Whole-genome resequencing using genomic DNA extracted from horsehair roots          for gene-doping control in horse sports

Tozaki, Teruaki; Ohnuma, Aoi; Kikuchi, Mio; Ishige, Taichiro; Kakoi, Hironaga; Hirota, Kei‐ichi; Hamilton, Natasha A.; Kusano, Kanichi; Nagata, Shun‐ichi

doi:10.1294/jes.31.75

Cited by 11 publications

(8 citation statements)

References 22 publications

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“…This indicates that a gene-editing test could be performed using the same sample collected for the standard parentage test. In a previous study, an average of 8.5 ng/μL (200 μL) of genomic DNA was extracted from 15 hair roots of horses older than one-year-old [ 39 ]. However, to streamline the testing of several samples, it may be better to use a small number of hair roots (i.e., five hair roots) to minimize the time spent cutting samples.…”

Section: Resultsmentioning

confidence: 99%

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Tozaki

Ohnuma

Nakamura

et al. 2022

Genes

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The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

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Section: Resultsmentioning

confidence: 99%

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Tozaki

Ohnuma

Nakamura

et al. 2022

Genes

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“…The same software was able to identify 12 additional equine transgenes in silico 98 . Although developed for use with equine whole blood, this method has also been proposed for detection of transgenic animals using hair follicle samples 99 . A database of rare and common genetic variants in Thoroughbred horses has been established for the purpose of non‐targeted detection of transgenic equids 100 .…”

Section: Introductionmentioning

confidence: 99%

Administration and detection of gene therapy in horses: A systematic review

Haughan

Ortved

Robinson

2022

Drug Testing and Analysis

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Gene therapy uses genetic modification of cells to produce a therapeutic effect.Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression.Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses.Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

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“…Several transgene detection methods have been developed and reported 5–9 . While some use next‐generation sequencing technology, 10,11 most transgene detection methods are based on polymerase chain reaction (PCR), real‐time PCR and digital PCR. These are currently more popular than next‐generation sequencing methods due to the high sensitivity, low cost and the simple procedures involved 6,7 …”

Section: Introductionmentioning

confidence: 99%

Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control

Tozaki

Ohnuma

Hamilton

et al. 2021

Drug Testing and Analysis

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Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected lowcopy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.

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Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports

Cited by 11 publications

References 22 publications

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Administration and detection of gene therapy in horses: A systematic review

Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control

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