• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.235-240
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• 2007

This study was conducted to examine the viability of Korean native striped cattle (Bos namadicus Falconer, Chikso) clone embryos after embryo transfer. Chikso somatic cell nuclear transfer (SCNT) embryos were produced by fusion of ear skin cells derived from a female Chikso with enucleated oocytes matured in vitro for 18-24 hr. After in vitro culture of SCNT embryos for 7 to 8 days, fresh or vitrified blastocysts derived from SCNT were transferred into a uterine horn of recipient cows. Fifteen of total 43 recipients were pregnant at Day 50 and 4 recipients were maintained to term. Three IVF-derived calves and 1 clone Chikso calf were born. Pregnancy rate was higher when fresh embryos were transferred to recipients compared to vitrified embryos, but development to term was not different between both groups. The clone Chikso calf died at 5 days after birth due to the fullness of amniotic fluid in rumen and the infection of umbilical cord. The result of the present study shows that clone Chikso calf can produced from the embryo transfer of SCNT embryos, however, solution of abortion problem is necessary to improve the cloning efficiency.

• IMMUNE NETWORK
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• v.16 no.5
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• pp.305-310
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• 2016

In this study, we compared two different tumor cell vaccines for their induction of anti-tumor immunity; one was a tumor cell clone expressing a membrane-bound form of IL-12 p35 subunit (mbIL-12 p35 tumor clone), and the other was a tumor clone expressing heterodimeric IL-12 as a single chain (mb-scIL-12 tumor clone). The stimulatory effect of mb-scIL-12 on the proliferation of ConA-activated splenocytes was higher than that of mbIL-12 p35 in vitro. However, the stimulatory effect of mbIL-12 p35 was equivalent to that of recombinant soluble IL-12 (3 ng/ml). Interestingly, both tumor clones (mbIL-12 p35 and mb-scIL-12) showed similar tumorigenicity and induction of systemic anti-tumor immunity in vivo, suggesting that tumor cell expression of the membrane-bound p35 subunit is sufficient to induce anti-tumor immunity in our tumor vaccine model.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.23-31
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• 2011

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

• Biomedical Science Letters
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• v.15 no.1
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• pp.55-60
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• 2009

Insect cells such as Spodoptera frugiperda Clone 9 (Sf9) cells are widely chosen as the host for heterologous expression of a mammalian sugar transport protein using the baculovirus expression system. Characterization of the expressed protein is expected to include assay of its function, including its ability to transport sugars and to bind inhibitory ligands such as cytochalasin B. It is therefore very important first to establish the transport characteristics and other properties of the endogenous sugar transport proteins of the host insect cells. However, very little is known of the transport characteristics of Sf9 cells, although their ability to grow on TC-100 medium strongly suggested the presence of endogenous glucose transport system. In order to investigate the substrate and inhibitor recognition properties of the Sf9 cell transporter, the ability of pentoses to inhibit 2-deoxy-D-glucose (2dGlc) transport was investigated by measuring inhibition constants $(K_i)$. To determine the time period over which of sugar into the Sf cells was linear, the uptake of 2dGlc 0.1mM extracellular concentration was measured over periods ranging from 30 seconds to 30 minutes. The uptake was linear for at least 2 minutes at the concentration, implying that uptake made over a 1 minute time course would reflect initial rates of the sugar uptake. The data have also revealed the existence of a saturable transport system for pentose uptake by the insect cells. The transport was inhibited by D-xylose and D-ribose, although not as effective as hexoses. However, L-xylose had a little effect on 2dGlc transport in the Sf9 cells, indicating that the transport is stereoselective. Unlike the human erythrocyte-type glucose transport system, D-ribose had a somewhat greater apparent affinity for the Sf9 cell transporter than D-xylose. It is therefore concluded that Sf9 cells contain an endogenous sugar transport activity that in some aspects resembled the human erythrocyte-type counterpart, although the Sf9 and human transport systems do differ in their affinity for cytochalasin B.

