• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.216-222
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• 2003

Human caspase-9, an essential apoptosis initiator protease, was excessively degraded when expressed in Escherichia coli under the conventional induction condition. To optimize the conditions for induction and develop a rapid purification method for obtaining significant amounts of wild-type procaspase-9, we expressed procaspase-9 as GST fusion in E. coli. The addition of 0.01 mM IPTG as an inducer to the bacterial culture and decreasing the culture temperature to 25oC improved the production of procasapse-9 protein by circumventing proteolytic degradation in E. coli. The wild-type procaspae-9 was purified to approximately 70% purity with relatively high yields using the method developed in this study. In addition, we found that GST-caspase-9 is autocatalytically cleaved after aspartic acid 315, which is the same site for processing in mammalian cells, during expression in E. coli.

• Journal of Life Science
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• v.22 no.11
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• pp.1545-1551
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• 2012

Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

• Korean Journal of Breeding Science
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• v.49 no.3
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• pp.170-179
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• 2017

Plant molecular farming has attracted a lot of attention lately in the field of mass production of industrially valuable materials by extending application of the plant as a kind of factory concept. Among them, protein expression system using rice(Oryza sativa L.) callus is a technology capable of mass culture and industrialization because of a high expression rate of a target protein. This study was carried out to develop an Agrobacterium-mediated transformation system to increase the utilization of rice callus. The transformation efficiency was improved by using the hand when seeds were de-husked for callus induction. Furthermore, we were possible induction of callus from 6 years old seed smoothly. Selection of the callus contained the target gene was required a cultivation period of at least 3 weeks, and the most efficient selection period was after 6 weeks of culture including one passage. This selection was confirmed that the gene was stably inserted into the genomic DNA of the plant cell by the southern blot analysis and progeny test. Such an efficient selection system of rice callus that can be cultured in the long term will be contribute to the industrialization of useful recombinant proteins using rice.

• Journal of Life Science
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• v.19 no.1
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• pp.123-128
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• 2009

Production of highly valuable immunotherapeutic proteins such as monoclonal antibodies and vaccines using plant biotechnology and genetic engineering has been studied as a popular research field. Plant expression system for mass production of such useful recombinant therapeutic proteins has several advantages over other existing expression systems with economical and safety issues. Immunotherapy of multiple monoclonal antibodies, which can recognize multiple targeting including specific proteins and their glycans highly expressed on the surface of cancer cells, can be an efficient treatment compared to a single targeting immunotherapy using a single antibody. In this study, we have established plant production system to express two different targeting monoclonal antibodies in a single transgenic plant through crossing fertilization between two different transgenic plants expressing anti-colorectal cancer mAbCO17-1A and anti-breast cancer mAbBR55, respectively. The F1 seedlings were obtained cross fertilization between the two transgenic parental plants. The presence, transcription, and protein expression of heavy chain (HC) and light chain (LC) genes of both mAbs in the seedlings were investigated by PCR, RT-PCR, and immunoblot analyses, respectively. Among all the seedlings, some seedlings did not carry or transcribe the HC and LC genes of both mAbs. Thus, the seedlings with presence and transcription of HC and LC genes of both mAbs were selected, and the selected seedlings were confirmed to have relatively stronger density of HC and LC protein bands compared to the transgenic plant expressing only each mAb. These results indicate that the F1 seedling plant with carrying both mAb genes was established. Taken together, plant crossing fertilization can be applied to generate an efficient production system expressing multiple monoclonal antibodies for immunotherapy in a single plant.

• Journal of Life Science
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• v.26 no.12
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• pp.1376-1382
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• 2016

Xylitol is widely used in the food and medical industry. It is produced by the reduction of xylose (lignocellulosic biomass) in the Saccharomyces cerevisiae strain, which is considered genetically safe. In this study, the expression system of the GRE3 (YHR104W) gene that encodes xylose reductase was constructed to efficiently produce xylitol in the S. cerevisiae strain, and the secretory production of xylose reductase was investigated. To select a suitable promoter for the expression of the GRE3 gene, pGMF-GRE3 and pAMF-GRE3 plasmid with GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter for secretory production. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$, and $SEY2102{\Delta}trp1$/pGMF- GRE3 and $SEY2102{\Delta}trp1$/pAMF-GRE3 transformants were selected. In the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, the total activity of xylose reductase reached 0.34 unit/mg-protein when NADPH was used as a cofactor; this activity was 1.5 fold higher than that in $SEY2102{\Delta}trp1$/pAMF-GRE3 with ADH1 as the promoter. The secretion efficiency was 91% in both strains, indicating that most of the recombinant xylose reductase was efficiently secreted in the extracellular fraction. In a baffled flask culture of the $SEY2102{\Delta}trp1$/pGMF-GRE3 strain, 12.1 g/l of xylitol was produced from 20 g/l of xylose, and ~83% of the consumed xylose was reduced to xylitol.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.11
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• pp.1600-1608
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• 2005

The study was carried out to evaluate the effect of exogenous bovine somatotropin on water metabolism in relation to mammary function in early lactation of crossbred Holstein cattle. Ten, 87.5% crossbred Holstein cattle were divided into two groups of 5 animals each. At day 60 of lactation, the control group was given placebo while animals in the experimental group were given recombinant bovine somatotropin (rbST) by subcutaneous injection with 500 mg of rbST (14-days prolonged-release rbST). In rbSTtreated animals, milk yield increased 19.8%, which coincided with a significant increase in water intake (p<0.01), while DM daily intake was not different when compared to the control animals. Water turnover rate as absolute values significantly increased (p<0.05), while the biological half-life of water did not change in rbST-treated animals. Total body water (TBW) and total body water space (TOH) as absolute values significantly increased (p<0.01) in rbST-treated animals, while it was decreased in the control animals. Absolute values of empty body water (EBW) markedly increased (p<0.05), which was associated with an increase in the extracellular fluid (ECF) volume. Absolute values of plasma volume and blood volume were also significantly increased (p<0.05) in rbST-treated animals. The increase in mammary blood flow in rbST-treated animals was proportionally higher than an increase in milk production. The plasma IGF-1 concentration was significantly increased (p<0.01) in rbST-treated animals when compared with those of control animals during the treatment period. Milk fat concentration increased during rbST treatment, while the concentrations of both protein and lactose in milk were not affected. The present results indicate that rbST exerts its effect on an increase in both TBW and EBW. An increased ECF compartment in rbST-treated animals might partly result from the decrease in fat mass during early lactation. The action of rbST on mammary blood flow might not be mediated solely by the action of IGF-1 for increase in blood flow to mammary gland. An elevation of body fluid during rbST treatment in early lactation may be partly a result of an increase in mammary blood flow in distribution of milk precursors to the gland.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1407-1416
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• 2007

Ten, first lactation, 87.5%HF dairy cattle were used to investigate effects of long-term administration of recombinant bovine somatotropin (rbST) on nutrient uptake by the mammary gland at different stages of lactation. Measurements of arterial plasma concentrations and arterial-venous differences of metabolites across the mammary gland were performed in combination with measurment of mammary blood flow to estimate the mammary uptake. Animals in experimental groups were injected subcutaneously every 14 days from day 60 of lactation with a prolonged-release formulation of 500 mg of rbST (POSILAC, Monsanto, USA) or with sterile sesame oil without rbST in the control group. During early lactation, the milk yield of rbST-treated animals was higher than that of the control animals (p<0.05). The peak milk yield in both groups of animals declined from the early period of lactation with progression to mid- and late-lactation. No significant changes were observed in the concentration of milk lactose, while the concentrations of milk protein significantly increased as lactation advanced to mid- and late-lactation in both groups. Milk fat concentrations were significantly higher in rbST-treated animals than in control animals, particularly in early lactation (p<0.05). Mammary blood flow (MBF) markedly increased during rbST administration and was maintained at a high level throughout lactation. The mean arterial plasma concentrations for glucose and acetate of rbST-treated animals were unchanged. The net mammary glucose uptake of rbST-treated animals increased approximately 20% during early lactation, while it significantly decreased (p<0.05), including the arteriovenous differences (A-V differences) and extraction ratio across the mammary gland, as lactation advanced to mid- and late-lactation. A-V differences, mammary extraction and mammary uptake for acetate increased during rbST administration and were significantly higher (p<0.05) than in the control animals in early and mid-lactation. Mean arterial plasma concentrations for ${\beta}$-hydroxybutyrate and free glycerol were unchanged throughout the experimental periods in both groups. A-V differences and extraction ratio of ${\beta}$-hydroxybutyrate across the mammary gland did not alter during rbST administration. Mean arterial plasma concentrations for free fatty acids ($C_{16}$ to $C_{18}$), but not for triacylglycerol, increased in rbST-treated animals and were significantly higher than in control animals during early lactation (p<0.01). These findings suggest that an increase in MBF during rbST administration would not be a major determinant in the mediation of nutrient delivery and uptake by the mammary gland for increased milk production. Local changes in biosynthetic capacity within the mammary gland would be a factor in the utilization of substrates resulting in the rate of decline in milk yield with advancing lactation.

• KSBB Journal
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• v.23 no.1
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• pp.44-47
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• 2008

Anthracycline antibiotics doxorubicin (DXR) is clinically important cancer therapeutic agent produced by Streptomyces peucetius. DXR result by further metabolism of rhodomycin D (RHOD) and require a deoxy-sugar component for their biological activity. In this study, production of TDP-L-daunosamine and its attachment to ${\varepsilon}$-rhodomycinone (RHO) to generate RHOD has been achieved by bioconversion in Streptomyces venezuelae that bears eleven genes. S. peucetius seven genes (dnmUTJVZQS) were transformed by plasmid and S. venezuelae two genes desIII, IV and two more S. peucetius drrA, B genes were integrated into chromosomal DNA. To generate the feeding substrate RHO, 6L S. peucetius grown on agar plate was harvested, extracted with organic solvent and then purified using preparative HPLC. Recombinant S. venezuelae grown on agar plate containing RHO was harvested and its n-butanol soluble components were extracted. The glycosylated product of aromatic polyketide RHO using heterologous host S. venezuelae presents the minimal information for TDP-L-daunosamine biosynthesis and its attachment onto aglycone. Moreover, the structure of auxiliary protein, DnrQ, was predicted by fold recognition and homology modeling in this study. This is a general approach to further expand of new glycosides of antitumor anthracycline antibiotics.

• Journal of Life Science
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• v.14 no.1
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• pp.121-125
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• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.