Detection of exogenous gene doping of IGF‐I by a real‐time quantitative PCR assay

Zhang, Jinju; Xu, Jianguo; YongWei, Shen; Ma, Shijiao; Zhang, Tingting; Meng, Qinglin; Lan, Wenxian; Zhang, Chun; Liu, Xiaomei

doi:10.1002/bab.1518

Cited by 16 publications

(17 citation statements)

References 19 publications

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“…It has been reported that human candidate genes at a high risk of gene doping include EPO , insulin-like growth factor-1 ( IGF-1 ), hypoxia inducible factor-1 ( HIF-1 ), vascular endothelial growth factor-a ( VEGF-A ), and follistatin ( FST ), etc. [20,21,22]. Gene doping may enhance their expression.…”

Section: Discussionmentioning

confidence: 99%

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Sugasawa

Aoki

Watanabe

et al. 2019

Genes

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With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

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Section: Discussionmentioning

confidence: 99%

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Sugasawa

Aoki

Watanabe

et al. 2019

Genes

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“…LAMP and PCR-based gene doping detection methods (like real-time PCR, nested PCR, droplet digital PCR and internal threshold PCR) target exon-exon junctions of transgenic gene constructs similarly to our NGS-based method [3,[5][6][7][8][9][10][11][12][13]. However, these methods need at least one wild-type copyDNA junction for detection of gene doping [6,8,9].…”

Section: Discussionmentioning

confidence: 99%

“…Currently published methods for detection of gene doping use PCR-based methods or loop-mediated isothermal amplification (LAMP) that target unique sequences in a doping gene corresponding to exon-exon junctions in the intron-less transgene [3,[5][6][7][8][9][10][11][12][13]. However, because the exon-exon junctions of doping genes are known and the short PCR primers are even interrupted by the slightest change of the sequence, it is relatively simple to evade detection using current PCR-based methods by modifying the doping gene with for example synonymous mutations, which will then give a false-negative result.…”

Section: Introductionmentioning

confidence: 99%

A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

et al. 2019

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Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon-exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted nextgeneration sequencing based assay for the detection of all exon-exon junctions of the potential doping genes, EPO, IGF1, IGF2, GH1, and GH2, which is resistant to tampering. Using this assay, all exon-exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmidderived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.

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“…Although not yet commonplace, research in the eld of gene therapy is constantly improving [34][35][36][37] and with every advancement in genetic methods intended for hereditary disease therapies, comes further opportunities for exploiting the technology for doping strategies [38][39][40][41][42]. Therefore, developing methodologies to combat this is necessary [43][44][45].…”

Section: Introductionmentioning

confidence: 99%

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

Hill

Porter

Neto

et al. 2022

Preprint

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BackgroundThoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C>T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype.ResultsPrimary myoblasts conditionally immortalized with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 in the diet. ConclusionsThe observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine.

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Detection of exogenous gene doping of IGF‐I by a real‐time quantitative PCR assay

Cited by 16 publications

References 19 publications

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR

A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

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