An ELISA-based method for rapid genetic
Advanced genetic screens in Drosophila have been instrumental in elucidating the cellular and molecular pathways that govern nearly every facet of biology.
However, current screening methods in Drosophila are either rapid, but limited in their specificity, or rely on imaging, requiring significant expertise, time and cost. We have developed a rapid GFP-based ELISA which, when combined with the wealth of genetic tools available in Drosophila, can be used to screen for regulators of many cell subpopulations, transcriptional programs and proteins.
Using this test, we have identified the genes necessary for the elimination of astrocyte synapses. This technique provides a rapid, accessible, and broadly applicable screening platform for many pathways and processes, making Drosophila an even more powerful screening platform.
Summary
Drosophila is a powerful model for performing genetic screens, but screening tests that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offers a comprehensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in many cell subpopulations. Thus, a tool capable of rapidly and quantitatively assessing GFP levels in Drosophila tissues would provide a broadly applicable screening platform.
We have developed a GFP-based enzyme immunoassay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues at all stages of Drosophila development. We demonstrate that this assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validate that it can identify genes that alter the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. . We found that this assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp.
This approach is able to detect changes in Brp-GFP signaling during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, we used the assay to perform a small-scale screen, which identified syntaxins as potential regulators of astrocyte-mediated synapse knockout. Together, these studies establish ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to analyze a wide range of fundamental biological questions.