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A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression Background: The tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse. Methods: To confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei. Results: Reverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells. Conclusions: The HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle. Keywords: Skeletal muscle-specific, Tetracycline-responsive Background we generated a transgenic mouse which uses the human Since the original description, the tetracycline-responsive skeletal muscle α-actin (HSA) promoter to drive skeletal system (Tet-ON/OFF) has proven to be a powerful tool in muscle-specific expression of the reverse-tetracycline biomedical research because of the ability to manipulate transactivator (rtTA) which we have designated as the gene expression within the mouse in both a temporal and HSA-rtTA mouse. To validate the HSA-rtTA mouse, we tissue-specific manner [1, 2]. Although a number of crossed it with the tetracycline-responsive histone H2B- skeletal muscle-specific Tet-ON/OFF mice have been green fluorescent protein (TRE-H2B-GFP) mouse to easily described, they have used promoters that drive primarily visualize and quantify myonuclear GFP expression follow- fast-twitch, type II gene expression; in addition, these mice ing doxycycline treatment [5]. As expected, rtTA mRNA are not readily available [3, 4]. To address these limitations, was highly expressed in skeletal muscle as > 95% of myo- nuclei were GFP-positive in both type I and type II mus- cles. Importantly, an extremely small number of satellite * Correspondence: jjmcca2@uky.edu cells appeared to be GFP-positive in soleus muscle Masahiro Iwata and Davis A. Englund contributed equally to this work. The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, cross-section, thus confirming the ability of the HSA-rtTA USA mouse to drive robust skeletal muscle-specific expression Department of Physiology, College of Medicine, University of Kentucky, 800 of a tetracycline-responsive gene of interest. Rose Street, Medical Science Building, Rm: MS-607A, Lexington, KY 40536, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Iwata et al. Skeletal Muscle (2018) 8:33 Page 2 of 8 Methods Analysis of rtTA gene expression Generating the HSA-rtTA transgenic mouse Total RNA was isolated from skeletal muscles (gastrocne- As previously described by us for the HSA-MerCreMer mius, plantaris, soleus, extensor digitorum longus (EDL), transgene, the promoter and first exon (− 2,000 to + 239 tibialis anterior (TA), diaphragm and heart, and non- relative to the transcription start site) of the human skeletal muscle tissue (brain, liver, lung, stomach, spleen, kidney, muscle α-actin (HSA) gene was amplified from human and fat) of HSA-GFP mice. Tissue was immediately frozen genomic DNA (Promega, Madison, WI, USA) and cloned in liquid nitrogen upon excision and subsequently homog- into the ClaI site of the SG5 expression vector (Agilent enized using a Bullet Blender (Next Advance Inc., Averill Technologies, Santa Clara, CA, USA) upstream of the Park, NY, USA) in Direct-zol (Zymo Research, Irvine, CA, β-globin intron II [6]. The rtTA cDNA was amplified from USA) according to the manufacturer’s instructions. Total the pCMV-Tet3G expression vector (Takara Bio, Mountain RNA concentration and quality were determined by nano- View, CA,USA)andclonedinto the EcoRI/BamHIsites of Vue spectrophotometer (GE Healthcare, USA). cDNA was the pSG5-HSA plasmid to generate the pSG5-HSA-rtTA; synthesized from 1 μg of total RNA using the SuperScript® the rtTA insert was subsequently sequenced for verifica- VILO IV™ (ThermoFisher Scientific, Waltham, MA, USA) tion. The HSA-rtTA transgene (Fig. 1) was released from according to the manufacturer’s instructions. PCR analysis the plasmid by HindIII/NsiI enzyme digestion, gel-purified of rtTA mRNA accumulation used the following primers: using the QIAquick Gel Extraction Kit according to the F, 5′- GAGGAACAGGAGC ATCAAGTAG-3′;R,5′-GT manufacturer’s directions (Qiagen, Valencia, CA, USA), CAGCAGGCAGCATATCA-3′ and generated a 270 bp and then provided to the University of Michigan Trans- product. genic Animal Model Core for microinjection. F1 generation pups were screened by PCR for the presence of the rtTA Single fiber analysis sequence using genomic DNA isolated from tail snips with GFP+ and GFP− myonuclei were counted on isolated the following primers: F, 5′ATGTCTAGACTGGACAAG single muscle fibers as previously described by us [7]. AGCA AAG-3′;R, 5′-TTACCCGGGGAGCATGTC-3′ Briefly, hind limb muscles were fixed in situ at resting generating a product of 747 bp. Eight F1 pups were positive length in 4% paraformaldehyde for 48 h. Fixed whole for the HSA-rtTA transgene and subsequently crossed to muscles were removed from the hind limb, dissected, the TRE-H2B-GFP mouse (The Jackson Laboratory, and dissociated in 40% sodium hydroxide with manual stock number 005104) to determine the ability to manipulation at room temperature. Isolated fibers were drive H2B-GFP expression following doxycycline then stained with DAPI and carefully pipetted on to treatment. Of the eight founder lines, line 6 was iden- glass slides and covered using Vectashield (Vector tified as driving robust H2B-GFP expression in both Laboratories, Burlingame, CA, USA). slow- and fast-twitch muscles of the lower hind limbs and was further characterized as described below. For Immunohistochemistry