Department of Health Sciences, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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Direct injection of plasmid DNA into muscle allows the study of promoters in a physiological environment. Because of the variability of reporter gene activity, attempts have been made to normalize activity to muscle plasmid uptake by coinjection of a second "control" plasmid whose reporter gene is constitutively expressed by a viral promoter. The purpose of this study was to evaluate the use of a control plasmid vs. Southern blot to normalize for differences in uptake of plasmids containing promoter fragments of the skeletal muscle-specific sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) gene. Results showed that the correlation of luciferase activity from control vs. SERCA1 plasmids is poor and that normalization by a virally driven control plasmid increased variability of SERCA1 luciferase activity. In several cases, the presence of a control plasmid inhibited SERCA1 reporter expression. When Southern blot analysis was used to normalize for differences in plasmid uptake there was less variability than with coinjection, and correlations between plasmid uptake and SERCA1 luciferase activity were better. Moreover, there were no inhibitory effects of a control plasmid allowing for optimization of injection conditions of the SERCA1 deletion constructs. The use of Southern analysis is suggested to determine whether plasmid uptake is differentially affected by physiological stimuli, muscle types, or plasmid sizes under study.
in vivo plasmid injection; coinjection; sarco(endo)plasmic reticulum calcium adenosinetriphosphatase
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INTRODUCTION |
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PROMOTER ANALYSIS OF muscle-specific genes in cell lines or primary cultures has revealed important information about transcriptional mechanisms. However, these experiments do not replicate physiological events in vivo (9). Transgenic animals have been used for in vivo promoter analysis, but integration of the transgene occurs in random locations throughout the genome. Random integrations may occur in multiple places, resulting in the need to control for copy number effects (8, 9), and they can disrupt or completely knock out the regulation and expression of genes lying in regions adjacent to integration sites (14). In addition, because the chromosomal positioning of the transgene is distinct from the endogenous gene, regulatory information contained in the chromosome due to three-dimensional topology, nuclear location, and/or packaging is also lost. This may contribute to the loss of fiber specificity seen in some transgenic animals expressing muscle-specific transgenes (22). Finally, producing transgenic animals with the number of constructs necessary to perform promoter analysis is expensive and time consuming.
An alternative to promoter analysis in cell culture or in transgenic animals is the direct injection of plasmid promoter constructs into muscle (for review see Ref. 11). Muscle is unique in its ability to take up plasmid DNA, with 2-35% of muscle fibers expressing the recombinant protein after injection (7, 8, 12, 23). Plasmid DNA is preferentially taken up by myofibers, as evidenced by electron microscopy (23). Because of this property, direct plasmid injection into muscle has become a widely used approach to study transcriptional regulation in a physiologically relevant system (4, 6, 11, 18, 21). Plasmid injection has also been used to overexpress a recombinant protein to examine its influence on in vivo physiology (2, 12). Injection of plasmid DNA has advantages over adenoviral or retroviral vectors for transfer of DNA into skeletal muscle because it does not provoke an immune response (7, 8). A drawback to plasmid injection is that, like all transient transfections, the DNA remains episomal, and thus any regulatory information contained in the chromosome is lost. This limitation can be somewhat minimized by comparing the expression of the reporter construct and the endogenous gene. Another drawback is that plasmid uptake efficiency is variable (4, 6, 21, 24). Therefore, to compare reporter activity from one set of muscles to another, attempts have been made to normalize activity to plasmid DNA uptake. This has involved the coinjection of a second "control" plasmid whose reporter gene is constitutively expressed by a viral promoter. The control plasmid is intended to account for conditions that influence plasmid uptake, so that expression of the control plasmid reflects expression of the experimental plasmid. This approach for normalization of reporter activity was adopted from its general application in cell culture.
The purpose of this paper is to present the problems encountered when reporter activity is normalized to the activity of a coinjected control plasmid and to suggest an alternative method of normalization. The experimental plasmids under study contain promoter fragments from the skeletal muscle-specific sarco- (endo)plasmic reticulum Ca2+-ATPase (SERCA1) gene. The results indicated that activity of the experimental plasmids (SERCA1-driven firefly luciferase) did not correlate well with activity of a control plasmid (virally driven Renilla luciferase). Furthermore, expression often became more variable when normalized to Renilla luciferase. The presence of the control plasmid abrogated expression of several of the experimental plasmids, suggesting interference between the two plasmids. In light of these observations we measured plasmid DNA uptake in slow- and fast-twitch skeletal muscle using Southern blot analysis. The correlations between firefly luciferase activity and DNA uptake were better overall, and interpretations were not affected by differences in the amount of DNA injected. This suggests that Southern blot analysis is a more accurate reflection of plasmid uptake efficiency than coinjection of a control plasmid.
