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bioRxiv · 10.1101/2021.04.21.440847

An ELISA-based method for rapid genetic screens in Drosophila

Jay, T. R.·Kang, Y.·Jefferson, A.·Freeman, M.
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Abstract

Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. To quantify GFP levels from Drosophila lysates, we developed a GFP-based ELISA assay. We demonstrate that this assay can detect membrane localized GFP in a variety of neuronal and glial cell populations and validate that it can identify genes that change the morphology of these cells. This assay was also able to detect STAT transcriptional activity after injury. We found that this assay can detect endogenously GFP-tagged proteins, including Draper and Cryptochrome, and it is able to report developmental and circadian changes in the expression of these proteins. Finally, we validated that the assay can be used to detect changes in synapse elimination upon genetic manipulation of astrocytes. We then used the assay to perform a small-scale screen, which identified Syntaxins as novel regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy and quantitative in vivo screening method to assay a wide breadth of fundamental questions in neurobiology. Significance StatementForward genetic screens in Drosophila have played an integral role in elucidating the cellular and molecular pathways that govern almost every facet of biology. However, current screening methods in Drosophila are either fast, but limited in their specificity for particular pathways or processes, or rely on imaging, which requires substantial expertise, time, and cost. We have developed a rapid GFP-based ELISA screening method that, when paired with the wealth of GFP-based genetic tools already available in Drosophila, can be used to screen for regulators of many subpopulations of cells, transcriptional programs and levels of thousands of different proteins. Using this assay, we have identified a novel family of genes required for astrocytes to mediate developmental synapse elimination. This technique provides a screening platform that is fast, accessible, and broadly applicable to many pathways and processes, making Drosophila an even more powerful screening tool.

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Jay, T. R., Kang, Y., Jefferson, A., Freeman, M.. 2021-04-22. An ELISA-based method for rapid genetic screens in Drosophila. https://doi.org/10.1101/2021.04.21.440847

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