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Li J.,Nanjing Agricultural University | Gao Y.,Copenhagen University | Gao Y.,University of Wisconsin - Madison | Petkov S.,Institute for Farm Animal Genetics | And 3 more authors.
Animal Reproduction Science | Year: 2014

Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages. In addition, the expression level of H3K27me3 functional methylase EZH2 increased while no changes were observed on H3K27me3 demethylase JMJD3 in relation to passage number. Moreover, the expression levels of both the H3K4me3 methylase MLL1 and the H3K4me3 demethylase RBP2 were increased at high passages. By using lower passage (numbers 3-5) EG cells as donor cells, the SCNT efficiency was significantly lower compared with use of fetal fibroblast donor cells. However, similar blastocyst rates were achieved when using higher passage (numbers 9-12) EG cells as donor cells. In conclusion, the present study suggests that the epigenetic status of EG cells change with increasing passage numbers, and that higher passage number EG cells are better primed for SCNT. © 2014 Elsevier B.V.

Petkov S.,Institute for Farm Animal Genetics | Hyttel P.,Copenhagen University | Niemann H.,Institute for Farm Animal Genetics
Cellular Reprogramming | Year: 2013

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells. By using different promoters to drive transgenic expression, we show that the efficiency of reprogramming varies with the promoter type. The cells transfected with two different vector systems under the control of doxycycline-induced tet operator (TetO) promoters failed to upregulate essential endogenous pluripotency genes and to maintain stable iPSC morphology, whereas with the Ef1a and CAG promoters the same vectors proved efficient in generating iPSC-like cells with high levels of endogenous pluripotency gene expression that could be maintained long term in vitro. Our results suggest that the choice of expression vector promoters could significantly influence the efficiency of iPSC production from porcine somatic cells. © 2013, Mary Ann Liebert, Inc.

Ellrichmann M.,University of Kiel | Dhar S.,University of Kiel | Hadeler K.-G.,Institute for Farm Animal Genetics | Seehusen F.,University of Veterinary Medicine Hannover | And 5 more authors.
Surgical Endoscopy and Other Interventional Techniques | Year: 2016

Background: Interventional endoscopies entail a risk of infection secondary to perforation of the luminal wall. Thereby, bacteria may be introduced into the sterile environment of the peritoneal cavity (PC). Limited data are available regarding the efficacy of prophylactic anti-infective treatments. The aim of the study was to examine the efficacy/safety of anti-infective means in the prevention of infection by interventional endoscopies in a randomized controlled animal trial. Methods: Forty pigs were randomized to: 1: control; 2: oral lavage; 3: gastric lavage; 4: oral/gastric lavage; 5: i.m. antibiotics. Lavage was performed with Octenisept prior to the operation. After gastric wall perforation, peritoneoscopy was performed. Before the procedure, after closure and prior to autopsy, intraabdominal lavage for bacterial culture was taken using mini-laparoscopy. At autopsy, macroscopic appearance of the PC was scored. Lavage fluids were grown to identify/quantify bacterial load. Concentration of intraperitoneal bacteria at autopsy was defined as main outcome parameter. Results: No major complications occurred in any of the procedures. Bacterial load of the PC at autopsy was significantly reduced with antibiotics compared to all other groups, whereas it did not differ between the lavage groups and control. Macroscopic scoring of the PC showed significant lower rate of intraabdominal abscesses in the antibiotic group compared to the lavage groups and control (p < 0.01). Conclusion: Only antibiotic prophylaxis is effective for the prevention of infection after iatrogenic perforation of the gastrointestinal wall. There was no difference between any form of lavage and the control group. Further studies in humans are required to prove these animal data. © 2015, Springer Science+Business Media New York.

Rehbock C.,Universitaetsstr 7 | Jakobi J.,Universitaetsstr 7 | Gamrad L.,Universitaetsstr 7 | van der Meer S.,Universitaetsstr 7 | And 5 more authors.
Beilstein Journal of Nanotechnology | Year: 2014

Due to the abundance of nanomaterials in medical devices and everyday products, toxicological effects related to nanoparticles released from these materials, e.g., by mechanical wear, are a growing matter of concern. Unfortunately, appropriate nanoparticles required for systematic toxicological evaluation of these materials are still lacking. Here, the ubiquitous presence of surface ligands, remaining from chemical synthesis are a major drawback as these organic residues may cause cross-contaminations in toxicological studies. Nanoparticles synthesized by pulsed laser ablation in liquid are a promising alternative as this synthesis route provides totally ligand-free nanoparticles. The first part of this article reviews recent methods that allow the size control of laser-fabricated nanoparticles, focusing on laser post irradiation, delayed bioconjugation and in situ size quenching by low salinity electrolytes. Subsequent or parallel applications of these methods enable precise tuning of the particle diameters in a regime from 4-400 nm without utilization of any artificial surface ligands. The second paragraph of this article highlights the recent progress concerning the synthesis of composition controlled alloy nanoparticles by laser ablation in liquids. Here, binary and ternary alloy nanoparticles with totally homogeneous elemental distribution could be fabricated and the composition of these particles closely resembled bulk implant material. Finally, the model AuAg was used to systematically evaluate composition related toxicological effects of alloy nanoparticles. Here Ag+ ion release is identified as the most probable mechanism of toxicity when recent toxicological studies with gametes, mammalian cells and bacteria are considered. © 2014 Rehbock et al.

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