Tozaki Teruaki, Ohnuma Aoi, Takasu Masaki, Nakamura Kotono, Kikuchi Mio, Ishige Taichiro, Kakoi Hironaga, Hirora Kei-Ichi, Tamura Norihisa, Kusano Kanichi, Nagata Shun-Ichi
Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya, Tochigi, 320-0851, Japan.
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu, 501-1193, Japan.
Gene Ther. 2021 Apr;28(3-4):199-205. doi: 10.1038/s41434-020-00185-y. Epub 2020 Aug 7.
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.
基因兴奋剂已引起人类和马术运动以及赛马行业的关注。在体育和赛马行业中，基因兴奋剂可能有两种类型：（1）对出生后的动物施用基因兴奋剂物质；（2）通过改造卵子培育基因工程动物。在本研究中，我们旨在通过全基因组重测序（WGR）识别基因工程动物，以进行基因兴奋剂控制。构建了转基因细胞系，即将促红细胞生成素基因（EPO）的cDNA形式插入马成纤维细胞基因组中，作为转基因马的模型。基于拆分读段和双末端算法的DELLY以及基于读段深度算法的Control-FREEC用于进行全基因组范围内非靶向转基因的筛选，以发现结构变异。通过DELLY的拆分读段算法，我们在WGR数据中检测到EPO转基因为内含子缺失。此外，通过WGR鉴定了EPO转基因中人工引入的单核苷酸多态性和插入/缺失。因此，即使目标未知，使用WGR进行全基因组筛选也有助于基因兴奋剂控制。这是第一项将转基因检测为内含子缺失以进行基因兴奋剂检测的研究。
