because
only differentiated muscle cells contain the necessary accessory
proteins to support the protein-protein interactions that are necessary
for electrical E-C coupling. RyR1 channels expressed in heterologous
cells not only lack most of the accessory proteins essential for normal muscle function, but are also missing the necessary structural organization of the junctional complex. Isolation of myoblasts from the
mdg (2) and dyspedic (15, 22) mouse have now given us the
opportunity to study the structure-function relationship of these
proteins in a homologous expression system without the interference of
wild-type protein expression. However, with current methods of
transfection [calcium phosphate, cationic lipids, electroporation (1, 6, 10)], the efficiency of introducing the very large cDNAs
of wild-type and mutated RyRs in myoblasts, or fully differentiated myotubes, is very low, rendering the myogenic models ineffective.
Recombinant herpes simplex virus type 1 (HSV-1) vectors have been used
to efficiently transduce many different cell types, including dividing
and postmitotic muscle cells (9, 11). However, as with all recombinant
virus vector systems, constructs are difficult to make, and
transduction with recombinant vectors has been accompanied by
significant cytotoxicity. To alleviate the need for isolating
recombinant viruses and to reduce viral gene-related toxicity, an
HSV-1-based plasmid amplicon vector has been developed that contains
only ~1% of the 152-kb HSV-1 virus genome. This vector contains only
the HSV-1 origin of DNA replication (ori) and a DNA
cleavage/packaging signal (pac) in addition to the desired
gene(s) and a plasmid backbone. The amplicon vector is as easy to
manipulate as any mammalian expression vector and its relatively small
size (4.8 kb) provides minimal limitation to the size of the desired
transgene. In the presence of helper virus genes, amplicons can be
efficiently packaged into HSV-1 virions (19). HSV-1 replication uses
the rolling circle mechanism, so that the products of amplicon
replication are serial head-to-tail concatamers that are cleaved at
pac signals into the ~152-kb unit length genome required to
fill the virus capsid. Consequently, each amplicon vector particle
delivers, depending on the size of the seed amplicon, a "pseudo
genome" with multiple copies of the gene(s) of interest and their
promoter(s). In the current study, 28 copies of the GFP amplicon and 6 copies of the RyR1 amplicon, arranged as head-to-tail concatemers, were
delivered into the target cells.
Helper functions for amplicon packaging were initially provided by
including a replication-defective HSV-1 helper virus (8). Although
replication-defective mutants of HSV-1 are not capable of producing
progeny virus in nonpermissive cells, gene expression from this
contaminating mutant genome still imparted cytopathic effects on
transduced cells. More recently, helper virus-free packaging systems
have been developed (7, 17, 20). These newer protocols allow the
production of amplicon-derived virions that are infectious, replication
deficient, and devoid of all viral genes, thereby eliminating toxicity.
The present paper describes the use of helper virus-free HSV-1 amplicon
vectors to transduce myoblasts and fully differentiated myotubes with
both a small reporter gene (1.2 kb, GFP) and the full-length (15.3-kb)
RyR1 cDNA at a high efficiency. This technique has solved the problem of low transfection efficiency in primary muscle cells and has virtually eliminated the need for "permanent transfectants" for RyR structure-function studies.
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MATERIALS AND METHODS |
Muscle Cell Lines
Primary murine skeletal muscle myoblasts were isolated from neonatal
C57BL10 mice, and primary dyspedic myoblasts were isolated from
neonates derived from a 129SVj/C57BL6 cross that was homozygous for an
insertion in the RyR1 gene that disrupted translation of the protein
(15). The latter neonates died at birth due to an inability to breathe.
1B5 myoblast cells were derived from homozygous "dyspedic"
embryonic stem cells, as described in detail elsewhere (14). Primary
myoblasts were grown in Ham's F-10 media supplemented with 20% FCS, 5 ng/ml basic fibroblast growth factor (Promega), 50 µ/ml
penicillin, and 50 µg/ml streptomycin. 1B5 cells were grown in DMEM
supplemented with 20% FCS, 50 µ/ml penicillin, and 50 µg/ml
streptomycin. Both cell types were induced to differentiate into
multinucleated myotubes by changing their respective growth media to
DMEM supplemented with 2% heat-inactivated horse serum (HIHS).
HSV-1 Amplicons and Preparation of Vector Stocks
The organization of the vector pHSVGN bearing GFP under the human
cytomegalovirus (CMV) promoter and a neomycin-resistant gene driven by
a SV40 promotor [provided by Dr. David Jacoby, Massachusetts
General Hospital (MGH)] is seen in Fig.
1A. For pHSVRyR1, a
15.3-kb Hind III fragment, which contained the RyR1 cDNA (14),
was isolated from plasmid pCMVRyR1 and inserted into the Hind
III site of the HSV-1 amplicon vector, pHSVprPUC (provided by Dr. H. Federoff, University of Rochester), which expresses RyR1 from the HSV-1
IE 4/5 promoter (see Fig. 1B). To facilitate the evaluation of
the transduction efficiency of RyR1 constructs, and to prevent
promiscuous RyR1 expression in myoblasts, we built a second RyR1
vector, pHSVGRyR1 (see Fig. 1C). This vector has both an eGFP
cassette driven by the HSV IE 4/5 promoter and an RyR1 cassette driven
by a myosin light-chain kinase promoter enhancer (from pMEX, provided
by Dr. Nadia Rosenthal, MGH). Helper virus-free stocks of HSVGN,
HSVRyR1, and HSVGRyR1 amplicon vectors were prepared as previously
described in detail (7), and the resulting vector stocks contained
106 to 107 transduction units/ml crude
supernatant. Briefly, amplicon DNA was cotransfected with DNA from
cosmid set C6
a48
a into 2-2 cells by using the Lipofectamine
protocol (GIBCO BRL). Cosmid set C6
a48
a represents the entire
HSV-1 genome, with the exception of the two packaging signals
(pac) in five overlapping clones that can form, via homologous
recombination, circular replication-competent virus genomes (5).
However, because the HSV-1 DNA cleavage/packaging signals (pac)
are deleted from the cosmid set, its reconstituted virus genomes cannot
be packaged. However, they still provide all the helper functions
required for the replication and packaging of cotransfected amplicon
DNA. The resulting stocks of packaged amplicon vectors are therefore
essentially free of contaminating helper virus. Viral titers are
calculated by infecting both BHK cells that are easily transduced by
these amplicons with high efficiency.

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Fig. 1.
Schematic of plasmids pHSVGN (A), pHSVRyR1 (B), and
pHSVGRyR1 (C) showing their critical elements: 1) HSV-1
oriS, IE 4/5 promoter, and pac (packaging) sequences;
2) full-length rabbit RyR1 cDNA; 3) green fluorescence
protein (GFP) expression cassette; and 4) SV40 or bHGH poly(A)
signals. MLC, myosin light chain; prom, promoter; ehnan, enhancer.
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Immunohistochemistry
For immunohistochemistry, cells grown on Thermanox (Nunc)
coverslips coated either with Matrigel (Becton Dickinson) or 1% collagen were differentiated as described above. Eighteen to
twenty-four hours after infection with HSVRyR1 amplicon vector or after
transfection with pCMVRyR1 using Lipofectamine, the cells were fixed
for 15 min to 1 h in cold (
20°C) methanol. The cells were
then incubated with PBS-5% goat serum for 2 h at room temperature (RT;
~24°C) to block nonspecific antibody binding sites. After
blocking, the cells were washed three times with PBS and then exposed
to the primary monoclonal anti-RyR antibody 34C (Developmental Studies Hybridoma Bank, Drs. J. Airey and J. Sutko, University of
Iowa), diluted 1:5 in PBS, for 2 h at RT. The primary
antibody was removed, and the cells were washed three times with PBS
and then exposed to the Cy3-conjugated goat anti-mouse
secondary antibody (Jackson Laboratories, Broomhall, PA) diluted 1:250
in PBS for 1 h. After a final wash three times with PBS, the cells were
examined with a Olympus epifluorescence microscope. Transfection was
done at a Lipofectamine/DNA ratio that had been previously determined empirically to be the optimum for transfection efficiency.
