Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, P.R. China.
Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, P.R. China.
Biosci Rep. 2021 Sep 30;41(9). doi: 10.1042/BSR20211525.
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.
在此，建立了 Broccoli/mCherry 和 EGFP/mCherry 双荧光报告系统，以在真核细胞中定量测定转录和翻译水平的启动子活性。基于这些系统，通过结合准确的蛋白质和 RNA 定量，在转录和翻译水平上准确评估了四个常用的启动子（Pol II 的 CMV 和 SV40 以及 Pol III 的 U6、H1）。此外，我们通过结合自我切割核酶和人工 poly(A) 尾，验证了 Pol III 启动子可以诱导蛋白质表达，而 Pol II 启动子可以用于表达具有定义长度的 RNA 分子。本文描述的双色荧光报告系统在评估其他基因表达调节剂用于基因治疗方面可能发挥重要作用。
