• Journal of Microbiology and Biotechnology
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• 제18권7호
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• pp.1342-1351
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• 2008

The feasibility of the use of Flp-mediated cassette exchange in the development of a CHO cell line, which produces erythropoietin (EPO) stably and largely, was investigated. A stable, high enhanced green fluorescence protein (EGFP)-producing clone was screened by extensive flow cytometric analysis. An EPO expression unit was targeted into the premarked locus of the stable parental clone by Flp-mediated cassette exchange and a correctly targeted clone (FC28T7) was obtained. The EPO production of FC28T7 was proven to be stable in long-term culture. Furthermore, the Flp-mediated cassette exchange did not alter the stable parental clone's characteristics concerning transgene expression level and stability. Taken together, the data obtained here indicated that the establishment of CHO cell lines stably producing a desired protein is achievable using Flp-mediated cassette exchange.

• 약학회지
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• 제51권3호
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• pp.214-218
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• 2007

In the previous reports, we developed the CO5 by introducing genes for the first and second urea cycle enzymes, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) into the IBE cell lines producing erythropoietin (EPO). The CO5 have been found out to have 15-20% higher cell growth rate and produce 2-times more EPO than the parental cell line, IBE. To investigate the role of CPS I in CO5 cell line for the cell growth and amount of EPO, we knock-downed CPS I gene expression via siRNA treatment. Expression level of EPO in cell lysate of CO5 was 3-5 fold higher than that of IBE. After siRNA treatment, the cell growth of CO5 was decreased 8-21% and the EPO productivity in the cell Iysate was significantly decreased. However, these changes of the cell growth and EPO productivity were not observed in IBE. These results indicate that CPS I gene expression is important for the increased cell growth and EPO productivity of CO5 cell line.

• Archives of Pharmacal Research
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• 제28권2호
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• pp.220-226
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• 2005

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcar-bamoylase (OTC). When supplements were added to the medium at $33\;^{\circ}C$, the growth of IBE and CO5 cells increased by $27\%\;and;26\%$, respectively and the maximum yield of EPO was increased by $40\%$ in both cell lines. The absolute EPO concentration in the CO5 cells was always $55{\sim}60\%$ higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at $33\;^{\circ}C$ until their growth rates approached those at $37\;^{\circ}C$, the growth rates of both IBE and CO5 cells increased by $54\%$ and their maximum EPO levels increased by up to $73\%\;and\;56\%$, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

• KSBB Journal
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• 제32권2호
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• pp.90-95
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• 2017

Mammalian cell cultures have been used extensively to produce proteins for therapeutic agent because of their ability to perform post-translational modification including glycosylation. To produce recombinant protein, many factors and parameter are considered such as media composition, host cell type, and culture process. In this study, recombinant human erythropoietin (rhEPO) producing cell line was established by using glutamine synthetase system. To reduce serum concentration in media, we compared direct adaptation with step adaptation. Cell growth was faster in step adaptation. In low-level serum media, there were insufficient glucose for cell growth. Thus, we added glucose in low-level serum media from 2 g/L to 4.5 g/L. Titer of rhEPO was higher than other conditions at 4.5 g/L of glucose. Additionally, N-methyl-D-aspartate (NMDA), 13-cis-retinal, and pluronic F-68 (PF-68) were added to enhance productivity in CHO cell cultures. In conclusion, we applied CHO cell producing rhEPO to low-level of serum in media using step-adaptation. Also, we confirmed positive effect of NMDA, 13-cis-retinal, and PF-68.