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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 C6Delta a48Delta a into 2-2 cells by using the Lipofectamine protocol (GIBCO BRL). Cosmid set C6Delta a48Delta 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.


View larger version (14K):
[in this window]
[in a new window]
 
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.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   FACS analysis of HSV-1 amplicon transduction efficiency

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).


View larger version (12K):
[in this window]
[in a new window]
 
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.

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.


View larger version (41K):
[in this window]
[in a new window]
 
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.

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.


View larger version (123K):
[in this window]
[in a new window]
 
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).

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).


View larger version (11K):
[in this window]
[in a new window]
 
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.

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).


View larger version (17K):
[in this window]
[in a new window]
 
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 (tau C1) = 0.289 ± 0.224 and tau C2 = 5.381 ± 0.283 and a mean open time constant (tau O1) = 3.928 ± 0.024 and tau 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.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effects of Ca2+ on single-channel activity of native and viral expressed RyR1


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Chang, D. C. Cell poration and cell fusion using an oscillating electric field. Biophys. J. 56: 641-652, 1989[Abstract].

2.   Chaudhari, N., and K. G. Beam. The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation. Dev. Biol. 133: 456-467, 1989[ISI][Medline].

3.   Chen, S. R., K. Ebisawa, X. Li, and L. Zhang. Molecular identification of the ryanodine receptor Ca2+ sensor. J. Biol. Chem. 273: 14675-14678, 1998[Abstract/Free Full Text].

4.   Chen, S. R. W., X. Li, K. Ebisawa, and L. Zhang. Functional characterization of the recombinant type 3 Ca2+ release channel (ryanodine receptor) expressed in HEK293 cells. J. Biol. Chem. 272: 24234-24246, 1997[Abstract/Free Full Text].

5.   Cunningham, C., and A. J. Davison. A cosmid-based system for constructing mutants of herpes simplex virus type 1. Virology 197: 116-124, 1993[ISI][Medline].

6.   Felgner, P. L., T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1987[Abstract].

7.   Fraefel, C., S. Song, F. Lim, P. Lang, L. Yu, Y. Wang, P. Wild, and A. I. Geller. Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J. Virol. 70: 7190-7197, 1996[Abstract].

8.   Geller, A. I., and X. O. Breakefield. A defective HSV-1 vector expresses Escherichia coli beta -galactosidase in cultured peripheral neurons. Science 241: 1667-1669, 1988[ISI][Medline].

9.   Glorioso, J. C., N. A. DeLuca, and D. J. Fink. Development and application of herpes simplex virus vectors for human gene therapy. Annu. Rev. Microbiol. 49: 675-710, 1995[ISI][Medline].

10.   Graham, F. L., and A. J. van der Eb. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456-467, 1973[ISI][Medline].

11.   Huard, J., D. Krisky, T. Oligino, P. Marconi, C. S. Day, S. C. Watkins, and J. C. Glorioso. Gene transfer to muscle using herpes simplex virus-based vectors. Neuromuscul. Disord. 7: 299-313, 1997[ISI][Medline].

12.   Ma, J., M. B. Bhat, and J. Zhao. Rectification of skeletal muscle ryanodine receptor mediated by FK506 binding protein. Biophys. J. 69: 2398-2404, 1995[Abstract].

13.   McPherson, P. S., and K. P. Campbell. The ryanodine receptor/Ca2+ release channel. J. Biol. Chem. 268: 13765-13768, 1993[Free Full Text].

14.   Moore, R. A., H. Nguyen, J. Galceran, I. N. Pessah, and P. D. Allen. A transgenic myogenic cell line lacking ryanodine receptor protein for homologous expression studies: reconstitution of Ry1R protein and function. J. Cell Biol. 140: 843-851, 1998[Abstract/Free Full Text].

15.   Nakai, J., R. T. Dirksen, H. T. Nguyen, I. N. Pessah, K. G. Beam, and P. D. Allen. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature 380: 72-75, 1996[ISI][Medline].

16.   Protasi, F., C. Franzini-Armstrong, and P. D. Allen. Structural characterization of a dyspedic cell line. J. Cell Biol. 140: 831-842, 1998[Abstract/Free Full Text].

17.   Saeki, Y., T. Ichikawa, A. Saeki, E. A. Chiocca, K. Tobler, M. Ackermann, X. O. Breakefield, and C. Fraefel. Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum. Gene Ther. 9: 2787-2794, 1998[ISI][Medline].

18.   Saito, A., S. Seiler, A. Chu, and S. Fleischer. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J. Cell Biol. 99: 875-885, 1984[Abstract].

19.   Spaete, R. R., and N. Frenkel. The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning-amplifying vector. Cell 30: 295-304, 1982[ISI][Medline].

20.   Stavropoulos, T. A., and C. A. Strathdee. An enhanced packaging system for helper-dependent herpes simplex virus vectors. J. Virol. 72: 7137-7143, 1998[Abstract/Free Full Text].

21.   Takeshima, H. Primary structure and expression from cDNAs of the ryanodine receptor. Ann. NY Acad. Sci. 707: 165-177, 1993[ISI][Medline].

22.   Takeshima, H., M. Iino, H. Takekura, M. Nishi, J. Kuno, O. Minowa, H. Takano, and T. Noda. Excitation-contraction uncoupling and muscular degeneration in mice lacking functional skeletal muscle ryanodine-receptor gene. Nature 369: 556-559, 1994[ISI][Medline].

23.   Van Deutekom, J. C., S. S. Floyd, D. K. Booth, T. Oligino, D. Krisky, P. Marconi, J. C. Glorioso, and J. Huard. Implications of maturation for viral gene delivery to skeletal muscle. Neuromuscul. Disord. 8: 135-148, 1998[ISI][Medline].


Am J Physiol Cell Physiol 278(3):C619-C626
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society