convenience, the HSA-rtTA/TRE-H2B-GFP mouse is For immunohistochemistry (IHC) analyses, the various referred to as the HSA-GFP mouse. hind limb muscles were covered in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Tor- Doxycycline treatment rance, CA, USA) and pinned at resting length to a cork To induce H2B-GFP expression, 3–10-month-old HSA- covered in aluminum foil. Muscles were frozen in liquid GFP mice were administered doxycycline (0.5 mg/mL) in nitrogen-cooled isopentane and stored at − 80 °C. Mus- drinking water supplemented with 2% sucrose for 3 weeks. cles were sectioned at the mid-belly on a cryostat at − Tissue was collected immediately upon completion of 23 °C. Frozen muscle sections (7 μm) were air-dried for doxycycline treatment. To determine the earliest time of at least 1 h and stored at − 20 °C. For Pax7/DAPI IHC, GFP induction, skeletal muscle was collected after 12 h or muscles were first fixed in 4% paraformaldehyde for 24 h following doxycycline administration. 7 min and then subjected to epitope retrieval. Following HSA promoter BGI Cre rtTA Mer Fig. 1 A schematic of the HSA-rtTA transgene. The promoter and first exon (− 2,000 to + 239 relative to the transcription start site) of the human skeletal muscle α-actin (HSA) gene regulates expression of an optimized reverse tetracycline transactivator (rtTA) gene which has been reported to be sevenfold more active and 100-fold more doxycycline sensitive than the original Tet-On system [8]. The β-globin intron ΙΙ (BGI) and poly(A) tail were incorporated into the transgene to ensure proper splicing and transcript stability, respectively. The positions of the PCR primers used for genotyping are indicated by half-arrows Iwata et al. Skeletal Muscle (2018) 8:33 Page 3 of 8 epitope retrieval in sodium citrate (10 mM, pH 6.5) for diaphragm fibers (n = 2) were counted representing a total 20 min at 92 °C, endogenous peroxidases were blocked for of 906 myonuclei, whereas 37 plantaris fibers (n =2) were 7 min with 3% hydrogen peroxide in phosphate-buffered counted representing a total of 1294 myonuclei. For IHC, saline (PBS), followed by 1 h with 1% Tyramide Signal images were captured at × 20 magnification using a Zeiss Amplification (TSA) blocking reagent (TSA kit, T20935, upright fluorescent microscope (Zeiss Axio Imager M1, Invitrogen) supplemented with Mouse-on-Mouse (MoM) Oberkochen, Germany). Whole muscle sections were ob- IgG blocking reagent (Vector Laboratories, Burlingame, tained using the mosaic function in Zeiss Zen 2.3 imaging CA, USA). Sections were washed in PBS and incubated software. Satellite cells (Pax7+/DAPI+) and GFP+ satellite overnight with mouse anti-Pax7 IgG1 antibody (1:100, cells were identified manually using Zen software tools. Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA) and chicken anti-GFP antibody (1:200, Results Abcam, Cambridge, MA, USA) diluted in 1% TSA block- Skeletal muscle-specific rtTA transgene ing reagent. It was necessary to use anti-GFP antibody to To generate the skeletal muscle-specific rtTA transgene detect GFP expression because the antigen retrieval for microinjection, we cloned downstream of the human process quenched the GFP signal. The following day, skeletal muscle α-actin (HSA) promoter a third gener- sections were washed with PBS, incubated for 70 min in ation rtTA gene that was reported to be sevenfold more goat anti-mouse IgG1 biotinylated secondary antibody active and 100-fold more doxycycline sensitive than the (1:1000, 115-065-205, Jackson ImmunoResearch, West original rtTA [8]. A schematic of the HSA-rtTA trans- Grove, PA, USA) and anti-chicken GFP secondary anti- gene is shown in Fig. 1. body, (1:250, Abcam), washed in PBS, incubated for 1 h in streptavidin-horseradish peroxidase (1:500, S-911, Invitro- Skeletal muscle-specific expression of rtTA mRNA gen) diluted in PBS, washed again in PBS, then incubated We determined by reverse transcription-PCR the expres- for 15 min in TSA Alexa Fluor 594 (1:500, TSA kit, Invi- sion of rtTA mRNA in several hind limb muscles, the trogen) in the supplied amplification diluents. Sections diaphragm, fat, and several other non-muscle organs. As were stained with DAPI (1:10,000 in PBS, D35471, Invitro- shown in Fig. 2, rtTA mRNA was highly expressed in all gen) for 5 min and mounted with VectaShield fluorescent of the hind limb muscles examined and to a lesser extent mounting media. in the diaphragm and heart. As expected, rtTA expression was not detectable in any non-muscle tissues. Image acquisition and quantification GFP+/DAPI+ and GFP−/DAPI+ myonuclei from ~ 10 iso- Effective labeling of myonuclei in hind limb skeletal lated fibers from four doxycycline-treated mice (two male muscles and two female) were counted for each muscle, resulting Having established that rtTA was highly enriched in skel- in a range of 202–452 myonuclei being analyzed across etal muscle, we sought to determine how effective the muscles. Twelve to 15 single fibers from two untreated HSA-rtTA transgene was in driving H2B-GFP expression mice (1 male and 1 female) were analyzed for each muscle, in response to doxycycline treatment. Given that resulting in a range of 254–600 myonuclei being analyzed H2B-GFP is nuclearly localized, we used the percentage of across muscles. For the time course analysis, 35 myonuclei that were GFP+ on single fibers as a measure Fig. 2 Skeletal muscle-specific expression of rtTA. PCR analysis of rtTA mRNA expression of different tissues from the HSA-GFP transgenic mouse showed high levels of expression in skeletal muscle (gastrocnemius, soleus, plantaris, tibialis anterior (TA), and extensor digitorum longus (EDL)), modest expression in the diaphragm, very low expression in the heart, and not detectable in non-muscle tissue (brain, fat, lung, liver, stomach, spleen, and kidney) Iwata et al. Skeletal Muscle (2018) 8:33 Page 4 of 8 of the effectiveness of the HSA-rtTA transgene to induce digitorum longus (EDL) of doxycycline-treated mice and expression of a tetracycline-responsive gene. Following then stained with DAPI to identify myonuclei. As shown fixation, single fibers were isolated from the plantaris, in Fig. 3a, b, greater than 95% (range of 96.4–97.9%) of gastrocnemius, soleus, tibialis anterior (TA), and extensor myonuclei were GFP+ across all muscles from Fig. 3 HSA-rtTA transgene drives robust myofiber expression of tetracycline-responsive H2B-GFP transgene. a Representative single fiber images of hind limb muscles taken from HSA-GFP mice (n = 4) treated with doxycycline. Single fiber images show robust myonuclear GFP expression in muscles composed of slow- and fast-twitch fibers. b Quantification of GFP+ myonuclei of single fibers from hind limb skeletal muscles (plantaris, gastrocnemius, soleus, tibialis anterior (TA), and extensor digitorum longus (EDL)) of HSA-GFP mice showed greater than 95% of all DAPI+ myonuclei within a fiber were GFP+. The gray bar represents the average percentage of GFP-positive myonuclei (n = 4) for each muscle Iwata et al. Skeletal Muscle (2018) 8:33 Page 5 of 8 doxycycline-treated HSA-GFP mice. We observed no GFP GFP labeling is highly specific to myonuclei + myonuclei in skeletal muscle single fibers of untreated To determine if the HSA-rtTA drove expression of the HSA-GFP mice demonstrating tight regulation of H2B-GFP transgene in satellite cells, we performed immu- tetracycline-responsive H2B-GFP gene (data not shown). nohistochemistry on both soleus and plantaris muscle These findings confirm the HSA-rtTA mouse is capable of cross-sections for DAPI, Pax7, and GFP. As shown in Fig. 4, driving robust expression of a tetracycline-responsive gene GFP labeling did not localize with Pax7 staining; however, in adult skeletal muscles composed of both slow- and in the soleus, of the 190 satellite cells counted, three Pax7+ fast-twitch fibers. cells appeared to be GFP+. These results demonstrate the Plantaris Soleus DAPI GFP Pax7 Fig. 4 GFP expression is specific to myonuclei in HSA-GFP mice. Representative muscle cross-section images of the plantaris and soleus muscles from HSA-GFP mice treated with doxycycline (n = 3). As indicated by white arrows, DAPI+/GFP+ myonuclei (green) did not show co-localization with DAPI+/Pax7+ satellite cells (pink). These results confirm that the HSA-rtTA transgene is able to drive myofiber-specific expression of a tetracycline-response gene Iwata et al. Skeletal Muscle (2018) 8:33 Page 6 of 8 HSA-rtTA drives highly myofiber-specific expression of a skeletal muscle α-actin (HSA) promoter was used to tetracycline-responsive transgene. drive skeletal muscle-specific expression of the reverse- tetracycline transactivator (rtTA), designated as the Rapid GFP labeling of myonuclei HSA-rtTA mouse. The HSA promoter contains 2,000 bp To determine the time course of GFP labeling of myo- of human skeletal α-actin 5′-flanking sequence plus the nuclei, skeletal muscle was collected from HAS-GFP first exon and 149 bp of the first intron and was first re- mice after 12 or 24 h of doxycycline treatment. As ported by Muscat and Kedes to promote robust, skeletal shown in Fig. 5a, b, approximately 90% of myonuclei in muscle-specific expression [9]. We choose to use the the plantaris muscle were GFP-positive after 24 h of HSA promoter because we previously showed it was able doxycycline treatment; in contrast, GFP expression was to drive effective Cre-mediated recombination in both not detected following 12 h of doxycycline administra- slow- and fast-twitch fibers with minimal expression in tion (data not shown). We also examined whether GFP the heart [6]. This is an important improvement over a expression followed the same time course of induction previous skeletal muscle-specific Tet-ON mouse (MCK- given the modest expression of rtTA mRNA in the rtTA) which only allowed over-expression of a gene of diaphragm (see Fig. 2). While GFP expression was not as interest in fast-twitch, type IIb fibers [10]. Together with robust as that observed in the plantaris, 60% of myonu- the HSA-CreER mouse, the HSA-rtTA mouse now pro- clei of the diaphragm were GFP-positive following 24 h vides the ability to perform loss- and gain-of-function of doxycycline treatment, consistent with the lower rtTA studies, respectively, to determine the in vivo function of mRNA expression (see Fig. 5a–b). a gene of interest in skeletal muscle fibers [6, 11]. The complement to these two inducible, skeletal muscle-spe- Discussion cific mice are the satellite cell-specific inducible Cre and The purpose of this study was to characterize a new Tet-ON mice; however, while the satellite cell-specific skeletal muscle-specific Tet-ON mouse. The human Cre mouse has been extensively used, to the best of our Fig. 5 Rapid GFP labeling of myonuclei. a Representative single fiber images of plantaris and diaphragm muscles taken from HSA-GFP mice (n = 2) treated with doxycycline for 24 h. b Quantification of GFP+ myonuclei showed ~ 90% of myonuclei were GFP-positive in myofibers from