title: Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells.
abstract: 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.
authors.affiliation: Institute of Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
authors.forename: Rima
authors.initials: R
authors.lastname: Matsuyama
authors.affiliation: Institute of Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Tsutsui
authors.affiliation: Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kyoung Ho
authors.initials: KH
authors.lastname: Lee
authors.affiliation: Institute of Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Onitsuka
authors.affiliation: Institute of Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan; Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: omasa@bio.eng.osaka-u.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000378:metabolism; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D016466:CHO Cells; D059447:Cell Cycle Checkpoints / Q000235:genetics*; D060846:Cell Engineering / Q000379:methods*; D002469:Cell Separation; D002999:Clone Cells / Q000378:metabolism; D003412:Cricetulus; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics; D020687:cdc25 Phosphatases / Q000235:genetics / Q000378:metabolism
chemical_list: D018033:Antibodies, Bispecific; D013762:Tetrahydrofolate Dehydrogenase; C045073:Cre recombinase; D019426:Integrases; D020687:cdc25 Phosphatases; D008727:Methotrexate
keywords: Bispecific antibody; Cell cycle; Chinese hamster ovary cells; Cre-loxP system; Gene amplification