• Title/Summary/Keyword: CHO/dhfr-

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Transcriptional Activation and Repression of Cell Cycle Regulatory Molecules by Trichostatin A (Trichostatin A 처리에 의하 세포주기 조절인자들의 전사활성화 및 불활성화)

  • Baek Jong-Soo;Lee Hee-Kyung;Cho Young-Su;Kim Sung-Young;Park Kwan-Kyu;Chang Young-Chae
    • Journal of Life Science
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    • v.15 no.6 s.73
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    • pp.994-1004
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    • 2005
  • The dihydrofolate reductase (dhfr) promoter contains cis-acting element for the transcription factors Spl and E2F. Transcription of dhfr gene shows maximal activity during the Gl/S phase of cell cycle. The member of the Spl transcriptional factor family can act as both negative and positive regulators of gene expression. There was a report that Spl-Rb and E2F4-pl30 complexes cooperate to establish stable repression of dhfr gene expression in CHOC400 cells. Here, we examined the role of HDAC in dhfr, cyclin E, and cyclin A gene regulation using the histone deacetylation inhibitor, trichostatin A (TSA) in U2OS and C33A cells, a Rb-positive human osteosarcoma cell line, and a Rb-negative cervical carcinoma cell line, respectively. When the dhfr promoter constructs were applied in U2OS cells, TSA markedly stimulated over 14-fold of dhfr promoter activity through dhfr-Spl sites by the deletion of an E2F element. In contrast, the deletion of dhfr-Spl binding sites completely abolished promoter stimulation by TSA. The dhfr promoter activity including dhfr-Spl sites increased only 2-fold in C33A cells. Promoter activity containing only dhfr-E2F site did not have much effect by the treatment of TSA in both U2OS and C33A cells. On the other hand, treatment with TSA induced significantly mRNA expression of dhfr and cyclin E, whereas levels of cyclin A decreased in U2OS cells, but had no effect in C33A cells. These results indicate that TSA have contradictory effect, activation of dhfr and cyclin E genes on Gl phase, and down-regulation of cyclin A on G2 phase through transcriptional regulation in U2OS cells.

Optimization of Gene Transfection Using Fluorescence-Activated Cell Sorter(FACS) Analysis of Green Fluorescent Protein(GFP) (Green Fluorescent Protein(GFP)의 Fluorescence-Activated Cell Sorter(FACS) 분석을 통한 유전자 이입의 최적화)

  • 김태경;박민태;이균민
    • 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.

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Osmoprotective Effect of Glycine Betaine on Foreign Protein Production in Hyperosmotic Recombinant Chinese Hamster Ovary Cell Cultures Differs among Cell Lines

  • Ryu, Jun-Su;Kim, Tae-Gyeong;Jeong, Ju-Yeong;Lee, Gyun-Min
    • 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.

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Production of tissue-type plasminogen activator from immobilized CHO cells introduced hypoxia response element

  • Bae, Geun-Won;Kim, Hong-Jin;Kim, Gi-Tae;Kim, Ik-Yeong
    • 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.

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Cryopreservation of CHO Cell using Serum-Free Media (무혈청 배지를 이용한 CHO 세포의 동결보존)

  • Kim, Yoo-Kang;Park, Hong-Woo;Choe, Tae-Boo
    • 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.

Expression and Purification of Recombinant Human Angiopoictin-2 and Its Analog in Chinese Hamster Ovary Cells

  • Hwang, Su-Jeong;Kim, In-Jun;Go, Gyu-Yeong;Lee, Gyun-Min
    • 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.

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Changes in Mitogen-activated Protein Kinase Activities During Acidification-induced Apoptosis in CHO Cells

  • Kim, Jin-Young;Jeong, Dae-Won;Roh, Sang-Ho;Min, Byung-Moo
    • International Journal of Oral Biology
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    • v.30 no.3
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    • pp.85-90
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    • 2005
  • Homeostatic pH is very important for various cellular processes, including metabolism, survival, and death. An imbalanced-pH might induce cellular acidosis, which is involved in many abnormal events such as apoptosis and malignancy. One of several factors contributing to the onset of metabolic acidosis is the production of lactate and protons by lactate dehydrogenase (LDH) in anaerobic glycolysis. LDH is an important enzyme that catalyzes the reversible conversion of pyruvate to lactate. This study sought to examine whether decreases in extracellular pH induce apoptosis of CHO cells, and to elucidate the role of mitogen-activated protein kinases (MAPKs) in acidification-induced apoptosis. To test apoptotic signaling by acidification we used CHO dhfr cells that were sensitive to acidification, and CHO/anti-LDH cells that are resistant to acidification-induced apoptosis and have reduced LDH activity by stable LDH antisense mRNA expression. In the present study, cellular lactic acid-induced acidification and the role of MAPKs signaling in acidification-induced apoptosis were investigated. Acidification, which is caused by $HCO{_3}^-$-free conditions, induced apoptosis and MAPKs (ERK, JNK, and p38) activation. However, MAPKs were slightly activated in acidic conditions in the CHO/anti-LDH cells, indicating that lactic acid-induced acidification induces activation of MAPKs. Treatment with a p38 inhibitor, PD169316, increased acidification-induced apoptosis but apoptosis was not affected by inhibitors for ERK (U0126) or JNK (SP600125). Thus, these data support the hypothesis that activation of the p38 MAPK during acidification-induced apoptosis contributes to cell survival.

