pmRi-ZsGreen1 Vector
pmRi-ZsGreen1 Vector
Brand: Takara Bio.
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pmRi-ZsGreen1 Vector
The pmRi-ZsGreen1 Vector is a Tet-inducible mammalian expression vector that allows the coexpression of a user-generated microRNA (miRNA) and a green fluorescent protein, ZsGreen1, to be controlled by a Tet System transactivator and doxycycline. The TRE-based promoter, PTight, regulates the expression of the ZsGreen1 mRNA transcript, which contains your miRNA precursor seq-uence embedded in its 3' UTR. Inducibility requires that a Tet System transactivator (e.g. Tet-On Advanced) also be expressed in the target cells. To select stable cell lines, the pmRi-ZsGreen1 Vector must be cotransfected with one of the provided linear selection markers.
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.
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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.
