Cathelicidin ΔPb-CATH4 derived from Python bivittatus accelerates the healing of Staphylococcus aureus-infected wounds in mice

The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects.

     

Alpha (1,2)-Fucosyltransferase M307A Polymorphism Improves Piglet Survival

To confirm the beneficial effects of alpha (1,2)-fucosyltransferase (FUT1) M307 ^A on piglet survival on commercial farms, we performed PCR-RFLP analysis of FUT1 M307 in successfully marketed (n = 245) and disease affected/deceased pigs during weaning (n = 252) at a commercial farm. We also evaluated the FUT1 genotypes of 190 healthy pigs from three different genetic backgrounds. The distribution of genotypes differed between the successfully marketed and disease affected/deceased pig groups. The frequency of the A allele, associated with resistance to edema and post-weaning diarrhea, was higher in the post-weaning survival group (0.21) than in the non-survival group (0.16, P < 0.05). The odds ratio for piglet survival between AA and GG genotypes was 1.98; thus, piglet survival for individuals with the AA genotype was almost two-fold greater than for GG individuals. The FUT1 gene polymorphism can be used as an effective marker for selection programs to improve post-weaning piglet survival.     

Retrovirus-mediated gene transfer and expression of EGFP in chicken

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.     

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice

Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.     

Production of transgenic chickens expressing a tetracycline-inducible GFP gene

There is much interest in using farm animals as ‘bioreactors’ to produce large quantities of biopharmaceuticals. However, uncontrolled constitutive expression of foreign genes have been known to cause serious physiological disturbances in transgenic animals. The objective of this study was to test the feasibility of the controllable expression of an exogenous gene in the chicken. A retrovirus vector was designed to express GFP (green fluorescent protein) and rtTA (reverse tetracycline-controlled transactivator) under the control of the tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter, respectively. G0 founder chickens were produced by infecting the blastoderm of freshly laid eggs with concentrated retrovirus vector. Feeding the chickens obtained with doxycycline, a tetracycline derivative, resulted in emission of green body color under fluorescent light, and no apparent significant physiological dysfunctions. Successful germline transmission of the exogenous gene was also confirmed. Expression of the GFP gene reverted to the pre-induction levels when doxycycline was removed from the diet. The results showed that a tetracycline-inducible expression system in transgenic animals might be a promising solution to minimize physiological disturbances caused by the transgene.     

Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.