ARTICLE | doi:10.20944/preprints202205.0083.v1
Subject: Life Sciences, Biochemistry Keywords: fasting; refeeding; skeletal muscle; zebrafish; mRNA-sequencing; gene length
Online: 6 May 2022 (14:31:07 CEST)
Recently, fasting has been spotlighted from a healthcare perspective. However, the de-tailed biological mechanisms and significance by which the effects of fasting confer health benefits are not yet clear. Due to certain advantages of zebrafish, as a vertebrate model widely utilized in biological studies, we used mRNA-sequencing and bioinformatics analysis to examine comprehensive gene expression changes in skeletal muscle tissues during fasting-refeeding. Our results produced a novel set of nutrition-related genes under a fasting-refeeding protocol. We found five dramatically upregulated genes in each fasting (for 24 hours) and refeeding (after 3 hours), exhibiting a rapid response to the provided conditional changes. The assessment of the gene length revealed, the gene set whose expression was elevated only after 3 hours of refeeding had a shorter length, suggesting that nutrition-related gene function is associated with gene length. Taken together, our results from bioinformatics analyses provide new insights into biological mechanisms induced by fasting-refeeding conditions within zebrafish skeletal muscle.
ARTICLE | doi:10.20944/preprints202005.0366.v1
Subject: Life Sciences, Genetics Keywords: gene doping; gene therapy; in vivo transfection; in vivo imaging
Online: 23 May 2020 (10:11:31 CEST)
The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the artificial regulation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. First, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan-qPCR assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.
ARTICLE | doi:10.20944/preprints202107.0034.v1
Subject: Life Sciences, Biochemistry Keywords: Gene doping; Gene therapy; Erythropoietin; Adenoviral vector; Sports; Athlete; RNA sequencing
Online: 1 July 2021 (14:30:04 CEST)
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.