Satellite cells were derived from muscular tissue in postnatal pig. Satellite cell is an important to growth and development in animal tissues or organs. However, the progress underlying induced differentiation is not clear. The aim of this study was to evaluate the morphologic and the transcriptome changes in porcine satellite cell (PSC) treated with insulin, rosiglitazone, or dexamethasone respectively. PSC was obtained from postnatal muscle tissue. In study 1, for study the effect of insulin and FBS on the differentiated satellite cells, cells were cultured at absence or presence of insulin treated with FBS. Total RNA was extracted for determining the expression levels of myo-genic PAX3, PAX7, Myf5, MyoD, and myogenin genes by real-time PCR. Myogenic genes decreased expression levels of mRNA in treated with insulin. In study 2, in order to clarify the relationship between rosiglitazone and lipid in differentiated satellite cells, we further examined the effect of FBS on lipid accumulation in the presence or absence of the rosiglitazone and lipid. Significant differences were observed between rosiglitazone and lipid by FBS. The mRNA of FABP4 and PPARγ increased in rosiglitazone treatment. In study 3, we examined the effect of dexame-thasone on osteogenic differentiation in PSC. The mRNA was increased osteoblasotgenic ALP and ON genes treated with dexamethasone in 2% FBS. Dexamethasone induces osteoblastogenesis in differentiated PSC. Taken together, in differentiated PSCs, FABP4 and PPARγ increased to rosiglitazone. Whereas, no differences to FBS and lipid. These results were not comparable with previous reports. Our results suggest that adipogenic, myogenic, and osteoblasto-genic could be isolated from porcine skeletal muscle, and identify culture conditions which optimize proliferation and differentiation formation of PSC.

Muscular satellite cell (SC), which is stem cell of postnatal pig, is an important for study of differentiation into adipogenesis, myogenesis, and osteoblastogenesis. In this study, we isolated and examined from pig muscle tissue to determine capacity in proliferate, differentiate, and expression of various genes. Porcine satellite cells (PSC) were isolated from semimembranosus (SM) muscles of 90∼100 days old pigs according to standard conditions. The cell proliferation increased in multi-potent cell by Masson’s, oil red O, and Alizarin red staining respectively. We per-formed the expression levels of differentiation related genes using real-time PCR. We found that the differentiation into adipocyte increased expression levels of both fatty acid binding protein 4 (FABP4) and peroxisome proliferator- acti-vated receptor gamma (PPARγ) genes (p<0.01). Myocyte increased the expression levels of the myosin heavy chain (MHC), myogenic factor 5 (Myf5), myogenic regulatory factor (MyoD), and Myogenic factor 4 (myogenin) (p<0.01). Osteo-blast increased the expression levels of alkaline phosphatase (ALP) (p<0.01). Finally, porcine satellite cells were indu-ced to differentiate towards adipogenic, myogenic, and osteoblastogenic lineages. Our results suggest that muscle satellite cell in porcine may influence cell fate. Understanding the progression of PSC may lead to improved strat-egies for augmenting meat quality.

Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.

Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Although somatic cell nuclear transfer (SCNT) has successfully been produced cloned animals in several species, the cloning efficiency is extremely low. It is generally believed that the low cloning efficiency is mainly attributed to faulty epigenetic modifications underlying the aberrant reprogramming of donor cell nuclei in recipient cytoplasm after SCNT. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be a key factor in improving the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expressions (Nanog, Oct3/4 and Sox2) in porcine cloned blastocysts. The porcine cloned embryos were treated with a 50 nM concentration of TSA or a 100 μm concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expressions in NT blastocysts. All data was analyzed by chi-square. Following 4-5 replicates (245, 200 and 222 for NT, TSA and CBHA treated NT embryos respectively) there was no difference between normal NT and CBHA treated NT embryos, whereas TSA treated NT embryos was significantly decreased for cleavage rate (p<0.05). The developmental competence to the blastocyst stage in CBHA treated NT embryos (18.9%) significantly increased than that of normal NT and TSA treated NT embryos (9.4% and 11.5%) (p<0.05). In addition, all of pluripotent transcription factors (Nanog, Oct3/4 and Sox2) were highly expressed in the CBHA treated NT embryos, however, Sox2 and Oct3/4 were expressed in TSA treated NT embryos and Sox2 was only expressed in normal NT embryos (p<0.05). In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency- related gene expressions (Nanog, Oct3/4 and Sox2) in NT blastocysts.

Methanol and hot water extracts of Suillus granulatus were investigated for its antioxidant activity, polyphenol content and tyrosinase inhibitory effect. The antioxidant activity was evaluated using 1, 1- dipheny l-2-picrylhydrazyl (DPPH) scavenging assay. The antioxidant activities of hot water and 80% methanol extracts of Suillus granulatus fruiting body were 51.49%. and 90.58% at 1.5mg/ml concentration. Total polyphenol content of hot water and 80% methanol extracts were 33.21㎍ GAEs/mg and 27.97㎍ GAEs/mg. Tyrosinase inhibitory activity of water extract was 7.8% while 80% methanol extract was 20.88% at 1mg/ml concentration.

Methanol and hot water extracts of Daedalea bien-nis were investigated for its antioxidant activity, poly- phenol content and tyrosinase inhibitory effect. The antioxidant activity was evaluated using 1, 1-diphenyl -2-picrylhydrazyl (DPPH) scavenging assay. The antioxidant activities of hot water and 80% methanol extracts of Daedalea biennis fruiting body were 52.06%. and 92.62% at 1.5mg/ml concentration. Total polyphenol content of hot water and 80% methanol extracts were 12.26㎍ GAEs/mg and 13.69㎍ GAEs/mg. Tyrosinase inhibitory activity of water extract was 19.70% while 80% methanol extract was 15.75% at 1mg/ml concentration.