Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7
Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7
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DOI：
10.1016/j.jbiotec.2010.12.005
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发表时间：
2011-01-20
影响因子：
4.1
通讯作者：
Clynes, M.
中科院分区：
文献类型：
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作者：
Barron, N.;Kumar, N.;Clynes, M.
在现代生物过程中，中国仓鼠卵巢（CHO）细胞高效生产重组蛋白通常通过使用增殖控制策略来增强。最常见的方法是将培养温度从37摄氏度改变到28-33摄氏度，尽管实现相同效果的遗传方法也很有趣。在这项工作中，我们使用基于qRT-PCR的表达谱，使用TLDA（TM）卡来鉴定在从37摄氏度到31摄氏度的温度转换（TS）后24小时显示差异表达的miRNA。发现六种miRNAs显著上调（mir-219、mir-518 d、mir-126、mir-30 e、mir-489和mir-345），四种下调（mir-7、mir-320、mir-101和mir-199）。此外，在有和没有TS的情况下，在6天分批培养中miR-7表达的qRT-PCR分析表明，在两种培养物中随着时间的推移表达降低，但在转移到较低培养温度的细胞中表达降低的程度显著更大。出乎意料的是，当在CHO-K1细胞中通过用miR-7模拟物转染而瞬时增加miR-7水平时，在37 ℃下的细胞增殖在96小时培养期内被有效阻断。另一方面，使用拮抗剂瞬时抑制内源性miR-7水平对细胞生长没有影响。miR-7的外源性过表达也导致在37 ℃下增加的标准化（每个细胞）产量。尽管产率低于在降低的温度下生长的细胞。这是第一份证明特定miRNA失调对分批培养中CHO细胞行为的功能影响的报告，并提供了这些分子在CHO生产克隆中的工程靶标方面可能具有的潜力的一些证据。最后，我们报告仓鼠特异性cgr-miR-7的克隆和测序。(C)2010 Elsevier B. V.保留所有权利。
The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bio-processes is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 degrees C to 28-33 degrees C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA (TM) cards to identify miRNAs displaying differential expression 24h after temperature-shift (TS) from 37 degrees C to 31 degrees C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 degrees C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 degrees C. though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7. (C) 2010 Elsevier B.V. All rights reserved.