Novel Mammalian Ubiquitous Promoter Isolated from Bovine MSTN Gene Promoter

Novel Mammalian Ubiquitous Promoter Isolated from Bovine MSTN Gene Promoter | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Novel Mammalian Ubiquitous Promoter Isolated from Bovine MSTN Gene Promoter Kyeong-Hyeon Eom, Dong-Hyeok Kwon, Young-Chai Kim, Gyeong-Min Gim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3851722/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract As the understandings about the biotechnology and the pathophysiology of diseases getting advanced, the genetic materials or genetically engineered cells have brought hopes on patients with inherited diseases. Among many congenital diseases, the muscular dystrophy has been globally one of the major subjects of genetic therap. To apply genetic therapy selectively in muscular tissue, the promoters which express genes specifically in muscle have been necessitated by researchers. In the current study, the promoter region of MSTN gene was postulated as candidate muscle-specific promoter for gene therapy, from the biological significance and muscle-specific distribution of the myostatin. Accordingly, we aimed to isolate a novel promoter for gene therapy from the MSTN gene promoter and trim it more suitable for the therapeutic applications. During the experiments, it was revealed that the MSTN promoter region have functionally distinguishable parts: the highly conserved core region and the region that react to myogenic differentiation. The core region of bovine MSTN gene promoter showed ubiquitous expression of marker gene in differentiated cell lines or cells with stemness, originated from human, mouse, and cattle. In conclusion, we suggest the proximal region of bovine MSTN gene promoter as novel ubiquitous promoter. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction As the understandings about the biotechnology and the pathophysiology of diseases getting advanced, the genetic materials or genetically engineered cells have brought hopes on patients with inherited diseases to which symptomatic therapies were only option of treatment in the past. Among many congenital diseases, the muscular dystrophy (e.g. Duchenne muscular dystrophy) has been globally one of the major subjects of genetic therapy, considering its severity of symptoms. To apply genetic therapy selectively in muscular tissue, while not affecting the other tissues, the promoters which express genes specifically in muscle have been necessitated by researchers. As such attempts, the promoter of genes which are expressed specifically in muscle tissue have been the candidate muscle-specific promoter 1 . And in the same line, we selected the promoter region of MSTN gene, which encodes a negative regulator of myogenesis, myostatin, or also known as growth-differentiation factor 8 (GDF-8) as a candidate muscle-specific promoter, considering its muscle-specific distribution in vivo . Since its first discovery, the MSTN gene has been of interest in biomedical fields as a biomarker or a potent target of drugs for muscular deficiency diseases 2 – 6 . Recently, from the functional and structural conservation, the MSTN gene has been targeted for inducing knockout mutation or knock-down in domestic livestock and this interspecies conservation of the coding sequences led us to postulate the conservation of promoter region 7 – 11 . The promoter regions of MSTN gene have been reviewed as a potential modulator the expression of myostatin 12 . Among many of binding sites that regulates the activity of the promoter, the enhancer box (E-box; CANNTG) motives has been highlighted in the MSTN gene promoter regions. Particularly, the E-box domains to which the major myogenesis-related factor, the MyoD protein binds were previously discovered in bovine MSTN gene promoter 13 . Accordingly, we aimed to isolate a novel promoter for gene therapy from the MSTN gene promoter and trim it more suitable for the therapeutic applications. 2. Results Proximal parts of the promoter regions of the MSTN gene were conserved in mammalian species. Before designing the studies of the promoter region of the MSTN gene, the positions of E-box motives were reviewed. When the E-box motif sequences were arranged based on the distance from the start codon in first exon of the coding sequences, total numbers of the E-boxes showed no consensus. However, the proximal three E-boxes seemed to be highly conservated in similar position among mammalian species, while the distal motives were arranged characteristically by the species (Fig. 1 ). Isolation of a core promoter region from the bovine MSTN promoter According to the conservation and the previous report by Spiller et al., the E-boxes were grouped in three parts in the next steps: E-boxes 1 and 2 were included as a proximal group, E-boxes 3, 4, 5, and 6 were grouped as a middle group. The residual E-boxes 7, 8, 9, and 10 were a distal group. As the first step, The C2C12 myoblast cell line transfected with GFP expression vector, with 1.7kb of bovine MSTN gene promoter as a promoter, was generated as a myogenic model cell line and a bovine fibroblast line was also employed. The three E-box groups were deleted respectively, using two guide RNAs targeting 5’ and 3’ ends of each group (Fig. 2 A). After inducing deletions in each group, the GFP expression was significantly decreased in the C2C12 cell line for all knockout groups compared to the untreated cell line: 28.06 ± 4.69% for the distal, 49.20 ± 20.63% for the middle, and 6.18 ± 1.54% for the proximal cluster knockout groups (Fig. 2 B). Representative images after inducing deletion and deletion efficiency are showed in the Supplementary Fig. 1. The expression level of MSTN was significantly decreased in bovine primary cells in all treated groups, with slightly different tendencies. The expression level decreased to 33.29 ± 25.13% for distal, 36.69 ± 24.77% for middle, and 5.30 ± 2.29% for proximal cluster knockouts compared with the untreated control group (Fig. 2 C). The E-boxes in the bovine MSTN promoter were functionally distinguishable. Subsequently, each E-box group was cloned into a transposon vector with a GFP expression sequence downstream. And the C2C12 cell lines were produced using the vectors possessing each cluster of bovine MSTN promoter (Fig. 3 A). The C2C12 cell’s GFP expression levels were measured before and after differentiation into myotubes (Fig. 3 B and C). The cell line containing the proximal E-box group showed the highest GFP expression level (Fig. 3 D and E). These results suggest that the proximal E-box group plays a core role in expression of bovine MSTN gene promoter. In contrast, the cell line with the middle cluster showed increased promoter activity after differentiation, as supported by the results of a previous study (Fig. 3 E) 13 . For further analysis, the middle E-box group was attached with EF1a promoter, which have been conventionally employed as a ubiquitous promoter (Fig. 3 F). The expression of the reporter gene expression was decreased in C2C12 cell, before differentiation (Fig. 3 G and H). However, 48h after exposure to the differentiation media, the expression level was increased with the middle E-box group, while the expression level with EF1a promoter alone was remained similar level (Fig. 3 I). Application of the proximal E-box group from bovine MSTN promoter as novel transgene promoter in various cell lines Consequently, it was examined if the proximal E-box group may induce marker gene expression in different cells from various species. The cell lines evaluated are listed in Supplementary Table 4. Before testing, the proximal E-box group (M243 promoter, hereon) was modified in two ways and the modified promoters were also included for comparison. For the first modification, the enhancer sequence from the cytomegalovirus (CMV) promoter was added to the 5′ end of the MP#1 to increase the expression level (named as ceM243), and the downstream 73 bp sequences after the transcription start site was deleted for the other modification (named as sM243). The M243 promoter and the modified ones were introduced in 10 cell lines from mouse, human, and cattle. And the expression level of marker GFP expression were compared with groups transfected with ubiquitous promoters; CAG promoter, human elongation factor-1 alpha short (EFS) promoter, and mouse PGK (mPGK) promoter. The GFP signal was detected using fluorescence microscopy and flow cytometry (Fig. 4 ), and the mean fluorescence intensity was presented as a heatmap (Supplementary Fig. 2). The M243 promoter had a consistent GFP signal in all the tested cell lines with a comparable fluorescence level to the mPGK promoter (Fig. 4 and supplementary Fig. 2). The two modifications applied to M243 showed opposite results. The ceM243 showed fluorescence comparable with the CAG and EFS promoters. In contrast, the sM243 showed the lowest fluorescence intensity, with GFP barely detected by fluorescence microscopy. Application of M243 and ceM243 promoter as novel transgene promoters for bovine embryo and human/ mouse embryonic stem cells. As a next step, the novel promoters were tested in human and mouse embryonic stem cells. In the same line with the results from cell lines, the M243 promoter and ceM243 promoter showed the marker gene expression (Fig. 5 A-F). Lastly, the activity of novel promoters was tested in bovine pre-implantation embryos. For bovine embryo, lentiviruses were packaged with either M243 or ceM243 with GFP sequences downstream and were injected into presumptive bovine zygotes. For the group injected with M243, during 3 times of microinjection, total of 217 zygotes was included and 16.19 ± 4.86% reached blastocyst at day 8 post-fertilization. Among embryos which reached blastocyst stage, 71.02 ± 10.81% showed GFP fluorescence. And for the ceM243 group, 178 zygotes were included during 4 times of repeats and 24.00 ± 6.93% of embryos reached blastocyst stage. Among blastocysts, 85.84 ± 6.27% showed GFP fluorescence. Representative images are displayed in Fig. 5 G and H. 3. Discussion In the current study, we aimed to isolate the novel muscle-specific promoter from the promoter region of the MSTN gene, which shows selectively high expression level in skeletal muscle. For the purposes, the E-box motives, which were previously focused as a target for transcription factor related to myogenesis, were reviewed first and the proximal E-boxes showed consensus among mammalian species. Based on this conservation, it could be postulated that the proximal cluster of the E-box motives may have a critical role in MSTN expression. Therefore, the promoter region from bovine species was selected for further experiments and the deletion experiment was conducted to verify the hypothesis. Consequently, the deletion experiment revealed that the proximal E-box group would critically affect the promoter activity in myogenic model cell line and non-myogenic cells. However, unlike gene coding sequences in which ‘on’ or ‘off’ is usually determined after a knockout, the randomity of the CRISPR-Cas9 system was an obstacle to the study’s progress in the case of the promoter (e.g., single-cell level analysis) and led us to separate each part and clone them for further analysis. Interestingly, the experiments with cloned vectors revealed that the proximal E-boxes showed the highest expression level, regardless of differentiation, while the middle cluster showed the highest change in expression before and after differentiation. In this step, the bovine MSTN promoter seemed to operate as coordination of sequences with different functions. To verify, the middle cluster was attached to the ubiquitous promoter and with the additional sequences, the expression level increased selectively in differentiated cells. These results showed that the promoter region of bovine MSTN gene is functioning with two separate parts: the short core region and the region reactive to myogenesis. This functional discrimination was unexpected, and we employed the proximal core region for further analysis, considering the small size would be more beneficial for gene therapy than the middle one. The proximal group of E-boxes were then named as M243 promoter and tested further in various cell lines originated from different tissues of human, mouse and cattle. However, about the proximal E-box group, the results between the previous report by Spiller et al. and the current study seemed to be contradictory, in aspect of E-boxes included. The hint to explain the differences is in the differences of expression level between M243 and sM243 promoter (Fig. 4 ). The 73bp after transcription start site was sM243 promoter and the expression level decreased dramatically and the sequences spanning E-box 1 and 2 in the study by Spiller et al. is closer to sM243 than M243, considering the size of sequences used in the experiments. Consequently, the M243 and ceM243 promoter were assumed as novel ubiquitous promoter. After validation in differently differentiated cell lines, the expression in cells with stemness and embryos were also tested, and stable expression was identified. These results led us to suggest the proximal region of the bovine MSTN gene promoter as novel ubiquitous promoter which can be employed in therapeutic genetic engineering or generation of transgenic animal model. We expect the novel promoters suggested in this study could be used extensively not only for its small size which can be packed with larger gene of interest in vectors with a limited cargo capacity like viral vectors, but also for its inter-species conservation which could possibly be an escapeway from silencing issue, when applied to mammalian species. 4. Materials & Methods Identification of E-box motif and cloning of the bovine MSTN promoter E-box regions and cloning Upstream sequences from first exon of MSTN gene from cattle (ARS-UCD 1.2), sheep (Oar_rambouillet_v1.0), goat (ARS1), pig (Sscrofa11.1), mouse (GRCm39) and human (GRCh38.p14) genome were analyzed for E-box motif CANNTG. Based on the results, the assumed bovine MSTN promoter region of 3 kb from the start codon in the MSTN exon 1 was cloned from bovine primary cells. The cloned region and GFP expression sequences were inserted into a vector with piggyBac transposon sequences using overlapping PCR-based cloning kits (Clontech, Cat. no. 639648) 14 . In addition, the E-box clusters were cloned from the 3 kb promoter region using automatically designed primer sets spanning each cluster. The cloned E-box clusters and GFP sequence were inserted into the piggyBac transposon vector. Sequences of promoters cloned in this study are listed in supplementary table 1 . Guide ribonucleic acid design and synthesis Guide ribonucleic acid (gRNA) target sites were suggested by RGEN Cas-Designer software ( http://www.rgenome.net/cas-designer/ ) to induce mutations in the bovine MSTN gene promoter region. Among the suggestions, double gRNA targeting sequences nearest each cluster’s 5′ and 3′ ends were selected to induce large deletion on each cluster. The gRNAs and primer sequences used to detect mutations are listed in supplementary tables 2 and 3. The selected guide RNAs were synthesized in vitro, using Precision gRNA synthesis Kit (Product no. A29377; Thermo Fisher Scientific Inc., Waltham, MA, USA), following the manufacturer’s protocol. The induced deletion mutations were detected by primers listed in Supplementary Table 3; the efficiency was estimated using relative intensity between bands with the expected intact product and shorter product with a larger deletion. The shorter bands were interpreted to possess the expected deletion using Sanger sequencing. Cell culture and transfection For non-stem cells, all cells were cultured in Dulbecco’s Modified Eagle Medium (Cat. no. 21068028) supplemented with 20% fetal bovine serum (Cat. no. GIB-16000–044), 1% penicillin/streptomycin (Cat. no. 15140148; all Gibco™, Thermo Fisher Scientific Inc., Waltham, MA, USA), at 37.5°C and 5% CO2 humidified atmosphere. Depending on the purpose of each experiment, vectors or the Cas9-gRNA complex (RNP) were introduced into cells using a NEON™ transfection kit (Cat. no. MPK1096; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The RNP was composed of 100ng/µl Cas9 protein and 200ng/µl of guide RNA. In case of the transposon vectors, 500 ng each of the vectors of interest and vectors coding transposase were co-transfected. Except for Cal-62 and ISD-0615, Cells used in the current study was purchased from the American Type Culture Collection (ATCC, USA) and the catalog numbers are listed as: C2C12; #CRL-1772, 3T3-L1; #CL-173, C127:LT; #CRL-1804, MCF7; #HTB-22, HeLa; #CCL-2, A549; #CCL-185, PC-3; #CRL-1435,and MDBK; #CCL-22. Cal-62 cells was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany, #ACC 448). ISD-0615 cell line is an bovine immortalized fibroblast cell line established in previous study 15 . In case of stem cells, mouse embryonic stem cells (mESCs) were maintained on 0.5% porcine gelatin-coated tissue culture dish under the following culture media - DMEM high glucose (Gibco, #11965) supplemented with 15% FBS (Gibco, #16000), 1X β-mercaptoethanol (Gibco, #21985), 1X glutaMAX (Gibco, #35050), 1X non-essential-amino-acid (Gibco, #11140), 0.1% Gentamicin (Gibco, #15710064), 1,000 U/ml mouse leukemia inhibitory factor (mLIF) (Millipore, Merck, #ESG1107), 1 µM PD0325901 (Peprotech, #3911091) and 3 µM CHIR99021(Peprotech, #2520691)- at 37°C and 5% CO2 incubating condition. Cells were passaged using Accutase (BD Biosciences, #561527). Human embryonic stem cells (hESCs) were maintained on 1X Matrigel (Corning, #354277) coated tissue culture dish under the following culture media - stemMACS (Miltenyi Biotec, #130-104-368) supplemented with 0.1% Gentamicin (Gibco, #15710064)- at 37°C and 5% CO2 incubating condition. Cells were passaged using 1X dispase (Gibco, #17105). For mESCs, 5 x 10 5 cells were seeded 1 day before the transfection. 