Grapevines are cultivated globally, and are also damaged by a large number of insect pests especially including leafhoppers. Leafhoppers are one of the important insect pests on various crops including grape. Among them, the genus Arboridia Zachvatkin (Hemiptera: Auchenorrhyncha: Cicadellidae: Typhlocybinae) was known as insect pests against grape farming. Eighty species were described in the genus in the world, and eight species were recorded in the Korean Peninsula so far, which are: Arboridia apicalis, A. kakogawana, A. koreacola, A. maculifrons, A. nigrigena, A. okamotonis, A. silvarum, A. suzukii. Among them, A. apicalis, A. kakogawana, A. maculifrons, A. nigrigena, A. suzukii have been known as important insect pests on grapes. In this talk, we propose a new species from the Korean Peninsula, and confirmed two new records: A. agrillacea and A. lunula. Additionally we present differential diagnoses of the Arboridia species damaging on grapevines for the field of applied biology.

Flexible transparent conducting films (TCFs) were fabricated by dip-coating single-wall carbon nanotubes (SWCNTs) onto a flexible polyethylene terephthalate (PET) film. The amount of coated SWCNTs was controlled simply by dipping number. Because the performance of SWCNT-based TCFs is influenced by both electrical conductance and optical transmittance, we evaluated the film performance by introducing a film property factor using both the number of interconnected SWCNT bundles at intersection points, and the coverage of SWCNTs on the PET substrate, in field emission scanning electron microscopic images. The microscopic film property factor was in an excellent agreement with the macroscopic one determined from electrical conductance and optical transmittance measurements, especially for a small number of dippings. Therefore, the most crucial factor governing the performance of the SWCNT-based TCFs is a SWCNT-network structure with a large number of intersection points for a minimum amount of deposited SWCNTs.

To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of polyhedrin fragments were investigated by fusion expression them with the enhanced green fluorescence protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the minimal region for self-assembly and the KRKK nuclear localization signal (NLS). The increase of EGFP production by fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production of EGFP was shown by the mutation of the NLS within the fused polyhedrin fragment. Among the fusion expressed protein in cytoplasm, the most hyper-expression was observed in the fusion of amino acids 32 to 59 of polyhedrin. Polyhedrin fragment fusion expression with classical swine fever virus E2 protein also resulted hyper-enhanced expression of E2 protein. However, the fusion expression of porcine circovirus ORF2 with polyhedrin fragment did not show significant enhance of ORF2 production. These results suggested that the enhancement of foreign protein production when fused with polyhedrin is caused by the enhanced stability of expressed protein.

Induced pluripotent stem cells (iPSCs), generated by the overexpression of transcription factors Oct4, Sox2, Klf4 and c‐Myc in somatic cells, are pluripotent. iPSCs reprogrammed from differentiated cells get through a epigenetic modification during reprogramming and finally have the similar epigenetic state to embryonic stem cells (ESCs). In this study, these epigenetic changes were observed in reprogramming of uni‐parental parthenogenetic somatic cells. Furthermore, we have shown that parthenogenetic pattern of imprinted genes were changed during pluripotential reprogramming. Parthenogenetic neural stem cells (pNSCs) containing only maternal alleles regain the biparental imprinting patterns after reprogramming. However, we have yet to define whether the changed imprinted genes are maintained or reverted to the parthenogenetic state when the reprogrammed cells are differentiated into specialized cell types. To address this question, we compared genome‐wide expression profiles of biparental female neural stem cells (fNSCs), parthenogenetic neural stem cells (pNSCs), and NSCs differentiated from parthenogenetic maternal iPSC (miPS‐NSCs). Furthermore, this study establishes the correlation between the alteration of genome methylation and activation of imprinting genes in the parthenogenetic cells and reports for the first time that the silenced PWS‐related imprinted genes are activated in miPS‐NSCs. Our data demonstrated that pluripotential reprogramming of parthenogenetic somatic cells were able to reset the parthenogenetic imprinting patterns; reprogrammed miPSCs showed erasure of maternal methylation imprints and acquisition of methylation in paternally imprinted genes. Furthermore, the changed imprinting patterns were maintained when the reprogrammed cells are differentiated into specialized cell type. * This work was supported by the Next‐Generation BioGreen 21 program (Grant PJ008- 009) funded by the Rural Development Administration, Republic of Korea.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Ski protein is a nuclear transcription factor that does not bind DNA directly. Due to its unique binding properties with multiple factors, Ski could perform various roles in the regulation of both cellular proliferation and differentiation. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vitro models. RT-PCR and real time PCR analysis respectively revealed that LH had no effect on c-Ski mRNA expression in the cultured granulosa cells regardless of LH treatment. Though Ski protein isabsent in granulosa cells of preovulatory follicle, its mRNA(c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally. Moreover, expression of mRNA of Arkadia, an E3 ubiquitin ligases, in luteinizing granulosa cells in vivo was assessed by real time-PCR. The levels of Arkadia mRNA expression were unchanged during follicular growth and post ovulatory luteinization. These findings suggest that Ski protein level may be regulated during luteinization at translational and/or post-translational level but not by Arkadia.

