Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells

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• 作者：Rima Matsuyama¹ ; Tomomi Tsutsui¹ ; Kyoung Ho Lee² ; ^§ ; ; Masayoshi Onitsuka¹ ; Takeshi Omasa¹ ; ² ; ^{omasa@bio.eng.osaka-u.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Chinese hamster ovary cells ; Gene amplification ; Bispecific antibody ; Cre-loxP system ; Cell cycle
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：120
• 期：6
• 页码：701-708
• 全文大小：1124 K

文摘

The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems.