Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.
Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.
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使用双色荧光报告系统结合蛋白质和 RNA 定量来评估启动子活性
DOI：
10.1042/bsr20211525
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发表时间：
2021-09-30
影响因子：
4
通讯作者：
Tang Z
中科院分区：
文献类型：
--
作者：
Peng Y;Huang X;Huang T;Du F;Cui X;Tang Z
本文建立了西兰花/mCherry和EGFP/mCherry双色荧光报告系统，用于定量真核细胞中转录和翻译水平的启动子活性。在此基础上，通过精确的蛋白质和RNA定量结合，在转录和翻译水平上准确评估了4个常用启动子（Pol II和U6的CMV和SV40， Pol III的H1）。此外，我们验证了Pol III启动子可以诱导蛋白质表达，并且Pol II启动子可以通过结合自切割核酶和人工聚(a)尾来表达具有一定长度的RNA分子。本文描述的双色荧光报告系统可以在评估基因治疗的其他基因表达调节因子方面发挥重要作用。
Herein, Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation levels in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy.