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Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay

1
Laboratory of Laboratory/Sports Medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Ibaraki, Japan
2
Doctoral Program in Sports Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Ibaraki, Japan
3
Master’s Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Ibaraki, Japan
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Genes 2020, 11(7), 750; https://doi.org/10.3390/genes11070750
Received: 21 May 2020 / Revised: 29 June 2020 / Accepted: 3 July 2020 / Published: 6 July 2020
(This article belongs to the Section Human Genomics and Genetic Diseases)
The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans. View Full-Text
Keywords: gene doping; gene therapy; in vivo transfection; in vivo imaging gene doping; gene therapy; in vivo transfection; in vivo imaging
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MDPI and ACS Style

Sugasawa, T.; Aoki, K.; Yanazawa, K.; Takekoshi, K. Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay. Genes 2020, 11, 750. https://doi.org/10.3390/genes11070750

AMA Style

Sugasawa T, Aoki K, Yanazawa K, Takekoshi K. Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay. Genes. 2020; 11(7):750. https://doi.org/10.3390/genes11070750

Chicago/Turabian Style

Sugasawa, Takehito, Kai Aoki, Kouki Yanazawa, and Kazuhiro Takekoshi. 2020. "Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay" Genes 11, no. 7: 750. https://doi.org/10.3390/genes11070750

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