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Team:MIT/miRNA
From 2013.igem.org
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<h1>Overview of miRNA repression</h1> | <h1>Overview of miRNA repression</h1> | ||
<p>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.</p> | <p>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.</p> | ||
| - | <p>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 | + | <p>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.</p> |
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<img src="https://static.igem.org/mediawiki/2013/9/95/MiRNAcircuit.png" width="300" height="450"> | <img src="https://static.igem.org/mediawiki/2013/9/95/MiRNAcircuit.png" width="300" height="450"> | ||
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| - | <p>The simplest receiver circuit is composed of constitutively expressed eYFP (under the hEF1a promoter), designed with target sites for either miR-451 | + | <p>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.</p> |
<p>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.</p> | <p>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.</p> | ||
| - | <img src="https://static.igem.org/mediawiki/2013/c/c7/MiRNA1.png" width=" | + | <img src="https://static.igem.org/mediawiki/2013/c/c7/MiRNA1.png" width="100%"></img> |
| - | <p>The above histogram | + | <p>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 affect of siRNA, regardless of target sequence. |
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Revision as of 05:05, 27 September 2013
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 affect of siRNA, regardless of target sequence.
Exosome Isolation and Co-Culturing
Cell-Cell Co-Culturing
