CRE DD Green Reporter System
CRE DD Green Reporter System
Brand: Takara Bio.
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CRE DD Green Reporter System
The CRE DD Green Reporter System is designed to monitor cAMP response element binding protein (CREB) activity in mammalian cells, with minimal background signal. It includes the pCRE-DD-ZsGreen1 Reporter vector and Shield1.
pCRE-DD-ZsGreen1 encodes a green fluorescent protein reporter tagged at its N-terminus with the ProteoTuner destabilization domain (DD), and under the control of the CRE promoter. The DD causes the DD-ZsGreen1 reporter to be rapidly targeted to and degraded by proteasomes. This minimizes background fluorescence from leaky promoters prior to promoter activation.
To monitor CREB activity, a candidate inducer is added to the medium simultaneously with the DD's stabilizing ligand, Shield1. This allows DD-ZsGreen1 to accumulate in response to CREB activation. As a result, only the reporter molecules expressed during CRE induction contribute to the fluorescence signal. This system provides a considerably higher signal-to-noise ratio than can be obtained with non-destabilized or constitutively destabilized reporter systems.
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD. HEK 293 cells were transfected with plasmids encoding the following reporters: CRE-tdTomato, CRE-DD-tdTomato, CRE-ZsGreen1, and CRE-DD-ZsGreen1. 24 hr later, the cells were stimulated with 10 μM forskolin and simultaneously treated with 1 μM Shield1. After 4.5 hr, fluorescence intensity was measured via flow cytometry, and fold induction was calculated. The tdTomato and ZsGreen1 reporters containing the DD had three- and six-fold greater fluorescence intensity respectively, than the versions without the DD, due to the latter’s increased background levels.
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization. A small destabilization domain (DD) is fused to the fluorescent protein reporter. The small membrane-permeant ligand Shield1 binds to the DD and protects the entire fusion protein from proteasomal degradation. Removal of Shield1, however, causes rapid degradation of the entire DD-fluorescent protein reporter. The default pathway for the DD-Fluorescent Protein Reporter Systems is degradation of the DD-fluorescent protein reporter, assuring low background, unless Shield1 is present, assuring low background.
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DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD
DD-Fluorescent Protein promoter reporters provide a much greater fold increase in signal intensity than traditional fluorescent protein reporters, which do not contain the DD. HEK 293 cells were transfected with plasmids encoding the following reporters: CRE-tdTomato, CRE-DD-tdTomato, CRE-ZsGreen1, and CRE-DD-ZsGreen1. 24 hr later, the cells were stimulated with 10 μM forskolin and simultaneously treated with 1 μM Shield1. After 4.5 hr, fluorescence intensity was measured via flow cytometry, and fold induction was calculated. The tdTomato and ZsGreen1 reporters containing the DD had three- and six-fold greater fluorescence intensity respectively, than the versions without the DD, due to the latter’s increased background levels.
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization
Ligand-dependent, targeted, and reversible fluorescent protein reporter stabilization. A small destabilization domain (DD) is fused to the fluorescent protein reporter. The small membrane-permeant ligand Shield1 binds to the DD and protects the entire fusion protein from proteasomal degradation. Removal of Shield1, however, causes rapid degradation of the entire DD-fluorescent protein reporter. The default pathway for the DD-Fluorescent Protein Reporter Systems is degradation of the DD-fluorescent protein reporter, assuring low background, unless Shield1 is present, assuring low background.
