Journal
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
Volume 123, Issue 3, Pages 382-389Publisher
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2016.09.006
Keywords
Chinese hamster ovary cells; Breast cancer 1; siRNA; Cell cycle checkpoint; Gene amplification; Histone modification; Chromatin remodeling; Recombinant protein production; Monoclonal antibody
Funding
- Ministry of Economy, Trade and Industry of Japan (METI)
- Japan Agency for Medical Research and Development (AMED)
- JSPS KAKENHI [JP26630433, JP26249125]
- Grants-in-Aid for Scientific Research [17H06157, 26249125] Funding Source: KAKEN
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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. BRCAl-downregulated cell clones CHO B1Sa and BISb 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 BISb clones. Moreover, B1Sa and BlSb clones at 20 passages had approximately 3.5-and 53-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. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.
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