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MATERIALS AND METHODS |
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Animals. Female Wistar rats, 8 wk old (~200 g), were used for all experiments. At all times the rats were treated in conformity with APS Guiding Principles for Research Involving Animals and Human Beings.
Plasmid construction. PCR was used to
create the clones SERCA1 (962)-pGL3 and SERCA1
(
612)-pGL3. Primers were designed (20, 25) with restriction
enzyme sites added to their 5' end
(Bgl II or
Hind III). The upstream primers for
962 to +172 and
612 to +172, which added a
Bgl II site, were
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A rat SERCA1 3636 to +172 clone was isolated using the
GenomeWalker Kit (Clontech). Downstream gene-specific primers were designed
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Control plasmids. The control plasmids, pRL-CMV, pRL-SV40, and pRL-TK (Promega) contain the Renilla reniformis (seapansy) luciferase reporter gene driven by the viral promoters cytomegalovirus (CMV), simian virus 40 (SV40), and herpes simplex virus thymidine kinase (TK), respectively. Control and experimental plasmid DNA was amplified using the Endotoxin-free Plasmid Maxiprep kit or the Endotoxin-free Plasmid Megaprep kit (Qiagen).
Plasmid injections: normalization by
coinjection. In an attempt to find the optimum
conditions for coinjection we varied the amount of SERCA1-pGL3 DNA
injected and the ratio of control to experimental plasmid injected
(i.e., pRL to SERCA1-pGL3). An appropriate amount of
Renilla control plasmid and SERCA1
plasmid (25, 50, 75, or 100 µg) was mixed to achieve a
ratio of 1:3, 1:10, 1:20, or 1:50 (see Table
1). The mixture was ethanol precipitated
overnight at 20°C with 0.1 M NaCl. The pellets
were resuspended in a 25% sucrose-1× PBS solution to the
appropriate concentration and placed at 4°C overnight. Preinjection
of muscle with sucrose, as previously used (7), did not reduce
luciferase variability in our hands.
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Animals were anesthetized with 45 mg/kg pentobarbital sodium. An incision was made on the lateral side of the leg, and the soleus and extensor digitorum longus (EDL) muscles were isolated. A 28-gauge 0.3-ml insulin syringe containing 50 µl of the plasmid mix was inserted near the distal myotendinous junction and pushed ~1 cm rostrally along the longitudinal axis of the muscle, toward the proximal myotendinous junction. Plasmid was injected evenly throughout the longitudinal axis of the muscle during syringe withdrawal. The fascia and skin were sutured and the animals were returned to their cages. Seven days after surgery animals were anesthetized (50 mg/kg pentobarbital sodium), and soleus and EDL muscles were extracted and frozen in isopentane cooled to dry ice. Anesthetized rats were killed by KCl injection to the heart.
One to four days after extraction, muscles were homogenized in 1 ml of
1× passive lysis buffer (PLB from Dual Luciferase Reporter Assay
Kit, Promega) and centrifuged at 5,500 g for 20 min. Supernatants were
removed and frozen at 80°C. Firefly and
Renilla luciferase activities were
determined on a Turner design 20/20 luminometer (Promega) using the
Dual Luciferase Reporter Assay Kit (Promega). Because all muscles were
homogenized in the same volume of buffer (1 ml) and luciferase
activities [relative light units (RLU)] were determined on the same
volume of homogenate (20 µl), the RLU reported reflects activity per muscle.
Plasmid injections: normalization by Southern blot
analysis. To prepare the DNA for plasmid injections,
SERCA1 plasmids (25, 50, 75, 100, or 125 µg) were ethanol
precipitated overnight at 20°C with 0.1 M NaCl. The pellets
were resuspended to the appropriate concentration in 25%
sucrose-1× PBS solution. The resuspended pellets were placed at
4°C overnight. In vivo DNA injections were carried out as described
above. Muscle homogenates were prepared as above, except that the
supernatants (1 ml for all muscles) were removed and frozen at
80°C while the pellets were resuspended in 9 ml of buffer G2
(Blood and Cell Culture DNA Maxi Kit, Qiagen) and frozen at 20°C
for 1-4 days. Luciferase activity was determined on 20 µl of the
supernatant fraction using the Dual Luciferase Reporter Assay Kit
(Promega) and reflects activity per whole muscle.
