Induction of the unfolded protein response (UPR) during Marek's disease virus (MDV) infection.

Marek's disease (MD) is a pathology of chickens associated with paralysis, immune suppression, and the rapid formation of T-cell lymphomas. MD is caused by the herpesvirus, Marek's disease virus (MDV). We examined endoplasmic reticulum (ER) stress and the activation of unfolded protein response (UPR) pathways during MDV infection of cells in culture and lymphocytes in vivo. MDV strains activate the UPR as measured by increased mRNA expression of GRP78/BiP with concomitant XBP1 splicing and induction of its target gene, EDEM1. Cell culture replication of virulent, but not vaccine MDVs, activated the UPR at late in infection. Pathotype-associated UPR activation was induced to a greater level by a vv + MDV. Discrete UPR activation was observed during MDV in vivo infection, with the level of UPR modulation being affected by the MDV oncoprotein Meq. Finally, ATF6 was found to be activated in vv + MDV-induced primary lymphomas, suggesting a possible role in tumor progression.

Human H1 promoter expressed short hairpin RNAs (shRNAs) suppress avian influenza virus replication in chicken CH-SAH and canine MDCK cells.

Influenza A virus represents a significant health and economic threat to both humans and animals worldwide. The polymerase III human H1 promoter was used to express nine short hairpin RNAs (shRNAs) targeting nucleocapsid protein (NP) or the acidic component of polymerase complex (PA) genes of avian influenza virus. Tested shRNAs led to a decrease of up to 400 fold in infective titre and up to 80% in the level of viral NP and PA mRNAs in chicken CH-SAH cells (p<0.05). In mammalian MDCK cells a decrease of up to 10(6)-fold in infective titre, and up to 90% in the level of viral mRNAs (p<0.05) was detected. While RNAi silencing played a major role in viral inhibition, transfection of any plasmid induced an antiviral interferon response in MDCK cells. In CH-SAH cells, no induction of IFN response was observed by constructs, and the IFN response was suppressed by influenza virus infection. Simultaneous induction of RNAi and IFN responses in CH-SAH cells resulted in 18,500-fold decrease in infective titre (p<0.05). We also identified novel efficient and conserved RNAi target site in the NP gene which can be used in antiviral cocktails of shRNAs to block the escape of influenza virus.

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (> or = 1.2-fold, p<0.05), but only one of them, GDP-dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender-related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.

Adverse effects induced by short hairpin RNA expression in porcine fetal fibroblasts.

RNA interference is a recent, gene silencing technique that could be extremely valuable in studying gene function, treating diseases, and developing novel animal models for human diseases. Here, we investigated the feasibility of applying shRNA-mediated RNA interference in fetal fibroblasts for silencing of the myostatin gene and investigate adverse effects of RNAi. We report that up to 97% silencing of myostatin mRNA was achieved using shRNA constructs in transiently and stably transfected fetal fibroblasts (p<0.05). At the same time we also demonstrate that high level of shRNA expression resulted in 10- to 1000-fold induction of interferon responsive genes (OAS1, IFN-beta) (p<0.05). In addition we also report novel adverse effect of shRNA expression in stably transfected cells-interference with microRNA processing/transport which led to 500-fold increase in the level of miR21 precursors (p<0.05). Reduction of these side effects will be essential to obtain long term stable RNAi silencing.

Analysis of Sus scrofa liver proteome and identification of proteins differentially expressed between genders, and conventional and genetically enhanced lines.

Porcine liver proteome iTRAQ analysis enabled the confident identification of 880 proteins with a rate of false positive identifications of less than 5%. Proteins involved in energy metabolism, catabolism, protein biosynthesis, electron transport, and other oxidoreductase reactions were highly enriched confirming the central role of liver as the major chemical and energy factory. Comparative analysis with human and mouse liver proteomes demonstrated that 80% of proteins were common to all three liver proteomes. In addition, it was also demonstrated that both sex of the animal and introduction of a novel phytase transgene into the genome each affected around 5% of total liver proteome. After controlling the false discovery rate (FDR

Transgene and mitochondrial DNA are indicators of efficient composting of transgenic pig carcasses.

Composting is an environmentally sound method for the disposal of on-farm livestock mortalities that generates material suitable for use as fertilizer; however, this method is not generally permitted for disposal of transgenic livestock mortalities during the research and development phase. This study has explored the application of the polymerase chain reaction (PCR) as a method for assessing the persistence of transgene and mitochondrial DNA markers during the composting of euthanized transgenic pig. There was at least a 10(7) fold reduction of genetic material to a level that not either transgene or mitochondrion markers were detectable. At the end of the composting period, only bone fragments that were completely demineralised and chalky were detected. Chemically the compost was similar to that from pig litter and poultry mortalities, except the copper content was lower. Based on these data, composting appears to be an appropriate method for the disposal of transgenic animals.

Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.