Western Blot Analysis
The methods used for Western blot analysis were described in detail
elsewhere (14). Briefly, with the following modifications, proteins
were denatured in reducing sample buffer placed in boiling water for 30 min, and 1-30 µg of protein were loaded onto 3-10% SDS-polyacrylamide gels and electrophoresed at constant voltage (200 V). The size-separated proteins were transferred onto polyvinylidene difluoride (PVDF) microporous membranes (Millipore, Bedford, MA) by
electroblotter (Mini Trans-Blot; Bio-Rad Laboratories, Hercules, CA)
overnight at 30 V (4°C) and for 1 h at 200 V. PVDF transfers were
incubated with TTBS [137 mM NaCl, 20 mM
Tris · HCl (pH 7.6), 0.05% Tween 20]
containing 5% nonfat milk for 1 h at 25°C, with gentle agitation.
The blots were then probed with 34C anti-RyR1 primary antibody (1:200
dilution; Developmental Studies Hybridoma Bank, University of Iowa).
The primary antibody was diluted in TTBS plus 1% BSA and incubated
with the blot for 1 h at 25°C, with gentle agitation. The
immunoblots were then rinsed three times for 10 min with TTBS and then
incubated with a horseradish peroxidase-conjugated sheep anti-mouse
antibody (A-5906; Sigma Chemical, St. Louis, MO) while shaking for 1 h.
Enhanced chemiluminescent techniques (New England Nuclear Life Science
Products, Boston, MA) were used to visualize the immunoblots after a
final rinse step (4 times for 10 min in TTBS).
Calcium Imaging
Differentiated 1B5 cells grown on glass coverslips were loaded with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) and 0.05% BSA (fraction
V) for 20 min at 37°C in a 10% CO2 incubator. The loading buffer consisted of (in mM) 125 NaCl, 5 KCl, 2 KH2PO4, 2 CaCl2, 25 HEPES, 6 glucose, and 1.2 MgSO4, pH 7.4. The glass coverslips were
then washed three times with 1 ml imaging buffer (loading buffer with
250 µM sulfinpyrazone). The cells were transferred to an
inverted-stage microscope (Nikon Diaphot, Melville, NY) equipped with a
thermostat-controlled plate set at 37°C. Fura 2 was excited
alternately at 340 and 380 nm with the use of the DeltaRam fluorescence
imaging system (Photon Technology International, Princeton, NJ).
Fluorescence emission was measured at 510 nm using a ×10 quartz
objective. Data were collected from 20-100 individual cells with
imaging equipment (IC-300 camera; Photon Technology International)
utilizing ImageMaster software (Photon Technology International) in
free run mode. The data were presented as the ratio of the emissions
obtained at 340 and 380 nm (340/380 ratio).
Microsomal Membrane Preparations
1B5 myotubes that had been differentiated in 5% HIHS/DMEM for 6 days
were infected with HSVRyR1. After 48 h in differentiation medium, the
cells were rinsed twice with ice-cold PBS and scraped from 100-mm
plates in the presence of 2 ml of harvest buffer (in mM: 137 NaCl, 3 KCl, 8 Na2HPO4, 1.5 KH2PO4, 1.5 EDTA, pH 7.4). Cells from 10 plates
were combined and centrifuged for 5 min at 100 g. Each cell
pellet was resuspended in ice-cold hypotonic lysis buffer (1 mM EDTA, 1 µM leupeptin, 250 µM phenylmethylsulfonyl fluoride, 10 µg/ml
pepstatin A, 10 µg/ml aprotinin, 10 mM HEPES, pH 7.4). The
resuspended pellet was homogenized using a PowerGen 700D, three times
for 5 s at 1,400 rpm (Fisher Scientific, Pittsburgh, PA). An equal
volume of ice-cold 20% sucrose buffer (10 mM HEPES, pH 7.4) was added,
and the tissue was further homogenized as above. This homogenate was
centrifuged at 110,000 g in a Beckman Ti80 rotor (Palo Alto,
CA) for 1 h at 4°C. The 110,000 g pellet was then
resuspended in 4 ml of buffer containing 10% sucrose and 10 mM HEPES,
pH 7.4. The pellet was dispersed in a glass Dounce homogenizer and
loaded on top of a two-step 27%/45% sucrose gradient (10 mM HEPES, pH
7.4). The sucrose gradient was centrifuged at 40,000 g (17,000 rpm) in a Beckman SW41 rotor for 1 h. The fraction at the 27%/45%
interface was collected and diluted in 10 mM HEPES, pH 7.4. This was
then pelleted at 110,000 g in a Beckman Ti80 rotor for 1 h at
4°C. The pellets were resuspended in 10% sucrose buffer, frozen in
liquid nitrogen, and stored at
80°C. For Western blot
control samples, junctional SR was isolated from rabbit skeletal muscle
using the method of Saito et al. (18).
Single-Channel Measurements
Microsomal membrane vesicles from RyR1 transfected 1B5 cells were fused
into a bilayer lipid membrane (BLM) made from a 5:3:2 mixture of
natural phosphatidylethanolamine (PE), phosphatidylserine (PS), and
phosphatidylcholine (PC) or a 5:2 mixture of synthetic PE and PC
suspended at 50 mg/ml in decane. The BLM was formed across a 200- to
250-µm hole in a polystyrene cup separating two chambers (cis
and trans) of 0.7 ml each. Microsomal membrane vesicles (0.1-5 µg protein) were added to the cis chamber in the
presence of 200 µM Ca2+. The cis and
trans chambers contained 500 mM CsCl, 20 mM HEPES (pH 7.4), and
100 mM CsCl, 20 mM HEPES (pH 7.4), respectively. After fusion, 300 µM
EGTA was added to the cis chamber to prevent any additional
fusion events. The cis chamber was then perfused with a
solution composed of 500 mM CsCl, 20 mM HEPES, pH 7.4 (asymmetrical conditions 5:1 cis:trans). A patch-clamp amplifier (Dagan,
model 3900) was used to measure currents through a single channel. The data were then filtered at 2 kHz (Digidata 1200; Axon Instruments,) and
stored on computer. Experimental reagents were added to the cis
chamber and stirred for 30 s. Subsequent channel-gating behavior was
recorded for 1-20 min using Axotape software (Axon Instruments, Burlingame, CA). Single-channel data from BLM experiments were analyzed
using pCLAMP software (pCLAMP version 6.0-7.0, Axon Instruments), and Fig. 6 was prepared using Origin 4.0 (Microcal, Northampton, MA).
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RESULTS |
Efficiency of Transduction
Transduction efficiency in myoblasts. Wild-type myoblasts
(106) were infected at a multiplicity of infection (MOI) of
1 and 5 with HSVGN vector. GFP expression was monitored 18 h after
infection by fluorescence-activated cell sorter (FACS) analysis using
GFP fluorescence as a marker; 72% or 93% of myoblasts expressed the transgene at MOI of 1 or 5, respectively (see Table
1). Theoretically, transduction efficiency is determined by the susceptibility of cells to
the specific virion, but the strength of promoter and other factors
that regulate the expression of a transgene may also play a significant
role in its detection. The size of the transgene should not be one of
those factors. This is because the total size of any and all
head-to-tail concatamers contained in any HSV-1 virion, despite the
size of the transgene, is between 143 and 162 kb in length (the limits
of the size of the genome that can be efficiently packaged into HSV-1
virion capsids) (17). To test this hypothesis, pHSVGRyR1, which carries
both eGFP and RyR1 transcription cassettes, was packaged and used to
infect dyspedic myoblasts at MOI 5. FACS analysis 48 h after the
infection demonstrated that 73% of myoblasts expressed GFP (see Table
1). The small difference between these results and those with the smaller GFP amplicon in these rapidly growing cells (80-100%/24 h) was most likely caused by the 24-h difference in the postinfection time before analysis, rather than the difference in the size of the
constructs. It is highly probable that the transduction efficiency was
~100% in both cultures, and the dividing myoblasts, which tend to
lose amplicon DNA, diluted the number of GFP-positive cells, making the
efficiency appear falsely low.
