Rotaviruses are enteric pathogens causing acute watery dehydrating diarrhea in humans and animals. The importance of group C rotavirus (GpC-RV) infections has not been established as the studies on the GpC-RV have been hampered by the lack of an in vitro culture system. However, diarrheal diseases associated with GpC-RV have been gradually increasing worldwide. In this study, VP6 gene of bovine GpC-RV Korean isolate was expressed, and monoclonal antibodies (mAbs) against VP6 were produced and characterized. The VP6 gene was cloned and expressed based on a baculovirus expression system. Indirect fluorescence antibody (IFA), polymer chain reaction (PCR), and Western blot assays were used to confirm expression of VP6 gene synthesized by the recombinant baculovirus. Eleven mAbs against VP6 were produced using expressed VP6. Cross-reactivity of the mAbs was assessed with recombinant VP6 proteins from porcine GpC-RV and human GpA-RV, or different serotypes of group A rotavirus strains by IFA test. Some mAbs reacted with intact porcine GpC-RV Cowden strain as well as bovine GpC-RV VP6 recombinant baculoviruses, but not with human and animal GpA-RV strains. The VP6-specific mAbs might be useful to develop immunodiagnostic tests such as rapid diagnostic kit, IFA and enzyme-linked immunosorbent assay (ELISA) for detection of GpC-RV.

Among laboratory animals, pigs are anatomically and physiologically closer to human. Transgenic (TG) pigs can be widely applied as models of human diseases. Many researchers created TG pigs which have specific modified genome under a constitutively active promoter. A constitutively active promoter is effective to express a target gene, but the uncontrollable expression often results in unwanted outcomes. In this study, as a way to solve these problems, we tried to regulate the expression of target genes by tetracycline (Tet) on/off system. We tested the operation of Tet on/off system in TG donor cells. Miniature porcine fetal fibroblasts were transfected with universal doxycycline- inducible vector and an enhanced green fluorescent protein (eGFP) was used as the target gene. The induced transgene expression by doxycycline was detected on fluorescence microscopy. On one day after 1 μg/ml doxycycline treatment, the fluorescence intensity for TG cells was increased. And we then performed Somatic Cell Nuclear Transfer (SCNT) to confirm the working of Tet on/off system in the porcine SCNT-TG embryos. Total 649 transgenic porcine SCNT embryos were made. From these, 64 of SCNT embryos used in invitroculturewith1 μg/mldoxycycline. Among these porcine SCNT-TG embryos, 39 embryos (60.9%) were cleaved. Finally, 15 transgenic porcine SCNT embryos developed blastocyst. Induced transgene expression was observed all of cleaved embryos and blastocysts. The remaining 585 embryos were transferred to 6 surrogates. On 25 days after embryo transfer, two surrogates were diagnosed as pregnant (pregnancy rate =33.3%). On day 113 (one day prior to delivery), we obtained six cloned TG piglets from first pregnant surrogate. Unfortunately, all TG piglets died because their surrogate died suddenly at delivery time. However, we could obtain the TG cell lines from the cloned TG piglets. Being analyzed by PCR, all piglets were found to be eGFP gene targeted. Now, second pregnant surrogate have maintained at 80 days after embryo transfer and shown more than three embryonic sacs. This data suggested that, Tet on/off system can control target gene expression in transgenic porcine SCNT embryos. This result has presented new possibilities of regulation of target gene expression in cloned TG pigs by Tet on/off system. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The study investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and gene expression. Three experiments were conducted. Firstly the trans-ε-viniferin was purified from the leaves and stems of the Vitis amurensis , a common wild grape found in Korea, Japan, and China. In the first experiment, a total of 594 cumulus oocytes complexes (COCs) were used for the evaluation of the nuclear maturation. COCs were matured with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM). After IVM 42 44 h, the nuclear maturation was evaluated. In the second experiment, a total of 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular GSH and ROS levels. In the third experiment, the gene expression of oocytes matured with trans-ε-viniferin (0.5 μM) and the untreated group were evaluated after IVM. As results, we observed that trans-ε- viniferin treatment during IVM did not improve the nuclear maturation. But significantly increased (p<0.05) intracellular GSH levels in 0.5 μM group (0 μM vs. 0.5 μM; 14.6 vs. 16.8 pmol/oocyte) and reduced ROS levels (0 μM vs. 0.5 μM and 50 μM; 174.6 vs. 25.7 and 23.8 pixel/oocyte). The trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (p<0.05) expression of Dnmt1 mRNA in 0.5 μM treatment group than in the control group. But, the other gene expressions (PCNA, OCT4, caspase3, BAK, BAX and sit1) did not significantly differ from the control. In conclusion, these results indicated that the trans-ε-viniferin treatment during porcine IVM increased the cytoplasmic maturation through increasing the intracellular GSH synthesis, reducing ROS levels and increasing the Dnmt1 gene expression.

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, gene expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). In the nuclear maturation after 44 h IVM, the groups of 0.1, 0.5, and 2.0 μM (83.0%, 84.1%, and 88.3%, respectively) had no significant difference compared to the control (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.

In this study, we examined the effects of porcine granulocyte-macrophage colonystimulating factor (pGM-CSF) on in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) at first time. The objective of present study was to verify effects of pGM-CSF on SCNT-derived blastocyst formation and evaluate gene expressions and qualities of the blastocyst formed after pGM-CSF treatment. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. A total 522 cloned embryos in 6 replicates were treated with 10 ng/ml concentration of pGM-CSF during in vitro culture (IVC). It was demonstrated that treatment of 10 ng/ml pGM-CSF could increase blastocyst formation and total cell number in blastocyst significantly (p<0.05) compared to the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no any effect on cleavage rate. It was found that the number of cells in the inner cell mass (ICM) and trophectoderm (TE) were significantly increased compared to the control (4.4 and 31.9, respectively) when cloned embryos were cultured with 10 ng/ml pGM-CSF (6.0 and 43.0, respectively). It was also found that treatment of 10 ng/ml pGM-CSF significantly (p<0.05) increased POU5F1 and Cdx2 mRNA expressions in blastocysts. In addition, Bcl-2 mRNA expression was found to be significantly (p< 0.05) up-regulated in blastocysts in the pGM-CSF supplemented group compared to the control. In conclusion, these results suggest that pGM-CSF may improve the quality and developmental viability of porcine cloned embryos by enhancing nuclear reprogramming via regulating transcription factors expression.