Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice
Background Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors contain...
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PeerJ Inc.
2021-10-01
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| Online Access: | https://peerj.com/articles/12285.pdf |
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| author | Koki Yanazawa Takehito Sugasawa Kai Aoki Takuro Nakano Yasushi Kawakami Kazuhiro Takekoshi |
| author_facet | Koki Yanazawa Takehito Sugasawa Kai Aoki Takuro Nakano Yasushi Kawakami Kazuhiro Takekoshi |
| author_sort | Koki Yanazawa |
| collection | DOAJ |
| description | Background Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>. Methods First, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. Results The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. Conclusions These results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>. |
| first_indexed | 2024-03-09T06:31:32Z |
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| id | doaj.art-de6065f8a1964e3e8bcccc062025ea11 |
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| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:31:32Z |
| publishDate | 2021-10-01 |
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| spelling | doaj.art-de6065f8a1964e3e8bcccc062025ea112023-12-03T11:05:03ZengPeerJ Inc.PeerJ2167-83592021-10-019e1228510.7717/peerj.12285Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in miceKoki Yanazawa0Takehito Sugasawa1Kai Aoki2Takuro Nakano3Yasushi Kawakami4Kazuhiro Takekoshi5Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, JapanLaboratory of Clinical Examination/Sports Medicine, Division of Clinical Medicines, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanLaboratory of Clinical Examination/Sports Medicine, Division of Clinical Medicines, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanGraduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, JapanLaboratory of Clinical Examination/Sports Medicine, Division of Clinical Medicines, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanLaboratory of Clinical Examination/Sports Medicine, Division of Clinical Medicines, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanBackground Gene doping is the misuse of genome editing and gene therapy technologies for the purpose of manipulating specific genes or gene functions in order to improve athletic performance. However, a non-invasive detection method for gene doping using recombinant adenoviral (rAdV) vectors containing human follistatin (hFST) genes (rAdV<hFST>) has not yet been developed. Therefore, the aim of this study was to develop a method to detect gene doping using rAdV<hFST>. Methods First, we generated rAdV<hFST> and evaluated the overexpression of the hFST gene, FST protein, and muscle protein synthesis signaling using cell lines. Next, rAdV<hFST> was injected intravenously or intramuscularly into mice, and whole blood was collected, and hFST and cytomegalovirus promoter (CMVp) gene fragments were detected using TaqMan-quantitative polymerase chain reaction (qPCR). Finally, to confirm the specificity of the primers and the TaqMan probes, samples from each experiment were pooled, amplified using TaqMan-qPCR, and sequenced using the Sanger sequencing. Results The expression of hFST and FST proteins and muscle protein synthesis signaling significantly increased in C2C12 cells. In long-term, transgene fragments could be detected until 4 days after intravenous injection and 3 days after intramuscular injection. Finally, the Sanger sequencing confirmed that the primers and TaqMan probe specifically amplified the gene sequence of interest. Conclusions These results indicate the possibility of detecting gene doping using rAdV<hFST> using TaqMan-qPCR in blood samples. This study may contribute to the development of detection methods for gene doping using rAdV<hFST>.https://peerj.com/articles/12285.pdfGene dopingFollistatinAdenovirus vector |
| spellingShingle | Koki Yanazawa Takehito Sugasawa Kai Aoki Takuro Nakano Yasushi Kawakami Kazuhiro Takekoshi Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice PeerJ Gene doping Follistatin Adenovirus vector |
| title | Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| title_full | Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| title_fullStr | Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| title_full_unstemmed | Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| title_short | Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| title_sort | development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice |
| topic | Gene doping Follistatin Adenovirus vector |
| url | https://peerj.com/articles/12285.pdf |
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