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.

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 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 A novel approach to identifying marker genes and estimating the cellular composition of whole blood from gene expression profiles

2016 ◽  
Author(s):  
Casey P. Shannon ◽  
Robert Balshaw ◽  
Virginia Chen ◽  
Zsuzsanna Hollander ◽  
Mustafa Toma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Whole Blood ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Gaussian Model ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Marker Genes ◽  
Sample Collection ◽  
Dynamic Heterogeneity

Measuring genome-wide changes in transcript abundance in circulating peripheral whole blood cells is a useful way to study disease pathobiology and may help elucidate biomarkers and molecular mechanisms of disease. The sensitivity and interpretability of analyses carried out in this complex tissue, however, are significantly affected by its dynamic heterogeneity. It is therefore desirable to quantify this heterogeneity, either to account for it or to better model interactions that may be present between the abundance of certain transcripts, some cell types and the indication under study. Accurate enumeration of the many component cell types that make up peripheral whole blood can be costly, however, and may further complicate the sample collection process. Many approaches have been developed to infer the composition of a sample from high-dimensional transcriptomic and, more recently, epigenetic data. These approaches rely on the availability of isolated expression profiles for the cell types to be enumerated. These profiles are platform-specific, suitable datasets are rare, and generating them is expensive. No such dataset exists on the Affymetrix Gene ST platform. We present a freely-available, and open source, multi-response Gaussian model capable of accurately predicting the composition of peripheral whole blood samples from Affymetrix Gene ST expression profiles. This model outperforms other current methods when applied to Gene ST data and could potentially be used to enrich the >10,000 Affymetrix Gene ST blood gene expression profiles currently available on GEO.

scholarly journals Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sunita Chopra ◽  
Maria Moroni ◽  
Jaleal Sanjak ◽  
Laurel MacMillan ◽  
Bernadette Hritzo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Total Body Irradiation ◽  
Whole Blood ◽  
Radiation Effects ◽  
Expression Profiles ◽  
Mass Casualty ◽  
Machine Learning Algorithms ◽  
Term Survival ◽  
Blood Gene Expression ◽  
Total Body

AbstractGottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1–7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

scholarly journals Predicting Host Immune Cell Dynamics and Key Disease-Associated Genes Using Tissue Transcriptional Profiles

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 301
Author(s):  
Muying Wang ◽  
Satoshi Fukuyama ◽  
Yoshihiro Kawaoka ◽  
Jason E. Shoemaker
Keyword(s):  
Gene Expression ◽  
Gene Expression Data ◽  
Immune Cell ◽  
Mean Squared Error ◽  
Expression Profiles ◽  
Statistical Tests ◽  
Critical Factor ◽  
Expression Data ◽  
Cell Dynamics ◽  
Cell Counts

Motivation: Immune cell dynamics is a critical factor of disease-associated pathology (immunopathology) that also impacts the levels of mRNAs in diseased tissue. Deconvolution algorithms attempt to infer cell quantities in a tissue/organ sample based on gene expression profiles and are often evaluated using artificial, non-complex samples. Their accuracy on estimating cell counts given temporal tissue gene expression data remains not well characterized and has never been characterized when using diseased lung. Further, how to remove the effects of cell migration on transcript counts to improve discovery of disease factors is an open question. Results: Four cell count inference (i.e., deconvolution) tools are evaluated using microarray data from influenza-infected lung sampled at several time points post-infection. The analysis finds that inferred cell quantities are accurate only for select cell types and there is a tendency for algorithms to have a good relative fit (R 2 ) but a poor absolute fit (normalized mean squared error; NMSE), which suggests systemic biases exist. Nonetheless, using cell fraction estimates to adjust gene expression data, we show that genes associated with influenza virus replication and increased infection pathology are more likely to be identified as significant than when applying traditional statistical tests.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

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