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

Sugasawa, Takehito; Aoki, Kai; Yanazawa, Kouki; Takekoshi, Kazuhiro

doi:10.3390/genes11070750

Cited by 13 publications

(10 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our previous studies have shown that whole-blood DNA contains high levels of viral genome DNA fragments as direct proof [5][6][7]; therefore, we performed the TaqMan qPCR assay with whole-blood DNA. For detection of direct proof of gene doping in wholeblood DNA in the short-and long-term experiments using the TaqMan qPCR assays, the primers and TaqMan probes were designed to target the EPO gene (2 types), TkpA (thymidine kinase polyA from herpes simplex virus), hexon (major virus capsid protein from adenovirus), and CMVp (cytomegalovirus promoter) to ensure specific amplification of the rAdV-hEPO genome using Primer-BLAST (NIH National Library of Medicine, Bethesda, MD, USA).…”

Section: Taqman Qpcr Assaymentioning

confidence: 99%

See 1 more Smart Citation

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

Sugasawa

Nakano

Fujita

et al. 2021

Genes

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Taqman Qpcr Assaymentioning

confidence: 99%

“…This list includes "gene doping" as a form of abuse of gene therapy technology. Therefore, our research group has been conducting research for more than five years to establish an examination method for gene doping [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

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

Sugasawa

Nakano

Fujita

et al. 2021

Genes

Self Cite

View full text Add to dashboard Cite

show abstract

“…Our previous studies have shown that whole blood DNA contains high levels of viral genome DNA fragments as direct proof [5][6][7]; therefore, we performed the TaqMan qPCR assay with whole blood DNA. For the TaqMan qPCR assays to detect direct proof of gene doping in whole blood DNA in the short-and long-term experiments, the primers and TaqMan probes were designed to target the EPO gene (2 types), TkpA, Hexon, and CMVp to ensure specific amplification of the rAdV-hEPO genome using Primer-BLAST (NIH National Library of Medicine, Bethesda, MD, USA).…”

Section: Taqman Qpcr Assaymentioning

confidence: 99%

“…This includes "gene doping" as abuse of gene therapy technology. Therefore, our research group has been conducting research for more five years to establish an examination method for gene doping [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

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

Sugasawa¹,

Nakano²,

Fujita³

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

The World Anti-Doping Agency (WADA) has prohibited gene doping in the context of progress in gene therapy. In addition, there is a risk of the EPO gene being applied in gene doping among athletes. Along with this, development of a gene-doping test has been underway in worldwide. Here, we had two purposes: to develop a robust gene doping mouse model using the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to prove gene doping using this model. The rAdV including the hEPO gene were injected intravenously to transfer the gene to the liver. After injection, the mice developed significantly increased red blood cell counts in whole blood and increased gene expressions of hematopoietic markers in the spleen, indicating successful development of the gene doping model. Next, we detected direct and indirect proof of gene doping in whole blood DNA and RNA using qPCR assay and RNA sequencing. Proof was detected in one drop of whole blood DNA and RNA over a long period; furthermore, the overall RNA expression profiles significantly changed. Therefore, we have advanced detection of hEPO gene doping in humans.

show abstract

“…The primary method used to detect the presence of transgene sequences has been via the polymerase chain reaction (PCR) to amplify a transgene‐specific region of DNA extracted from an appropriate biological sample matrix (typically plasma or whole blood). Existing studies have explored real‐time qPCR, 6,7 droplet digital PCR , 8,9 microfluidic qPCR, 10 and massively parallel sequencing, 11,12 which all have their uses as screening and/or confirmatory methods. The quantitative nature of digital PCR and qPCR, coupled with a high specificity and sensitivity of the hydrolysis probe‐based reagents and instruments, makes them particularly suited to confirmatory investigations where limits of detection can be calculated.…”

Section: Introductionmentioning

confidence: 99%

Direct sequence confirmation of qPCR products for gene doping assay validation in horses

Maniego¹,

Pesko²,

Hincks³

et al. 2022

Drug Testing and Analysis

View full text Add to dashboard Cite

The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated. To fully validate a qPCR experiment, it is highly desirable to confirm the identity of the amplicon. Although post-PCR techniques such as melt curve and fragment size analysis can provide strong evidence that the amplicon is as expected, sequence identity confirmation may be beneficial as part of regulatory proceedings. We present here our investigation into two alternative processes for the direct assessment of qPCR products for five genes using next-generation sequencing: ligation of sequence-ready adapters to qPCR products and qPCR assays performed with primers tailed with Illumina flow cell binding sites. To fully test the robustness of the techniques at concentrations required for gene doping detection, we also calculated a putative limit of detection for the assays. Both ligated adapters and tailed primers were successful in producing sequence data for the qPCR products without further amplification. Ligated adapters are preferred, however, as they do not require re-optimisation of existing qPCR assays.

show abstract

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

Cited by 13 publications

References 19 publications

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

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

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

Direct sequence confirmation of qPCR products for gene doping assay validation in horses

Contact Info

Product

Resources

About