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

Abstract

BackgroundFluorescent proteins such as GFP (Green Fluorescent Protein) and DsRED (Discosoma sp.Red Fluorescent Protein) are often used as reporter molecules for transgene expression in Drosophila and other species. We have recently reported methods that allow simultaneous tracking of animal movement and GFP expression in real time, however the assay was limited to single animals and a single transgene. Numerous studies would be facilitated by methods that allow for assay of multiple animals and multiple transgenes.FindingsHere 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.ConclusionThe 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.

Drosophila Small Heat Shock Proteins: An Update on Their Features and Functions

“…CG14207 , l ( 2 ) efl , hsp22 , CG7409 and hsp23 are all induced by different oxidative stressors while hsp26 and CG4461 are up-or down-regulated according to the stressor (Grover et al 2009 ;Gruenewald et al 2009 ;Hirano et al 2012 ;Landis et al 2004Landis et al , 2012Yang and Tower 2009 ;Zou et al 2000 ). Interestingly, the regulation of hsp67Ba , hsp27 and hsp67Bc expression is not affected by any kind of oxidative stress suggesting their involvement in different regulatory processes (Landis et al 2012 ).…”

“…Indeed, using the hsp22 promoter to drive the expression of GFP or DsRed, a spike of reporter expression was consistently observed in the hours preceding and overlapping the death of the fl ies (Grover et al 2009 ). This was also true for hsp70 -reporter (Grover et al 2009 ;Yang and Tower 2009 ), suggesting that both Hsp22 and Hsp70 are aging biomarkers. Moreover, it Wang et al ( 2004 ) No data were available for CG43851, CG13133 and Hsp67Ba ND not determined G. Morrow and R.M.…”

“…Hsp22 is the sHsp preferentially expressed during aging and its level of expression is partially predictive of longevity in individual fl ies (Grover et al 2009 ;King and Tower 1999 ;Yang and Tower 2009 ). Indeed, using the hsp22 promoter to drive the expression of GFP or DsRed, a spike of reporter expression was consistently observed in the hours preceding and overlapping the death of the fl ies (Grover et al 2009 ).…”

Finding Behavioral Motifs in Fly Trajectories

“…In our previous work, we extended the real-time tracking system by exploiting fluorescent reporters for simultaneous tracking of movement and tissue-specific gene expression [4,5]. Since DsRED and GFP are used extensively as reporters for transgene expression in Drosophila and other organisms, methods to correlate movement and behaviors with levels of, and changes in, gene expression in vivo should provide insight into regulatory pathways.…”

Determination of the Spontaneous Locomotor Activity in <em>Drosophila melanogaster</em>

“…A three-dimensional video monitoring protocol uses flies expressing GFP and multiple cameras fitted with filters allowing for detection of fluorescence to determine fly mobility 9 . Flies in this protocol tend to exhibit cylindrical flight patterns, which is potentially due to the shape of Drosophila culture vials 10 . This method was improved by using a dome that allows measuring spontaneous movement of two flies 11 .…”