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.
Highlights
Heterologous gene expression is critical to cell biology, in gene therapy and cell therapy [1], through which exogenous gene encoding proteins or functional RNA molecules could express in the target organism
The results demonstrated that the expression efficiency of the CMV promoter was the highest and the Pol III promoters (U6 and H1) were able to induce the expression of EGFP, which is consistent with the observed results of the EGFP/mCherry dual-color fluorescent reporting system
Four commonly used promoters were evaluated by combining accurate protein and RNA quantification, and we drew the following conclusions: (i) both Pol II and Pol III promoters can induce the protein expression, and the order of activity of the four promoters at the translational level was CMV > SV40 >> U6 > H1, showing the Pol II promoters are ten-times more efficient than Pol III promoters; (ii) but, at the transcriptional level, the efficiency differences between them are not so significant, only several times; (iii) the highest RNA expression was reached 24 h after transfection for all four promoters, while the protein expression reached the highest expression 48 h after transfection
Summary
Heterologous gene expression is critical to cell biology, in gene therapy and cell therapy [1], through which exogenous gene encoding proteins or functional RNA molecules could express in the target organism. RNA polymerases (Pol I, II, and III) are responsible for transcribing the distinct subsets of genes, synthesizing different classes of transcripts [4]. Pol II synthesizes mRNAs and some small nuclear (sn)RNAs, Pol II promoters are widely used for protein expression. Because Pol III promoters have defined transcription start and termination sites, they are mainly used for the expression of exogenous RNA molecular tools like ribozyme in gene knockdown and short hairpin RNA (shRNA) in RNAi applications [6]. Pol III promoter activity has been analyzed indirectly by expressing RNA-silenced target proteins such as GFP and p53 [9]. The currently used methods to evaluate promoter activity only focus on either the transcriptional or translational level. Several important questions have not yet been answered, such as whether the protein can express by Pol III promoters, whether the Pol II promoters can express non-coding RNA, and when the highest RNA and protein expression can be obtained by transient transfection
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