• KSBB Journal
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• v.14 no.3
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• pp.377-379
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• 1999

In order to improve the transfection efficiency of CHO/dhfr- cells using cationic lipid, optimal concentrations of the cationic lipid($LipofectAmine^{TM}$) and DNA(pEGFP-C1) need to be determined. The use of green fluorescent protein(GFP) gene as a reporter gene facilitated the quantification of transfection efficiency. The green fluorescence intensity of each cell transfected at various lipid-DNA concentrations was measured using fluorescence-activated cell sorter(FACS) analysis. A combination of $2.0{\mu}L$ cationic lipid and 0.4{$\mu}g$ DNA in a well resulted in the highest trasfection efficiency. Taken together, the method using FACS analysis of GFP is simple and fast, facilitating the optimization of transfection.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.57-63
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• 1996

The biliary excretion kinetics of the active folate derivatives were examined after an intravenous injection of methotrexate at doses of 0.3 and 10mg/kg to clarify the mechanism of the acute decrease in the plasma folate by the dihydrofolate reductase inhibitors. Even at a higher dose than used in the clinical therapy, methotrexate did not cause any acute depletion of folate denvatives in the excreted bile. Therefore, the decrease in the plasma folate appeared not to be related with the biliary excretion process of folates. A factor responsible for the plasma folate depletion by DHFR inhibitors may be due to the malabsorption of folate derivatives excreted into the small intestine.

• Parasites, Hosts and Diseases
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• v.32 no.1
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• pp.19-26
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• 1994

An in uipo culturing to examine the cyst stage of ToxopLQsma gondii (ME49 strain) was Investigated using murine peritoneal macrophages, and we also examined the effect of CAMP or DHFR Inhibitors on the growth of bradyzoltes. For experiments ICR mice were Injected 1.p. with 1,500 brain cysts. At 1, 3, 5 and 7 days, peritoneal exudates were Isolated and then adherent peritoneal macrophages were cultured for 1,3,5 and 10 days. Growth pattern of bradyzoltes was measured by (3H)-uracil uptake assay and morphological pattern of pseudocysts formed in macrophages was observed Uth Glemsa stain. Mostly bradyzoites were observed In the macrophages extracted at 3 and S days post Infection. After 3 days in vitro, a number of pseudocysts were formed in the macrophages and the size of pseudocysts was increased during further 5 and 10 days in vitro culture. CAMP stimulated the growth of bradyzoltes when in uiuo 3 and 5 days and then in vitro 5 and 10 days conditions were applied. In case of.DHFR Inhibitors, pynmethamlne produced a linearly decremental effect with a cont.-dependent mode but methotrexate was not effective against Intracellular bradyzoltes or pseudocysts In this system. It was suggested that cyst-forming strain of T gondii (ME49 strain) could be maintained and cultivated in uitro by use of murine peritoneal macrophages. In uivo 3 and 5 days and then in uiko 5 and 10 days conditions appeared to be suitable for culturing of bradyzoltes. CAMP and pyrimethamine had an effect of stimulation and inhibition on the growth of bradyzolte, respectively.

• Korean Journal of Veterinary Service
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• v.32 no.3
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• pp.227-239
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• 2009

At the present study, it was aimed to detect virulence genes and antimicrobial resistance genes among 102 strains of 12 Salmonella serotypes isolated from pigs and cattle. In polymerase chain reaction (PCR), invA was detected from all strains of Salmonella spp., spvC was detected from Salmonella enterica serotype Enteritidis (S. Enteritidis) (100%), S. Bradenburg (75%), and S. Typhimurium (20.4%). Drug resistance related genes of 12 types were detected from all strains. TEM ($bla_{TEM}$) gene was detected from 51 (92.7%) of 55 $\beta$-lactams (54 ampicillin or 1 amoxicillin) resistance strains. 55 (100%) of 55 chloramphenicol resistance strains, 3 (100%) of 3 gentamicin resistance strains and 5 (100%) of 5 kanamycin resistance strains did contain cml, aadB, and aphA1-Iab, respectively. strB (89.9%), strA (88.4%), aadA2 (84.1%) and aadA1 (72.5%) were detected from 69 streptomycin resistance strains. sulII and dhfrXII were detected from 49 (100%) of 49 sulfamethoxazole/trimethoprim resistance strains, but sulI was not detected. tetA (97.9%) and tetB (21.6%) were detected from 97 tetracycline resistance strains. int gene was detected from 58 (56.9%) of 102 strains. 54 S. Typhimurium of 102 Salmonella spp. were attempted to detect drug resistance genes. TEM was detected from 44 (95.7%) of 46 $\beta$-lactams (45 ampicillin or 1 amoxicillin) resistance strains. cmlA was detected from 51 (100%) of 51 chloramphenicol resistance strains. aadA2 (100%), strA (100%), strB (100%), and aadA1 (79.6%) were detected from 54 streptomycin resistance strains. sulII (100%) and dhfrXII (100%) were detected from 49 sulfamethoxazole/trimethoprim resistance strains. tetA was detected from 54 (100%) of 54 tetracycline resistance strains. int gene was detected from 54 (100%) of 54 strains. The major drug resistance pattern and resistance gene profile were ampicillin, chloramphenicol, streptomycin, sulfamethoxazole/trimethoprim and tetracycline (ACSSuT) and TEM, cmlA, aadA1, aadA2, strA, strB, sulII, dhfrXII, tetA and int, respectively.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.313-316
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• 2000