the plantaris with 60% of myonuclei GFP-positive in myofibers isolated from the diaphragm Iwata et al. Skeletal Muscle (2018) 8:33 Page 7 of 8 knowledge, the satellite cell-specific Tet-ON mouse has Funding This work was supported by NIH grants AG049806 and AR060701 to JJM and yet to be fully characterized [12–15]. Collectively, these CAP and AR071753 to KAM. inducible, skeletal muscle-, and satellite cell-specific mice provide powerful tools to manipulate in vivo gene Availability of data and materials The datasets used and/or analyzed during the current study are available expression to identify and better understand the mecha- from the corresponding author on request. The HSA-rtTA mouse is available nisms regulating skeletal muscle biology in health and upon request. disease. While 3 weeks of doxycycline treatment was able to in- Declarations N.A. duce > 95% GFP labeling of myonuclei, we wanted to know the earliest time point when GFP expression could be Authors’ contributions detected following doxycycline administration. As shown in JJM cloned the transgene. MI, DAE, YW, CMD, KAM, IJ, CBM, CAP, and JJM Fig. 5, single fiber analysis revealed myonuclear GFP assisted in developing the study design and characterizing the mouse. DAE and JJM wrote the manuscript. All authors read and approved the final expression was observed as early as 24 h post-doxycycline manuscript. exposure; in contrast, we observed no GFP expression at 12 h post-doxycycline treatment (data not shown). These Ethics approval and consent to participate results demonstrate the HSA-rtTA mouse is very respon- All animal procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals as approved by the sive to doxycycline and will provide the ability to study the Animal Care and Use Committee of the University of Kentucky. relative early (~ 24 h) effects of gene activation on a given biological process. For example, the HSA-rtTA mouse Consent for publication N.A. could be used to study how the early (~ 24 h) activation of Akt1 (using the tetracycline-responsive, constitutively active Competing interests Akt1 mouse, TRE-myrAkt1) affects the hypertrophic The authors declare that they have no competing interests. response in skeletal muscle as Akt1 is typically not activated until 48 h [16, 17]. Publisher’sNote In contrast to the strong rtTA expression in hind limb Springer Nature remains neutral with regard to jurisdictional claims in muscles, there was modest, but detectable, expression of published maps and institutional affiliations. rtTA mRNA in the diaphragm. We do not know the rea- Author details son why rtTA expression is lower in the diaphragm 1 The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, compared to hind limb muscles, but it may reflect a USA. Department of Rehabilitation Sciences, College of Health Sciences, University of Kentucky, Lexington, KY 40536, USA. Department of limitation of the HSA promoter to drive high levels of Physiology, College of Medicine, University of Kentucky, 800 Rose Street, expression in the diaphragm. We also found compara- Medical Science Building, Rm: MS-607A, Lexington, KY 40536, USA. tively lower expression of Cre in the diaphragm of the Department of Rehabilitation, Faculty of Health Sciences, Nihon Fukushi University, 26-2 Higashihaemi-cho, Handa 475-0012, Japan. HSA-CreER mouse, consistent with the idea that the HSA promoter is not as robust in the diaphragm as it is Received: 27 June 2018 Accepted: 16 October 2018 in other muscles. Despite low rtTA expression, we still observed ~ 60% GFP labeling of diaphragm myonuclei References after only 24 h of doxycycline treatment. This result in- 1. Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, et al. Temporal dicates the HSA-rtTA mouse is a useful tool for investi- control of gene expression in transgenic mice by a tetracycline-responsive gators studying the diaphragm. promoter. Proc Natl Acad Sci U S A. 1994;91(20):9302–6. 2. Das AT, Tenenbaum L, Berkhout B. Tet-On systems for doxycycline-inducible The HSA-rtTA transgenic mouse allows for inducible, gene expression. Curr Gene Ther. 2016;16(3):156–67. myofiber-specific gene expression in both slow- and 3. Ghersa P, Gobert RP, Sattonnet-Roche P, Richards C, Pich EM, Van fast-twitch muscles. The HSA-rtTA mouse will provide Huijsduijnen RH. Highly controlled gene expression using combinations of a tissue-specific promoter, recombinant adenovirus and a tetracycline- researchers with a powerful tool to reversibly induce regulatable transcription factor. Gene Ther. 1998;5(9):1213. gene expression in an effort to better understand skeletal 4. Grill MA, Bales MA, Fought AN, Rosburg KC, Munger SJ, Antin PB. muscle biology. The HSA-rtTA mouse will be freely Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice. Transgenic Res. 2003;12(1):33–43. available upon request. 5. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science. 2004;303(5656):359–63. 6. McCarthy JJ, Srikuea R, Kirby TJ, Peterson CA, Esser KA. Inducible Cre Abbreviations transgenic mouse strain for skeletal muscle-specific gene targeting. Skelet dox: Doxycycline; EDL: Extensor digitorum longus; GFP: Green fluorescent Muscle. 2012;2(1):8. protein; H2B: Histone H2B; HSA: Human skeletal muscle α-actin; 7. Murach KA, White SH, Wen Y, Ho A, Dupont-Versteegden EE, McCarthy JJ, et al. IHC: Immunohistochemistry; rtTA: Reverse tetracycline transactivator; Differential requirement for satellite cells during overload-induced muscle TA: Tibialis anterior; Tet-ON/OFF: Tetracycline-responsive system hypertrophy in growing versus mature mice. Skelet Muscle. 