Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay
Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay
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DOI：
10.3390/genes11070750
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发表时间：
2020-05
期刊：
影响因子：
3.5
通讯作者：
Takeito Sugasawa;Kai Aoki;Koki Yanazawa;Ryo Hagino;Shinsuke Tamai;Koichi Watanabe;T. Takemasa;Yoshinori Takeuchi;Yuichi Aita;N. Yahagi;Y. Yoshida;T. Kuji;Yasuhiro Shishikura;Seiko Ono;Takuro Nakano;Ban Kai;K. Takeuchi;Yasushi Kawamai;K. Takekoshi
中科院分区：
文献类型：
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作者：
Takeito Sugasawa;Kai Aoki;Koki Yanazawa;Ryo Hagino;Shinsuke Tamai;Koichi Watanabe;T. Takemasa;Yoshinori Takeuchi;Yuichi Aita;N. Yahagi;Y. Yoshida;T. Kuji;Yasuhiro Shishikura;Seiko Ono;Takuro Nakano;Ban Kai;K. Takeuchi;Yasushi Kawamai;K. Takekoshi
世界反兴奋剂机构在基因治疗取得进展的背景下禁止使用基因兴奋剂。使用质粒扩增基因有可能被用于基因兴奋剂。然而，还没有建立检测这一点的金标准方法。在这里，我们的目的是开发一种方法来检测多个转基因片段作为基因兴奋剂的证据。首先，通过腹腔注射含有聚乙烯亚胺（PEI）的萤火虫荧光素酶质粒，建立了基因转染小鼠模型。结果证实了模型的成功建立，在体内成像时具有足够的发光。接下来，使用TaqMan-定量实时PCR（qPCR）测定在血浆无细胞（cf）DNA、血细胞级分DNA和粪便DNA中检测模型中的多个转基因片段，其中在血浆cfDNA中具有最高水平。仅使用来自模型的一滴全血，我们还尝试了长期检测。结果表明，直到第11天才检测到多个转基因片段。这些发现表明，血浆cfDNA或仅一滴全血与TaqMan-qPCR测定的组合对于检测基于质粒-PEI的基因掺杂是可行的。我们的发现可以加速检测人类基因兴奋剂的方法的发展。
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.