The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

Kai Aoki; Takehito Sugasawa; Kouki Yanazawa; Koichi Watanabe; Tohru Takemasa; Yoshinori Takeuchi; Yuichi Aita; Naoya Yahagi; Yasuko Yoshida; Tomoaki Kuji; Nanami Sekine; Kaoru Takeuchi; Haruna Ueda; Yasushi Kawakami; Kazuhiro Takekoshi

doi:10.7717/peerj.8595

The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

PeerJ. 2020 Feb 25:8:e8595. doi: 10.7717/peerj.8595. eCollection 2020.

Authors

Affiliations

¹ Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
² Laboratory of Laboratory/Sports medicine, Division of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
³ Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba, Japan.
⁴ Nutrigenomics Research Group, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
⁵ Department of Medical Technology, Faculty of Health Sciences, Tsukuba International University, Tsuchiura, Japan.
⁶ Laboratory of Environmental Microbiology, Division of Basic Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

^# Contributed equally.

Abstract

Background: With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping.

Methods: Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR.

Results: In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected.

Conclusions: These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

Keywords: Erythropoietin; Gene doping; Plasmid.

Grants and funding

This work was supported by a grant from the promotional business of doping prevention activities, Japan Sports Agency (JSA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.