用于在 HEK293 细胞中控制表达重组基因的 CMV 启动子工程,Biotechnology Journal

当前位置： X-MOL 学术 › Biotechnol. J. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

用于在 HEK293 细胞中控制表达重组基因的 CMV 启动子工程
Biotechnology Journal ( IF 3.2 ) Pub Date : 2022-04-28 , DOI: 10.1002/biot.202200062
Yusuf B Johari ₁ , Joseph M Scarrott ₁ , Thilo H Pohle ₁ , Ping Liu ₂ , Ayda Mayer ₂ , Adam J Brown _{1,

3} , David C James _{1,

3}

Affiliation

HEK293 细胞中重组基因的表达经常用于生产重组蛋白和病毒载体。这些系统经常使用巨细胞病毒 (CMV) 启动子来驱动重组基因转录。然而，CMV 介导的 HEK293 细胞中转录激活的机制基础尚不清楚，因此没有策略来设计 CMV 以控制重组基因的表达。对人类 CMV 序列中的转录因子调节元件 (TFRE) 和 HEK293 转录组中的转录因子 mRNA 进行的广泛生物信息学分析揭示了 80 种可能的调节相互作用。通过使用含有离散 TFRE 或 CMV 缺失变体的报告构建体的体外功能测试，我们确定了 CMV 序列内的关键 TFRE 组件和 TFRE 簇（顺式调节模块）。我们的数据表明，HEK293 细胞中的 CMV 活性是启动子各种组成 TFRE 的函数，包括 AhR:ARNT、CREB、E4F、Sp1、ZBED1、JunB、c-Rel 和 NF-κB。我们还确定了有效转录所需的转录起始位点附近的关键 Sp1 依赖性上游激活因子以及介导反式抑制的 YY1 和 RBP-Jκ 结合位点。我们的研究首次表明这部小说，

"点击查看英文标题和摘要"

Engineering of the CMV promoter for controlled expression of recombinant genes in HEK293 cells

Expression of recombinant genes in HEK293 cells is frequently utilized for production of recombinant proteins and viral vectors. These systems frequently employ the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. However, the mechanistic basis of CMV-mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis-regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c-Rel, and NF-κB. We also identified critical Sp1-dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP-Jκ binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV-derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild-type counterpart.

更新日期：2022-04-28

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南