Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
by
Takehito Sugasawa 1,†,
Kai Aoki 2,†,
Koichi Watanabe 3,
Koki Yanazawa 2,
Tohru Natsume 4,
Tohru Takemasa 3
,
Kaori Yamaguchi 3,
Yoshinori Takeuchi 5,
Yuichi Aita 5,
Naoya Yahagi 5,
Yasuko Yoshida 6,
Katsuyuki Tokinoya 2,7,
Nanami Sekine 2,
Kaoru Takeuchi 8,
Haruna Ueda 2,
Yasushi Kawakami 1,
Satoshi Shimizu 3 and
Kazuhiro Takekoshi 1,*
Takehito Sugasawa 1,†,
Kai Aoki 2,†,
Koichi Watanabe 3,
Koki Yanazawa 2,
Tohru Natsume 4,
Tohru Takemasa 3,
Kaori Yamaguchi 3,
Yoshinori Takeuchi 5,
Yuichi Aita 5,
Naoya Yahagi 5,
Yasuko Yoshida 6,
Katsuyuki Tokinoya 2,7,
Nanami Sekine 2,
Kaoru Takeuchi 8,
Haruna Ueda 2,
Yasushi Kawakami 1,
Satoshi Shimizu 3 and
Kazuhiro Takekoshi 1,*
1
Laboratory of Laboratory/Sports medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8577, Japan
2
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
3
Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
4
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 100-8921, Japan
5
Nutrigenomics Research Group, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan
6
Department of Medical Technology, Faculty of Health Sciences, Tsukuba International University, 6-20-1 Manabe, Tsuchiura, Ibaraki 300-0051, Japan
7
Japan Society for the Promotion of Science; Kojimachi Business Center Building, Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japan
8
Laboratory of Environmental Microbiology, Division of Basic Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2019, 10(6), 436; https://doi.org/10.3390/genes10060436
Received: 11 April 2019 / Revised: 15 May 2019 / Accepted: 4 June 2019 / Published: 8 June 2019
(This article belongs to the Section Human Genomics and Genetic Diseases)
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.
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Keywords:
gene doping; gene therapy; droplet digital PCR; adenoviral vector
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MDPI and ACS Style
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.; Takekoshi, K. Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR. Genes 2019, 10, 436.
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