• Journal of Microbiology and Biotechnology
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• 제17권6호
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• pp.1036-1040
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• 2007

Sodium butyrate (NaBu) has been used to enhance protein expression levels in mammalian cell culture. To determine the clonal variability of recombinant Chinese hamster ovary (rCHO) cells in response to NaBu addition regarding specific antibody productivity $(q_{Ab})$, three rCHO clones were subjected to different concentrations of NaBu. For all three clones, NaBu addition inhibited cell growth and decreased cell viability in a dose-dependent manner. On the other hand, the enhancing effect of NaBu on $q_{Ab}$ varied significantly among the clones. NaBu addition enhanced the antibody production of only one clone. RT-PCR analysis revealed that the changes in $q_{Ab}$ correlated linearly with those of the mRNA transcription level. Thus, it was concluded that the different enhancing effects of NaBu on protein expression in rCHO cell clones resulted from their different mRNA transcription levels.

• Journal of Ginseng Research
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• 제12권2호
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• pp.104-113
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• 1988

여러가지 농도의 cholesterol을 함유한 배지에서 배양한 CHO 세포내로 흡입된 cholesterol 양을 조사한 결과 흡입량이 배지의 cholesterol 농도에 비례하였으므로 cholesterol 흡입은 확산에 의한 것으로 생각된다. $^{125}I$으로 표지된 저밀도 지방단백질($^{125}I$-LDL)을 이용하여 여러가지 농도의 cholesterol을 함유한 배지에서 배양한 CHO 세포와 cholesterol이 없는 정상배지에서 배양한 CHO 세포에서의 LDL 수용체의 합성양상을 조사한 결과 배지에 가해준 cholesterol이 LDL 수용체 합성을 억제함을 확인하였다. Cholesterol의 LDL 수용체 생합성 억제작용에 미치는 ginsenoside의 영향을 조사하기 위해 ginsenoside와 cholesterol을 함께 함유한 배지에서 배양한 CHO 세포(시험군)과 cholesterol만을 포함한 배지에서 배양한 CHO 세포(대조군)에서의 LDL 수용체의 활성양상을 분석한 결과 대조군에 비해 시험군에서의 LDL 수용체 활성이 크게 증가하였다. RNA 및 단백질 합성도 시험군이 대조군보다 증가하였음을 관찰하였다. 그러나 이와 같은 실험조건하에서의 대조군과 시험군의 cholesterol농도를 측정한 결과 시험군의 cholesterol 농도가 대조군보다 훨씬 저하되고 있었다. Ginsenoside의 cholesterol농도 저하작용을 관찰하기 위해 CHO세포에서의 cholesterol의 steroid hormone(estradiol, progesterone)으로의 전환에 미치는 ginsenoside의 영향을 조사한 결과 ginsenoside는 cholesterol의 hormone으로의 전환을 촉진하였음이 확인되었다. 위와 같은 실험결과로 볼 때 ginsenoside는 CHO 세포내부에서의 cholesterol의 LDL 수용체 합성억제를 완화시켜 주는 것이라고 생각된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권4호
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• pp.319-324
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• 2006

Although the sera used in animal cell culture media provide the macromolecules, nutrients, hormones, and growth factors necessary to support cell growth, it could also be an obstacle to the production of recombinant proteins in animal cell culture systems used in many sectors of the biotechnology industry. For this reason, many research groups, including our laboratory, have been trying to develop serum-free media (SFM) or serum-supplemented media (SSM) for special or multi-purpose cell lines. The Chinese hamster ovary (CHO) cell, for example, is frequently used to produce proteins and is especially valuable in the large-scale production of pharmaceutically important proteins, yet information about its genome is lacking. Also, SFMs have only been evaluated by comparing growth patterns for cells grown in SFMs with those grown in SSM or by measuring the titer of the target protein obtained from cells grown in each type of medium. These are not reliable methods of obtaining the type of information needed to determine whether an SFM should be replaced with an SSM. We carried out a cDNA microarray analysis to evaluate MED-3, an SFM developed in our laboratory, as a CHO culture medium When CHO cells were cultured in MED-3 instead of an SSM, several genes associated with cell growth were down-regulated, although this change diminished over time. We found that the insulin-like growth factor (IGF) gene was representative of the proteins that were down-regulated in cells cultured in MED-3. When several key supplements - including insulin, transferrin, ethanolamine, and selenium - were removed from MED-3, the IGF expression was consistently down- regulated and cell growth decreased proportionately. Based on these results, we concluded that when an SFM is used as a culture medium, it is important to supplement it with substances that can help the cells maintain a high level of IGF expression. The data presented in this study, therefore, might provide useful information for the design and development of SFM or SSM, as well as for the design of genome-based studies of CHO cells to determine how they can be used optimally for protein production in pharmaceutical and biomedical research.

• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1116-1122
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• 2013

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERT-S132A-based secretion engineering could be an effective strategy for enhancing recombinant t-PA production in CHO cells.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2011년도 춘계학술대회 논문집
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• pp.1405-1406
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• 2011

• 한국에너지공학회:학술대회논문집
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• 한국에너지공학회 2004년도 춘계 학술발표회 논문집
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• pp.103-108
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• 2004

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.121-129
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• 2002

Using recombinant Chinese hamster ovary (CHO) cells, strategies for developing high producers for the recombinant human Transforming Growth $Factor-{\beta}1$ ($TGF-{\beta}1$) protein are proposed and their physiological characteristics in cell cultures were investigated. $TGF-{\beta}1$ is a pleiotrophic polypeptide involved in various biological activities, including cell growth, differentiation, and deposition of extracellular matrix proteins. The CHO cells included human $TGF-{\beta}1$ cDNA in conjunction with a dihydrofolate reductase (DHFR) gene, which was cotransfected into the cells to amplify the transfected $TGF-{\beta}1$ cDNA. As a first-round screening of the transfected cells, a relatively high $TGF-{\beta}1$-producing cell line was selected, and then, it acquired a resistance to increasing concentrations of methotrexate (MTX) up to $60{\mu}M$,resulting in a significant improvement in its $TGF-{\beta}1$ biosynthetic ability. After applying a monoclonal selection strategy to the MTX-resistant cells, more productive cells were screened, including the APP-3, App-5, and App-8 cell lines. These high producers were compared with two other cell lines (AP-l cell line without amplification of transfected $TGF-{\beta}1$ cDNA and nontransfectant of $TGF-{\beta}1$ cDNA) in terms of cell growth, $TGF-{\beta}1$ productivity, sugar uptake, and byproduct formation, in the presence or absence of MTX in the culture medium. Consequently, both monoclonal selection as well as an investigation of the physiological characteristics were found to be needed for the efficient screening of higher $TGF-{\beta}1$ producers, even after the transfection and amplification of the transfected gene.

• 한국환경성돌연변이발암원학회지
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• 제16권2호
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• pp.117-123
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• 1996

This study was carried out to examine the mechanism of Arsenic cytotoxicity through several in vitro test systems. Dose-dependent decrease of cell survival by Arsenic was observed by colony forming assay. Arsenic was weak mutagenic in inducing HGPRT point mutation in CHO cells. The frequency of chromosomal aberrations increased in a dose-dependent manner and the most frequent type of chromosomal aberrations induced by Arsenic were chromatid type deletions. U!trafiltrates of culture media from CHO cells treated with Arsenic induced sister chromatid exchanges(SCE) in CHO cells and Arsenic was able to induce lipid peroxidation in CHO cells. The results suggested that the ultrafiltrates of media from CHO cells treated with Arsenic contain clastogenic factor(CF) and Iipid peroxidation might be involved in the formation of CF.

• 대한의생명과학회지
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• 제14권4호
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• pp.203-210
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• 2008

One of cell surface receptor proteins, human folate receptor (hFR) involves in the uptake of folates through cell membrane into cytoplasm, and is anchored to the plasma membrane by a fatty acid linkage, which has been identified in some cells as a glycosylphosphatidylinositol (GPI)-tailed protein with a molecular mass of about 40 kDa. The hFR is released by phosphatidylinositol phospholipase C (PI-PLC) because it contains fatty acids and inositol on the GPI tail. Caveolin decorates the cytoplasmic surface of caveolae and has been proposed to have a structural role in maintaining caveolae. It is unknown whether caveolin is involved in targeting, and is necessary for the function of GPI-tailed proteins. To compare the ability of folic acid binding, internalization and expression of hFR, and the effect of caveolin at the both apical and basolateral side of cell surfaces in Chinese hamster ovary (CHO) clone cells overexpressed the hFR and/or caveolin. Our present results suggest a possibility that the overexpression of caveolin does not be involved in expression of hFR, but plays a role as a factor in PI-PLC releasing kinetics, and for a regulation of formation, processing and function of hFR in CHO clone cells overexpressed cavcolin.

• Journal of Microbiology and Biotechnology
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• 제16권5호
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• pp.695-703
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• 2006

The dissolved oxygen level of any cell culture environment has a critical effect on cellular metabolism. Specifically, hypoxia condition decreases cell viability and recombinant protein productivity. In this work, to develop CHO cells producing recombinant protein with high productivity, mammalian expression vectors containing a human tissue-type plasminogen activator (t-PA) gene with hypoxia response element (HRE) were constructed and stably transfected into CHO cells. CHO/2HRE-t-PA cells produced 2-folds higher recombinant t-PA production than CHO/t-PA cells in a $Ba^{2+}-alginate$ immobilized culture, and 16.8-folds in a repeated batch culture. In a non-aerated batch culture of suspension-adapted cells, t-PA productivity of CHO/2HRE/t-PA cells was 4.2-folds higher than that of CHO/t-PA cells. Our results indicate that HRE is a useful tool for the enhancement of protein productivity in mammalian cell cultures.