We have tested the two eGFP reporters that we designed, and have validated their efficiency. In general, the ATG/ACG reporters have shown high efficiency in terms of the difference in the level of expression between the wild type and mutated version, while the β-globin reporters showed significantly less difference. The exact efficiency of these reporters are shown below:
ATG/ACG eGFP Reporters
We transfected HEK293T cells grown in 6-well plates, fixed the cells to slides, and obtained images via microscopy (details). For each chosen field, two images were obtained in order to perform calculation: one image for green fluorescence (Figure 1a), and one for red fluorescence (Figure 1b). The red fluorescence produced by the constitutively expressed RFP gene (mCherry) serves as an indicator for successful transfection, as well as a baseline for measuring the amount of green fluorescence. The fluorescence was quantified using ImageJ, which measures the amount and intensity of the fluorescence by grey scale.
Figure 1a & 1b: eGFP positive control, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
The positive control transfected cells showed that the transfection method functioned properly for both the eGFP and the mCherry RFP plasmids. Additionally, when compared visually, the RFP fluoresced significantly less than the eGFP, but was expressed at about the same level as the eGFP. This was noticed because the cells shown to have slightly more RFP were also the cells that fluoresced green to a higher extent when filtered for eGFP.
HEK293T cells from the same cell line were also transfected with a combination of our custom ATG GFP plasmid and the same mCherry RFP plasmid. These cells were placed under the same microscope and used the same exposure times to take pictures of the fluorescing cells. Two examples of the ATG eGFP plus mCherry reporters were recorded in Figure 2a and Figure 2b.
In Figure 2a, the ATG reporter seems to produce less green fluorescence than the positive control. The amount of red fluorescence is similar in the positive control and the ATG reporter. It was also noted again that the same cells that showed high levels of green fluorescence also showed high levels of red fluorescence. To ensure that the high levels of RFP did not influence measurements of eGFP, when calculating green fluorescence, the value for green was divided by the fluorescence value of red.
Background fluorescence was also taken into account through the creation of a negative control. This negative control lacked an eGFP plasmid and was created from the same line of cells as the other samples. The averaged value of the green fluorescence of the negative control was subtracted from the other samples as a way to remove background fluorescence. The negative control cells under green and red fluorescence filters were recorded in Figure 3a and Figure 3b.
Figure 3a & 3b: ACG eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
In Figure 3a, the only visible cells show a very low level of GFP expression, while most cells cannot be seen. There is a visible difference between Figure 2a and Figure 3a, which suggests that the mutated start codon did have an effect on the translation of the eGFP.
The quantified data indicates relatively high efficiency in the ATG/ACG reporters. According to the microscopy data (Figure 4), the ATG reporter showed 70.57% of fluorescence compared to the positive control, while the ACG reporter only gave 4.21%. Therefore, the mutated start codon is proven to be able to produce the difference between the two versions of the reporter.
Figure 4: Efficiency of ATG/ACG reporters compared to positive control. Error bars represent standard error between normalized results from 3 biological replicates of the experiment. (Standard error <0.05)
The results were further validated using flow cytometry as part of the collaboration with BostonU Wetlab. The results show that the ATG reporter has a similar level of fluorescence compared to the positive control, while the ACG reporter shows much less (7.21%), similar to the microscopy results.
Figure 5: ATG/ACG reporters efficiency confirmation by flow cytometry. The difference between the wild type and mutant reporter is consistent with the microscopy data.
The wild type used for this experiment was a sample of HEK293T cells that was not transfected. The data collected from the flow cytometry showed that the ATG eGFP cells had slightly higher levels of fluorescence when compared to the positive control. It was also noted that the wild type cells had slightly higher levels of fluorescence than the negative control. However, the slight difference and the percent of error for the wild type indicated that the wild type and the negative control most likely had about the same levels of fluorescence.
WT/PTC β-globin eGFP Reporters
The validation of the β-globin reporters followed the same procedures as the ATG/ACG reporters. The β-globin reporters had a similar design to the ATG/ACG reporters. With the β-glbin, the mutated version had a premature stop codon just before an exon junction in the globin sequence that was fused to the eGFP. The "functional" version of the globin reporter also had the globin sequence fused to the eGFP sequence, but without the premature stop codon.
Figure 6a & 6b: WT β-globin eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
In Figure 6a, fewer glowing cells are seen, and the fluorescence is visibly weaker than the ATG eGFP reporter. However, the PTC β-globin reporter, in Figure 7a, showed even less expression of the GFP. This difference between the WT and PTC reporters was consistently seen in all replicates of the experiment.
Figure 7a & 7b: PTC β-globin eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
According to the data (Figure 8), the wild type β-globin reporter showed a much lower level of expression (14.77% of positive control) compared to the ATG reporter. Therefore, the difference in the level of expression between the WT and PTC reporter is much smaller, which means lower reporter efficiency. The standard error, however, indicates that the difference that is present is very valid, and the premature termination codon is likely to have an actual impact on the expression of the GFP.
Figure 8: Efficiency of WT/PTC β-globin reporters compared to positive control. Error bars represent standard error between normalized results from 3 biological replicates of the experiment. (Standard error <0.01)
* The complete experiment (RNA editing with APOBEC/ADAR) has been attempted, but no data is available at this point.