2017;7(1):14. 8. Zhou X, Vink M, Klaver B, Berkhout B, Das AT. Optimization of the Tet-On Acknowledgements system for regulated gene expression through viral evolution. Gene Ther. N.A. 2006;13(19):1382–90. Iwata et al. Skeletal Muscle (2018) 8:33 Page 8 of 8 9. Muscat GE, Kedes L. Multiple 5′-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression. Mol Cell Biol. 1987;7(11):4089–99. 10. Izumiya Y, Hopkins T, Morris C, Sato K, Zeng L, Viereck J, et al. Fast/Glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese mice. Cell Metab. 2008;7(2):159–72. 11. Schuler M, Ali F, Metzger E, Chambon P, Metzger D. Temporally controlled targeted somatic mutagenesis in skeletal muscles of the mouse. Genesis. 2005;41(4):165–70. 12. Nishijo K, Hosoyama T, Bjornson CR, Schaffer BS, Prajapati SI, Bahadur AN, et al. Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J. 2009;23(8):2681–90. 13. Lepper C, Conway SJ, Fan CM. Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements. Nature. 2009;460(7255):627–31. 14. Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development. 2011;138(17):3625–37. 15. Lee SJ, Huynh TV, Lee YS, Sebald SM, Wilcox-Adelman SA, Iwamori N, et al. Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway. Proc Natl Acad Sci U S A. 2012;109(35):E2353–60. 16. Shiojima I, Sato K, Izumiya Y, Schiekofer S, Ito M, Liao R, et al. Disruption of coordinated cardiac hypertrophy and angiogenesis contributes to the transition to heart failure. J Clin Invest. 2005;115(8):2108–18. 17. Miyazaki M, McCarthy JJ, Fedele MJ, Esser KA. Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling. J Physiol. 2011;589(Pt 7):1831–46. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: The tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse. Methods: To confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei. Results: Reverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells. Conclusions: The HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle. Keywords: Skeletal muscle-specific, Tetracycline-responsive Background we generated a transgenic mouse which uses the human Since the original description, the tetracycline-responsive skeletal muscle α-actin (HSA) promoter to drive skeletal system (Tet-ON/OFF) has proven to be a powerful tool in muscle-specific expression of the reverse-tetracycline biomedical research because of the ability to manipulate transactivator (rtTA) which we have designated as the gene expression within the mouse in both a temporal and HSA-rtTA mouse. To validate the HSA-rtTA mouse, we tissue-specific manner [1, 2]. Although a number of crossed it with the tetracycline-responsive histone H2B- skeletal muscle-specific Tet-ON/OFF mice have been green fluorescent protein (TRE-H2B-GFP) mouse to easily described, they have used promoters that drive primarily visualize and quantify myonuclear GFP expression follow- fast-twitch, type II gene expression; in addition, these mice ing doxycycline treatment [5]. As expected, rtTA mRNA are not readily available [3, 4]. To address these limitations, was highly expressed in skeletal muscle as > 95% of myo- nuclei were GFP-positive in both type I and type II mus- cles. Importantly, an extremely small number of satellite * Correspondence: jjmcca2@uky.edu cells appeared to be GFP-positive in soleus muscle Masahiro Iwata and Davis A. Englund contributed equally to this work. The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, cross-section, thus confirming the ability of the HSA-rtTA USA mouse to drive robust skeletal muscle-specific expression Department of Physiology, College of Medicine, University of Kentucky, 800 of a tetracycline-responsive gene of interest. Rose Street, Medical Science Building, Rm: MS-607A, Lexington, KY 40536, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Iwata et al. Skeletal Muscle (2018) 8:33 Page 2 of 8 Methods Analysis of rtTA gene expression Generating the HSA-rtTA transgenic mouse Total RNA was isolated from skeletal muscles (gastrocne- As previously described by us for the HSA-MerCreMer mius, plantaris, soleus, extensor digitorum longus (EDL), transgene, the promoter and first exon (− 2,000 to + 239 tibialis anterior (TA), diaphragm and heart, and non- relative to the transcription start site) of the human skeletal muscle tissue (brain, liver, lung, stomach, spleen, kidney, muscle α-actin (HSA) gene was amplified from human and fat) of HSA-GFP mice. Tissue was immediately frozen genomic DNA (Promega, Madison, WI, USA) and cloned in liquid nitrogen upon excision and subsequently homog- into the ClaI site of the SG5 expression vector (Agilent enized using a Bullet Blender (Next Advance Inc., Averill Technologies, Santa Clara, CA, USA) upstream of the Park, NY, USA) in Direct-zol (Zymo Research, Irvine, CA, β-globin intron II [6]. The rtTA cDNA was amplified from USA) according to the manufacturer’s instructions. Total the pCMV-Tet3G expression vector (Takara Bio, Mountain RNA concentration and quality were determined by nano- View, CA,USA)andclonedinto the EcoRI/BamHIsites of Vue spectrophotometer (GE Healthcare, USA). cDNA was the pSG5-HSA plasmid to generate the pSG5-HSA-rtTA; synthesized from 1 μg of total RNA using the SuperScript® the rtTA insert was subsequently sequenced for verifica- VILO IV™ (ThermoFisher Scientific, Waltham, MA, USA) tion. The HSA-rtTA transgene (Fig. 1) was released from according to the manufacturer’s instructions. PCR analysis the plasmid by HindIII/NsiI enzyme digestion, gel-purified of rtTA mRNA accumulation used the following primers: using the QIAquick Gel Extraction Kit according to the F, 5′- GAGGAACAGGAGC ATCAAGTAG-3′;R,5′-GT manufacturer’s directions (Qiagen, Valencia, CA, USA), CAGCAGGCAGCATATCA-3′ and generated a 270 bp and then provided to the University of Michigan Trans- product. genic Animal Model Core for microinjection. F1 generation pups were screened by PCR for the presence of the rtTA Single fiber analysis sequence using genomic DNA isolated from tail snips with GFP+ and GFP− myonuclei were counted on isolated the following primers: F, 5′ATGTCTAGACTGGACAAG single muscle fibers as previously described by us [7]. AGCA AAG-3′;R, 5′-TTACCCGGGGAGCATGTC-3′ Briefly, hind limb muscles were fixed in situ at resting generating a product of 747 bp. Eight F1 pups were positive length in 4% paraformaldehyde for 48 h. Fixed whole for the HSA-rtTA transgene and subsequently crossed to muscles were removed from the hind limb, dissected, the TRE-H2B-GFP mouse (The Jackson Laboratory, and dissociated in 40% sodium hydroxide with manual stock number 005104) to determine the ability to manipulation at room temperature. Isolated fibers were drive H2B-GFP expression following doxycycline then stained with DAPI and carefully pipetted on to treatment. Of the eight founder lines, line 6 was iden- glass slides and covered using Vectashield (Vector tified as driving robust H2B-GFP expression in both Laboratories, Burlingame, CA, USA). slow- and fast-twitch muscles of the lower hind limbs and was further characterized as described below. For Immunohistochemistry convenience, the HSA-rtTA/TRE-H2B-GFP mouse is For immunohistochemistry (IHC) analyses, the various referred to as the HSA-GFP mouse. hind limb muscles were covered in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Tor- Doxycycline treatment rance, CA, USA) and pinned at resting length to a cork To induce H2B-GFP expression, 3–10-month-old HSA- covered in aluminum foil. Muscles were frozen in liquid GFP mice were administered doxycycline (0.5 mg/mL) in nitrogen-cooled isopentane and stored at − 80 °C. Mus- drinking water supplemented with 2% sucrose for 3 weeks. cles were sectioned at the mid-belly on a cryostat at − Tissue was collected immediately upon completion of 23 °C. Frozen muscle sections (7 μm) were air-dried for doxycycline treatment. To determine the earliest time of at least 1 h and stored at − 20 °C. For Pax7/DAPI IHC, GFP induction, skeletal muscle was collected after 12 h or muscles were first fixed in 4% paraformaldehyde for 24 h following doxycycline administration. 7 min and then subjected to epitope retrieval. Following HSA promoter BGI Cre rtTA Mer Fig. 1 A schematic of the HSA-rtTA transgene. The promoter and first exon (− 2,000 to + 239 relative to the transcription start site) of the human skeletal muscle α-actin (HSA) gene regulates expression of an optimized reverse tetracycline transactivator (rtTA) gene which has been reported to be sevenfold more active and 100-fold more doxycycline sensitive than the original Tet-On system [8]. The β-globin intron ΙΙ (BGI) and poly(A) tail were incorporated into the transgene to ensure proper splicing and transcript stability, respectively. The positions of the PCR primers used for genotyping are indicated by half-arrows Iwata et al. Skeletal Muscle (2018) 8:33 Page 3 of 8 epitope retrieval in sodium citrate (10 mM, pH 6.5) for diaphragm fibers (n = 2) were counted representing a total 20 min at 92 °C, endogenous peroxidases were blocked for of 906 myonuclei, whereas 37 plantaris fibers (n =2) were 7 min with 3% hydrogen peroxide in phosphate-buffered counted representing a total of 1294 myonuclei. For IHC, saline (PBS), followed by 1 h with 1% Tyramide Signal images were captured at × 20 magnification using a Zeiss Amplification (TSA) blocking reagent (TSA kit, T20935, upright fluorescent microscope (Zeiss Axio Imager M1, Invitrogen) supplemented with Mouse-on-Mouse (MoM) Oberkochen, Germany). Whole muscle sections were ob- IgG blocking reagent (Vector Laboratories, Burlingame, tained using the mosaic function in Zeiss Zen 2.3 imaging CA, USA). Sections were washed in PBS and incubated software. Satellite cells (Pax7+/DAPI+) and GFP+ satellite overnight with mouse anti-Pax7 IgG1 antibody (1:100, cells were identified manually using Zen software tools. Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA) and chicken anti-GFP antibody (1:200, Results Abcam, Cambridge, MA, USA) diluted in 1% TSA block- Skeletal muscle-specific rtTA transgene ing reagent. It was necessary to use anti-GFP antibody to To generate the skeletal muscle-specific rtTA transgene detect GFP expression because the antigen retrieval for microinjection, we cloned downstream of the human process quenched the GFP signal. The following day, skeletal muscle α-actin (HSA) promoter a third gener- sections were washed with PBS, incubated for 70 min in ation rtTA gene that was reported to be sevenfold more goat anti-mouse IgG1 biotinylated secondary antibody active and 100-fold more doxycycline sensitive than the (1:1000, 115-065-205, Jackson ImmunoResearch, West original rtTA [8]. A schematic of the HSA-rtTA trans- Grove, PA, USA) and anti-chicken GFP secondary anti- gene is shown in Fig. 1. body, (1:250, Abcam), washed in PBS, incubated for 1 h in streptavidin-horseradish peroxidase (1:500, S-911, Invitro- Skeletal muscle-specific expression of rtTA mRNA gen) diluted