An additional 10 ml of buffer G2 were added to the resuspended pellets, and DNA was isolated following the instructions of the manufacturer with one modification (Qiagen). During the last step of the protocol the DNA pellets were washed in 2 ml of 70% ethanol instead of 4 ml, and the DNA was transferred to 2-ml screw-top tubes. Pellets were resuspended in 1× Tris/EDTA and vigorously shaken overnight at 4°C. DNA concentrations were determined by spectrophotometry.
Twenty micrograms of total DNA per muscle were digested with Hae III for 14-18 h and loaded onto a double-tiered 0.8% agarose gel. In addition to the muscle samples, eight standard lanes, containing known quantities of Hae III-digested pGL3-basic (12.5, 25, 50, 100, 125, 150, 200, and 300 pg), were loaded onto the gels. Downward alkaline transfers to HybondN+ membrane (Amersham) were carried out according to standard protocols (1). The gels and membranes were examined on a transilluminator for the presence of ethidium bromide after transfers were disassembled. In all cases the gels showed an absence of ethidium bromide stain and the membranes showed clear and consistent staining in all lanes, indicating that the transfers were complete. Prehybridization and hybridization steps used Rapid Hybridization Buffer (Amersham), and conditions were per the instructions of the manufacturer. Digestion of pGL3-basic with Sca I yielded a 444-bp fragment of firefly luciferase coding region. This fragment was radioactively labeled (32P) in a random priming reaction (New England Biolabs) and used as probe. Incubation of the membrane with this probe detects a 1595-bp fragment of the Hae III-digested SERCA1-pGL3 reporter construct or pGL3-basic. Blots were washed for 20 min with 0.2× sodium chloride-sodium citrate (SSC) + 0.1% SDS preheated to 65°C and for 20 min with 0.1× SSC + 0.1% SDS preheated to 65°C after a 2-h hybridization.
The blots were exposed to film for ~18 h. This exposure time gave signals in a linear range for quantitation using laser densitometry. A regression equation was determined for each Southern blot based on densitometry of the standard lanes, and plasmid DNA uptake was determined for each muscle. The DNA uptake value from densitometry reflects the number of copies of plasmid taken up by the muscle in 20 µg of total muscle DNA. Luciferase activity (RLU) per whole muscle was divided by plasmid uptake per whole muscle to give total RLU/total plasmid uptake.
Statistics. An unpaired Student's t-test was used to determine statistical significance at P < 0.05. Regression equations, generated from the pGL3-basic plasmid DNA standards, were used to calculate uptake of SERCA1-pGL3 plasmids by soleus and EDL muscles (SigmaPlot 4.0).
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RESULTS AND DISCUSSION |
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Optimization of plasmid injection
conditions. Dose-response curves were performed to
determine the optimal amount of SERCA1-pGL3 construct to inject (Fig.
1). In soleus muscles, luciferase activity peaks when 75 µg of the SERCA1 (962)-pGL3 construct is
injected. Similar dose-response curves were seen in soleus
muscles with SERCA1 (
3636)-pGL3 and SERCA1 (
612)-pGL3
(not shown). Coinjection of pRL-CMV and SERCA1 (
962)-pGL3
inhibited SERCA1-pGL3 luciferase activity particularly when 75 µg
were injected, indicating that the presence of a pRL-CMV plasmid has
inhibitory effects (Fig. 1). Varying the amount of DNA injected or the
ratio at which the SERCA1 and Renilla
plasmids were injected did not reveal a pattern in which coinjection
conditions could be optimized (Fig. 1, Table 1, and not shown). In
addition, there was no pattern observed between the length of the
SERCA1-pGL3 construct and the degree of inhibition by the control
plasmid. Differential sensitivity of SERCA1-pGL3 deletion constructs in
the presence of the control plasmid may be due to differences in
cis-elements among the SERCA1-pGL3 constructs (not shown).
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In comparison, the dose-response curves for EDL muscles peak when
between 100 and 125 µg plasmid DNA are injected (not shown). This is
most likely due to less plasmid DNA uptake by EDL vs. soleus muscles.
Even when 75 µg of SERCA1-pGL3 are injected into soleus muscles and
125 µg into EDL muscles, the number of copies of plasmid taken up is
38% lower in the EDL as determined by Southern blot analysis. However,
within a muscle type there is no relationship between the size of a
plasmid and copy number uptake [e.g., SERCA1 (3636)-pGL3 and SERCA1 (
330)-pGL3 have similar
uptake]. As in soleus muscles, expression from SERCA1
(
962)-pGL3 was inhibited in the presence of the control plasmid
pRL-CMV (1:20 ratio; not shown), and the extent of inhibition was
dependent on the SERCA1-pGL3 plasmid that was coinjected. Thus the
conditions for coinjection of the control and experimental plasmids
could not be optimized due to the differential inhibition of the
SERCA1-pGL3 deletion plasmids by the control vector.