Transduction efficiency in myotubes. In a second set of
experiments, dyspedic myoblasts were plated in 35-mm
plates. When 50% confluence was reached, the cells were
forced to withdraw from the cell cycle by replacing growth medium with
differentiation medium. After 5 days, the cells were fully
differentiated into myotubes and were infected with 105
transduction units of xRyR1 vector, which corresponds to an MOI of
~2. Twenty-four hours postinfection, the myotubes were fixed with
cold (
20°C) methanol for immunohistochemical studies using an anti-RyR antibody, 34C. In this experiment ~90% of myotubes expressed RyR1, suggesting that the transduction in myoblasts and
myotubes is the same. The transduced myotubes appeared normal morphologically (Fig. 2C). In a
parallel experiment done in an adjacent well using
Lipofectamine-mediated RyR1 transfection only <1% of dyspedic
myotubes express RyR1 (Fig. 2B).

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Fig. 2.
Immunohistochemistry of transfected and transduced cells: ×10
images of untransfected (A), optimized Lipofectamine-assisted
RyR1 cDNA transfected (B), and HSVRyR1 virion transduced cells
(C) at multiplicity of infection (MOI) 2 using 34C primary
antibody and Cy3 conjugated goat anti-mouse secondary
antibody.
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RyR1 Protein Expressed From Virally Transduced Myotubes by Western
Blot Analysis
RyR1 expressed in these myotubes using either technique was not
different from that found in rabbit skeletal muscle junctional SR (Fig.
3). The amount of RyR protein/total protein
that could be detected by Western blot from 10 virally transduced
100-mm plates was similar to what was seen in rabbit junctional SR. We have never been able to obtain sufficient RyR1 protein to obtain a
Western blot signal using Lipofectamine transfection, even from the
total protein recovered from 60 Lipofectamine-transfected plates.

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Fig. 3.
Western blot analysis of native, transfected and virally transduced
sarcoplasmic reticulum (SR). Lane 1, 1 µg junctional SR;
lane 2, 30 µg untransfected control 1B5 cells; lane
3, 30 µg RyR1 viral transduced 1B5 cells (MOI 2); lane 4,
30 µg RyR1 Lipofectamine-transfected 1B5 cells.
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Localization of RyR1 by Immunohistochemistry
Immunolocalization of RyR1 after infection of fully differentiated
myotubes was the same as when the RyR1 cDNA was delivered by standard
Lipofectamine transfection (Fig. 4) (16).
In both cases the staining was punctate and near the surface of the
cell, consistent with the SR/surface membrane junctions
that are characteristic of these cells (16). The only difference
between the two techniques was the huge increase in efficiency when the
virus was used.

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Fig. 4.
Oil immunocytochemistry (34C primary antibody, Cy3 conjugated goat
anti-mouse secondary antibody) images [×20 (A) and
×100 (B)] of differentiated 1B5 cells transfected with
HSVRyR1 virions at an MOI of 1. Note that in the ×100 image the
immunopositive staining is punctate, indicating that the RyRs are being
expressed as Ca2+-release units (21).
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Functional Characterization of Transduced RyR1 in Dyspedic Muscle
Cells
Ca2+ transients.
Native 1B5 myotubes lack responsiveness to K+
depolarization and known agonists of the ryanodine receptor including
caffeine and ryanodine (14). After infection with HSVRyR1,
intracellular Ca2+ release induced by 40 mM caffeine and
200 µM ryanodine was restored in 1B5 myotubes (Fig.
5A). The time course and magnitude
of these responses did not differ significantly from Ca2+
responses reconstituted previously by liposome-assisted transfection of
RyR1 cDNA. However, the efficiency of transduction was >10-fold higher. In addition, HSVRyR1-infected 1B5 myotubes exhibited E-C coupling, since 40 mM KCl could evoke Ca2+ release in these
cells. The type of E-C coupling seen was skeletal, since these
transients persisted even when the cells were challenged in the absence
of extracellular Ca2+ (Fig. 5B).

