A novel reporter system where gene knockdown is used to assay intercellular siRNA transfer

We have used a tetracycline controlled transcriptional activation system, known as tet-off, as a novel reporter system that demonstrates the capacity of Mesenchymal stem cells (MSCs) to transfer siRNA. Traditionally, knockdown systems using siRNA are designed to look for a decrease in expression of the target gene. The tet-off system would have allowed us to study expression levels from a different aspect, in that we were looking for positive signal due to siRNA knockdown that would have enabled us to accurately explore the potential of MSCs to transfer siRNA intercellularly. The siRNA was designed to target a tetracycline- controlled transactivator (tTA) driving GFP expression in the U87 and SHSY5Y cell lines. In a controlled transcriptional activation system, the tTA binds to DNA on the tet-operator to activate a promoter, and initiates gene transcription. In the presence of doxycycline, the tTA can no longer bind to the promoter, and the gene cannot be transcribed. Due to the failure of the tet-off system to maintain GFP expression in the presence of doxycycline after transduction with sh-tTA, we adapted our study for the tet-on system. The tet-on system was characterized by transducing U87s and SHSY5Ys with a tet-on 4dGFP vector. GFP expression was examined in the SHSY5Y and U87 target cells before and during treatment with doxycycline with microscopy and flow cytometry. In addition to assaying the effect of doxycycline alone, multiple sh-tTA were studied (323, 325 and 527). The efficacy of tTA knockdown by siRNA was determined by examining GFP expression levels by a variety of quantitative methods including flow cytometry, microscopy, and qPCR. Our studies show that our sh-tTAs, 527 in particular, show potential in partially silencing GFP expression in the presence of doxycycline in the tet-on system.