Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector

Sugasawa, Takehito; Nakano, Takuro; Fujita, Shin‐ichiro; Matsumoto, Yuki; Ishihara, Genki; Aoki, Kai; Yanazawa, Koki; Ono, S.; Tamai, Shinsuke; Manevich, Lev; Ueda, Hiroshi; Ishibashi, Noriyo; Tamai, Kenshirou; Kanki, Yasuharu; Yoshida, Yasuko; Watanabe, Koichi; Takemasa, Tohru; Kawakami, Yasushi; Takekoshi, Kazuhiro

doi:10.3390/genes12081249

Cited by 11 publications

(35 citation statements)

References 53 publications

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“…Western blotting reported in many papers shows different sizes and shapes of the bands compared with our bands [80][81][82][83][84][85]. The important part of the WADA recommendation is the use of the antibody clone AE7A5 [82].…”

Section: Deglycosylation-coupled Western Blottingmentioning

confidence: 60%

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

et al. 2023

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Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

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Section: Deglycosylation-coupled Western Blottingmentioning

confidence: 60%

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

et al. 2023

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“…Typically, the injected transgenes (e.g., local injection to muscle tissue or systemic intravenous administration) could be detected in human plasma . Previous reports demonstrated that the transgene concentration in blood could be in the range around 0.1–1 × 10 3 copy/μL (i.e., 0.15–1.5 × 10 –15 M) after intramuscular injection of the target plasmid. , To mimic a GD scenario, four low concentrations of hEPO plasmids (0, 10 –16 , 10 –13 , and 10 –10 M) were spiked in human plasma and measured after direct RPA without pretreatment (Figure a). The whole assay took ∼40 min, including 20 min of RPA and 20 min for CasGDP detection.…”

Section: Resultsmentioning

confidence: 99%

Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping

Yan

Zhou

et al. 2022

Anal. Chem.

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Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence-and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.

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“…Total RNA-Seq was performed to identify the novel RNA markers, that is, fluctuated expressions dependent on the blood storage period. The method was similar to procedures in our previous study [ 23 , 24 ]. Preparation of the libraries and NGS run was conducted at the Department of Sports Medicine in the Organization for Open Facility Initiatives, University of Tsukuba (Tsukuba, Ibaraki, Japan).…”

Section: Methodsmentioning

confidence: 99%

Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

Sugasawa

Kanki

Komine

et al. 2022

Genes

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The World Anti-Doping Agency (WADA) has prohibited the use of autologous blood transfusion (ABT) as a doping method by athletes. It is difficult to detect this doping method in laboratory tests, and a robust testing method has not yet been established. We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect ABT doping within red blood cells (RBCs) as a pilot study before human trials. This study used whole blood samples from Wistar rats. The whole blood samples were mixed with a citrate–phosphate–dextrose solution with adenine (CPDA) and then stored in a refrigerator at 4 °C for 0 (control), 10, or 20 days. After each storage period, total RNA-Seq and bioinformatics were performed following RNA extraction and the purification of the RBCs. In the results, clear patterns of expression fluctuations were observed depending on the storage period, and it was found that there were large numbers of genes whose expression decreased in the 10- and 20-day periods compared to the control. Moreover, additional bioinformatic analysis identified three significant genes whose expression levels were drastically decreased according to the storage period. These results provide novel insights that may allow future studies to develop a testing method for ABT doping.

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Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector

Cited by 11 publications

References 53 publications

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue

Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping

Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion

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