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Fig. 5.
Calcium imaging of RyR1-expressing 1B5 myotubes. A:
Ca2+ responses of HSVRyR1-infected 1B5 cells were observed
after addition of 40 mM caffeine (n = 54 cells) and 200 µM
ryanodine (n = 46 cells). B: chemical-induced
depolarization of 1B5 cells infected with HSVRyR1.
Ca2+ transients were evoked by 40 mM KCl in either the
presence (2 mM CaCl2, n = 14 cells) or absence (2 mM EGTA, n = 10 cells) of extracellular Ca2+, as
well as by 40 mM caffeine (n = 13 cells). No responses to 40 mM
KCl, 40 mM caffeine, or 200 µM ryanodine were observed in 1B5
myotubes that were not exposed to virus. Scale: vertical bar, change in
340/380 fura 2 ratio [arbitrary units (AU)]; horizontal
bar, time in seconds.
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Single channels. Microsomal membranes prepared from 1B5
myotubes infected with HSVRyR1, but not native 1B5 myotubes, produced large-conductance channels with rapid, full-conductance gating transitions characteristic of native RyR1 from rabbit skeletal muscle
junctional SR preparations (Fig.
6A). These RyR1 channels were
sensitive to Ca2+ in a manner almost identical to what is
seen in native channels (Table
2). In addition, channels
obtained from 1B5 myotubes infected with HSVRyR1 display a linear
current-voltage relationship, with a unitary conductance for the
channel shown of 409 pS (Fig. 6B). The open probability of the
reconstituted channel was also highly dependent on the concentration of
Ca2+ in the cis chamber of the BLM (Table 2). The
addition of 10 µM ryanodine to the cis chamber of the BLM
produced a modified channel with a long-lived ~50% subconductance
(Fig. 6C). Subsequently, the addition of 10 µM ruthenium red
was able to completely block the Cs+ current (Fig.
6C).

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Fig. 6.
Expressed RyR1 single channels. A: continuous 2.5-s trace of
RyR1 from infected 1B5 myotubes in the presence of 100 µM
Ca2+ shows a large-conductance channel with rapid,
predominantly full-conductance gating transitions at a holding
potential of +20 mV. This channel had a mean open probability of 0.867 ± 0.149 with a mean closed time constant ( C1) = 0.289 ± 0.224 and C2 = 5.381 ± 0.283 and a mean open time
constant ( O1) = 3.928 ± 0.024 and
O2= 70.105 ± 0.117. B: these channels also
display a linear current-voltage (I-V) relationship with a
unitary conductance for the channel shown of 409 pS. C: First
two traces represent control gating in the presence of 50 µM
Ca2+ at a holding potential of +30 mV. Addition of 10 µM
ryanodine to the cis chamber converts the channel to an ~50%
subconductance state (S1) that can be completely blocked by the
addition of 10 µM ruthenium red. Each panel represents a different
single channel, and all 3 measurements were performed in asymmetric 5:1
CsCl solutions.
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DISCUSSION |
Low transfection efficiencies have made it difficult to almost
impossible to study transient transgene expression in skeletal myoblasts and myotubes. This is especially true when trying to express
large cDNAs such as the RyRs or dystrophin. Viral vectors are more
efficient than most other gene delivery vehicles because of their high
transduction efficiency and, therefore, are used broadly in culture and
in vivo. Among the commonly used viral vectors, only adenovirus-based
vectors and HSV-1-based vectors are suitable for RyR or dystrophin gene
delivery, because the size of the cDNA of these genes exceeds the
packaging limits of other common viral systems (e.g., adeno-associated
virus, retrovirus, lente virus). We believe that the
HSV-1 amplicon vector has advantages over adenoviral vectors because
1) they are easy to manipulate (which is very important when
many constructs need to be made), 2) the size of the transgenes
that can be delivered are theoretically limited only by the ~150-kb
capacity of the HSV-1 virion (this provides broad limits for packaging
large transgenes), and 3) one viral particle can deliver
multiple copies of a transgene. In addition to these advantages, HSV-1
amplicon vectors, like all other HSV-1-based vectors, but unlike
adenoviral vectors, have an equal ability to infect both myoblasts and
myotubes. This is because there is no HSV-1 viral receptor loss during
the maturation of muscle cells (23). Furthermore, when HSV-1 amplicon
vectors are packaged using a helper-virus-free packaging system, the
only infectious particles in the virus stock are amplicon virions (7). Thus all the wild-type viral genes and their associated cytotoxicity are precluded.
