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Identification of a novel temperature sensitive promoter in cho cells

机译：鉴定cho细胞中新型温度敏感启动子

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Background The Chinese hamster ovary (CHO) expression system is the leading production platform for manufacturing biopharmaceuticals for the treatment of numerous human diseases. Efforts to optimize the production process also include the genetic construct encoding the therapeutic gene. Here we report about the successful identification of an endogenous highly active gene promoter obtained from CHO cells which shows conditionally inducible gene expression at reduced temperature. Results Based on CHO microarray expression data abundantly transcribed genes were selected as potential promoter candidates. The S100a6 (calcyclin) and its flanking regions were identified from a genomic CHO-K1 lambda-phage library. Computational analyses showed a predicted TSS, a TATA-box and several TFBSs within the 1.5 kb region upstream the ATG start signal. Various constructs were investigated for promoter activity at 37°C and 33°C in transient luciferase reporter gene assays. Most constructs showed expression levels even higher than the SV40 control and on average a more than two-fold increase at lower temperature. We identified the core promoter sequence (222 bp) comprising two SP1 sites and could show a further increase in activity by duplication of this minimal sequence. Conclusions This novel CHO promoter permits conditionally high-level gene expression. Upon a shift to 33°C, a two to three-fold increase of basal productivity (already higher than SV40 promoter) is achieved. This property is of particular advantage for a process with reduced expression during initial cell growth followed by the production phase at low temperature with a boost in expression. Additionally, production of toxic proteins becomes feasible, since cell metabolism and gene expression do not directly interfere. The CHO S100a6 promoter can be characterized as cold-shock responsive with the potential for improving process performance of mammalian expression systems.
机译：背景技术中国仓鼠卵巢（CHO）表达系统是生产用于治疗多种人类疾病的生物药物的领先生产平台。优化生产过程的努力还包括编码治疗基因的遗传构建体。在这里我们报告成功鉴定出从CHO细胞获得的内源性高活性基因启动子，该启动子在降低的温度下可有条件地诱导基因表达。结果基于CHO微阵列表达数据，选择大量转录的基因作为潜在的启动子候选物。从基因组CHO-K1λ噬菌体文库中鉴定出S100a6（钙环蛋白）及其侧翼区域。计算分析显示，ATG起始信号上游1.5 kb区域内有一个预测的TSS，一个TATA盒和几个TFBS。在瞬时荧光素酶报告基因测定中，研究了各种构建体在37℃和33℃的启动子活性。大多数构建体显示的表达水平甚至比SV40对照更高，并且在较低温度下平均增加超过两倍。我们鉴定了包含两个SP1位点的核心启动子序列（222 bp），并可能通过重复此最小序列显示出活性的进一步提高。结论该新型CHO启动子允许条件性高水平基因表达。转变为33°C时，基本生产力提高了2到3倍（已经高于SV40启动子）。对于在初始细胞生长期间随后表达降低的方法，然后在低温下具有表达增强的生产阶段，该特性特别有利。另外，由于细胞代谢和基因表达不会直接干扰，因此有毒蛋白质的生产变得可行。 CHO S100a6启动子可以被表征为冷休克反应，具有改善哺乳动物表达系统过程性能的潜力。

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