Somatic Cell and Molecular Genetics, Vol. 21, No. 4, 1995, pp. 233-240

Tetracycline-Regulated Gene Expression Following Direct Gene Transfer into Mouse Skeletal Muscle Jyotsna Dhawan, 1,3 T h o m a s A. Rando, 1,3 Sarah L. Eison, 3 H e r m a n n Bujard, 2 and Helen M. Blau 3 JPresent address: Department of Neurology, Stanford University School of Medicine, Stanford, California 94305; "-Department of Molecular Biology, University of Heidelberg, Heidelberg, Germany," and 3Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5322 Received 7 August 1995--Final 7 August 1995

AbstractmFor most experimental and therapeutic appfications of gene transfer, regulation of the

timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammafian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it & dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally admin&tered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-eontrolled expression of genes in vivo, suggesting applications to developmental studies and gene therapy.

INTRODUCTION Although delivery of genes in vivo has therapeutic and experimental applications in the treatment of inherited and acquired diseases and in the study of developmental regulators, both would benefit from the ability to regulate gene expression (1). Most gene transfer studies to date have involved strong viral promoters and resulted in constitutive expression of the delivered gene. Previously, when regulation of transgenes was attempted in mammals, eukaryotic promoters that were activated by heavy metals (2) or steroid hormones (3, 4) were frequently used. These inducible systems often suffered both from relatively high basal levels

of expression of the transferred gene in the un-induced state and from pleiotropic effects of the inducer on endogenous gene expression. By contrast, binary systems such as those responsive to isopropyl-13-D-thiogalactopyranoside (IPTG) (5), tet (6) and the progesterone antagonist RU-486 (7), allow for highly specific regulatable expression of genes introduced into mammalian cells. Each of these systems uses bacterial or yeast control sequences in conjunction with chimeric transactivator proteins. In the tetresponsive system, a chimeric tet-controlled transactivator (tTA) encoded by one plasmid binds to the tet operator (tetO) sequence of a second plasmid inducing expression of the gene of interest under the control of a

233 0740-7750/95/0700-0233507.50/0 ~ 1995 PLenum Publishing Corporation

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minimal CMV promoter (6). Tet disrupts the this report, we demonstrate that the expresbinding oftTA to tetO sequences, where tTA sion of tet-responsive plasmids directly inis a hybrid molecule consisting of the jected into skeletal muscle can be reversed DNA-binding tet repressor protein fused to and modulated over a range of two orders of the transcriptional activation domain of the magnitude by varying the dose of orally HSV-VP16 protein. Regulation of gene administered tet. Our findings should faciliexpression by tet is rapid, reversible, and can tate studies of development and gene therapy, be modulated over several orders of magni- both of which will benefit from methods for tude. Using this system, tet-controlled expres- controlling the levels and timing of gene sion has been demonstrated for cell cycle expression. regulatory proteins in cultured cells (8), reporter genes in plants (9) and transgenic MATERIALS AND METHODS mice (10) and reporter genes directly injected into the heart (11), but it has not been Animals. Adult (4-7 week old) male previously demonstrated in skeletal muscle, C3H/HeKal mice were obtained from the one of the best characterized and easily Department of Laboratory Animal Medicine targeted tissues for gene delivery in mam- at Stanford University and handled in accormals. dance with guidelines of the Administrative In this report, we assess the ability of the Panel on Laboratory Animal Care of Stanbipartite tet-regulatable system to function ford University. in skeletal muscle by direct injection of Plasmids. Plasmids used in this study naked plasmid DNA into the tibialis anterior have been described previously (6) and were (TA) muscles of adult mice. Skeletal muscle prepared by the Qiagen method (Qiagen is an attractive target tissue for gene delivery. Inc., Chatsworth, California). Prior to injecBy contrast with many other tissues, in tion into muscle, plasmids were tested by skeletal muscle stable constitutive expression transfection into myogenic C2C12 cells. of genes has been achieved by transplanta- Luciferase activity from transiently introtion of genetically engineered myoblasts duced or stably integrated plasmids in these (12-16), infection with adenoviral vectors cells could be suppressed up to two orders of (17-19), and direct injection of DNA (20- magnitude by the addition of 1 ixg/ml tet to 22). DNA injection, which is both rapid and the culture medium (data not shown). Plassimple, is an approach that is largely re- mid DNA was dissolved in normal saline for stricted to skeletal (20-22) and cardiac (23) injection into muscle. muscle. In these tissues, the DNA appears to Injection of DNA into Skeletal Muscle. persist extrachromosomally and exhibits long Mice were anaesthetized with pentobarbital term expression albeit in relatively few (60 mg/kg, intraperitoneally). The TA muscle myofibers. Although heart and skeletal was exposed by a single incision in the muscle can both serve as targets for direct overlying skin and DNA injected into the gene delivery, skeletal muscle has several belly of the muscle using a Hamilton syringe advantages over heart: (a) it is a readily as described for cell injections (16). Each accessible tissue, comprising a major portion injection delivered 10 Ixg of each plasmid in a of the body mass; (b) injections entail less volume of 10 fxl. The efficiency of DNA risk to the host; and (c) non-muscle gene injection was initially monitored by histoproducts derived from genetically engi- chemical examination of [3-gal activity in neered muscle tissue are secreted into the frozen sections of muscle tissue using the circulation for periods of months to years chromogenic substrate 5-chloro, 4-bromo, (12-15, 19), often at physiological levels. In 3-indoyl 13-D-galactoside (X-gal) as de-