Our data show that helper-virus-free HSV-1 amplicon vectors can
transduce both murine myoblasts and multinucleated myotubes with very
high efficiencies (~90% at MOIs of 2-5). This exceeds the
highest efficiency (5% with myoblasts) that we have ever observed for
cationic lipid-aided transfection protocols using small (~5 kb) cDNA
constructs, by a factor of ~20-fold. Several commercially available
products were tested, including calcium phosphate Dextran and LipoTAXI
(Stratagene); Transfectam and Tfx-50 (Promega); FuGENE6 (Roche);
TransIT-LT1, LT2, and 100 (Panvera); and DIMRE-C and Lipofectamine
(GIBCO BRL). Lipofectamine was the most efficient in this cell type
(data not shown). From our titering experiments we have shown that we
are also able to transduce the nonmuscle BHK and vero 2-2 cells
with similar or higher efficiency. Unlike lipid-aided transfection of
DNA, the efficiency of helper-virus-free HSV-1 amplicon vector-mediated
gene transfer was not affected by the size of the cDNA insert in the
amplicon because all HSV-1 viral particles carry the same 152-kb viral
genome or pesudogenome. This was not the case with lipid-aided
transfection, where efficiency of transfection was substantially
reduced when RyR constructs (~15+ kb) were used compared with GFP
(1.5 kb). The only difference between large and small amplicons is how
many copies of the cDNA are carried into the cell in each virion (30 for GFP and 5 for RyR).
When multinucleated myotubes are transduced with RyR1 amplicon virions,
these myotubes produce fully functional RyR1 protein. We are confident
of proper intracellular targeting of this protein, both because of the
pattern of immunostaining and because of the full rescue of the
wild-type phenotype, when the dyspedic cells are transduced with
wild-type RyR1. We conclude that the helper-virus-free HSV-1 amplicon
system can be used as an adjuvant to efficiently study
structure-function relationships of RyR1 in skeletal myotubes. However,
these data suggest that this system would also be effective in the
delivery of any gene to primary muscle myoblasts or myotubes. The
increased efficiency of this system makes it a simple matter to do
structure-function studies of any cDNA in skeletal muscle cells, and,
in most circumstances, this increased efficiency of transduction will
be sufficient to obviate the necessity for creating permanent
transfectant clones. As the theoretical capacity of HSV-1 amplicon
vectors is up to 152 kb (17), this system also would make the
possibility of inserting large complete genes feasible, using a
bacterial artificial chromosome rather than a plasmid as the vector
backbone. This would allow the study of the transduced gene under the
control of its own promotor and enhancer(s), rather than under the
control of a heterologous promoter such as the viral CMV promoter.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grants AR-43140 and AR-44750 (to P. D. Allen and I. N. Pessah) and National Institute of Neurological Disorders and Stroke Grant NS-24279 (to X. Breakefield), the American Heart Association, California Affiliate, Grant 97-403 (to R. A. Moore), Swiss National Science Foundation Grant 823A-046649, and an
American Liver Foundation grant (to C. Fraefel). The monoclonal antibody 34C was developed by Dr. J. Airey and Dr. J. Sutko and was
obtained from the Developmental Studies Hybridoma Bank developed under
the auspices of the National Institute of Child Health and Human
Development and maintained by the Department of Biological Sciences,
The University of Iowa, Iowa City, IA 52242.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. D. Allen,
Dept. of Anesthesia, Brigham and Women's Hospital, Boston, MA 02115 (E-mail: Allen{at}zeus.bwh.harvard.edu).
Received 10 August 1999; accepted in final form 28 September 1999.
 |
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