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Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control

Gene Therapy volume 28, pages 199–205 (2021)Cite this article

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Abstract

Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.

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Fig. 1: Detection of the horse erythropoietin (EPO) transgene as an intron deletion by DELLY.
Fig. 2: Detection concept as an intron deletion of a transgene by the split-read algorithm.

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Data availability

Sequencing data of transgenic cell lines will be provided upon request to the corresponding author because of gene doping control in the horseracing industry.

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Acknowledgements

We acknowledge Yuko Miyatake and Miyuki Kubokawa at Amelieff Corporation, Japan, for providing technical support and helpful discussions. We also thank Noriko Tanaka for her assistance with this study and the Japan Racing Association (JRA) for approving and supporting this study through a grant-in-aid (2017–2019).

Funding

This research was funded by the Japan Racing Association (2017–2019).

Author information

Author notes

  1. These authors contributed equally: Teruaki Tozaki, Aoi Ohnuma

Authors and Affiliations

  1. Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan

    Teruaki Tozaki, Aoi Ohnuma, Mio Kikuchi, Taichiro Ishige, Hironaga Kakoi, Kei-ichi Hirora & Shun-ichi Nagata

  2. Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu, 501-1193, Japan

    Masaki Takasu & Kotono Nakamura

  3. Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke, Tochigi, 329-0412, Japan

    Norihisa Tamura

  4. Equine Department, Japan Racing Association, 6-11-1, Roppongi, Minato, Tokyo, 106-8401, Japan

    Kanichi Kusano

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  1. Teruaki Tozaki
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Contributions

TT and AO conceived and designed the experiments; TT and AO performed the experiments; MK, TI, HK, KH; KK and SN performed the animal experiments; MT, KN, and NT generated transgenic cells; KK provided critical comments and contributed to the discussion of the results and TT and AO drafted the paper. All authors reviewed and revised the paper.

Corresponding author

Correspondence to Teruaki Tozaki.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Animal experiments in this study were approved by the Animal Care Committee of the Equine Research Institute of the Japan Racing Association (approval number 19-9 on January 31, 2019). Animal experiments were conducted at the facilities of the Equine Research Institute, Japan Racing Association (Shimotsuke, Japan).

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Tozaki, T., Ohnuma, A., Takasu, M. et al. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control. Gene Ther 28, 199–205 (2021). https://doi.org/10.1038/s41434-020-00185-y

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