2024-03-29T10:31:54Z
https://tsukuba.repo.nii.ac.jp/oai
oai:tsukuba.repo.nii.ac.jp:02001691
2022-04-27T09:52:48Z
2780:8053
2780:924
3:62:5589:7668
Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay
菅澤, 威仁
スガサワ, タケヒト
SUGASAWA, Takehito
Aoki, Kai
Yanazawa, Kouki
竹越, 一博
タケコシ, カズヒロ
TAKEKOSHI, Kazuhiro
open access
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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.
MDPI
2020-07
eng
journal article
http://hdl.handle.net/2241/0002001691
https://tsukuba.repo.nii.ac.jp/records/2001691
32640671
https://doi.org/10.3390/genes11070750
2073-4425
Genes
11
7
750
https://tsukuba.repo.nii.ac.jp/record/2001691/files/Genes_11-7.pdf
application/pdf
3.6 MB
2021-10-26