When 3 recombinant Chinese hamster ovary (rCHO) cell lines, CHO/dhfr-B-22-4, $CS13-1.00^{\ast}$ and $CSl3-0.02^{\ast}$, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality. Glycine betaine was found to have a strong osmoprotective effect on all 3 rCHO cell lines. Inclusion of 15 mM glycine betaine in hyperosmolar medim enabled rCHO cell lines to grow at 557-573 mOsm/kg where they could not grow in the absence of glycine betaine. However, effect of glycine betaine inclusion in hyperomolar medium on foreign protein production differed among rCHO cell lines. CHO/dhfr-B22-4 cells retained enhanced specific human thrombopoietin (hTPO) productivity in the presence of glycine betaine, and thereby, the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg). On the other hand, enhanced specific antibody productivity of $CSl3-1.00^{\ast}$ and $CSl3-0.02^{\ast}$ at elevated osmolality decreased significantly in the presence of glycine betaine at a cost of the recovery of cell growth. As a result, the maximum antibody titer at 557 mOsm/kg was similar to that obtained in the control culture with physiological osmolality. Taken together, efficacy of the simultanous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO cell lines.

• Parasites, Hosts and Diseases
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• v.28 no.2
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• pp.79-84
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• 1990

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro·dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses do novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reject the specific proliferation of Texoplasma. It can be exploited to characterize the elects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis. Key words: Toxoplasma gondii, uracil salvage, dihydrofolate reductase, thymidylate synthase TMP biosynthesis.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.257-260
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• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• Journal of Plant Biotechnology
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• v.2 no.3
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• pp.143-149
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• 2000

An Efficient in vitro regeneration system and an optimized Agrobacterium mediated transformation protocol are described, based on the use of young seedling cotyledons of Capsicum annuum L. Optimal regeneration efficiency can be obtained by cultivating cotyledon explants on media containing 4 mg/L benzyladenine and 0.1 mg/L indolacetic acid. The effect of antibiotics used to eliminate Agrobacteria, as well as the toxic level of some generally used selection agents (kanamycin, geneticin, hygromycin, phosphinotricin and methotrexate) in regenerating pepper tissues were determined. To enable the comparison of different selection markers in identical vector background, a set of binary vectors containing the marker genes for NPTII, HPT, DHFR and BAR respectively, as well as the CaMV 35S promoter/enhancer-GUS chimaeric gene was constructed and introduced into four different Agrobacterium host strains.

• KSBB Journal
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• v.21 no.2
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• pp.110-117
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• 2006

During routine maintenance, animal cell lines are commonly cryopreserved in growth medium containing serum with 10% DMSO. But, in case of bioprocess under the serum-free conditions, including cultivation of cell lines and producing of pharmaceuticals, the cryopreservation should be executed without serum to prevent a cross-contamination. This experiments were performed to investigate the effects of the serum-free cryopreservation on the CHO cells. To improve the survival rates of the cryopreserved CHO cells in serum-free condition, first, the effects of permeable and non-permeable additives for substitute serum on cell viability were investigated. The combination of 10% DMSO and 0.03 M raffinose in MEM-${\alpha}$ without serum indicated 76% of cell viability. However, it did not reach the survival rates(more than 95%) of the conventional cryopreservation. In the second, to evaluate the cryopreservative ability of the serum-free medium(SFM) we compared viability of the CHO cells cryopreserved in the SFMs(Sigma C5467, C4726, and C1707, JBI SF486 and PF486), the cryoprotectant(Genenmed CAN-1000) and the MEM-${\alpha}$ with serum. All solution contained 10% DMSO. As a result of the comparison, cryopreserved cells in the SFMs showed over 95% of viability and appeared predominant viability better than cryoprotectant CAN-1000. Finally, we assessed the stability of the CHO cells in the long-term cryopreservation(LTC) using SFM. Every three months, the cryopreserved CHO cells were thawed to estimate the cell viability and the recovery rates. Then, real-time RT-PCR analyzed the inserted CHO DHFR gene. All results for the LTC appeared the same stability as the serum containing cryopreservation. In the conclusion, it could be seen that the LTC in the SFM can substitute for serum using methods in the bioprocess proceeded by CHO cells for more than 18 months.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.459-462
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• 2001

Angiopoietin-2 (Ang2) is a naturally occurring antagonist for angiopoietin-l (Angl) and its Tie2 receptor during vasculogenesis, Although angiopoietins have been expressed in several mammalian cell lines, their expression levels are low. Recombinant Chinese hamster ovary (CHO) cell lines expressing a high level of human Ang2 or its analog, human $Ang2_{443}$, with an amino-terminal FLAG-tag were constructed by transfecting the expression vectors into dhfr-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate level. Secreted Ang2 or human $Ang2_{443}$ were purified from the cultured medium using an anti-FLAG- agarose affinity chromatography, The purified Ang2 and $Ang2_{443}$ migrated on SOS-PAGE as a broad band, characteristic of glycosylated protein. Their biological activity in vitro was demonstrated in a serum deprivation-induced apoptosis assay. Ang2 at high concentration, like AngI, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor.