On-demand reporter vectors are the next generation of promoter reporters. With these reporters systems, you can compensate effectively for reporter background without compromising your assay’s signal intensity. Low background and a bright signal are no longer mutually exclusive.
The Challenges: Overcoming High Background & Low Signal Intensity
Traditional promoter reporter assays generally struggle with the fact that most promoters are not very “tight.” As a result, your promoter of interest may drive reporter expression even without being activated—for example, during the time between transfection and the start of your experiment. These preexisting reporter molecules (the background) are the main cause of a low signal-to-noise ratio after promoter induction during the actual experiment.
A previous approach to this problem was to modify reporters for very quick, constitutive degradation. However, because these reporters are constitutively degraded as soon as they are made, it is impossible to accumulate a large quantity of reporter molecules inside the cell even upon promoter activation. As a result, only a fraction of the reporter molecules are present long enough to be measured, and this type of assay has low signal intensity.
The Solution: Reporters On-Demand
The On-Demand Living Colors Fluorescent Protein Reporter Systems meet the challenge by providing both a low background and a broad dynamic range. This versatility is possible because they use a combination of technologies: each system includes a bright fluorescent protein reporter (AmCyan1, tdTomato, or ZsGreen1) for high signal intensity, coupled with ligand-dependent ProteoTuner protein stabilization/destabilization technology to eliminate background.
In these systems, the fluorescent protein reporter is expressed as a fusion protein tagged on its N-terminus with a ligand-dependent destabilization domain (DD). The DD rapidly targets the reporter protein for proteasomal degradation, guaranteeing a low reporter background signal at the start of your experiment. However, when the small, membrane-permeant ligand Shield1 is added to the sample, it binds to the DD and protects the reporter from degradation, so that it can accumulate.
By adding Shield1 simultaneously with your candidate inducer, you can effectively stabilize the reporter protein when it is synthesized in response to promoter activation. The majority of the fluorescent protein reporter molecules expressed during promoter activation will contribute to your readout, allowing for a considerably higher dynamic range and drastically improved signal-to-noise ratio compared to other types of reporter systems.
|On-Demand Fluorescent Protein Reporter Systems ||Traditional Reporter Systems |
|Background||Uniformly low ||Promoter-dependent; may be high |
|Signal||Bright||Reporter-dependent. If background is low, signal is usually dim|
|Signal-to-noise ratio ||High; due to bright signal and low background ||Often low, especially when background is low |
|Studying timely promoter activity by eliminating unwanted reporter molecules ||Easy—simply remove Shield1 reagent ||Difficult; depends on reporter's natural lifespan|
High Signal, Low Background
In order to demonstrate the high signal-to-noise ratio and wide dynamic range of the DD-Fluorescent Protein Reporter Systems, we compared the fold induction achieved using the DD-fluorescent protein reporters with that achieved using regular (non-destabilized) fluorescent proteins. The DD-tagged reporters stabilized by Shield1 had a much wider dynamic range, and therefore a much larger fold increase in the signal than the untagged versions of the same reporters. For the untagged versions, we observed high background fluorescence from reporter molecules that accumulated prior to induction, which drastically reduced the fold increase in signal intensity.
Choose Your On-Demand Reporter System
Our On-Demand Reporter Systems each consist of the necessary vectors (red, green, or cyan) plus Shield1.
Use the DD Fluorescent Protein Reporter Systems to monitor any promoter of interest—just insert your promoter of interest upstream of the DD reporter. Choose from plasmid or lentiviral vector formats.