in PBS, washed again in PBS, then incubated We determined by reverse transcription-PCR the expres- for 15 min in TSA Alexa Fluor 594 (1:500, TSA kit, Invi- sion of rtTA mRNA in several hind limb muscles, the trogen) in the supplied amplification diluents. Sections diaphragm, fat, and several other non-muscle organs. As were stained with DAPI (1:10,000 in PBS, D35471, Invitro- shown in Fig. 2, rtTA mRNA was highly expressed in all gen) for 5 min and mounted with VectaShield fluorescent of the hind limb muscles examined and to a lesser extent mounting media. in the diaphragm and heart. As expected, rtTA expression was not detectable in any non-muscle tissues. Image acquisition and quantification GFP+/DAPI+ and GFP−/DAPI+ myonuclei from ~ 10 iso- Effective labeling of myonuclei in hind limb skeletal lated fibers from four doxycycline-treated mice (two male muscles and two female) were counted for each muscle, resulting Having established that rtTA was highly enriched in skel- in a range of 202–452 myonuclei being analyzed across etal muscle, we sought to determine how effective the muscles. Twelve to 15 single fibers from two untreated HSA-rtTA transgene was in driving H2B-GFP expression mice (1 male and 1 female) were analyzed for each muscle, in response to doxycycline treatment. Given that resulting in a range of 254–600 myonuclei being analyzed H2B-GFP is nuclearly localized, we used the percentage of across muscles. For the time course analysis, 35 myonuclei that were GFP+ on single fibers as a measure Fig. 2 Skeletal muscle-specific expression of rtTA. PCR analysis of rtTA mRNA expression of different tissues from the HSA-GFP transgenic mouse showed high levels of expression in skeletal muscle (gastrocnemius, soleus, plantaris, tibialis anterior (TA), and extensor digitorum longus (EDL)), modest expression in the diaphragm, very low expression in the heart, and not detectable in non-muscle tissue (brain, fat, lung, liver, stomach, spleen, and kidney) Iwata et al. Skeletal Muscle (2018) 8:33 Page 4 of 8 of the effectiveness of the HSA-rtTA transgene to induce digitorum longus (EDL) of doxycycline-treated mice and expression of a tetracycline-responsive gene. Following then stained with DAPI to identify myonuclei. As shown fixation, single fibers were isolated from the plantaris, in Fig. 3a, b, greater than 95% (range of 96.4–97.9%) of gastrocnemius, soleus, tibialis anterior (TA), and extensor myonuclei were GFP+ across all muscles from Fig. 3 HSA-rtTA transgene drives robust myofiber expression of tetracycline-responsive H2B-GFP transgene. a Representative single fiber images of hind limb muscles taken from HSA-GFP mice (n = 4) treated with doxycycline. Single fiber images show robust myonuclear GFP expression in muscles composed of slow- and fast-twitch fibers. b Quantification of GFP+ myonuclei of single fibers from hind limb skeletal muscles (plantaris, gastrocnemius, soleus, tibialis anterior (TA), and extensor digitorum longus (EDL)) of HSA-GFP mice showed greater than 95% of all DAPI+ myonuclei within a fiber were GFP+. The gray bar represents the average percentage of GFP-positive myonuclei (n = 4) for each muscle Iwata et al. Skeletal Muscle (2018) 8:33 Page 5 of 8 doxycycline-treated HSA-GFP mice. We observed no GFP GFP labeling is highly specific to myonuclei + myonuclei in skeletal muscle single fibers of untreated To determine if the HSA-rtTA drove expression of the HSA-GFP mice demonstrating tight regulation of H2B-GFP transgene in satellite cells, we performed immu- tetracycline-responsive H2B-GFP gene (data not shown). nohistochemistry on both soleus and plantaris muscle These findings confirm the HSA-rtTA mouse is capable of cross-sections for DAPI, Pax7, and GFP. As shown in Fig. 4, driving robust expression of a tetracycline-responsive gene GFP labeling did not localize with Pax7 staining; however, in adult skeletal muscles composed of both slow- and in the soleus, of the 190 satellite cells counted, three Pax7+ fast-twitch fibers. cells appeared to be GFP+. These results demonstrate the Plantaris Soleus DAPI GFP Pax7 Fig. 4 GFP expression is specific to myonuclei in HSA-GFP mice. Representative muscle cross-section images of the plantaris and soleus muscles from HSA-GFP mice treated with doxycycline (n = 3). As indicated by white arrows, DAPI+/GFP+ myonuclei (green) did not show co-localization with DAPI+/Pax7+ satellite cells (pink). These results confirm that the HSA-rtTA transgene is able to drive myofiber-specific expression of a tetracycline-response gene Iwata et al. Skeletal Muscle (2018) 8:33 Page 6 of 8 HSA-rtTA drives highly myofiber-specific expression of a skeletal muscle α-actin (HSA) promoter was used to tetracycline-responsive transgene. drive skeletal muscle-specific expression of the reverse- tetracycline transactivator (rtTA), designated as the Rapid GFP labeling of myonuclei HSA-rtTA mouse. The HSA promoter contains 2,000 bp To determine the time course of GFP labeling of myo- of human skeletal α-actin 5′-flanking sequence plus the nuclei, skeletal muscle was collected from HAS-GFP first exon and 149 bp of the first intron and was first re- mice after 12 or 24 h of doxycycline treatment. As ported by Muscat and Kedes to promote robust, skeletal shown in Fig. 5a, b, approximately 90% of myonuclei in muscle-specific expression [9]. We choose to use the the plantaris muscle were GFP-positive after 24 h of HSA promoter because we previously showed it was able doxycycline treatment; in contrast, GFP expression was to drive effective Cre-mediated recombination in both not detected following 12 h of doxycycline administra- slow- and fast-twitch fibers with minimal expression in tion (data not shown). We also examined whether GFP the heart [6]. This is an important improvement over a expression followed the same time course of induction previous skeletal muscle-specific Tet-ON mouse (MCK- given the modest expression of rtTA mRNA in the rtTA) which only allowed over-expression of a gene of diaphragm (see Fig. 2). While GFP expression was not as interest in fast-twitch, type IIb fibers [10]. Together with robust as that observed in the plantaris, 60% of myonu- the HSA-CreER mouse, the HSA-rtTA mouse now pro- clei of the diaphragm were GFP-positive following 24 h vides the ability to perform loss- and gain-of-function of doxycycline treatment, consistent with the lower rtTA studies, respectively, to determine the in vivo function of mRNA expression (see Fig. 5a–b). a gene of interest in skeletal muscle fibers [6, 11]. The complement to these two inducible, skeletal muscle-spe- Discussion cific mice are the satellite cell-specific inducible Cre and The purpose of this study was to characterize a new Tet-ON mice; however, while the satellite cell-specific skeletal muscle-specific Tet-ON mouse. The human Cre mouse has been extensively used, to the best of our Fig. 5 Rapid GFP labeling of myonuclei. a Representative single fiber images of plantaris and diaphragm muscles taken from HSA-GFP mice (n = 2) treated with doxycycline for 24 h. b Quantification of GFP+ myonuclei showed ~ 90% of myonuclei were GFP-positive in myofibers from the plantaris with 60% of myonuclei GFP-positive in myofibers isolated from the diaphragm Iwata et al. Skeletal Muscle (2018) 8:33 Page 7 of 8 knowledge, the satellite cell-specific Tet-ON mouse has Funding This work was supported by NIH grants AG049806 and AR060701 to JJM and yet to be fully characterized [12–15]. Collectively, these CAP and AR071753 to KAM. inducible, skeletal muscle-, and satellite cell-specific mice provide powerful tools to manipulate in vivo gene Availability of data and materials The datasets used and/or analyzed during the current study are available expression to identify and better understand the mecha- from the corresponding author on request. The HSA-rtTA mouse is available nisms regulating skeletal muscle biology in health and upon request. disease. While 3 weeks of doxycycline treatment was able to in- Declarations N.A. duce > 95% GFP labeling of myonuclei, we wanted to know the earliest time point when GFP expression could be Authors’ contributions detected following doxycycline administration. As shown in JJM cloned the transgene. MI, DAE, YW, CMD, KAM, IJ, CBM, CAP, and JJM Fig. 5, single fiber analysis revealed myonuclear GFP assisted in developing the study design and characterizing the mouse. DAE and JJM wrote the manuscript. All authors read and approved the final expression was observed as early as 24 h post-doxycycline manuscript. exposure; in contrast, we observed no GFP expression at 12 h post-doxycycline treatment (data not shown). These Ethics approval and consent to participate results demonstrate the HSA-rtTA mouse is very respon- All animal procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals as approved by the sive to doxycycline and will provide the ability to study the Animal Care and Use Committee of the University of Kentucky. relative early (~ 24 h) effects of gene activation on a given biological process. For example, the HSA-rtTA mouse Consent for publication N.A. could be used to study how the early (~ 24 h) activation of Akt1 (using the tetracycline-responsive, constitutively active Competing interests Akt1 mouse, TRE-myrAkt1) affects the hypertrophic The authors declare that they have no competing interests. response in skeletal muscle as Akt1 is typically not activated until 48 h [16, 17]. Publisher’sNote In contrast to the strong rtTA expression in hind limb Springer Nature remains neutral with regard to jurisdictional claims in muscles, there was modest, but detectable, expression of published maps and institutional affiliations. rtTA mRNA in the diaphragm. We do not know the rea- Author details son why rtTA expression is lower in the diaphragm 1 The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, compared to hind limb muscles, but it may reflect a USA. Department of Rehabilitation Sciences, College of Health Sciences, University of Kentucky, Lexington, KY 40536, USA. Department of limitation of the HSA promoter to drive high levels of Physiology, College of Medicine, University of Kentucky, 800 Rose Street, expression in the diaphragm. We also found compara- Medical Science Building, Rm: MS-607A, Lexington, KY 40536, USA. tively lower expression of Cre in the diaphragm of the Department of Rehabilitation, Faculty of Health Sciences, Nihon Fukushi University, 26-2 Higashihaemi-cho, Handa 475-0012, Japan. HSA-CreER mouse, consistent with the idea that the HSA promoter is not as robust in the diaphragm as it is Received: 27 June 2018 Accepted: 16 October 2018 in other muscles. Despite low rtTA expression, we still observed ~ 60% GFP labeling of diaphragm myonuclei References after only 24 h of doxycycline treatment. This result in- 1. Furth PA, St Onge L, Boger H, Gruss P, Gossen M, Kistner A, et al. 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Journal

Skeletal MuscleSpringer Journals

Published: Oct 27, 2018

References