identification-homologous-recombination-events-mouse-embryonic-stem

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Hunan Agricultural University (HUNAU)

Virus-like particles (VLPs) can be used as transfer vehicles carrying foreign proteins or antigen epitopes to produce chimeric VLPs for bivalent or multivalent vaccines. Based on the crystal structure of porcine circovirus type 2 (PCV2) capsid protein (Cap), in addition to alignment of the Cap sequences collected from various isolates of PCV2 and PCV1, we predicted that Loop CD of the PCV2 Cap should tolerate insertion of foreign epitopes, and furthermore that such an insertion could be presented on the surface of PCV2 VLPs. To validate this, the GP5 epitope B of porcine reproductive and respiratory syndrome virus (PRRSV) was inserted into Loop CD of the PCV2 Cap. The 3D structure of the recombinant PCV2 Cap (rCap) was simulated by homology modeling; it appeared that the GP5 epitope B was folded as a relatively independent unit, separated from the PCV2 Cap backbone. Furthermore, based on transmission electron microscopy, the purified PCV2 rCap self-assembled into chimeric VLPs which entered PK-15 cells. In addition, PCV2 chimeric VLPs induced strong humoral (neutralizing antibodies against PCV2 and PRRSV) and cellular immune responses in mice. We concluded that the identified insertion site in the PCV2 Cap had great potential to develop PCV2 VLPs-based bivalent or multivalent vaccines; furthermore, it would also facilitate development of a nano-device to present a functional peptide on the surface of the VLPs that could be used for therapeutic purposes. 

Generation and immunogenicity of porcine circovirus type 2 chimeric virus-like particles displaying porcine reproductive and respiratory syndrome virus GP5 epitope B.

Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.

Evidence of natural co-infection with PCV2b subtypes in vivo.

T-2 and HT-2 toxins can cause cytotoxicity and oxidative stress in animals, while DL-Selenomethionine plays an important role in preventing oxidative stress and improving cell viability. However, the role of DL-Selenomethionine in T-2/HT-2 toxins-induced cell damage is still unknown. In this study, we investigated whether DL-Selenomethionine plays a protective role against T-2/HT-2-induced cytotoxicity and oxidative stress in primary hepatocytes. Our results demonstrated that T-2/HT-2 toxins-exposed broiler hepatocytes exhibited significantly decreased cell viability and intracellular glutathione (GSH) concentration while increased Lacate dehydrogenase (LDH) leakage, intracellular reactive oxygen species (ROS), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and catalase (CAT) levels, as well as elevated expression levels of genes related to oxidative stress, in a toxin dose-dependent manner (P < 0.05). However, the application of DL-Selenomethionine into T-2/HT-2 treated hepatocytes effectively alleviated the adverse effects of T-2/HT-2, as demonstrated by increased cell viability, decreased LDH leakage, declined intracellular ROS and MDA levels, increased expression of oxidative stress-related genes, as well as accordingly enhanced activities of GSH, GSH-PX, SOD and CAT as compared to the control groups (P < 0.05). Therefore, our in vitro data demonstrate that DL-Selenomethionine can function as an effectively protective agent against T-2/HT-2-induced cytotoxicity and oxidative stress.

Aibing Wang

Lab of Animal Models and Functional Genomics (LAMFG),
The Key Laboratory of Animal Vaccine & Protein Engineering,
College of Veterinary Medicine,
Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine

Hunan Agricultural University (HUNAU)

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction