An ELISA-based method for rapid genetic screens in Drosophila.
An ELISA-based method for rapid genetic screens in Drosophila.
复制标题
一种基于 ELISA 的果蝇快速遗传筛选方法。
DOI：
10.1073/pnas.2107427118
复制
发表时间：
2021
影响因子：
11.1
通讯作者：
Freeman,MarcR
中科院分区：
文献类型：
--
作者：
Jay,TaylorR;Kang,Yunsik;Jefferson,Amanda;Freeman,MarcR
果蝇是一种进行基因筛选的强大模型，但是既快速又可用于检查各种细胞和分子途径的筛选分析是有限的。果蝇提供了一个广泛的工具箱，包括基于GFP的转录报告，GFP标记的蛋白质和驱动系，可用于在许多细胞亚群中表达GFP。因此，一种能够快速定量评估果蝇组织中GFP水平的工具将提供一个广泛适用的筛选平台。我们开发了一种基于GFP的酶联免疫吸附试验（ELISA），可以检测从果蝇发育的所有阶段的整个动物和解剖组织中收集的果蝇分析物中的GFP。我们证明，这种方法可以检测各种神经元和神经胶质群体中膜定位的GFP，并验证它可以识别改变这些细胞形态的基因，以及STAT和JNK转录活性的变化。我们发现这种方法可以检测内源性gfp标记的蛋白质，包括Draper， Cryptochrome和突触标记Brp。这种方法能够检测发育突触重塑过程中Brp-GFP信号的变化，并且可以使用这种ELISA方法鉴定已知的胶质突触吞噬的遗传调节因子。最后，我们使用该方法进行了小规模筛选，确定了Syntaxins作为星形胶质细胞介导的突触消除的潜在调节因子。总之，这些研究建立了ELISA作为一种快速，简单，定量的体内筛选方法，可用于分析广泛的基础生物学问题。
Drosophilais 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.Drosophilaoffer 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 inDrosophilatissue would provide a broadly applicable screening platform. We developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP inDrosophilalysates collected from whole animals and dissected tissues across all stages ofDrosophiladevelopment. 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 change 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 signal 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 elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions.