中国仓鼠卵巢细胞
DNA甲基化
分子生物学
甲基化
生物
细胞培养
单克隆抗体
基因表达
DNA
基因
抗体
遗传学
作者
Yuansheng Yang,Steven C L Ho,Janet Chusainow,Miranda G.S. Yap
标识
DOI:10.1016/j.jbiotec.2010.04.004
摘要
Production instability currently limits the use of mammalian cells for industrial production of therapeutic proteins. We have previously reported that the loss of productivity in recombinant monoclonal antibody producing Chinese Hamster Ovary (CHO-mAb) cell lines is mainly due to a decrease in heavy chain (HC) and light chain (LC) transcripts. Molecular analysis indicates that the decreased mRNA levels are not due to a loss in gene copies and change of integration sites. In this work, we further demonstrate that impaired trans-acting factors and spontaneous mutations to the DNA are not responsible for the reduced HC and LC transcription. Examination of two CpG sites by methyl-assisted quantitative real-time PCR assay revealed an increase in methylation of the human cytomegalovirus major immediate-early enhancer and promoter (hCMV-MIE) controlling the expression of LC and HC in cells which exhibited loss in productivity. Treatment of these cells with a DNA methylation inhibitor, 5-Aza-2′-deoxycytidine, partially restored the lost specific mAb productivity. The increase in productivity correlated to the increase in mRNA levels of HC and LC and the demethylation of hCMV-MIE promoter. This finding, which indicates that DNA methylation contributes to production instability, will be beneficial for generation of high-producing cell lines with stable productivity.
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