iGEM 2012


  • Project Overview

miRNA Signal

  • Overview
  • siRNA Characterization
  • Exosome Isolation and Co-Culturing
  • Cell-Cell Co-Culturing

Protein Signals

  • Overview
  • GFP
  • rtTA3
  • Cre
  • L7Ae
  • Cas9-VP16

Novel DNA Sensor: Cas9 Split Venus Fusion

  • Overview
  • Leucine Zipper Fusion
  • DNA Sensing

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  • Attributions

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 [1]. It has been shown that exosomes are naturally enriched in miR-451 [2]. For this reason we chose miR-451 as our signal, since it would allow us to use unengineered cells as our senders. We then engineered a receiver circuit which could detect miR-451. We chose to use Jurkat T cells as sender cells and chose HEK293 cells as our receiver cells because they are well characterized, easy to culture, and easy to transfect.

eYFP-target Characterization

The 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 (BBa_K1179072). In addition, we include constitutively expressed tagBFP (BBa_K1179068) 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 synthesized siRNA corresponding to miR-451. As a negative control we also synthesized siRNA corresponding to miR-503 because its different sequence will not complement miR-451 target sites. 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 miR-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. This effect is caused by the specific interaction of the siRNA and RISC with the target sites, confirmed because the control with a "scrambled" siRNA (siRNA-503) shows levels of fluorescence nearly exactly equal to the levels without any siRNA. The repression is not simply caused by a general effect of siRNA independent of the target sequence, but is dependent on siRNA repression.

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 then demonstrated 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 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 (given by +/- 1.96*SE). As the two confidence intervals do not overlap, we know 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.

We simultaneously tested (as a control) HEK293 cells transfected with normal eYFP and then treated with the same exosomes. As shown in this graph, we do not see the same decrease in yellow fluorescence. In fact, the exosome treatment has slightly increased the level of yellow fluorescence relative to blue. This graph represents the 95% confidence intervals with errorbars. Again, this control shows that the repression we have seen is not caused by non-specific effects of exosomes on the receiver cells, but is dependent on miRNA repression.

Cell-Cell Co-Culturing

The following graph compares populations of HEK293 cells transfected with eYFP-4x451 that were and were not co-cultured with Jurkat T cells. There is an evident decrease in yellow fluorescence in the population co-cultured with 2 million Jurkats relative to populations co-cultured with fewer or no Jurkats. This is a preliminary but exciting result suggesting that cell-cell communication mediated by exosomes is feasible, and can be detected by its affects on fluorescent proteins. this work we be continued to cement this result, to increase the signal to noise ratio and to create more complex circuits.