Competition between injected promoters for limited transcription factors may explain why the presence of a second virally driven plasmid inhibits expression from the SERCA1-driven plasmids. Viral promoters and enhancers may bind factors more strongly than endogenous promoters and enhancers, thereby reducing their expression. Other groups have shown good correlations between two coinjected reporter plasmids when both are driven by viral promoter and/or enhancer sequences (12, 19, 21). Thus, when transcription of both experimental and control plasmids is under viral control, reporter expression seems to be related. These findings strengthen the argument that the poor correlation between the SERCA1-pGL3 and Renilla plasmids is due to competition between the viral and muscle-specific promoter for limited transcription factors.
Correlation of SERCA1-pGL3 and Renilla luciferase
activity. In an effort to find an amount and type of
control plasmid that did not cause inhibition of luciferase activity
from the experimental plasmid, different SERCA1 constructs were
coinjected with different types and amounts of virally driven control
plasmids in soleus and EDL muscles. The data show that there
was often a poor correlation between the SERCA1-driven firefly
luciferase activity and the Renilla
luciferase activity (Table 1). This occurred regardless of the SERCA1
or viral promoter used, the amount of DNA injected, or the ratio of the
Renilla to SERCA1-pGL3 plasmid
coinjected. These results suggest that the
Renilla plasmid does not reflect uptake of the SERCA1-pGL3 plasmid, violating the basic premise for use
of a control plasmid for normalization. Even in cases where the
correlation between firefly and
Renilla luciferase was good, there
remained the problem that luciferase activity from the same virally
driven Renilla plasmid gave a poor
correlation when coinjected with a different SERCA1 promoter construct.
One example is that while correlation of the luciferase activities was
high when pRL-SV40 and SERCA1 (612)-pGL3 were coinjected at a
1:3 ratio (r = 0.97), there was an
unacceptable correlation when pRL-SV40 and SERCA1 (
330)-pGL3
were coinjected at the same ratio (r =
0.66).
Southern blot of injected plasmid DNA.
To avoid the problems associated with coinjection, deletion analysis of
the SERCA1 promoter was performed without a control plasmid. Plasmid
uptake was determined by measuring the presence of SERCA1-pGL3 in
injected muscles using Southern blot (Fig.
2). SERCA1-pGL3 plasmid copy number uptake
per whole muscle was determined, and this value was used to normalize
total luciferase activity (RLU). We were confident that all the plasmid
DNA in the muscle was isolated from the pellet of the muscle homogenate
because Southern analysis detected no plasmid DNA in the supernatant of
these samples (not shown).
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Overall, the correlation of SERCA1 luciferase activity and muscle plasmid uptake, as determined by Southern analysis, was better than the correlation of firefly to Renilla luciferase activity (Table 1). This suggests that Southern analysis is a more accurate way to normalize for plasmid uptake efficiency. However, the correlation coefficients were not always as high as expected. One explanation may be that in some cases plasmid DNA is distributed evenly among nuclei along the injection tract and in others the plasmid DNA enters many of the same nuclei along the injection tract. As a result of the latter, the presence of a large amount of DNA in one nucleus may saturate the transcriptional apparatus, thereby decreasing the amount of luciferase protein produced per unit of plasmid DNA in that muscle. A second explanation is that the DNA entering a myocyte may not always localize to a nucleus. This would result in different luciferase activities despite similar plasmid uptake from muscle to muscle. If operative, normalization by coinjection is also subject to these scenarios. Additionally, even though the correlation coefficients are not always as high as expected, normalization by Southern analysis does not have the problems associated with the presence of a second inhibitory plasmid.
Normalization of SERCA1 (962)-pGL3 activity by
coinjection vs. Southern analysis. An important
characteristic of Southern analysis to normalize for plasmid uptake is,
unlike normalization by coinjection, that it is tolerant to differences
in the amount of plasmid DNA injected. This is of crucial importance,
as it can change the interpretation of the experiment. For instance, when 50 or 75 µg of SERCA1 (
962)-pGL3 (Fig.
3A,
bottom) were injected into soleus
muscles, there was no difference in the normalized expression of
luciferase activity. A similar result was found with injection of 50 and 75 µg of SERCA1 (
3636)-pGL3 (not shown). However, when
soleus muscles were coinjected with pRL-CMV and SERCA1
(
962)-pGL3 at a 1:50 ratio, normalized luciferase activity was
different at 50 vs. 75 µg (Fig. 3B,
bottom). The same is true for
coinjection with pRL-TK (Fig. 3D,
bottom). Thus, with normalization by
coinjection, the outcome of the experiment may be altered by a change
in the amount of plasmid injected. In addition, injection of the same
amount of experimental plasmid (i.e., 50 µg) but with a different
ratio of experimental to control plasmid can effect the experimental
interpretation (Fig. 3, B vs.
C). Although normalization using
coinjection with pRL-SV40 decreased variability and showed consistent
normalized expression between 50 and 75 µg (Fig.
3E), this was not the case with all
SERCA1-pGL3 constructs coinjected with pRL-SV40 (not shown).
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Fiber-type-specific expression of SERCA1-pGL3 using
coinjection. Normalization of the SERCA1-pGL3 plasmids
by coinjection did not establish a fiber-type-specific expression.
Endogenous SERCA1 is expressed at sixfold greater levels in EDL
compared with soleus muscles (17, 25). When SERCA1 (962)-pGL3
luciferase activity was normalized using coinjection of the pRL-SV40
control plasmid, the EDL muscles showed greater SERCA1 expression (Fig. 3E,
bottom). Yet, when luciferase
activity from the same SERCA1-pGL3 construct was normalized to the
pRL-CMV (Fig. 3, B and
C,
bottom) or the pRL-TK (Fig.
3D,
bottom) control plasmids, the
outcome varied. Depending on the injection, the soleus muscles
exhibited greater expression, lesser expression, or no difference
compared with the EDL muscles. The difficulty in establishing
fiber-type specificity may be due to differential suppression of SERCA1
luciferase expression in the presence of the viral plasmid in soleus
and EDL muscles. Alternatively, the
Renilla luciferase protein may be
expressed at different levels in soleus and EDL muscles, despite the
same uptake of control plasmid DNA. This may be the result of a
different milieu of transcription factors in soleus vs. EDL muscles.
Fiber-type-specific expression of SERCA1-pGL3 using
Southern analysis. When Southern analysis was used to
examine SERCA1-pGL3 fiber-type specificity, neither SERCA1
(3636)-pGL3, SERCA1 (
962)-pGL3, SERCA1 (
612)-pGL3,
nor SERCA1 (
330)-pGL3 expressed the endogenous fiber-specific
pattern in soleus vs. EDL muscles (Fig. 4).
Thus the first 3636 bp of the SERCA1 5' flanking region does not
contain all of the cis-elements
necessary for fiber-type-specific expression (Fig. 4). However, the
SERCA1 (
3636)-pGL3 construct is responsive to muscle unloading
and, therefore, contains the region(s) necessary for upregulation of
the SERCA1 gene by unloading (17). The most likely explanation for the
lack of fiber-specific expression is that pertinent fiber-type-specific
cis-elements are not contained between
3636 and +172, but rather are found in intronic regions as shown
for other muscle genes (3, 5, 10, 13, 15, 16). Alternatively, the
chromosomal context in which the endogenous SERCA1 gene is found may
contain the information necessary to direct fiber-specific expression.
If this is the case, then whenever the SERCA1 regulatory region is
removed from its endogenous chromosomal configuration, be it an
episomal plasmid or an integrated transgene, there will be loss of
fiber-type-specific expression.
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Direct plasmid injection is a powerful approach for performing in vivo deletion analysis. Because the amount of DNA taken up by muscle is variable and can be differentially influenced by experimental conditions, reporter activity should be normalized by the best estimate of DNA uptake. The use of Southern blot analysis for the normalization of plasmid reporter activity is a more valid and reliable approach than normalization by coinjection of a control plasmid. This is because the variability of normalized luciferase activity is lower, the correlations of DNA uptake with RLU are better, and there are no inhibitory effects of a second plasmid. This technique is reproducible, and the experimental outcome is not altered by changes in the amount of plasmid injected.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41705.
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FOOTNOTES |
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S. C. Kandarian is an Established Investigator of the American Heart Association.
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: S. C. Kandarian, Boston Univ., Dept. of Health Sciences, 635 Commonwealth Ave., 4th Floor, Boston, MA 02215 (E-mail: skandar{at}bu.edu).
Received 10 June 1999; accepted in final form 2 August 1999.
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