pmRi-ZsGreen1 Vector

Tetracycline-inducible microRNA

Tetracycline-inducible microRNA. In the presence of doxycycline (Dox), the Tet-On Advanced protein binds to the inducible promoter, PTight, and elicits high levels of transcription of a composite mRNA that encodes a fluorescent protein (mCherry or ZsGreen1) and your miRNA sequence in its 3’ UTR. Cells that fluoresce also express your microRNA.

Tetracycline-inducible miRNA

Tetracycline-inducible miRNA. The RNAiMonitor System was used to create vectors that express a luciferase reporter containing miR-1 or miR-9 cognate target sequences in their 3’ UTRs (3 each). These reporters were then co-transfected with pmRi-ZsGreen1 vectors containing miR-1 or miR-9 miRNA inserts into MCF-7 Tet-On Advanced cells. The parent pmRi-ZsGreen1 vector provided a negative control for each experiment. Inducing miR-1 or miR-9 expression with Dox resulted in >90 reduction in normalized luciferase activity

Tetracycline-inducible shRNA in MCF-7 cells

Tetracycline-inducible shRNA in MCF-7 cells. MCF-7 Tet-On Advanced cells expressing an miR-1 miRNA construct from the pmRi-mCherry and pmRi-ZsGreen1 vectors, exhibit bright red and green fluorescence, respectively, but only in the presence of doxycycline.