2µg of piggy-bac vectors and 2µg of Transposase vectors were co-transfected into adherent cells using Lipofectamine 3000 reagent (Invitrogen, #L3000-001). For hESCs, 2µg of piggy-bac vectors and 2µg of Transposase vectors were co-transfected into 1 x 10 6 cells through electroporation at 750V for 2.5sec using NEPA-21 (NEPA GENE). Quantitative real-time polymerase chain reaction The total RNA was extracted from cells treated with vectors or RNPs after a minimum of 7 days of culture using RNeasy Mini Kit (Cat. no. 74106; Qiagen GmbH, Hilden, Germany) to measure expression levels Complementary deoxyribonucleic acid (cDNA) was synthesized from 2 µg of RNA using the RNA to cDNA EcoDry™ Premix (Cat. no. 639543; Takara Bio Inc., Kusatsu, Shiga, Japan). Gene expression assay was conducted using SYBR™ Green (Thermo Fisher Scientific Inc., Waltham, MA, USA) on a QuantStudio™ 3 real-time polymerase chain reaction (PCR) system (Cat. no. A28132; Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA), and relative cycle threshold (Ct) values were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of corresponding species (e.g., mouse GAPDH for C2C12 samples and bovine GAPDH for bovine fibroblast samples). Flow cytometry The fluorescence of the transfected cells was measured using a FACSAria™ II flow cytometer (Becton, Dickinson and Company [BD], Franklin Lakes, NJ, USA). Culture medium was removed, and cells were washed with phosphate buffered saline (PBS) (Gibco, Cat. no. 10010023). The culture medium was removed, and cells were washed with Gibco™ phosphate-buffered saline (PBS) (Cat. no. 10010023; Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were detached from the culture dishes using Gibco™1X TrypLE™ Express Enzyme (Cat. no. 12605010; Thermo Fisher Scientific Inc., Waltham, MA, USA); the reaction was stopped with fresh culture media containing FBS. Cells were then collected and centrifuged at 2,000 rpm for 5 min at room temperature. The supernatant was discarded, and the remaining cells were washed three times using PBS. The fluorescence intensity of samples containing at least 10,000 cells was measured by flow cytometry at 488 nm wavelength laser, and the mean fluorescence intensity was compared between the samples. In vitro production of bovine embryos Immature oocytes were collected in the ovaries from slaughterhouses and matured in TCM based medium for in vitro maturation and motile spermatozoa were selected using the Percoll gradient method after 24h of maturation. The maturation and sperm refinement were performed as previous described 7 . Briefly, frozen-thawed semen from F0 bull at 35 ℃ was filtered by centrifugation on a Percoll discontinuous gradient (45–90%) at 366 x g for 15 min. To produce the 45% Percoll solution, 1 mL of capacitation-Tyrode’s albumin lactate pyruvate (TALP) medium was added to 1 mL of 90% Percoll. The sperm pellet was washed twice by adding 3 mL of the capacitation-TALP medium and centrifuged at 366 x g for 5 min. The washed motile spermatozoa were used for in vitro fertilization. Spermatozoa (1–2 × 10 6 sperm /mL) were incubated with mature oocytes for 18 h in 50 µL micro drops of IVF-TALP medium covered with mineral oil (Cat. no. NO-100; NidaCon International AB, Mölndal, Sweden) in a humidified atmosphere of 5% CO2 at 38.5 ℃. After 18 h of co-incubation, cumulus cells were removed from the presumptive zygotes. The zygotes were cultured in a two-step chemically defined culture media that was covered in mineral oil in an atmosphere of 5% O 2 , 5% CO 2 , and 90% N 2 at 38.5 ◦ C 7 , 14 . Sub-zonal injection of lentivirus into bovine embryos To induce expression of exogenous gene by promoters used in current study, the promoters and following GFP sequence were packaged into lentivirus by commercial service (request no. VB230712-1243tqu and VB230712-1244phu; Vectorbuilder Inc., Chicago, IL, USA). The resultant titer of > 10 8 transducing units (TU)/ml of each strain was guaranteed by manufacturer. Following 20–22 hours of in vitro fertilization, the presumptive zygotes were denuded and used for the subsequent experiments. Five microliters of each lentivirus was loaded without concentration dilution and the loaded materials were injected into perivitelline space of zygotes. The one-third point of the bottom of each zygote was targeted for injection to prevent cytoplasmic injection. Injection was performed using Eppendorf™ FemtoJet™ injection pump (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 20–30 s at 100–150 hPa of constant pressure (Pc), causing the embryo to swell 1.5 times. Declarations Acknowledgements This study was financially supported by the Research Institute of Veterinary Science and the BK21 Four for Future Veterinary Medicine Leading Education and Research Center, Seoul National University (SNU) grant (#550e2020005), and by the Technology Innovation Program (20023353) funded By the Ministry of Trade, Industry & Energy (MOTIE, Korea). Author contributions K.H.E., H.J.C., and G.J. conceived the study. K.H.E., G.M.G., and S.Y.Y. designed the methodology and provided data validation. K.H.E., Y.C.K., D.H.K., and S.M.K. conducted experiments. K.H.E. and S.M.K. conducted data analysis, imaging. K.H.E. wrote the manuscript. G.G.M. and S.Y.Y. contributed to the review and editing process. G.J. supervised the whole process. Data availability The datasets generated and/or analyzed during this study are available from the corresponding author on reasonable request. The authors declared no potential conflicts of interest for the research, authorship, and/or publication of this article. References Skopenkova, V. V., Egorova, T. V. & Bardina, M. V. Muscle-Specific Promoters for Gene Therapy. Acta Naturae 13 , 47-58 (2021). https://doi.org/10.32607/actanaturae.11063 Burch, P. M. et al. Reduced serum myostatin concentrations associated with genetic muscle disease progression. J Neurol 264 , 541-553 (2017). https://doi.org/10.1007/s00415-016-8379-6 Campbell, C. et al. Myostatin inhibitor ACE-031 treatment of ambulatory boys with Duchenne muscular dystrophy: Results of a randomized, placebo-controlled clinical trial. Muscle Nerve 55 , 458-464 (2017). https://doi.org/10.1002/mus.25268 Kerschan-Schindl, K. et al. Myostatin and markers of bone metabolism in dermatomyositis. 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Supplementary Files SupplementaryFig.1.pdf SupplementaryFIg.2.pdf SupplementaryTable1.pdf SupplementaryTable2.pdf SupplementaryTable3.pdf SupplementaryTable4.pdf Cite Share Download PDF Status: Published Journal Publication published 12 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Mar, 2024 Reviews received at journal 19 Mar, 2024 Reviewers agreed at journal 13 Mar, 2024 Reviews received at journal 25 Feb, 2024 Reviewers agreed at journal 19 Feb, 2024 Reviewers invited by journal 02 Feb, 2024 Editor assigned by journal 22 Jan, 2024 Editor invited by journal 15 Jan, 2024 Submission checks completed at journal 15 Jan, 2024 First submitted to journal 10 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3851722","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267185146,"identity":"a53bc376-b986-4675-bf40-3b2ce2775cc1","order_by":0,"name":"Kyeong-Hyeon Eom","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Kyeong-Hyeon","middleName":"","lastName":"Eom","suffix":""},{"id":267185147,"identity":"3f1c2f49-e925-427b-9edf-4e43a11453aa","order_by":1,"name":"Dong-Hyeok Kwon","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hyeok","middleName":"","lastName":"Kwon","suffix":""},{"id":267185148,"identity":"b190ca60-3c23-4558-8b54-e24ac111e9e3","order_by":2,"name":"Young-Chai Kim","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Young-Chai","middleName":"","lastName":"Kim","suffix":""},{"id":267185149,"identity":"7cfe2b5d-3b07-4201-9b4f-cab2c7466b93","order_by":3,"name":"Gyeong-Min Gim","email":"","orcid":"","institution":"LART Bio Inc","correspondingAuthor":false,"prefix":"","firstName":"Gyeong-Min","middleName":"","lastName":"Gim","suffix":""},{"id":267185150,"identity":"77dac19a-b598-45c2-ad6c-ad1f549342f3","order_by":4,"name":"Soo-Young Yum","email":"","orcid":"","institution":"LART Bio Inc","correspondingAuthor":false,"prefix":"","firstName":"Soo-Young","middleName":"","lastName":"Yum","suffix":""},{"id":267185151,"identity":"32c723e1-2c44-4186-9b1b-0f1fc1fa294d","order_by":5,"name":"Seong-Min Kim","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Seong-Min","middleName":"","lastName":"Kim","suffix":""},{"id":267185152,"identity":"21895c4e-cbd5-403e-8882-6fd9ee9171ac","order_by":6,"name":"Hyuk-Jin Cha","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Hyuk-Jin","middleName":"","lastName":"Cha","suffix":""},{"id":267185153,"identity":"0622714b-c705-4140-9c72-0a944efc69d7","order_by":7,"name":"Goo Jang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYJACAyCWQ+YQp8WYNC0gkNhAtBb52T0GBT931KbPBzIYftQwGJs3ENBicOeMgWHvmeO5G4AMxp5jDGYyBwhpkcgxMOBtO5a7Achg4G1gsJEg6LAZOQaGf9uOpYMYjH+J0cJwI8fAmLetJgHEYAbaYkZQi8GNtAJj2bYDhhuAjMMyxySMiXBY8jbDt2118kDGxodvamwMZxB0GAMDGzAqDoNZBxgYCPsEBJgfMDDUEaVyFIyCUTAKRigAACESOuy+CuPZAAAAAElFTkSuQmCC","orcid":"","institution":"Seoul National University","correspondingAuthor":true,"prefix":"","firstName":"Goo","middleName":"","lastName":"Jang","suffix":""}],"badges":[],"createdAt":"2024-01-10 23:29:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3851722/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3851722/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-76937-2","type":"published","date":"2024-11-12T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49766318,"identity":"3889f798-c11e-4d1c-ae47-110db8780aa4","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38814,"visible":true,"origin":"","legend":"\u003cp\u003ePositions of E-box motives in mammalian species. Positions of E-box motives was described proportionally, from first ATG codon in first exon of myostatin gene to upstream 1.7kb in human and domestic mammals.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/96121c66e8f30064c45885fc.png"},{"id":49766981,"identity":"d60e8865-7904-42b0-82c9-4ce2071f97e8","added_by":"auto","created_at":"2024-01-17 17:07:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227449,"visible":true,"origin":"","legend":"\u003cp\u003ePromoter activity after inducing large deletion in Bovine \u003cem\u003eMSTN\u003c/em\u003e promoter. (A) Experimental scheme of knockout study. Guide ribonucleic acid (gRNA) of red, blue, and green were used to induce large deletions of E-box’s distal, middle, and proximal clusters, respectively. (B) Green fluorescent protein (GFP) expression levels in a C2C12 model cell line after inducing large deletions. Values were normalized with same deletion efficiency. (C) Myostatin (\u003cem\u003eMSTN\u003c/em\u003e) expression after inducing a large deletion in bovine immortalized fibroblasts. Values were normalized with same deletion efficiency.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/83d5317294512b0e1609d909.png"},{"id":49767428,"identity":"e8fbfd1f-c27c-4c01-abb8-3a1c8c84c745","added_by":"auto","created_at":"2024-01-17 17:15:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":958366,"visible":true,"origin":"","legend":"\u003cp\u003eGreen fluorescent protein (GFP) expression in C2C12 cell lines transfected with E-box vectors.\u003cstrong\u003e \u003c/strong\u003e(A) Experimental scheme and GFP vector construction used. Each group was transfected using vectors with proximal cluster (a and a’), middle cluster (b and b’), or distal cluster (c and c’). Bright and fluorescent images of cells before exposure to differentiation medium (B) and after exposure (C) Scale bar = 275μm. (D) Relative GFP expression from each experimental group were determined by quantitative real-time polymerase chain reaction. (qPCR). (E) Fold change of GFP expression between proliferating cells and differentiated cells. Calculated from results shown in Fig. 3D. (F) GFP vector construction for functional analysis of middle E-box group. (G) Representative images of C2C12 cells transfected with the illustrated vectors. (a and a’) EF1a promoter-GFP vector, (b and b’) EF1a-promoter-GFP vector with middle E-box group attached at 5’ end. Scale bar = 750 μm. (H) Flow cytometry results of cells in Fig. 3G. (a) cells with EF1a promoter only, (b) cells with middle E-box and EF1a promoter. (I) GFP expression level before and 48 hours after differentiation into myotubes. Only cells with GFP signal were used.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/5d61def77422ec1ba4bdd683.png"},{"id":49766324,"identity":"6b0d436b-abfc-4aa5-a194-add64ba78e19","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2211556,"visible":true,"origin":"","legend":"\u003cp\u003eGreen fluorescent protein (GFP) expression in cell lines originating from human, mouse, and cattle tissues using this study’s vector. Scale bar = 275μm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/79a57ed2f61d8923ba15c0ed.png"},{"id":49766327,"identity":"bded0c7a-706c-45be-9ec7-e4be89c5e006","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1870220,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of marker gene in mouse embryonic stem cell (mESC) (A-C) and human embryonic stem cell (hESC) (D-F). (A, D) Representative time-dependent images after transfection on mESC. The fluorescence images were merged with brightfield images. (B, E) fluorescence analysis results by flow cytometry. (C, F) Mean fluorescence intensity in fluorescence positive cells. Scale bar = 500μm. (G-H) Green fluorescent protein (GFP) expression in bovine pre-implantation embryos.\u003cstrong\u003e \u003c/strong\u003eBright and fluorescence image of embryos injected with lentivirus with M243 promoter (G) or ceM243 promoter (H). Scale bar = 100μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/5edd9e85d5bfbae814e8b7ec.png"},{"id":69286120,"identity":"41b51ad3-661a-418d-bbfe-bb2466c1906a","added_by":"auto","created_at":"2024-11-18 19:29:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6643580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/ad1b99c5-208a-472b-9437-75ba92e8318d.pdf"},{"id":49766985,"identity":"0e6834bc-62c6-4a8a-a807-2771163ab2ee","added_by":"auto","created_at":"2024-01-17 17:07:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":174714,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/9220402fe59263340d93dc97.pdf"},{"id":49766982,"identity":"f478f4b3-db80-49dd-a844-3f341f09b850","added_by":"auto","created_at":"2024-01-17 17:07:49","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":47995,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFIg.2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/8520cc65ee9a9d989836f7f0.pdf"},{"id":49767427,"identity":"cec70410-a484-4d5a-8761-f825e849becc","added_by":"auto","created_at":"2024-01-17 17:15:49","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":69429,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/272dee01a8853c6a48f72a3d.pdf"},{"id":49766320,"identity":"ed4ad87d-b09a-4a87-b627-8857b6cb0733","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11020,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/d4fccb062e2f0a6d5b68beaf.pdf"},{"id":49766323,"identity":"3b50c13c-a2c3-4279-9922-e6725b1b23ce","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11120,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/4b15b5a491e210e8c5639be9.pdf"},{"id":49766329,"identity":"81fbabf4-0785-4d81-a175-dd0d627a43ce","added_by":"auto","created_at":"2024-01-17 16:59:49","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15134,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3851722/v1/a5aec8d9b4f505268ffe95c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel Mammalian Ubiquitous Promoter Isolated from Bovine MSTN Gene Promoter","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs the understandings about the biotechnology and the pathophysiology of diseases getting advanced, the genetic materials or genetically engineered cells have brought hopes on patients with inherited diseases to which symptomatic therapies were only option of treatment in the past. Among many congenital diseases, the muscular dystrophy (e.g. Duchenne muscular dystrophy) has been globally one of the major subjects of genetic therapy, considering its severity of symptoms. To apply genetic therapy selectively in muscular tissue, while not affecting the other tissues, the promoters which express genes specifically in muscle have been necessitated by researchers.\u003c/p\u003e \u003cp\u003eAs such attempts, the promoter of genes which are expressed specifically in muscle tissue have been the candidate muscle-specific promoter\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. And in the same line, we selected the promoter region of \u003cem\u003eMSTN\u003c/em\u003e gene, which encodes a negative regulator of myogenesis, myostatin, or also known as growth-differentiation factor 8 (GDF-8) as a candidate muscle-specific promoter, considering its muscle-specific distribution \u003cem\u003ein vivo\u003c/em\u003e. Since its first discovery, the \u003cem\u003eMSTN\u003c/em\u003e gene has been of interest in biomedical fields as a biomarker or a potent target of drugs for muscular deficiency diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recently, from the functional and structural conservation, the \u003cem\u003eMSTN\u003c/em\u003e gene has been targeted for inducing knockout mutation or knock-down in domestic livestock and this interspecies conservation of the coding sequences led us to postulate the conservation of promoter region\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe promoter regions of \u003cem\u003eMSTN\u003c/em\u003e gene have been reviewed as a potential modulator the expression of myostatin\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Among many of binding sites that regulates the activity of the promoter, the enhancer box (E-box; CANNTG) motives has been highlighted in the \u003cem\u003eMSTN\u003c/em\u003e gene promoter regions. Particularly, the E-box domains to which the major myogenesis-related factor, the MyoD protein binds were previously discovered in bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Accordingly, we aimed to isolate a novel promoter for gene therapy from the \u003cem\u003eMSTN\u003c/em\u003e gene promoter and trim it more suitable for the therapeutic applications.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cem\u003eProximal parts of the promoter regions of the MSTN gene were conserved in mammalian species.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBefore designing the studies of the promoter region of the \u003cem\u003eMSTN\u003c/em\u003e gene, the positions of E-box motives were reviewed. When the E-box motif sequences were arranged based on the distance from the start codon in first exon of the coding sequences, total numbers of the E-boxes showed no consensus. However, the proximal three E-boxes seemed to be highly conservated in similar position among mammalian species, while the distal motives were arranged characteristically by the species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIsolation of a core promoter region from the bovine MSTN promoter\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAccording to the conservation and the previous report by Spiller et al., the E-boxes were grouped in three parts in the next steps: E-boxes 1 and 2 were included as a proximal group, E-boxes 3, 4, 5, and 6 were grouped as a middle group. The residual E-boxes 7, 8, 9, and 10 were a distal group. As the first step, The C2C12 myoblast cell line transfected with GFP expression vector, with 1.7kb of bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter as a promoter, was generated as a myogenic model cell line and a bovine fibroblast line was also employed. The three E-box groups were deleted respectively, using two guide RNAs targeting 5\u0026rsquo; and 3\u0026rsquo; ends of each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After inducing deletions in each group, the GFP expression was significantly decreased in the C2C12 cell line for all knockout groups compared to the untreated cell line: 28.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69% for the distal, 49.20\u0026thinsp;\u0026plusmn;\u0026thinsp;20.63% for the middle, and 6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54% for the proximal cluster knockout groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Representative images after inducing deletion and deletion efficiency are showed in the Supplementary Fig.\u0026nbsp;1. The expression level of \u003cem\u003eMSTN\u003c/em\u003e was significantly decreased in bovine primary cells in all treated groups, with slightly different tendencies. The expression level decreased to 33.29\u0026thinsp;\u0026plusmn;\u0026thinsp;25.13% for distal, 36.69\u0026thinsp;\u0026plusmn;\u0026thinsp;24.77% for middle, and 5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29% for proximal cluster knockouts compared with the untreated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eThe E-boxes in the bovine MSTN promoter were functionally distinguishable.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSubsequently, each E-box group was cloned into a transposon vector with a GFP expression sequence downstream. And the C2C12 cell lines were produced using the vectors possessing each cluster of bovine \u003cem\u003eMSTN\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The C2C12 cell\u0026rsquo;s GFP expression levels were measured before and after differentiation into myotubes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). The cell line containing the proximal E-box group showed the highest GFP expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). These results suggest that the proximal E-box group plays a core role in expression of bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter. In contrast, the cell line with the middle cluster showed increased promoter activity after differentiation, as supported by the results of a previous study (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For further analysis, the middle E-box group was attached with EF1a promoter, which have been conventionally employed as a ubiquitous promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The expression of the reporter gene expression was decreased in C2C12 cell, before differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H). However, 48h after exposure to the differentiation media, the expression level was increased with the middle E-box group, while the expression level with EF1a promoter alone was remained similar level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eApplication of the proximal E-box group from bovine MSTN promoter as novel transgene promoter in various cell lines\u003c/em\u003e \u003c/p\u003e \u003cp\u003eConsequently, it was examined if the proximal E-box group may induce marker gene expression in different cells from various species. The cell lines evaluated are listed in Supplementary Table\u0026nbsp;4. Before testing, the proximal E-box group (M243 promoter, hereon) was modified in two ways and the modified promoters were also included for comparison. For the first modification, the enhancer sequence from the cytomegalovirus (CMV) promoter was added to the 5\u0026prime; end of the MP#1 to increase the expression level (named as ceM243), and the downstream 73 bp sequences after the transcription start site was deleted for the other modification (named as sM243). The M243 promoter and the modified ones were introduced in 10 cell lines from mouse, human, and cattle. And the expression level of marker GFP expression were compared with groups transfected with ubiquitous promoters; CAG promoter, human elongation factor-1 alpha short (EFS) promoter, and mouse PGK (mPGK) promoter. The GFP signal was detected using fluorescence microscopy and flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and the mean fluorescence intensity was presented as a heatmap (Supplementary Fig.\u0026nbsp;2). The M243 promoter had a consistent GFP signal in all the tested cell lines with a comparable fluorescence level to the mPGK promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and supplementary Fig.\u0026nbsp;2). The two modifications applied to M243 showed opposite results. The ceM243 showed fluorescence comparable with the CAG and EFS promoters. In contrast, the sM243 showed the lowest fluorescence intensity, with GFP barely detected by fluorescence microscopy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eApplication of M243 and ceM243 promoter as novel transgene promoters for bovine embryo and human/ mouse embryonic stem cells.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAs a next step, the novel promoters were tested in human and mouse embryonic stem cells. In the same line with the results from cell lines, the M243 promoter and ceM243 promoter showed the marker gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). Lastly, the activity of novel promoters was tested in bovine pre-implantation embryos. For bovine embryo, lentiviruses were packaged with either M243 or ceM243 with GFP sequences downstream and were injected into presumptive bovine zygotes. For the group injected with M243, during 3 times of microinjection, total of 217 zygotes was included and 16.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86% reached blastocyst at day 8 post-fertilization. Among embryos which reached blastocyst stage, 71.02\u0026thinsp;\u0026plusmn;\u0026thinsp;10.81% showed GFP fluorescence. And for the ceM243 group, 178 zygotes were included during 4 times of repeats and 24.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.93% of embryos reached blastocyst stage. Among blastocysts, 85.84\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27% showed GFP fluorescence. Representative images are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn the current study, we aimed to isolate the novel muscle-specific promoter from the promoter region of the \u003cem\u003eMSTN\u003c/em\u003e gene, which shows selectively high expression level in skeletal muscle. For the purposes, the E-box motives, which were previously focused as a target for transcription factor related to myogenesis, were reviewed first and the proximal E-boxes showed consensus among mammalian species. Based on this conservation, it could be postulated that the proximal cluster of the E-box motives may have a critical role in \u003cem\u003eMSTN\u003c/em\u003e expression. Therefore, the promoter region from bovine species was selected for further experiments and the deletion experiment was conducted to verify the hypothesis. Consequently, the deletion experiment revealed that the proximal E-box group would critically affect the promoter activity in myogenic model cell line and non-myogenic cells.\u003c/p\u003e \u003cp\u003eHowever, unlike gene coding sequences in which \u0026lsquo;on\u0026rsquo; or \u0026lsquo;off\u0026rsquo; is usually determined after a knockout, the randomity of the CRISPR-Cas9 system was an obstacle to the study\u0026rsquo;s progress in the case of the promoter (e.g., single-cell level analysis) and led us to separate each part and clone them for further analysis. Interestingly, the experiments with cloned vectors revealed that the proximal E-boxes showed the highest expression level, regardless of differentiation, while the middle cluster showed the highest change in expression before and after differentiation. In this step, the bovine \u003cem\u003eMSTN\u003c/em\u003e promoter seemed to operate as coordination of sequences with different functions. To verify, the middle cluster was attached to the ubiquitous promoter and with the additional sequences, the expression level increased selectively in differentiated cells. These results showed that the promoter region of bovine \u003cem\u003eMSTN\u003c/em\u003e gene is functioning with two separate parts: the short core region and the region reactive to myogenesis. This functional discrimination was unexpected, and we employed the proximal core region for further analysis, considering the small size would be more beneficial for gene therapy than the middle one.\u003c/p\u003e \u003cp\u003eThe proximal group of E-boxes were then named as M243 promoter and tested further in various cell lines originated from different tissues of human, mouse and cattle. However, about the proximal E-box group, the results between the previous report by Spiller et al. and the current study seemed to be contradictory, in aspect of E-boxes included. The hint to explain the differences is in the differences of expression level between M243 and sM243 promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 73bp after transcription start site was sM243 promoter and the expression level decreased dramatically and the sequences spanning E-box 1 and 2 in the study by Spiller et al. is closer to sM243 than M243, considering the size of sequences used in the experiments. Consequently, the M243 and ceM243 promoter were assumed as novel ubiquitous promoter. After validation in differently differentiated cell lines, the expression in cells with stemness and embryos were also tested, and stable expression was identified. These results led us to suggest the proximal region of the bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter as novel ubiquitous promoter which can be employed in therapeutic genetic engineering or generation of transgenic animal model. We expect the novel promoters suggested in this study could be used extensively not only for its small size which can be packed with larger gene of interest in vectors with a limited cargo capacity like viral vectors, but also for its inter-species conservation which could possibly be an escapeway from silencing issue, when applied to mammalian species.\u003c/p\u003e"},{"header":"4. Materials \u0026 Methods","content":"\u003cp\u003e \u003cem\u003eIdentification of E-box motif and cloning of the bovine MSTN promoter E-box regions and cloning\u003c/em\u003e \u003c/p\u003e \u003cp\u003eUpstream sequences from first exon of \u003cem\u003eMSTN\u003c/em\u003e gene from cattle (ARS-UCD 1.2), sheep (Oar_rambouillet_v1.0), goat (ARS1), pig (Sscrofa11.1), mouse (GRCm39) and human (GRCh38.p14) genome were analyzed for E-box motif CANNTG. Based on the results, the assumed bovine \u003cem\u003eMSTN\u003c/em\u003e promoter region of 3 kb from the start codon in the \u003cem\u003eMSTN\u003c/em\u003e exon 1 was cloned from bovine primary cells. The cloned region and GFP expression sequences were inserted into a vector with piggyBac transposon sequences using overlapping PCR-based cloning kits (Clontech, Cat. no. 639648) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, the E-box clusters were cloned from the 3 kb promoter region using automatically designed primer sets spanning each cluster. The cloned E-box clusters and GFP sequence were inserted into the piggyBac transposon vector. Sequences of promoters cloned in this study are listed in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGuide ribonucleic acid design and synthesis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eGuide ribonucleic acid (gRNA) target sites were suggested by RGEN Cas-Designer software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rgenome.net/cas-designer/\u003c/span\u003e\u003cspan address=\"http://www.rgenome.net/cas-designer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to induce mutations in the bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter region. Among the suggestions, double gRNA targeting sequences nearest each cluster\u0026rsquo;s 5\u0026prime; and 3\u0026prime; ends were selected to induce large deletion on each cluster. The gRNAs and primer sequences used to detect mutations are listed in supplementary tables 2 and 3. The selected guide RNAs were synthesized in vitro, using Precision gRNA synthesis Kit (Product no. A29377; Thermo Fisher Scientific Inc., Waltham, MA, USA), following the manufacturer\u0026rsquo;s protocol. The induced deletion mutations were detected by primers listed in Supplementary Table\u0026nbsp;3; the efficiency was estimated using relative intensity between bands with the expected intact product and shorter product with a larger deletion. The shorter bands were interpreted to possess the expected deletion using Sanger sequencing.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell culture and transfection\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor non-stem cells, all cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (Cat. no. 21068028) supplemented with 20% fetal bovine serum (Cat. no. GIB-16000\u0026ndash;044), 1% penicillin/streptomycin (Cat. no. 15140148; all Gibco\u0026trade;, Thermo Fisher Scientific Inc., Waltham, MA, USA), at 37.5\u0026deg;C and 5% CO2 humidified atmosphere. Depending on the purpose of each experiment, vectors or the Cas9-gRNA complex (RNP) were introduced into cells using a NEON\u0026trade; transfection kit (Cat. no. MPK1096; Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;s instructions. The RNP was composed of 100ng/\u0026micro;l Cas9 protein and 200ng/\u0026micro;l of guide RNA. In case of the transposon vectors, 500 ng each of the vectors of interest and vectors coding transposase were co-transfected. Except for Cal-62 and ISD-0615, Cells used in the current study was purchased from the American Type Culture Collection (ATCC, USA) and the catalog numbers are listed as: C2C12; #CRL-1772, 3T3-L1; #CL-173, C127:LT; #CRL-1804, MCF7; #HTB-22, HeLa; #CCL-2, A549; #CCL-185, PC-3; #CRL-1435,and MDBK; #CCL-22. Cal-62 cells was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany, #ACC 448). ISD-0615 cell line is an bovine immortalized fibroblast cell line established in previous study\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn case of stem cells, mouse embryonic stem cells (mESCs) were maintained on 0.5% porcine gelatin-coated tissue culture dish under the following culture media - DMEM high glucose (Gibco, #11965) supplemented with 15% FBS (Gibco, #16000), 1X β-mercaptoethanol (Gibco, #21985), 1X glutaMAX (Gibco, #35050), 1X non-essential-amino-acid (Gibco, #11140), 0.1% Gentamicin (Gibco, #15710064), 1,000 U/ml mouse leukemia inhibitory factor (mLIF) (Millipore, Merck, #ESG1107), 1 \u0026micro;M PD0325901 (Peprotech, #3911091) and 3 \u0026micro;M CHIR99021(Peprotech, #2520691)- at 37\u0026deg;C and 5% CO2 incubating condition. Cells were passaged using Accutase (BD Biosciences, #561527). Human embryonic stem cells (hESCs) were maintained on 1X Matrigel (Corning, #354277) coated tissue culture dish under the following culture media - stemMACS (Miltenyi Biotec, #130-104-368) supplemented with 0.1% Gentamicin (Gibco, #15710064)- at 37\u0026deg;C and 5% CO2 incubating condition. Cells were passaged using 1X dispase (Gibco, #17105). For mESCs, 5 x 10\u003csup\u003e5\u003c/sup\u003e cells were seeded 1 day before the transfection. 2\u0026micro;g of piggy-bac vectors and 2\u0026micro;g of Transposase vectors were co-transfected into adherent cells using Lipofectamine 3000 reagent (Invitrogen, #L3000-001). For hESCs, 2\u0026micro;g of piggy-bac vectors and 2\u0026micro;g of Transposase vectors were co-transfected into 1 x 10\u003csup\u003e6\u003c/sup\u003e cells through electroporation at 750V for 2.5sec using NEPA-21 (NEPA GENE).\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuantitative real-time polymerase chain reaction\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe total RNA was extracted from cells treated with vectors or RNPs after a minimum of 7 days of culture using RNeasy Mini Kit (Cat. no. 74106; Qiagen GmbH, Hilden, Germany) to measure expression levels Complementary deoxyribonucleic acid (cDNA) was synthesized from 2 \u0026micro;g of RNA using the RNA to cDNA EcoDry\u0026trade; Premix (Cat. no. 639543; Takara Bio Inc., Kusatsu, Shiga, Japan). Gene expression assay was conducted using SYBR\u0026trade; Green (Thermo Fisher Scientific Inc., Waltham, MA, USA) on a QuantStudio\u0026trade; 3 real-time polymerase chain reaction (PCR) system (Cat. no. A28132; Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA), and relative cycle threshold (Ct) values were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of corresponding species (e.g., mouse GAPDH for C2C12 samples and bovine GAPDH for bovine fibroblast samples).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlow cytometry\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe fluorescence of the transfected cells was measured using a FACSAria\u0026trade; II flow cytometer (Becton, Dickinson and Company [BD], Franklin Lakes, NJ, USA). Culture medium was removed, and cells were washed with phosphate buffered saline (PBS) (Gibco, Cat. no. 10010023). The culture medium was removed, and cells were washed with Gibco\u0026trade; phosphate-buffered saline (PBS) (Cat. no. 10010023; Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were detached from the culture dishes using Gibco\u0026trade;1X TrypLE\u0026trade; Express Enzyme (Cat. no. 12605010; Thermo Fisher Scientific Inc., Waltham, MA, USA); the reaction was stopped with fresh culture media containing FBS. Cells were then collected and centrifuged at 2,000 rpm for 5 min at room temperature. The supernatant was discarded, and the remaining cells were washed three times using PBS. The fluorescence intensity of samples containing at least 10,000 cells was measured by flow cytometry at 488 nm wavelength laser, and the mean fluorescence intensity was compared between the samples.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro production of bovine embryos\u003c/em\u003e \u003c/p\u003e \u003cp\u003eImmature oocytes were collected in the ovaries from slaughterhouses and matured in TCM based medium for \u003cem\u003ein vitro\u003c/em\u003e maturation and motile spermatozoa were selected using the Percoll gradient method after 24h of maturation. The maturation and sperm refinement were performed as previous described \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Briefly, frozen-thawed semen from F0 bull at 35 ℃ was filtered by centrifugation on a Percoll discontinuous gradient (45\u0026ndash;90%) at 366 x g for 15 min. To produce the 45% Percoll solution, 1 mL of capacitation-Tyrode\u0026rsquo;s albumin lactate pyruvate (TALP) medium was added to 1 mL of 90% Percoll. The sperm pellet was washed twice by adding 3 mL of the capacitation-TALP medium and centrifuged at 366 x g for 5 min. The washed motile spermatozoa were used for \u003cem\u003ein vitro\u003c/em\u003e fertilization. Spermatozoa (1\u0026ndash;2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e sperm /mL) were incubated with mature oocytes for 18 h in 50 \u0026micro;L micro drops of IVF-TALP medium covered with mineral oil (Cat. no. NO-100; NidaCon International AB, M\u0026ouml;lndal, Sweden) in a humidified atmosphere of 5% CO2 at 38.5 ℃. After 18 h of co-incubation, cumulus cells were removed from the presumptive zygotes. The zygotes were cultured in a two-step chemically defined culture media that was covered in mineral oil in an atmosphere of 5% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 90% N\u003csub\u003e2\u003c/sub\u003e at 38.5 \u003csup\u003e◦\u003c/sup\u003eC \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSub-zonal injection of lentivirus into bovine embryos\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo induce expression of exogenous gene by promoters used in current study, the promoters and following GFP sequence were packaged into lentivirus by commercial service (request no. VB230712-1243tqu and VB230712-1244phu; Vectorbuilder Inc., Chicago, IL, USA). The resultant titer of \u0026gt;\u0026thinsp;10\u003csup\u003e8\u003c/sup\u003e transducing units (TU)/ml of each strain was guaranteed by manufacturer. Following 20\u0026ndash;22 hours of \u003cem\u003ein vitro\u003c/em\u003e fertilization, the presumptive zygotes were denuded and used for the subsequent experiments. Five microliters of each lentivirus was loaded without concentration dilution and the loaded materials were injected into perivitelline space of zygotes. The one-third point of the bottom of each zygote was targeted for injection to prevent cytoplasmic injection. Injection was performed using Eppendorf\u0026trade; FemtoJet\u0026trade; injection pump (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 20\u0026ndash;30 s at 100\u0026ndash;150 hPa of constant pressure (Pc), causing the embryo to swell 1.5 times.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Research Institute of Veterinary Science and the BK21 Four for Future Veterinary Medicine Leading Education and Research Center, Seoul National University (SNU) grant (#550e2020005), and by the Technology Innovation Program (20023353) funded By the Ministry of Trade, Industry \u0026amp; Energy (MOTIE, Korea).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.H.E., H.J.C., and G.J. conceived the study. K.H.E., G.M.G., and S.Y.Y. designed the methodology and provided data validation. K.H.E., Y.C.K., D.H.K., and S.M.K. conducted experiments. K.H.E. and S.M.K. conducted data analysis, imaging. K.H.E. wrote the manuscript. G.G.M. and S.Y.Y. contributed to the review and editing process. G.J. supervised the whole process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eThe authors declared no potential conflicts of interest for the research, authorship, and/or publication of this article.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSkopenkova, V. V., Egorova, T. V. \u0026amp; Bardina, M. V. Muscle-Specific Promoters for Gene Therapy. \u003cem\u003eActa Naturae\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 47-58 (2021). https://doi.org/10.32607/actanaturae.11063\u003c/li\u003e\n\u003cli\u003eBurch, P. M.\u003cem\u003e et al.\u003c/em\u003e Reduced serum myostatin concentrations associated with genetic muscle disease progression. \u003cem\u003eJ Neurol\u003c/em\u003e \u003cstrong\u003e264\u003c/strong\u003e, 541-553 (2017). https://doi.org/10.1007/s00415-016-8379-6\u003c/li\u003e\n\u003cli\u003eCampbell, C.\u003cem\u003e et al.\u003c/em\u003e Myostatin inhibitor ACE-031 treatment of ambulatory boys with Duchenne muscular dystrophy: Results of a randomized, placebo-controlled clinical trial. \u003cem\u003eMuscle Nerve\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 458-464 (2017). https://doi.org/10.1002/mus.25268\u003c/li\u003e\n\u003cli\u003eKerschan-Schindl, K.\u003cem\u003e et al.\u003c/em\u003e Myostatin and markers of bone metabolism in dermatomyositis. \u003cem\u003eBMC Musculoskelet Disord\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 150 (2021). https://doi.org/10.1186/s12891-021-04030-0\u003c/li\u003e\n\u003cli\u003eYamada, S.\u003cem\u003e et al.\u003c/em\u003e Factors Associated with the Serum Myostatin Level in Patients Undergoing Peritoneal Dialysis: Potential Effects of Skeletal Muscle Mass and Vitamin D Receptor Activator Use. \u003cem\u003eCalcif Tissue Int\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 13-22 (2016). https://doi.org/10.1007/s00223-016-0118-6\u003c/li\u003e\n\u003cli\u003eZhao, C.\u003cem\u003e et al.\u003c/em\u003e Myostatin serum concentrations are correlated with the severity of knee osteoarthritis. \u003cem\u003eJ Clin Lab Anal\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e (2017). https://doi.org/10.1002/jcla.22094\u003c/li\u003e\n\u003cli\u003eGim, G. M.\u003cem\u003e et al.\u003c/em\u003e Production of MSTN-mutated cattle without exogenous gene integration using CRISPR-Cas9. \u003cem\u003eBiotechnol J\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e2100198 (2022). https://doi.org/10.1002/biot.202100198\u003c/li\u003e\n\u003cli\u003eLi, R.\u003cem\u003e et al.\u003c/em\u003e Precise editing of myostatin signal peptide by CRISPR/Cas9 increases the muscle mass of Liang Guang Small Spotted pigs. \u003cem\u003eTransgenic Res\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 149-163 (2020). https://doi.org/10.1007/s11248-020-00188-w\u003c/li\u003e\n\u003cli\u003eRen, H.\u003cem\u003e et al.\u003c/em\u003e Myostatin regulates fatty acid desaturation and fat deposition through MEF2C/miR222/SCD5 cascade in pigs. \u003cem\u003eCommun Biol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 612 (2020). https://doi.org/10.1038/s42003-020-01348-8\u003c/li\u003e\n\u003cli\u003eWang, X.\u003cem\u003e et al.\u003c/em\u003e CRISPR/Cas9-mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass. \u003cem\u003eAnim Genet\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 43-51 (2018). https://doi.org/10.1111/age.12626\u003c/li\u003e\n\u003cli\u003eXu, K.\u003cem\u003e et al.\u003c/em\u003e Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e (2020). https://doi.org/10.3390/ijms21072584\u003c/li\u003e\n\u003cli\u003eGrade, C. V. C., Mantovani, C. S. \u0026amp; Alvares, L. E. Myostatin gene promoter: structure, conservation and importance as a target for muscle modulation. \u003cem\u003eJ Anim Sci Biotechnol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 32 (2019). https://doi.org/10.1186/s40104-019-0338-5\u003c/li\u003e\n\u003cli\u003eSpiller, M. P.\u003cem\u003e et al.\u003c/em\u003e The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD. \u003cem\u003eMol Cell Biol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 7066-7082 (2002). https://doi.org/10.1128/MCB.22.20.7066-7082.2002\u003c/li\u003e\n\u003cli\u003eYum, S. Y.\u003cem\u003e et al.\u003c/em\u003e Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 27185 (2016). https://doi.org/10.1038/srep27185\u003c/li\u003e\n\u003cli\u003eChoi, W.\u003cem\u003e et al.\u003c/em\u003e Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells. \u003cem\u003ePrion\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 278-291 (2015). https://doi.org/10.1080/19336896.2015.1071459\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3851722/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3851722/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs the understandings about the biotechnology and the pathophysiology of diseases getting advanced, the genetic materials or genetically engineered cells have brought hopes on patients with inherited diseases. Among many congenital diseases, the muscular dystrophy has been globally one of the major subjects of genetic therap. To apply genetic therapy selectively in muscular tissue, the promoters which express genes specifically in muscle have been necessitated by researchers. In the current study, the promoter region of \u003cem\u003eMSTN\u003c/em\u003e gene was postulated as candidate muscle-specific promoter for gene therapy, from the biological significance and muscle-specific distribution of the myostatin. Accordingly, we aimed to isolate a novel promoter for gene therapy from the \u003cem\u003eMSTN\u003c/em\u003e gene promoter and trim it more suitable for the therapeutic applications. During the experiments, it was revealed that the \u003cem\u003eMSTN\u003c/em\u003e promoter region have functionally distinguishable parts: the highly conserved core region and the region that react to myogenic differentiation. The core region of bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter showed ubiquitous expression of marker gene in differentiated cell lines or cells with stemness, originated from human, mouse, and cattle. In conclusion, we suggest the proximal region of bovine \u003cem\u003eMSTN\u003c/em\u003e gene promoter as novel ubiquitous promoter.\u003c/p\u003e","manuscriptTitle":"Novel Mammalian Ubiquitous Promoter Isolated from Bovine MSTN Gene Promoter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 16:59:44","doi":"10.21203/rs.3.rs-3851722/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-26T04:30:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-19T04:28:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5a57a1a8-8df6-469e-876b-dd9861227926","date":"2024-03-13T20:33:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-25T12:24:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9b0bc97e-c042-43ad-9364-ae014e6deb84","date":"2024-02-19T05:27:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-02T06:30:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-22T11:08:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-15T13:13:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-15T07:19:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-10T23:20:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"963621ec-e715-40db-aaf1-92a996b0aff0","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T19:27:13+00:00","versionOfRecord":{"articleIdentity":"rs-3851722","link":"https://doi.org/10.1038/s41598-024-76937-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-12 15:57:59","publishedOnDateReadable":"November 12th, 2024"},"versionCreatedAt":"2024-01-17 16:59:44","video":"","vorDoi":"10.1038/s41598-024-76937-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-76937-2","workflowStages":[]},"version":"v1","identity":"rs-3851722","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3851722","identity":"rs-3851722","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}