Part:BBa_K1179072
Hef1a_eYFP-4x451
This construct generates an eYFP transcript which can be repressed when miRNA451 is present.
Overview of miRNA repression
miRNA are short (22-24 nt) strands of RNA known to regulate gene expression through repression of mRNA. mRNA that contain the complementary sequence to the miRNA are targeted by the RNA-induced silencing complex and are selectively degraded, thus repressing protein production.
Our goal is to use miRNA as a signal for cell-cell communication. We believe this can be accomplished by the packaging of miRNA into exosomes, which would then carry the miRNA signal to a receiver cell. Further, certain miRNA seem to be selectively targeted to exosomes, through a mechanism which is poorly understood. It has been shown that in Jurkat T cells, exosomes are naturally enriched in miR-451. For this reason, we chose to use Jurkat cells as our sender cells and to design a receiver circuit which could detect this miRNA. For our receiver cells, we have chosen HEK293 cells because they are well characterized, easy to culture, and easy to transfect.
eYFP-target Characterization
The simplest receiver circuit is composed of constitutively expressed eYFP (under the hEF1a promoter), designed with target sites for either miR-451 in the 3' UTR. This part is called eYFP-4x451. In addition, we include constitutively expressed tagBFP (also under the hEF1a promoter) as a control for transfection efficiency. This allows us to distinguish cells showing repression from cells that were simply not transfected efficiently.
To test the receiver circuit, we designed and had synthesized siRNA corresponding to miR-451 and miR-503. Co-transfection of these siRNA and the receiver circuit in the same cells allows us to characterize the sensitivity of the eYFP reporter and demonstrate that we can detect the silencing affect. It also gives us confidence that the reporter will be sensitive to natural miRNA-451 as well.
The above histogram shows the results of the siRNA experiment. We can see that eYFP-4x451 expressed in cells co-transfected with siRNA-451 is substantially repressed compared to eYFP-4x451 expressed in cells without any siRNA. We are confident that this affect is caused by the specific interaction of the siRNA and RISC with the target sites because the control with a "scrambled" siRNA (siRNA-503) shows levels of fluorescence nearly exactly equal to the levels without any siRNA. This is evidence that the repression is not simply caused by a general effect of siRNA independent of the target sequence.
This is another representation of the above histogram, here plotting each cell as a point in a scatter plot. The x-axis is the level of blue fluorescence, and the y-axis is the level of yellow fluorescence for each cell. This graph allows us to separate individual cells by their transfection efficiency, and thus directly compare the fluorescence levels of individual cells. Here we see the two populations of cells from before: the orange population is HEK293 cells co-transfected with hEF1a_eYFP-4x451 and siRNA-451, and the green population is HEK293 cells co-transfected with hEF1a_eYFP-4x451 and siRNA-503. This dramatically demonstrates the repression of the eYFP-4x451 in the presence of siRNA-451.
In this same graph we also represent the data a third way, by separating the population into 'bins', and then plotting the median yellow fluorescence among the cells at each level of blue fluorescence. This allows us to condense a scatter plot into a single line plot, making it much easier to compare populations.
Exosome Isolation and Co-Culturing
Once we confirmed that the reporter was functioning as designed, we began attempts to show that miRNA could be transferred from Jurkat T cells to HEK293 receiver cells. Our first approach was to isolate the exosomes produced by large numbers of Jurkat T cells using the Total Exosome Isolation reagent from Invitrogen. One of the advantages of this procedure is that it concentrates the exosomes, allowing us to treat the receiver cells with a much higher effective ratio of sender cells to receiver cells than would otherwise be possible. The other main advantage is that in the future, isolating exosomes from the media will also allow us to directly assay their cargoes, which will provide supporting evidence that any affects we observe are in fact caused by the engineered exosomal cargoes.
In this experiment, Jurkat T cells were grown in 100mm dishes to a concentration of 1 million cells per mL, at which point then the media was harvested and exosomes were isolated using the invitrogen protocol. The exosome suspension was then mixed with HEK293 receiever cells immediately after they were transfected with hEF1a_eYFP-4x451 and hEF1a_tagBFP. Again, tagBFP serves as an indirect measure of transfection efficiency, which should be independent of miRNA interference effects.
As before, this graph is a representation of a scatter plot, generated by splitting the population into bins of different levels of blue fluorescence, but in this case plotting the mean yellow fluorescence in each bin. Here we see a difference in yellow fluorescence in the population of cells treated with exosomes derived from Jurkat T cells. In order to test the statistical significance of this difference, we not only plotted the mean of each bin, but also the 95% confidence interval around the mean within each bin (defined as +/- 1.96*SE). As the two confidence intervals do not overlap, we are in fact confident that the difference between the two populations is statistically significant. However, further testing is required before we will be confident in saying that the difference was caused by the exosomal transfer of miRNA that we expected.
In data not shown here we have also tested the control with HEK293 cells treated with the same exosomes immediately after being transfected with normal eYFP. In those tests we have either not seen the above decrease in yellow fluorescence or have seen a slight increase in yellow fluorescence. This control makes us confident that non-specific effects of exosomes on the receiver cells are not causing the repression we have seen.
Cell-Cell Co-Culturing
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2073
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Illegal PstI site found at 1434 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2073
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Illegal PstI site found at 1434 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2073
Illegal BglII site found at 569
Illegal BamHI site found at 1197
Illegal XhoI site found at 968 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2073
Illegal SpeI site found at 2049
Illegal PstI site found at 315
Illegal PstI site found at 820
Illegal PstI site found at 1434 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2073
Illegal SpeI site found at 2049
Illegal PstI site found at 315
Illegal PstI site found at 820
Illegal PstI site found at 1434
Illegal NgoMIV site found at 703
Illegal AgeI site found at 81 - 1000COMPATIBLE WITH RFC[1000]
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