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Home / Equine Research Bank / Gene Ther / Detection of non-targeted transgenes by whole-genome reseque...
Gene therapy2020; 28(3-4); 199-205; doi: 10.1038/s41434-020-00185-y

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

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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Publication Date: 2020-08-07 PubMed ID: 32770095DOI: 10.1038/s41434-020-00185-yGoogle Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research discusses a method for identifying genetically engineered animals in sports and horse racing, where gene doping is a concern. Using whole-genome resequencing (WGR), the authors were able to screen for non-targeted transgenes and detect artificially inserted genes.

Objective

The objective of the research was to detect the presence of gene doping in animals used in sports and racing. Gene doping is the act of manipulating an animal’s genetic makeup to enhance its performance. There are two methods through which gene doping can take place:

  • Administering a gene-doping substance to postnatal animals
  • Modifying the eggs to create a genetically engineered animal

The Method

The researchers used transgenic cell lines as models for their study, where the erythropoietin gene (EPO) in its cDNA form was inserted into the genome of horse fibroblasts. Using WGR, the researchers then performed a genome-wide screening for non-targeted transgenes. The screening involved looking for structural variations in the genome. Two tools were used for this purpose:

  • DELLY: Using split-read and paired-end algorithms provided by this tool, the researchers were able to detect EPO transgene as an intron deletion in the WGR data.
  • Control-FREEC: This tool uses a read-depth algorithm.

Findings

Besides detecting EPO transgene as an intron deletion, the researchers were able to identify single-nucleotide polymorphisms and insertions/deletions of the EPO transgene that had been artificially introduced.

Significance

The findings of this study are significant because they demonstrate that genome-wide screening using WGR can effectively contribute to the control of gene doping even if the targets are unknown. This research is the first of its kind to detect transgenes as intron deletions for gene-doping detection. With this method, it becomes possible to reliably detect genetic manipulation in animals used in sports and racing industries, thereby enabling better enforcement of anti-doping measures.

Cite This Article

APA
Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI. (2020). Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control. Gene Ther, 28(3-4), 199-205. https://doi.org/10.1038/s41434-020-00185-y

Publication

Gene therapy
ISSN: 1476-5462
NlmUniqueID: 9421525
Country: England
Language: English
Volume: 28
Issue: 3-4
Pages: 199-205

Researcher Affiliations

Tozaki, Teruaki
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan. ttozaki@lrc.or.jp.
Ohnuma, Aoi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Takasu, Masaki
  • Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu, 501-1193, Japan.
Nakamura, Kotono
  • Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu, 501-1193, Japan.
Kikuchi, Mio
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Ishige, Taichiro
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Kakoi, Hironaga
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Hirora, Kei-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Tamura, Norihisa
  • Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke, Tochigi, 329-0412, Japan.
Kusano, Kanichi
  • Equine Department, Japan Racing Association, 6-11-1, Roppongi, Minato, Tokyo, 106-8401, Japan.
Nagata, Shun-Ichi
  • Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.

MeSH Terms

  • Algorithms
  • Animals
  • Animals, Genetically Modified
  • Doping in Sports
  • Erythropoietin / genetics
  • Horses
  • Transgenes

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Citations

This article has been cited 6 times.
  1. Lu Y, Yan J, Ou G, Fu L. A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis.. Molecules 2023 Jul 18;28(14).
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  2. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.. J Equine Sci 2023 Jun;34(2):21-27.
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  3. Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Nagata SI. Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.. Genes (Basel) 2022 Sep 4;13(9).
    doi: 10.3390/genes13091589pubmed: 36140757google scholar: lookup
  4. Dahlgren AR, Knych HK, Arthur RM, Durbin-Johnson BP, Finno CJ. Transcriptomic Markers of Recombinant Human Erythropoietin Micro-Dosing in Thoroughbred Horses.. Genes (Basel) 2021 Nov 24;12(12).
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  6. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Kusano K, Nagata SI. Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports.. J Equine Sci 2020;31(4):75-83.
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