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Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.

ABSTRACT: 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.

SUBMITTER: Peng Y

PROVIDER: S-EPMC8433482 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Combining protein and RNA quantification to evaluate promoter activity by using dual-color fluorescent reporting systems.

Peng Yan Y Huang Xin X Huang Tianfang T Du Feng F Cui Xin X Tang Zhuo Z

Bioscience reports 20210901 9

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 prote ...[more]

PMID: 34467968

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