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 and miR-503. For this reason, we chose to use Jurkat cells as our sender cells and to design a receiver circuit which could detect these two miRNA.

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 or miR-503 in the 3' UTR. 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.

Exosome Isolation and Co-Culturing

Cell-Cell Co-Culturing