Simultaneous tracking of movement and gene expression in multiple Drosophila melanogaster flies using GFP and DsRED fluorescent reporter transgenes

Here we report an improved fly video tracking system that allows multiple transgenic flies to be tracked simultaneously using visible light, GFP fluorescence and DsRED fluorescence. The movement of multiple flies could be accurately tracked at real time rates, while simultaneously assaying the expression level of two different transgenes marked with GFP and DsRED. The individual flies could be accurately tracked and distinguished even during periods when transgene fluorescence was undetected. For example, characteristic patterns of hsp70 and hsp22 transgene induction could be simultaneously quantified and correlated with animal movement in aging flies, and as groups of flies died due to dessication/starvation.The improved methods allow for more efficient assay of the correlation between gene expression, behavior, aging and mortality: multiple animals can be assayed with simultaneous quantification of multiple transgenes using GFP and DsRED fluorescence. These methods should allow for increased flexibility in experimental designs. For example, in the future it should be possible to use gene expression levels to predict remaining life span more accurately, and to quantify gene expression changes caused by interactions between animals in real time.The use of autofluorescent proteins such as Green Fluorescent Protein (GFP) has caused a revolution in biology and the study of gene expression [1,2]. Because the proteins require no added substrate in order to fluoresce, they require no invasive procedures for their assay, and serve as ideal reporters for the expression of transgenes in cells and whole animals. In addition, protein activity, protein-protein interactions, and protein sub-cellular locations can be assayed using fluorescence [3,4].Video tracking is increasingly being used in studies of movement, behavior, and aging across species [5-8]. Examples include detailed studies of movement behavior in Drosophila[9,10] and C. elegans [11]. We have recently reported a vi