Validation of the System using c-Myc 3'UTR + miR-145

The cytotoxic screen was performed using control (no 3'UTR) and c-Myc 3'UTR miR-Selection lentivectors. Both stable cell lines were transduced with equal amounts of Lenti-miR-145 virus (GFP marker). The cells were treated for 4 days with 1x Ctx cytotoxin drug.

The 3’UTR of the human c-Myc gene was cloned into the miR-Selection vector and transduced into 293 cells. If microRNAs bind to the 3’ UTR being tested, then the expression levels of both luciferase and the Ctx sensors will be greatly reduced. Lowering the amount of the Ctx sensor is what will enable the cells to survive in the presence of the Ctx drug. This interaction between microRNAs and the 3’ UTR is key to the selection system and is what is being measured during the screen. Measurements of the luciferase (Fire) activities of the +/- c-Myc 3’ UTR in the miR-Selection vector infected with or without Lenti-miR-145 virus without Ctx selection show that the levels of luciferase were unchanged in the No UTR controls as expected. A pronounced 63% reduction in luciferase activity in the c-Myc 3’UTR plus Lenti-miR-145 cells was observed in experimental cells when compared to the controls.