Laboratory of Laboratory/Sports Medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Ibaraki, Japan.
Doctoral Program in Sports Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Ibaraki, Japan.
Genes (Basel). 2020 Jul 6;11(7):750. doi: 10.3390/genes11070750.
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.
世界反兴奋剂机构已在基因治疗进展的背景下禁止基因兴奋剂。使用质粒增强基因可能被用于基因兴奋剂。然而，尚未建立检测这种兴奋剂的金标准方法。在这里，我们旨在开发一种方法来检测多个转基因片段，以证明基因兴奋剂的存在。首先，通过将萤火虫荧光素酶质粒与聚乙烯亚胺（PEI）注射到腹腔中，创建了基因传递模型小鼠，以模拟基因兴奋剂。结果证实该模型成功建立，活体成像显示出足够的发光。接下来，使用 TaqMan-定量实时 PCR(qPCR) 检测模型中的多个转基因片段在血浆无细胞（cf）DNA、血细胞部分 DNA 和粪便 DNA 中的存在，其中血浆 cfDNA 中的水平最高。使用模型中仅一滴全血，我们还尝试了长期检测。结果表明，直到第 11 天，仍可检测到多个转基因片段。这些发现表明，使用 TaqMan-qPCR 检测与血浆 cfDNA 或仅一滴全血结合，是检测基于质粒-PEI 的基因兴奋剂的可行方法。我们的发现可能会加速人类基因兴奋剂检测方法的发展。
