title: Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1.
abstract: The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400 nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation.
authors.affiliation: Graduate School of Advanced Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan.
authors.forename: Rima
authors.initials: R
authors.lastname: Matsuyama
authors.affiliation: Graduate School of Bioscience and Bioindustry, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Yamano
authors.affiliation: Graduate School of Advanced Technology and Science, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, Japan.
authors.forename: Namiko
authors.initials: N
authors.lastname: Kawamura
authors.affiliation: Graduate School of Bioscience and Bioindustry, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8513, 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; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D019313:BRCA1 Protein / Q000172:deficiency* / Q000235:genetics*; D016466:CHO Cells; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000235:genetics*; D005784:Gene Amplification / Q000187:drug effects / Q000235:genetics; D020868:Gene Silencing; D019398:Genes, BRCA1; D006657:Histones / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D008727:Methotrexate / Q000494:pharmacology; D008745:Methylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D019313:BRCA1 Protein; D006657:Histones; D034741:RNA, Small Interfering; D008727:Methotrexate
keywords: Breast cancer 1; Cell cycle checkpoint; Chinese hamster ovary cells; Chromatin remodeling; Gene amplification; Histone modification; Monoclonal antibody; Recombinant protein production; siRNA