To demonstrate that a single nucleotide mutation in the start codon could result in ON to OFF change in EGFP expression in cells, we cloned both the EGFP-T2A-mCherry-WT (BBa_K2807012) and the EGFP-T2A-mCherry-ACG mutant (BBa_K2807013) into C1 mammalian expression vector for expression in HEK293 cells. We transfected HEK293 cells with both wild type (WT) reporter and ACG mutated (ACG) reporter and thereafter, imaged them via microscopy and harvested them for flow cytometry analysis.
As shown in Figure 1, the cells transfected with EGFP-T2A-mCherry-WT reporter expressed both GFP and mCherry proteins in the same cells, showing that T2A self cleavage peptide is effective. We then further quantified the level of fluorescence by flow cytometry and our results showed that at least 36.6% of the transfected cells are both EGFP+/mCherry+ (Figure 3). There are about 7.9% of the transfected cells with EGFP signal only. This is expected because it is possible for the ribosome to fall off from the mRNA when it encounters the T2A signal peptide and cannot continue with translation. As a result, only EGFP protein is produced but not mCherry.
On the other hand, HEK293 cells transfected with EGFP-T2A-mCherry-ACG mutant showed red fluorescence expression, with very low or undetectable green fluorescent protein levels as visualised via microscopy (Figure 2). Our experiment showed that the mutation of the start codon in EGFP is indeed effective in abolishing the expression of EGFP without affecting the expression of the mCherry protein. Compared to the cells transfected with WT reporter, the percentage of EGFP+/mCherry+ positive cells in cells transfected with the EGFP-T2A-mCherry-ACG mutant is almost undetectable (Figure 3). In addition, the mean fluorescence intensity for mCherry in cells transfected with EGFP-T2A-mCherry-WT and cells transfected with EGFP-T2A-mCherry-ACG mutant are similar, while there is significant reduction in EGFP expression between the two groups of cells.
Team:NUS Singapore-Sci/Dual Reporter
Dual Reporter
Validation of our designed EGFP-T2A-mCherry Reporter system in live cells
1) Mutation of the start codon ATG to ACG in EGFP can significantly block EGFP expression without affecting mCherry expression