Tetracycline-Regulated Gene Expression in Mouse Skeletal Muscle

scribed (13): the injection of 10 ~xg of pSV21acZ plasmid DNA resulted in 3-10 dark blue [3-gal positive myofibers (data not shown). Tetracycline TreatmenL The drug was orally administered by dissolving tet (tetracycline hydrochloride, Sigma Chemical Co., St. Louis Missouri) in drinking water (acid water pH 2.7 was used since tet is relatively insoluble at neutral pH at a dose of 2.2 mg/ml). Drinking water containing tet was changed daily as the antibiotic is both light sensitive and labile in solution. The daily water consumption of tet-treated mice did not differ from untreated controls and averaged ~25 ml/day per cage of 5 mice. Parenteral administration of tet was achieved by daily IP injection at a dose of 100 mg/kg. Preparation of Muscle Extracts. Seven to fifteen days after DNA injection, the TA was dissected out, minced finely and frozen in liquid nitrogen. Extracts were prepared by homogenizing the muscle in a total of 1 ml of 0.1 M potassium phosphate extraction buffer containing protease inhibitors and 0.1% Triton X-100. Extracts were cleared by centrifugation, stored at 4~ and used for biochemical analysis within 48 hours of preparation.

BIOCHEMICAL ANALYSES

Luciferase Assay. 20 txl aliquots of each muscle extract were assayed for luciferase activity (24) in a Monolight 2001 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI), using the integral mode (10 sec). Luciferase activity is expressed in relative light units (rlu) in Figure 1 and as rlu/txg protein in Figures 2-4. Luciferase activity in extracts from control uninjected muscle ranged from 0-0.17 rlu/~g protein. [3-galactosidase Assay. 20 I~1 of each extract were used in a chemiluminescent assay for [3-gal activity using AMPGD (Tropix Inc., Bedford Massachusetts) as the sub-

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strate (25). [3-gal activity was measured in a luminometer using the integral mode (10 sec) and is expressed in rlu or rlu/txg protein. Unlike luciferase, the endogenous [3-gal activity of control uninjected muscle was significant and ranged from 55.8-71 rlu/ixg protein. Therefore, background 13-gal activity was subtracted in each case to determine the [3-gal activity resulting from the expression of the injected pSV21acZ plasmid. Protein Assay. Total protein content of muscle extracts used in Figures 2-4 was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, California). RESULTS To assess the ability of the bipartite tet-responsive system to function in skeletal muscle, the two plasmids were directly injected into the tibialis anterior (TA) muscles of adult mice. One plasmid encoded the tTA and the other included tandem tetO sequences and a minimal CMV promoter upstream of the luciferase reporter gene. As shown in Figure 1, reporter gene expression in skeletal muscle is dependent on coinjection of the two pIasmids. Luciferase expression from the target plasmid injected alone was close to, although detectably above that of control, uninjected muscle (Figure 1A). By contrast, co-injection of the plasmid encoding the hybrid tet-transactivator resulted in a 2,000 fold induction of luciferase expression. In these experiments, in which tet was not present, the bipartite plasmid system yielded high levels of luciferase expression on a par with pRSVlux (24), a single plasmid in which luciferase expression is driven by a strong constitutive promoter. To control for reproducibility of the injection procedure, a plasmid, pSV21acZ, that uses a strong constitutive promoter to drive expression of 13-galactosidase ([3-gal) was included in the injections with the tet-responsive plasmids. In biochemical as-

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w h e t h e r tet, a p o t e n t r e v e r s i b l e i n h i b i t o r o f t T A b i n d i n g to t e t O (6), c o u l d s u p p r e s s r e p o r t e r g e n e a c t i v i t y to b a s a l levels in muscle, two routes of antibiotic delivery were compared. Whereas muscle extracts from untreated mice exhibited high 'levels of l u c i f e r a s e activity, b o t h p a r e n t e r a l and o r a l d e l i v e r y o f tet s u p p r e s s e d e x p r e s s i o n o f t h e l u c i f e r a s e r e p o r t e r in i n j e c t e d m u s c l e by two o r d e r s o f m a g n i t u d e ( F i g u r e 2). T h a t t h e d i f f e r e n c e s w e r e d u e to t e t a n d n o t d u e to d i f f e r e n c e s in t h e p l a s m i d i n j e c t i o n s was c l e a r f r o m t h e c o n s t a n t levels o f [3-gal

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Fig. 1. Expression of luciferase from the tet-operator plasmid pUHC13-3 is dependent on co-injection of the CMV-tet-transactivator plasmid pUHD15 1 in mouse skeletal muscle. The TA muscles of adult C3H mice were injected (see Methods) with 10 pA of PBS containing either 10 ~g of pRSVlux (positive control) or 10 txg each of pUHD15 1 (a plasmid encoding the chimeric tet-controlled transactivator) and pUHCI3-3 (a plasmid including heptamerized tet operator sequences and a minimal CMV promoter driving the luciferase gene). As a control for injection procedure, each injection also contained 10 ~tg of pSV21acZ. (A) 20 ~xl aliquots of each extract were assayed for luciferase activity. Luciferase activity is expressed in relative light units (rlu). In different experiments, protein content of muscle extracts obtained from mice of the same age was measured, and found to be 44.2 _+ 10.4 gg/mg tissue (n = 46). (B) 20 pA of the same extracts as in (A) were assayed for [3-gal activity (expressed in rlu). The background of endogenous 13-gal activity in uninjected muscle was subtracted in each case. Each data point represents the mean _+ SD (n = 8). says, [3-gal activity w a s s i m i l a r in t h e p r e s e n c e a n d a b s e n c e o f t T A ( F i g u r e 1B). T h u s , w h e r e a s [3-gal e x p r e s s i o n r e m a i n e d c o n s t a n t , r e l a t i v e l y low b a s a l a n d h i g h m a x i m a l l e v e l s

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Fig. 2. Tetracycline suppresses gene activation in mouse skeletal muscle. Adult mice were injected with a mixture of pUHD15-1, pUHC13-3 and pSV21acZ (10 ~g each plasmid in a volume of 10 r per injection) as described in Methods. Group 1 received tet by IP injection daily at a dose of 100 mg/kg bodyweight. Group 2 received tet at a dose of 2.2 mg/ml in the drinking water. Group 3 (controls) received standard acid drinking water, as did Group 1. The volume of water ingested per day did not differ among the three groups. Luciferase activity in muscle extracts was assayed as described and normalized to protein content. Each data point represents the mean _+ S.D. (n = 10 for groups 1 and 2; n = 14 for group 3). Expression of the co-injected [3-gal plasmid was determined as described in Methods and did not differ significantly between tet-treated and untreated mice (105 _+ 47.3 and 42.6 _+ 43.4 rlu/ixg protein, respectively).

Tetracycline-Regulated Gene Expression in Mouse Skeletal Muscle

resulting from the coinjected pSV21acZ plasmid. Expression of Injected Plasmids is Dependent on the Dose of Oral Tet. To test whether tet-regulated gene activity could be modulated in a dose-dependent manner, the concentration of tet in the drinking water was altered. The highest oral dose used was 2.2 mg/ml, the limit of aqueous solubility of tet (26) and a dose that is nontoxic even upon chronic administration (27). Suppression of normalized luciferase activity was evident in the dose range of 0.05 to 2.2 mg/ml tet (Figure 3). Greater suppression of luciferase activity by IP injections of tet was not observed. Thus, maximal suppression of gene expression appears to be feasible with nontoxic doses of orally administered tet. Suppression of Gene Expression by Tet is Reversible. To determine whether tet-mediated suppression of reporter gene expression in vivo was reversible, mice were injected with the two tet plasmids, provided with drinking water containing maximum doses of

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tet for 7 days and subsequently given standard drinking water without tet for an additional 8 days. In control animals (no tet administration), mean luciferase activity was stable between 8 and 15 days after DNA injection (Figure 4, dashed line), consistent with published reports (20). By contrast, in tet-treated mice that had been maximally suppressed by tet for 7 days, luciferase activity was detectably increased within 6 hours of tet removal (Figure 4). The timing of derepression of luciferase expression is consistent with the half-life of tet in serum which is approximately 10 hours (28). By 48 hours after tet withdrawal, luciferase expression increased to the level of expression of untreated control mice i.e., 100-fold basal levels. DISCUSSION

In this report, we demonstrate that the expression of tet-responsive genes directly A 1000 1

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Fig. 3. Gene expression in mouse skeletal muscle is responsive to the dose of oral tetracycline. Adult mice were all injected with a mixture of pUHD15-1 and p U H C I 3 - 3 as described in Figure 2. Groups of mice (n = 6 for each group) were maintained on different doses of oral tetracycline ranging from 0.005 to 2.2 mg/ml for the course of the experiment. One group of mice (n = 4) received tet by IP injection as described in Figure 2. Seven days after D N A injection, the T A was removed, muscle extracts prepared and luciferase activity measured and normalized to total protein as described.

Fig. 4. Time course of gene activation after tetracycline withdrawal. Mice injected with the two plasmids were divided into two groups: one group was treated with oral tet at a dose of 2.2 mg/ml, whereas the control group did not receive tet. Eight days after D N A injection, antibiotic was eliminated from the drinking water of the tet-treated group and animals were maintained on standard drinking water. Animals from both groups were sacrificed at different times after drug withdrawal and muscle extracts assayed for luciferase activity and normalized to protein content (n = 6 at each time point). The dashed line represents the m e a n luciferase activity of untreated control mice for the total 15 days of the experiment.

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injected into skeletal muscle can be regulated over two orders of magnitude in response to oral delivery of a systemic modulator, tetracycline. The data show that the difference in luciferase expression between groups of tet-treated and untreated mice is due to the presence of tet and is not a result of variable success of DNA injection. Variability within groups may reflect the mechanism of DNA uptake which remains largely unknown although T tubules (20), caveolae (29) and transient microdisruptions of the myofiber plasma membrane (30) may be routes of plasmid entry. An understanding of this mechanism could greatly enhance the efficiency of direct gene transfer into a greater proportion of muscle fibers which is currently well below that observed with myoblast-mediated gene delivery (16) or adenoviral delivery (18). A significant effort is warranted to overcome this challenging biological problem, since direct DNA injection into skeletal muscle is currently by far the simplest and most rapid method of gene delivery and overcomes potential problems associated with viral vectors. Although suppression of luciferase activity by tet was dramatic it was not complete. Basal levels of luciferase activity in tettreated mice were approximately 10 fold higher than uninjected muscles. For certain applications and gene products not only the extent of regulation, but also absolute levels of either basal or maximal expression may be critical and depend in part on the half-life of the particular gene product. However, different promoters and altered ratios of the two plasmids may serve to achieve lower basal levels. Alternatively, analogs of tet with higher affinity for tTA may result in greater suppression. Modulation of transgene expression by tet is rapid and reversible and, because of the bacterial origin of the control elements, unlikely to have pleiotropic effects on endogenous mammalian genes. Similarly, the RU486-inducible gene switch has been demon-

D h a w a n et al.

strated to yield rapid, reversible and specific regulation in genetically modified cells reimplanted into mice (7). Although hybrid transactivator proteins based on the bacterial lac repressor allow specific induction of lac operator-containing transgenes by IPTG in cultured mammalian cells, this system is of limited utility in vivo due to the temperaturesensitivity of the transactivator and the toxicity of the inducer (5). Whereas chronic exposure to high doses of tet are known to have effects on bone development,' the lack of toxicity associated with doses of tet used in this study has been established in rodents (27) and in humans (31). Synthetic tet analogs which have lower antibiotic activity and higher affinity for tTA have been identified (32) but remain to be assessed in vivo and may further the applications of this system. Eventually this system may allow regulation of expression of multiple transgenes in a single host. Recently, it has become possible to control the location, the time of onset of expression, or the targeted inactivation of genes using tissue-specific promoters (33, 34) and site-specific recombinases such as Cre (35) or FLP (36). However, the ability to induce gene expression reversibly and to modulate the levels of the gene product of interest over time should be of greater utility, especially when overexpression or inactivation of the gene of interest results in embryonic lethality. The tet-regulated expression of genes implicated in human disease could be used to generate mouse models in adult transgenic animals without affecting embryonic development. Although relatively few fibers take up injected DNA, the ability to engineer developing or mature fibers in situ allows the generation of a mosaic tissue and a direct comparison of genetically altered and neighboring normal myofibers within a single host muscle. Thus, regulated expression of genes directly injected into skeletal muscle would allow an evaluation of the role of regulators of growth and differen-

Tetracycline-Regulated Gene Expression in Mouse Skeletal Muscle

tiation in the d e v e l o p m e n t both of muscle and tissues induced by muscle (37). Regulated gene expression also has therapeutic applications, since constitutive expression is often not physiological and may even lead to deleterious effects (38). In addition, constitutive delivery of proteins such as h o r m o n e s could lead to a progressive decline in their effectiveness, for example, by receptor down-regulation due to persistent exposure to the ligand (39). T h e ability to modulate the expression of genes using tet is likely to increase therapeutic efficacy and safety and extend the applicability of gene therapy to diseases in which pulsatile delivery is desirable. Finally, a n u m b e r o f gene therapy strategies designed to deliver either muscle-specific or systemic proteins may benefit from the ability to stringently regulate the timing and m a g n i t u d e of gene expression in a readily accessible tissue such as muscle,

ACKNOWLEDGMENTS We are grateful to our colleagues W.A. Mohler, M.J. C o n b o y and Dr. M. Springer, for critical discussions of the manuscript. J. D h a w a n was the recipient of a postdoctoral fellowship from the Muscular Dystrophy Association. T.A. R a n d o was the recipient of a D a n a F o u n d a t i o n Fellowship and a H o w a r d H u g h e s Medical Institute Physician Research Fellowship. This work was s u p p o r t e d by grants from the National Institutes of Health, the Muscular Dystrophy Association and the M a r c h of Dimes Birth Defects F o u n d a t i o n to H.M. Blau.

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