Sloan-Kettering virus gene product of a cellular pro-oncogene c-Ski is an unique nuclear pro-onco protein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of this study was, by means of immunohistochemical methods, to locate Ski protein in the rat ovaries during ovulation and corpora lutea(CL) formation to predict the possible involvement of Ski in luteinization. In addition, to examine whether the initiation of luteinization with luteinizing hormone(LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vivo models. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone(LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum(CL). These results indicate that Ski is profoundly expressed in the luteinized granulosa cells and luteal cells of CL during luteinization, and suggest that Ski may play a role in luteinization of granulosa cells.

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at — 7℃ to — 35℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen- thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5 ml, and 43.6 % frozen at 20embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski)was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously. The frequency of true hermaphroditism is higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymes, penes, and uteri. Histomorphologically, the testicular tissues consisted of Sertoli cells that were devoid of spermatogenic germ cells and showed proliferation of interstitial cells. However, the uteri were of normal architecture and had well-developed uterine endometrial glands. The samples were 38, XX female karyotype without the sex-determining region Y (SRY) gene. The findings of this study could contribute to the understanding of true hermaphroditism in the Korean pig industry. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

Neural stem cells (NSCs) are self-renewing tripotent cell populations and have capacity of neuronal (neurons) and glial (astrocytes and oligodendrocytes) differentiation. Many researchers have reported that NSCs have therapeutic effects in neurological disease by transplantation. However, it is not easy to obtain NSCs in vitro. Recently, Yamanaka and colleagues showed that somatic cells could be reprogrammed into pluripotent state by enforcing reprogramming factors. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. Direct differentiation into a specialized cell types from iPS cells hold considerable promise for regenerative medicine as well as basic research. Here, we induced differentiation of iPS cells into NSCs in vitro and in vivo, which were compared with embryonic stem (ES) cell-derived NSCs and brain derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from brain derived NSCs and stained positive for NSCs markers Nestin and Sox2. ES cells derived NSCs were transcriptionally distinguishable from brain derived NSCs. However, global gene expression pattern were similar but distinct between iPS derived NSCs and brain derived NSCs. Moreover, iPS derived NSCs were spontaneously aggregated upon passaging, formed ES cell like colonies, and finally reactivated Oct4-GFP. The spontaneously reverted GFP-positive cells (iPS-NSC-iPS) expressed similar levels of pluripotency markers (Oct4,Nanog) to ES and iPS cells, and could form germ line chimera. One possible explanation for this phenomenon is that spontaneously re-reprogramming was associated with transgene re-activation when iPS cells were differentiated into NSCs. However, NSCs from dox-inducible iPScells could not be reprogrammed into pluripotent state without doxycycline. Taken together, iPS derived NSCs were morphologically and similar to brain derived NSCs, but differ in gene expression pattern and maintenance. * This work was supported by the Next Generation Bio-Green21 Program funded by the Rural Development Administration (Grant PJ008009).

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

The objective of the current study was to describe in vitro embryo production in Hanwoo, analyzing oocytes yield and embryo production. The effects of oocytes production and the number of OPU procedures per animal on embryo production were also evaluated. OPU was done every 3～4 days during experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, we compared the recovery rate of oocytes based on OPU session (Experiment 1). The average of collected oocytes was calculated from every 10 session. The average number of total oocytes recovered per animalonsessionwas 5.16 (mean). Second, we compared the recovery rate base on collection period of OPU (Experiment 2). The following results show the difference of the number of recovered oocytes in every month during the procedure between the months of session. Every animal shows the constant number of recovered oocytes for the first 5 months. However, the recovery rate of oocytes was decreased from month 6 to 8. Third, we compared the developmental rate to blastocyst in two groups (Experiment 3). Oocytes by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24～48 h after in vitro fertilization (IVF) was 75.8% and blastocyst development rate was 18.8% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (61.1%), blastocyst development rate was higher compared with needle puncture group (28.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio- technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being.

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at —7 ℃ to —35 ℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen-thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5ml, and 43.6 % frozen at 20 embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.