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.121-126
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• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Archives of Pharmacal Research
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• v.20 no.5
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• pp.465-470
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• 1997

The human cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT), was isolated from a .gamma.gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. The two clones had 74% nlicleotide sequence identities in the coding region. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. In order to establish substrate specificity, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. Sixty potential substrates were tested using cells transfected with pUDPGTh2. The order of relative substrate activity was as follows: 4-hydroxyestrone > estriol >2-hydroxyestriol > 4-hydroxyestradiol > $6{\alpha}$-hydroxyestradiol >$5{\alpha}$-androstane-$3{\alpha}$, $11{\beta}$, $17{\beta}$-triol=5${\beta}$-androstane-$3{\alpha}$ ${\beta}$, $17{\beta}$-triol. There were only trace amounts of gulcuronidation of 2-hydroxyestradiol and 2-hydroxyestrone, and in contrast to other cloned transferase, no gulcuronidation of either the primary estrogens and androgens (estrone, $17{\beta}$estradiol/testosterone, androsterone) or any of the exogenous substrates tested was detected. A lineweaver-Burk plot of the effect of 4-hydroxystrone concentration on the velocity of glucuronidation showed an apparent Km of $13{\mu}M$. The unique specificity of this transferase might play an important role in regulating the level and activity of these potent and active estrogen metabolites.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Animal cells and systems
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• v.12 no.4
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• pp.193-201
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• 2008

The eventual goal of tumor immunotherapy is to develop a vaccine inducing a specific anti-tumor immunity. Cytokine gene therapy is an effective way at least in animal models, but limited efficacy and various side effects obstruct clinical applications. In this study, we developed a tumor vaccine expressing a membrane-bound form of IL-2(mbIL-2) and SDF-1 in B16F10 melanoma cells. The tumor clones expressing mbIL-2 showed reduced tumorigenicity, and additional expression of SDF-1 to mbIL-2 expressing tumor cells caused more severe reduction in tumorigenicity. However, expression of the SDF-1 alone did not affect on the tumorigenicity, probably because of limited production of SDF-1 in the SDF-1 transfected clones. When the mice once rejected mbIL-2/SDF-1 expressing tumor clone were re-challenged with wild type B16F10 tumor cells, all of the mice survived. This result suggests that mbIL-2/SDF-1 tumor clone is effective in inducing systemic anti-tumor immunity against wild type B16 melanoma. Furthermore, culture supernatant of tumor clones expressing SDF-1 induced lymphocyte migration in vitro. These results, all together, suggest that expression of mbIL-2 and SDF-1 in tumor cells enhances anti-tumor immune responses through different roles; the secreted SDF-1 may function as a chemoattractant to recruit immune cells to tumor vaccine injection site, and the mbIL-2 on tumor cells may provide costimulatory signal for CTL activation in physical contacts.

• The Korea Journal of Herbology
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• v.22 no.4
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• pp.87-94
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• 2007

Objectives : We examined three parts of Nelumbo nucifera that might be useful for skin-whitening effect. Each parts of leaves, flowers and stamen were extracted with water or 70% ethanol. Methods : This study investigated inhibitory effects of each extracts on tyrosinase activity. Extracts were tested for cytotoxicity on clone M-3 melanocyte cells, melanin inhibitory activities were further assessed. Results : The water extract of Nelumbo nucifera stamen. ethanol and water extracts of flowers exhibited inhibitory effects on tyrosinase (IC50 values 726.16, 1063, and 1732.36 ug/mL, respectively). The water extract of Nelumbo nucifera stamen, ethanol extract of flowers, both ethanol and water extracts of leaves showed relatively lower cytotoxicity, with cell viability above 80% with a concentration of 20 ${\mu}g/mL$. In addition, The water extract of Nelumbo nucifera stamen inhibited the melanin production in clone M-3 melanocyte cells. Conclusions : Among each parts of Nelumbo nucifera. the water extract of stamen was the strongest candidates for skin-whitening cosmetic application.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.1-8
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• 2015

In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7^th codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.