Development of a dCas9-Based High-Throughput Gene Doping Analysis (HiGDA) for Human Erythropoietin

2021 ◽  
Author(s):  
Joon-Yeop Yi ◽  
Minyoung Kim ◽  
Byung Gee Kim ◽  
Junghyun Son ◽  
Changmin Sung
Keyword(s):  
High Throughput ◽  
Doping Analysis ◽  
Gene Doping ◽  
Human Erythropoietin

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

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.

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

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8595 ◽  
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.

scholarly journals Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy

Gene Therapy ◽  
2016 ◽  
Vol 23 (10) ◽  
pp. 708-717 ◽  
Author(s):  
A Baoutina ◽  
S Bhat ◽  
M Zheng ◽  
L Partis ◽  
M Dobeson ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Reference Material ◽  
Gene Doping ◽  
Human Erythropoietin

New application of the CRISPR‐Cas9 system for site‐specific exogenous gene doping analysis

2020 ◽  
Author(s):  
Joon‐Yeop Yi ◽  
Minyoung Kim ◽  
Hophil Min ◽  
Byung‐Gee Kim ◽  
Junghyun Son ◽  
...  
Keyword(s):  
Doping Analysis ◽  
Site Specific ◽  
Gene Doping ◽  
Exogenous Gene

Transgene traceability in transgenic mice: a bioanalytical approach for potential gene-doping analysis

Bioanalysis ◽  
2011 ◽  
Vol 3 (22) ◽  
pp. 2523-2531 ◽  
Author(s):  
Patrizia Bogani ◽  
Maria Michela Spiriti ◽  
Stefano Lazzarano ◽  
Annarosa Arcangeli ◽  
Marcello Buiatti ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Doping Analysis ◽  
Gene Doping

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses

2020 ◽  
Author(s):  
Hiu Wing Cheung ◽  
Kin‐Sing Wong ◽  
Venus Y.C. Lin ◽  
Terence S.M. Wan ◽  
Emmie N.M. Ho
Keyword(s):  
Qpcr Assay ◽  
Control Gene ◽  
Gene Doping ◽  
Human Erythropoietin

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

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.

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

Genes ◽  
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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