{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T04:16:19Z","timestamp":1780546579988,"version":"3.54.1"},"reference-count":25,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2019,6,8]],"date-time":"2019-06-08T00:00:00Z","timestamp":1559952000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.<\/jats:p>","DOI":"10.3390\/genes10060436","type":"journal-article","created":{"date-parts":[[2019,6,10]],"date-time":"2019-06-10T03:16:51Z","timestamp":1560136611000},"page":"436","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR"],"prefix":"10.3390","volume":"10","author":[{"given":"Takehito","family":"Sugasawa","sequence":"first","affiliation":[{"name":"Laboratory of Laboratory\/Sports medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Aoki","sequence":"additional","affiliation":[{"name":"Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koichi","family":"Watanabe","sequence":"additional","affiliation":[{"name":"Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koki","family":"Yanazawa","sequence":"additional","affiliation":[{"name":"Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tohru","family":"Natsume","sequence":"additional","affiliation":[{"name":"Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 100-8921, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9591-2250","authenticated-orcid":false,"given":"Tohru","family":"Takemasa","sequence":"additional","affiliation":[{"name":"Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaori","family":"Yamaguchi","sequence":"additional","affiliation":[{"name":"Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshinori","family":"Takeuchi","sequence":"additional","affiliation":[{"name":"Nutrigenomics Research Group, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuichi","family":"Aita","sequence":"additional","affiliation":[{"name":"Nutrigenomics Research Group, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoya","family":"Yahagi","sequence":"additional","affiliation":[{"name":"Nutrigenomics Research Group, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuko","family":"Yoshida","sequence":"additional","affiliation":[{"name":"Department of Medical Technology, Faculty of Health Sciences, Tsukuba International University, 6-20-1 Manabe, Tsuchiura, Ibaraki 300-0051, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katsuyuki","family":"Tokinoya","sequence":"additional","affiliation":[{"name":"Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"},{"name":"Japan Society for the Promotion of Science; Kojimachi Business Center Building, Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nanami","family":"Sekine","sequence":"additional","affiliation":[{"name":"Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaoru","family":"Takeuchi","sequence":"additional","affiliation":[{"name":"Laboratory of Environmental Microbiology, Division of Basic Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haruna","family":"Ueda","sequence":"additional","affiliation":[{"name":"Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasushi","family":"Kawakami","sequence":"additional","affiliation":[{"name":"Laboratory of Laboratory\/Sports medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satoshi","family":"Shimizu","sequence":"additional","affiliation":[{"name":"Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiro","family":"Takekoshi","sequence":"additional","affiliation":[{"name":"Laboratory of Laboratory\/Sports medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,6,8]]},"reference":[{"key":"ref_1","unstructured":"Japan Anti-Doping Agency (JADA) (2019, March 12). What Is Anti-Doping?. Available online: https:\/\/www.playtruejapan.org\/about\/."},{"key":"ref_2","unstructured":"The World Anti-Doping Agency (WADA) (2019, March 12). Who We Are?. Available online: https:\/\/www.wada-ama.org\/en\/who-we-are."},{"key":"ref_3","unstructured":"The World Anti-Doping Agency (WADA) (2016). Anti-Doping Textbook, WADA."},{"key":"ref_4","unstructured":"The World Anti-Doping Agency (WADA) (2018). World Anti-Doping Code with International Standard, Prohibited List, WADA."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2337","DOI":"10.1016\/j.ymthe.2018.07.011","article-title":"Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy","volume":"26","author":"Duan","year":"2018","journal-title":"Mol. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"R36","DOI":"10.1093\/hmg\/ddv475","article-title":"Adeno-associated viral vectors for the treatment of hemophilia","volume":"25","author":"High","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s40259-017-0234-5","article-title":"Adeno-Associated Virus (AAV) as a Vector for Gene Therapy","volume":"31","author":"Naso","year":"2017","journal-title":"BioDrugs"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1147","DOI":"10.1136\/bjophthalmol-2016-309975","article-title":"Leber congenital amaurosis\/early-onset severe retinal dystrophy: Clinical features, molecular genetics and therapeutic interventions","volume":"101","author":"Kumaran","year":"2017","journal-title":"Br. J. Ophthalmol."},{"key":"ref_9","unstructured":"Sharif, W., and Sharif, Z. (2017). Leber\u2019s congenital amaurosis and the role of gene therapy in congenital retinal disorders. Int. J. Ophthalmol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"421","DOI":"10.2174\/1566523213666131125095046","article-title":"Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy","volume":"13","author":"Wold","year":"2014","journal-title":"Curr. Gene Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.gendis.2017.04.001","article-title":"Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine","volume":"4","author":"Lee","year":"2017","journal-title":"Genes Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1089\/hum.2017.206","article-title":"Treatment of Uterine Sarcoma with rAd-p53 (Gendicine) Followed by Chemotherapy: Clinical Study of TP53 Gene Therapy","volume":"29","author":"Xia","year":"2018","journal-title":"Hum. Gene Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1089\/hum.2017.218","article-title":"The First Approved Gene Therapy Product for Cancer Ad- p53 (Gendicine): 12 Years in the Clinic","volume":"29","author":"Zhang","year":"2018","journal-title":"Hum. Gene Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"29189159","DOI":"10.2174\/1568009618666171129221503","article-title":"Oncorine, The World First Oncolytic Virus Medicine and its Update in China","volume":"18","author":"Liang","year":"2018","journal-title":"Curr. Cancer Drug Targets"},{"key":"ref_15","unstructured":"The Journal of Gene Medicine (2019, March 11). Charts and Tables, Vectors. Available online: http:\/\/www.abedia.com\/wiley\/vectors.php."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1016\/j.cmet.2011.08.012","article-title":"Imaging cytosolic NADH-NAD + redox state with a genetically encoded fluorescent biosensor","volume":"14","author":"Hung","year":"2011","journal-title":"Cell Metab."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.bbrc.2007.08.165","article-title":"In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression","volume":"363","author":"Takeuchi","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2373","DOI":"10.1016\/j.celrep.2016.07.069","article-title":"KLF15 Enables Rapid Switching between Lipogenesis and Gluconeogenesis during Fasting","volume":"16","author":"Takeuchi","year":"2016","journal-title":"Cell Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1006\/viro.2002.1406","article-title":"Evaluation of accuracy and precision of adenovirus absorptivity at 260 nm under conditions of complete DNA disruption","volume":"295","author":"Sweeney","year":"2002","journal-title":"Virology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1186\/s13104-018-3815-6","article-title":"Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection","volume":"11","author":"Tozaki","year":"2018","journal-title":"BMC Res. Notes"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1002\/bab.1518","article-title":"Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay","volume":"64","author":"Zhang","year":"2016","journal-title":"Biotechnol. Appl. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Brzezia\u0144ska, E., Doma\u0144ska, D., and Jegier, A. (2014). Gene doping in sport\u2014Perspectives and risks. Biol. Sport, 251\u2013259.","DOI":"10.5604\/20831862.1120931"},{"key":"ref_23","first-page":"294","article-title":"Fundamental study of detection of muscle hypertrophy-oriented gene doping by myostatin knock down using RNA interference","volume":"11","author":"Takemasa","year":"2012","journal-title":"J. Sports Sci. Med."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Neuberger, E.W.I., and Simon, P. (2017). Gene and Cell Doping: The New Frontier - Beyond Myth or Reality. Med. Sports Sci., 91\u2013106.","DOI":"10.1159\/000465456"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1038\/nbt.3758","article-title":"Robotic crowd biology with Maholo LabDroids","volume":"35","author":"Yachie","year":"2017","journal-title":"Nat. Biotechnol."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/10\/6\/436\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:56:58Z","timestamp":1760187418000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/10\/6\/436"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,6,8]]},"references-count":25,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2019,6]]}},"alternative-id":["genes10060436"],"URL":"https:\/\/doi.org\/10.3390\/genes10060436","relation":{"has-preprint":[{"id-type":"doi","id":"10.20944\/preprints201904.0285.v1","asserted-by":"object"}]},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,6,8]]}}}