1. Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR.
- Author
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Sugasawa T, Aoki K, Watanabe K, Yanazawa K, Natsume T, Takemasa T, Yamaguchi K, Takeuchi Y, Aita Y, Yahagi N, Yoshida Y, Tokinoya K, Sekine N, Takeuchi K, Ueda H, Kawakami Y, Shimizu S, and Takekoshi K
- Subjects
- Adenoviridae genetics, Animals, Gene Transfer Techniques, Genetic Therapy methods, Genetic Vectors genetics, Humans, Mice, Adenoviridae isolation & purification, Doping in Sports, Genetic Vectors isolation & purification, Transgenes genetics
- Abstract
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.
- Published
- 2019
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