Development of recombinant human chorionic gonadotropin (hCG) using high-density culture technique of suspension-adapted chinese hamster ovary (CHO) cells

  • Na, Kyu-Heum;Kim, Seung-Chul;Seo, Kwang-Seok;Lee, Sung-Hee;Kang, Soo-Hyung
    • 한국생물공학회:학술대회논문집
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    • 2005.04a
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    • pp.37-37
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    • 2005
  • Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of non-covalently linked two subunits, the ${\alpha}$ and ${\beta}$ subunit. It has been used as a infertility drug for ovulation to mimic luteinizing hormone $(LH).^{1)}$ A stable cell line was established by transfection of Rc/CMV-i-dhfr-hCG, expression vector containing hCG ${\alpha}-$ and ${\beta}-genes$, into dihydrofolate reductase-deficient CHO cells and subesquent methotrexate-mediated gene amplification. Anchorage-dependent CHO cells were adapted into a serum-free and/or animal component-free suspension medium through gradual serum weaning for the hCG production. The established cell line showed typical morphological characteristics and growth profile of CHO cells, and could produce FSH with passage-to-passage consistency. The high density perfusion culture of the CHO cells was carried out in Celligen Plus bioreactor equipped with a spin-filter as a internal cell retention device. The cell density reached up to $>1x10^{7}$ cells/ml in less than 7 days and a perfusion-control strategy based on cellular consumption rates of glucose was $established.^{2)}$ Biologically active recombinant hCG was purified by a series of chromatographic steps including anion exchange chromatography and hydrophobic interaction chromatography to homogeneity. The highly purified recombinant hCG was characterized for physicochemical, immunological and biological properties.

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High-Level Expression and Characterization of Single Chain Urokinase-type Plasminogen Activator(scu-PA) Produced in Recombinant Chinese Hamster Ovary(CHO) Cells

  • Kim, Jung-Seob;Min, Mi-Kyung;Jo, Eui-Cheol
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.2
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    • pp.117-127
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    • 2001
  • The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.

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Development of Recombinant Chinese Hamster Ovary Cell Lines Producing Human Thrombopoietin or Its Analog

  • Chung, Joo-Young;Ahn, Hae-Kyung;Lim, Seung-Wook;Sung, Yun-Hee;Koh, Yeo-Wook;Park, Seung-Kook;Lee, Gyun-Min
    • Journal of Microbiology and Biotechnology
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    • v.13 no.5
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    • pp.759-766
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    • 2003
  • Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level of human thrombopoietin (hTPO) or its analog, TPO33r, were obtained by transfecting expression vectors into dihydrofolate reductase-deficient (dhfr) CHO cells and subsequent gene amplification in media containing stepwise increments in methotrexate (MTX) level such as 20, 80, and 320 nM. The parental clones with a hTPO expression level $>0.40\;{\mu}g/ml$ (27 out of 1,200 clones) and the parental clones with a TPO33r expression level $>0.20\;{\mu}g/ml$ (36 out of 400 clones) were subjected to 20 nM MTX. The clones that displayed an increased expression level at 20 nM MTX were subjected to stepwise increasing levels of MTX such as 80 and 320 nM. When subjected to 320 nM MTX, most clones did not display an increased expression level, since the detrimental effect of gene amplification on growth reduction outweighed its beneficial effect of specific TPO productivity ($q_{TPO}$) enhancement at 320 nM MTX. Accordingly, the highest producer subclones ($1-434-80^{*}$ for hTPO and $2-3-80^{*}$ for TPO33r), whose $q_{TPO}$ was 2- to 3-fold higher than that of their parental clones selected at 80 nM MTX, were isolated by limiting dilution method and were established as rCHO cel1 lines. The $q_{TPO}$ of $1-434-80^{*}\;and\;2-3-80^{*}\;was\;5.89{\pm}074\;and\;1.02{\pm}0.23\;{\mu}g/10^6$ cells/day, respectively. Southern and Northern blot analyses showed that the enhanced $q_{TPO}$ of established rCHO cell lines resulted mainly from the increased TPO gene copy number and subsequent increased TPO mRNA level. The hTPO and TPO33r produced from the established rCHO cell lines were biologically active in vivo, as demonstrated by their ability to elevate platelet counts in treated mice.