The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

Background With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. Methods Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. Results In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. Conclusions These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

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The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay

10.7287/peerj.preprints.27925 ◽

2019 ◽

Author(s):

Kai Aoki ◽

Takehito Sugasawa ◽

Kouki Yanazawa ◽

Koichi Watanabe ◽

Tohru Takemasa ◽

...

Keyword(s):

Whole Blood ◽

Plasmid Vector ◽

Sybr Green ◽

Gene Doping ◽

Human Erythropoietin ◽

World Anti Doping Agency ◽

Total Dna ◽

Stool Dna ◽

Tail Tip ◽

Taqman Qpcr

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

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

10.20944/preprints201904.0285.v1 ◽

2019 ◽

Author(s):

Takehito Sugasawa ◽

Kai Aoki ◽

Koichi Watanabe ◽

Koki Yanazawa ◽

Tohru Natsume ◽

...

Keyword(s):

Blood Cell ◽

Quantitative Pcr ◽

Whole Blood ◽

Digital Pcr ◽

Cell Fraction ◽

Detection Methods ◽

Gene Doping ◽

World Anti Doping Agency ◽

Injection Methods ◽

Tail Tip

With the rapid progress of genetic engineering and gene therapy, World Anti-Doping Agency has alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here we aimed to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. rAdV vectors containing 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 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 directly be 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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Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay

Genes ◽

10.3390/genes11070750 ◽

2020 ◽

Vol 11 (7) ◽

pp. 750

Author(s):

Takehito Sugasawa ◽

Kai Aoki ◽

Kouki Yanazawa ◽

Kazuhiro Takekoshi

Keyword(s):

Whole Blood ◽

Abdominal Cavity ◽

Plasmid Vector ◽

Firefly Luciferase ◽

Qpcr Assay ◽

Gold Standard Method ◽

Gene Doping ◽

Taqman Qpcr Assay ◽

Taqman Qpcr

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the augmentation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. Firstly, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan- quantitative real-time PCR(qPCR) assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

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

Genes ◽

10.3390/genes10060436 ◽

2019 ◽

Vol 10 (6) ◽

pp. 436 ◽

Cited By ~ 3

Author(s):

Takehito Sugasawa ◽

Kai Aoki ◽

Koichi Watanabe ◽

Koki Yanazawa ◽

Tohru Natsume ◽

...

Keyword(s):

Blood Cell ◽

Quantitative Pcr ◽

Whole Blood ◽

Digital Pcr ◽

Cell Fraction ◽

Detection Methods ◽

Gene Doping ◽

World Anti Doping Agency ◽

Injection Methods ◽

Tail Tip

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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Detection of Multiple Transgene Fragments in a Mouse Model of Gene Doping Based on Plasmid Vector Using TaqMan-qPCR Assay

10.20944/preprints202005.0366.v1 ◽

2020 ◽

Author(s):

Takeito Sugasawa ◽

Kai Aoki ◽

Koki Yanazawa ◽

Ryo Hagino ◽

Shinsuke Tamai ◽

...

Keyword(s):

Whole Blood ◽

Abdominal Cavity ◽

Plasmid Vector ◽

Firefly Luciferase ◽

Qpcr Assay ◽

Gold Standard Method ◽

Gene Doping ◽

Taqman Qpcr Assay ◽

Taqman Qpcr

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the artificial regulation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. First, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan-qPCR assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

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

Genes ◽

10.3390/genes12081249 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1249

Author(s):

Takehito Sugasawa ◽

Takuro Nakano ◽

Shin-ichiro Fujita ◽

Yuki Matsumoto ◽

Genki Ishihara ◽

...

Keyword(s):

Mouse Model ◽

Whole Blood ◽

Recombinant Adenovirus ◽

Expression Profiles ◽

Marker Genes ◽

Gene Doping ◽

Human Erythropoietin ◽

Dna And Rna ◽

Cell Counts ◽

In Doping

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

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Blood transcriptional signature of recombinant human erythropoietin administration and implications for antidoping strategies

Physiological Genomics ◽

10.1152/physiolgenomics.00108.2015 ◽

2016 ◽

Vol 48 (3) ◽

pp. 202-209 ◽

Cited By ~ 25

Author(s):

Jérôme Durussel ◽

Diresibachew W. Haile ◽

Kerli Mooses ◽

Evangelia Daskalaki ◽

Wendy Beattie ◽

...

Keyword(s):

Whole Blood ◽

Recombinant Human Erythropoietin ◽

Transcriptional Profiling ◽

Rebound Effect ◽

Moderate Altitude ◽

Blood Doping ◽

Transcriptional Signature ◽

Human Erythropoietin ◽

World Anti Doping Agency ◽

Transcript Signature

Recombinant human erythropoietin (rHuEPO) is frequently abused by athletes as a performance-enhancing drug, despite being prohibited by the World Anti-Doping Agency. Although the methods to detect blood doping, including rHuEPO injections, have improved in recent years, they remain imperfect. In a proof-of-principle study, we identified, replicated, and validated the whole blood transcriptional signature of rHuEPO in endurance-trained Caucasian males at sea level ( n = 18) and Kenyan endurance runners at moderate altitude ( n = 20), all of whom received rHuEPO injections for 4 wk. Transcriptional profiling shows that hundreds of transcripts were altered by rHuEPO in both cohorts. The main regulated expression pattern, observed in all participants, was characterized by a “rebound” effect with a profound upregulation during rHuEPO and a subsequent downregulation up to 4 wk postadministration. The functions of the identified genes were mainly related to the functional and structural properties of the red blood cell. Of the genes identified to be differentially expressed during and post-rHuEPO, we further confirmed a whole blood 34-transcript signature that can distinguish between samples collected pre-, during, and post-rHuEPO administration. By providing biomarkers that can reveal rHuEPO use, our findings represent an advance in the development of new methods for the detection of blood doping.

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