High Levels of Histone H3 Acetylation at the CMV Promoter Are Predictive of Stable Expression in Chinese Hamster Ovary Cells
High Levels of Histone H3 Acetylation at the CMV Promoter Are Predictive of Stable Expression in Chinese Hamster Ovary Cells
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DOI：
10.1002/btpr.2271
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发表时间：
2016-05-01
影响因子：
2.9
通讯作者：
Goepfert, Ulrich
中科院分区：
文献类型：
--
作者：
Moritz, Benjamin;Woltering, Laura;Goepfert, Ulrich
中国仓鼠卵巢细胞（CHO）广泛用于生产糖基化治疗性蛋白质，如抗体。在扩增和维持期间，CHO细胞系易于产生不稳定性，这可能由启动子沉默、转基因拷贝丢失或转录后效应引起。重组基因的沉默可能伴随着DNA甲基化和组蛋白修饰。先前，我们证明了人巨细胞病毒主要立即早期启动子/增强子（hCMV-MIE）的胞嘧啶甲基化可用于预测产生抗体的CHO细胞系的不稳定性。然而，高的错误预测率激发了对稳定启动子活性的进一步标记的搜索。为此，我们将hCMV-MIE附近的各种组蛋白修饰与随时间的生产稳定性相关联。我们的研究结果表明，组蛋白H3（H3 ac）的乙酰化是一个更有效的指标生产稳定性比DNA甲基化。选择具有最高CMV启动子H3 ac水平的细胞系富集稳定表达子并提高生产细胞系的平均稳定性。对于组蛋白H3乙酰化测量，我们采用了基于染色质免疫沉淀（ChIP）的方法。在目前的形式下，该方法适合在几天内评估10-20个细胞系。我们建议一旦候选细胞系的数量根据生产率和产品质量缩小，就确定H3乙酰化。(C)2016年美国化学工程师学会生物技术。
Chinese hamster ovary cells (CHO) are widely used in the production of glycosylated therapeutic proteins such as antibodies. During expansion and maintenance, CHO cell lines are prone to production instability, which may be caused by promoter silencing, loss of transgene copies, or post-transcriptional effects. Silencing of recombinant genes may be accompanied by DNA methylation and histone modification. Previously, we demonstrated that cytosine methylation of the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) can be used to predict instability of antibody-producing CHO cell lines. However, the high rate of false prediction motivates the search for further markers of stable promoter activity. To this end, we correlated a variety of histone modifications in the vicinity of hCMV-MIE with production stability over time. Our results suggest that acetylation of histone H3 (H3ac) is a more effective indicator of production stability than DNA methylation. Selecting cell lines with highest CMV promoter H3ac levels enriches stable expressors and improves the average stability of production cell lines. For histone H3 acetylation measurement we employed a method based on chromatin immunoprecipitation (ChIP). In its current form, the method is suitable to evaluate 10-20 cell lines within a few days. We propose to determine H3 acetylation once the number of candidate cell lines has been narrowed based on productivity and product quality. (C) 2016 American Institute of Chemical Engineers Biotechnol.