The miR-Selection lentivector features a dual reporter system with firefly luciferase (Fire) and a cytotoxic sensor (Ctx). The miR-Selection platform captures the 3’ UTR to microRNA binding event using a survival screen by modulating the reduction of the cytotoxic sensor. Quantitative validation is made simple using the built-in luciferase reporter. This powerful and elegant technology finally enables the accurate identification of microRNA targets and enables high-throughput target identification screens.
Clone your 3'UTR into the vector - easily identify the microRNAs that bind
miR-Selection Lentivector details
• Total size of the lentivector = 10.2Kb • Sequence of the Multiple Cloning Site (MCS) 5'-GAATTCGCTAGCTGAGGCAGCAGAGACCGGCCGGCCGGTCTCTCTCT-GTTCGAGGATCCCTCGAGGTCGACGCGGCCGC-3'
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.
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.
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