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

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.

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

“…Therefore, if a primer-probe is designed on an exon-exon junction, amplification can be expected to be transgene-specific. These findings have been clarified in previous studies [ 18 , 24 , 25 ]. However, this specific PCR amplification could be avoided by codon modifications of the gene body.…”

“…In some studies on gene doping using animals injected with plasmids, the gene transfer efficiency into the targeted tissues was not measured [ 16 , 17 , 18 ]. Generally, when transfection is performed using plasmid alone, gene transfer efficiency is very low.…”

“…We previously reported results of another study similar to this one. This previous study showed that, when only naked plasmid of human EPO (hEPO) with CMV promoter was injected into the abdominal cavity of mice, the transgene fragments of the hEPO were significantly detected at a median of 9.18 copies/μL in whole blood (about one drop) at 48 h after the injection [ 18 ]. On the other hand, in this study, the transgene fragments of Fluc, Amp, and CMVp were significantly detected at medians of 97.5, 99.9, and 77.8 copies/μL in whole blood (about one drop) at 48 h from the injection of the plasmid-PEI complex.…”

EPO transgene detection in dried blood spots for antidoping application

“…9 Aoki et al recently studied EPO transgene detection in mice using Syber Green assay and Taqman assay and concluded on the much better results of the Taqman assay. 10 As this type of technology Note: NA: No Cq value determined (Cq > 40). DBS were analyzed 2 days after preparation.…”

Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control

“…Several transgene detection methods have been developed and reported 5–9 . While some use next‐generation sequencing technology, 10,11 most transgene detection methods are based on polymerase chain reaction (PCR), real‐time PCR and digital PCR.…”