The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay
- Published
- Accepted
- Subject Areas
- Genetics, Molecular Biology, Kinesiology, Medical Genetics
- Keywords
- gene doping, Erythropoietin, Plasmid
- Copyright
- © 2019 Aoki et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay. PeerJ Preprints 7:e27925v1 https://doi.org/10.7287/peerj.preprints.27925v1
Abstract
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.
Author Comment
This is a preprint submission to PeerJ Preprints.
Supplemental Information
Fig1. Overview of animal experiments carried out in this study
Fig2. Amplification plot of blood sample and human cells
Specific amplification was detected in blood samples. There were no amplification in human cells and distilled water (DW).
Table2. Detection of transgene fragments in blood samples by TaqMan qPCR and SYBR Green qPCR
Transgene fragments were detected in all samples in TaqMan qPCR. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of the hEPO coding naked plasmid. **: p < 0.01 vs Pre in each group. *: p < 0.05 vs Pre in each group.
Fig 5. Confirmation of accuracy and specificity in TaqMan qPCR from stool samples using Sanger sequence methods
Single peak was detected only in IV sample, but concordance between hEPO-reference and amplicon sequences in all samples was confirmed. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. Red lines indicatethe matching sequences between hEPO-reference and amplicon sequences.
Table3. Detection of transgene fragments in stool samples by TaqMan qPCR and SYBR Green qPCR
Transgene fragments were detected in all samples in TaqMan qPCR. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. **: p < 0.01 vs Pre in each group. *: p < 0.05 vs Pre in each group.
Fig4. Confirmation of the accuracy and specificity of TaqMan qPCR from blood samples using Sanger sequence methods
Single peak was detected from all samples and concordances between hEPO-reference and amplicon sequences in all samples was high. IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. Red lines indicatethe matching sequences between hEPO-reference and amplicon sequences.
Fig3. Confirmation of the accuracy and specificity in TaqMan qPCR using agarose gel electrophoresis
A single band was detected from blood samples, whereas two bands were detected from stool samples. There was no band in the negative control (human cells). IV is intravenous injection, IM is intramuscular injection, and IP is intraperitoneal injection of hEPO coding naked plasmid. HK: HK-2 cells (10 ng/μL), HuH7: HuH-7 cells (10 ng/μL), HEF: Human embryonic fibroblasts cells (10 ng/μL), 293: Human embryonic kidney 293 cells (10 ng/μL), HMS: Human mesenchymal stem cells (10 ng/μL), DW: Distilled water.