The Dual Sensor Technology for Survival Screens

The cytotoxic screening assay is a unique approach where multiple microRNA binding targets within the 3’UTR of interest can be easily identified in a single experiment. In this assay, the cytotoxic sensor is constitutively expressed via a CMV promoter and its expression becomes lethal to the cell when the cytotoxic drug (Ctx) is added to the medium. In a screen where multiple microRNAs are overexpressed, only the cells expressing those microRNAs that interact with the 3’UTR, and thus down-regulate the sensors, will survive the drug selection. These effectors microRNAs can be easily identified by PCR analysis from those surviving cells genomic DNA.

Cytotoxic and Luciferase Sensors

The dual sensors for Luciferase and Ctx are under the direct expression control of the 3’UTR inserted behind the markers. Addition of one or more microRNAs in combination with supplying the Ctx drug to cell culture media begins the selection screen. If there is no binding site in the 3’ UTR clone for the microRNA being tested, the cells do not survive the selection (skull and bones) but will thrive if microRNAs bind to the 3’ UTR in the construct (happy face). Quantitative validation is made simple using the built-in luciferase reporter.