Preprint Article Version 1 This version is not peer-reviewed

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Version 1 : Received: 24 April 2019 / Approved: 25 April 2019 / Online: 25 April 2019 (12:45:49 CEST)

A peer-reviewed article of this Preprint also exists.

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. 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.

Journal reference: Genes 2019, 10, 436
DOI: 10.3390/genes10060436

Abstract

With the rapid progress of genetic engineering and gene therapy, World Anti-Doping Agency has alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here we aimed to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. rAdV vectors containing 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 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 directly be 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.

Subject Areas

gene doping; gene therapy; droplet digital PCR; adenoviral vector

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