title: Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering.
abstract: The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.
authors.affiliation: Department of Biotechnology, 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.forename: Masayoshi
authors.initials: M
authors.lastname: Onitsuka
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D003412:Cricetulus; D015870:Gene Expression; D020868:Gene Silencing; D017930:Genes, Reporter; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D019076:Transgenes
chemical_list: D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins