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KMID : 0545120020120010121
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 1 p.121 ~ p.129
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Screening of High-Productivity Cell Lines and Investigation of Their Physiology in Chinese Hamster Ovary (CHO) Cell Cultures for Transforming Growth Factor-β1 Production
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Chun, Gie Taek
Lee, Joo Buom/Nam, Sang Uk/Lee, Se Won/Jeong, Yeon Ho/Choi, Eui Yul/Kim, Ik Hwan
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Abstract
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Using recombinant Chinese hamster ovary (CHO) cells, strategies for developing high producers for the recombinant human Transforming Growth Factor-β1 (TGF-β1) protein are proposed and their physiological characteristics in cell cultures were investigated. TGF-β1 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-β1 cDNA in conjunction with a dihydrofolate reductase (DHFR) gene, which was cotransfected into the cells to amplify the transfected TGF-β1 cDNA. As a first-round screening of the transfected cells, a relatively high TGF-β1-producing cell line was selected, and then, it acquired a resistance to increasing concentrations of methotrexate (MTX) up to 60μM, resulting in a significant improvement in its TGF-β1 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-1 cell line without amplification of transfected TGF-β1 cDNA and nontransfectant of TGF-(31 cDNA) in terms of cell growth, TGF-β1 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-β1 producers, even after the transfection and amplification of the transfected gene.
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