Image showing the timing of a gene’s activity in cells of the developing fly brain using the TransTimer reporter system. Image credit: Li He (CC BY 4.0)
Fruit flies and other animals have complex body plans containing many different types of cells. To make and maintain these body plans, individual genes must be switched on and off at specific times in particular cells to control how the animal grows. Some of these genes may be switched on for long periods of time, while others may be rapidly switched on and off on repeated occasions.
Fluorescent reporter proteins have been extensively used to study gene activity in cells. Typically, this involves linking the gene encoding the fluorescent reporter to a gene of interest, so that when the gene is switched on a fluorescent protein will also be produced. The fluorescent protein emits light of a particular color and measuring this light provides a way to monitor a gene’s activity.
Unfortunately, fluorescent proteins tend to break down slowly, and the level of fluorescence emitted cannot fluctuate quickly enough to reflect rapid changes in gene behavior. One way to overcome this limitation is to use destabilized fluorescent proteins that degrade more rapidly inside cells. However, current strategies for creating these proteins cause them to emit less light, making fluorescence more difficult to detect.
To address this issue, He et al. developed a new green destabilized protein, adding elements that increase production of the protein so a greater amount of light can be emitted. The green destabilized protein was then combined with a red fluorescent reporter that degrades more slowly to develop a new tool called TransTimer. When the gene linked to the reporter switches on, the green destabilized protein turns on before the red reporter turns on. But, as the gene switches off, the destabilized protein will degrade until only the red signal remains. This allows the ratio of green to red color emitted from the TransTimer to indicate the timing of a gene’s activity.
Using this tool, He et al. uncovered new details about the patterns of activity of two signals, known as Notch and STAT, that were largely missed by studies using traditional fluorescent reporters. Further experiments demonstrated that TransTimer can be used to carry out large-scale screens in living fruit flies, which have not been possible with more time consuming live-cell imaging techniques.
The fluorescent reporter developed by He et al. will be a useful tool to understand when and where genes are switched on during the lives of fruit flies. In the future, TransTimer could be adapted for use in other model animals or plants.
