Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
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DOI：
10.3390/genes10060436
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发表时间：
2019-06-01
期刊：
影响因子：
3.5
通讯作者：
Takekoshi, Kazuhiro
中科院分区：
文献类型：
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作者：
Sugasawa, Takehito;Aoki, Kai;Takekoshi, Kazuhiro
随着基因工程和基因治疗的快速发展，世界反兴奋剂机构已经对基因兴奋剂发出了警报，并禁止在体育运动中使用基因兴奋剂。然而，目前还没有标准的方法来检测重组腺病毒(RAdV)载体携带的转基因。在这里，我们的目标是在模拟基因掺杂的小鼠模型中建立一种检测rAdV载体携带的转基因的方法。这些含有mCherry基因的重组腺病毒载体通过静脉注射或局部肌肉注射在小鼠体内传递。5天后，采集粪便和全血样本，提取总DNA。作为额外的实验，从注射rAdv载体开始的15天内，也从小鼠的尾尖采集全血。用半定量聚合酶链式反应(QPCR)、定量聚合酶链式反应(QPCR)和滴状数字聚合酶链式反应(DdPCR)对不同DNA样品的转基因片段进行分析。结果表明，根据标本类型和注射方法的不同，qPCR和ddPCR可以直接从血细胞组分DNA、血浆无细胞DNA和粪便DNA中检测到转基因片段。我们观察到，血细胞分数DNA和ddPCR的组合比该模型中使用的其他组合更敏感。这些结果可能会加速基因兴奋剂检测方法的发展。
With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.