Resources for functional studies in Drosophila

项目摘要/摘要 最近从基于scRNAseq的研究中获得的知识为 细胞类型的表征和表型的描述。此外，从scRNAseq获得的信息 可以帮助开发对功能研究有用的新试剂。SCRNAseq研究中的主要挑战之一 要解决的是scRNAseq簇与解剖学的映射。虽然这可以相对容易地完成，如果一个集群 是由单个差异表达基因(DEG)的唯一表达定义的，任务更多 如果一组单元格只能使用DEG的组合来定义，则很复杂。确定以下各项的组合 其表达定义细胞簇的基因，我们在目标1中建议开发一种算法来确定 定义一个簇的最小、唯一的一组标记基因。许多方法和资源可以 用于将标记基因的表达与解剖学相关联；然而，在大多数情况下， 需要多个基因才能正确地将簇映射到解剖结构，而现有的试剂有限。作为 Split-Gal4方法，其中Gal4的DNA结合域和转录激活域是 在两个不同增强子的控制下独立表达，可扩展用于检测多个 基因，在目标2中，我们建议在我们已建立的Trip/DrSc平台上构建并生成一个集合 1012个Split-Gal4系将被用来定位在果蝇中存在的估计450个细胞团。 重要的是，从这些研究中产生的分裂Gal4线将使我们能够解决 果蝇领域，即现有Gal4线的特异性，正如我们和其他人观察到的那样 绝大多数所谓的组织特异性的Gal4驱动系也表达在额外的、不想要的细胞中 种类或纸巾。最后，由于目前的Split-Gal4系统与时间控制不兼容，防止了 使用Split-Gal4系进行需要UAS驱动的转基因的阶段特异性激活的实验，在 目标3，我们将开发一种健壮的Split-Gal4方法，该方法允许时间控制并生成 收集200个可诱导的Split-Gal4系，将允许产生100个可控组织 特殊的Gal4图案，覆盖大部分幼虫和成虫组织。这些资源加在一起将提供 拥有急需的试剂的社区，一旦他们被提供并分发 由布鲁明顿果蝇库存中心制作。

PROJECT SUMMARY / ABSTRACT Recent knowledge gained from studies based on scRNAseq provides new opportunities for the characterization of cell types and description of phenotypes. In addition, information gained from scRNAseq can help develop new reagents useful for functional studies. One of the main challenges in scRNAseq studies to address is the mapping of scRNAseq clusters to anatomy. While this can be relatively easily done if a cluster is defined by the unique expression of a single differentially expressed gene (DEG), the task is more complicated if a group of cells can only be defined using a combination of DEGs. To identify combinations of genes whose expression define cell clusters, we propose in Aim 1 to develop an algorithm that determines the smallest unique set of marker genes that define a cluster. A number of approaches and resources can be used to associate the expression of marker genes to the anatomy; however, in most cases co-expression of multiple genes is required to properly map clusters to the anatomy, and existing reagents are limited. As the split-Gal4 method, whereby the DNA-binding domain of Gal4 and a transcriptional activator domain are expressed independently under the control of two different enhancers, is scalable for the detection of multiple genes, in Aim 2, we propose to build on our established TRiP/DRSC platform and generate a collection of 1012 split-Gal4 lines that will be used to map the estimated 450 cell clusters present in the fly. Importantly, the split-Gal4 lines generated from these studies will allow us to address a long-standing issue in the Drosophila field, namely the specificity of existing Gal4 lines, as we and others have observed that the large majority of so-called “tissue-specific” Gal4 driver lines are also expressed in additional, unwanted cell types or tissues. Finally, as the current split-Gal4 system is not compatible with temporal control, preventing the use of split-Gal4 lines for experiments requiring stage specific activation of the UAS driven transgene, in Aim 3, we will develop a robust split-Gal4 method that allows temporal control and generate a collection of 200 inducible split-Gal4 lines that will allow the generation of 100 controllable tissue specific Gal4 patterns, covering most larval and adult tissues. Together, these resources will provide the community with much needed reagents that will be made available and distributed as soon as they are produced by the Bloomington Drosophila Stock Center.