Resources for functional studies in Drosophila

果蝇功能研究资源

• 批准号: 10597005
• 负责人: NORBERT PERRIMON
• 金额: $ 55.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597005
• 关键词: Address; Adult; Algorithms; Anatomy; Antibodies; Atlases; Brain; Cells; Collection; Communities; DNA Binding Domain; Data; Data Analyses; Data Set; Detection; Development; Drosophila genus; Engineering; Enhancers; Funding Opportunities; Gene Combinations; Gene Expression; Gene Expression Profile; Generations; Genes; Genomics; In Situ Hybridization; Knowledge; Larva; Letters; Maps; Methods; Molecular Profiling; Nucleic Acid Regulatory Sequences; Organ; Output; Pattern; Phenotype; Proteins; Proxy; R24; Reagent; Reporter; Research Personnel; Research Project Grants; Resources; Sampling; Specificity; System; TNFSF5 gene; Technology; Testing; Time; Tissues; Transcription Coactivator; Transgenes; United States National Institutes of Health; Validation; Visualization; animal model development; body map; cell type; cooking; differential expression; experimental study; fly; human disease; human model; prevent; response; single-cell RNA sequencing; tool

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.

项目概要/摘要 从基于 scRNAseq 的研究中获得的最新知识为 细胞类型的表征和表型的描述。此外，从 scRNAseq 获得的信息 可以帮助开发可用于功能研究的新试剂。 scRNAseq 研究的主要挑战之一 要解决的是 scRNAseq 簇到解剖学的映射。虽然如果集群的话这可以相对容易地完成 由单个差异表达基因（DEG）的独特表达来定义，任务更多 如果一组细胞只能使用 DEG 的组合来定义，那么情况就会变得复杂。识别组合 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 定义簇的最小的独特标记基因集。许多方法和资源可以 用于将标记基因的表达与解剖结构相关联；然而，在大多数情况下，共同表达 需要多个基因才能将簇正确地映射到解剖结构，并且现有试剂是有限的。作为 split-Gal4 方法，其中 Gal4 的 DNA 结合结构域和转录激活结构域是 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，将用于绘制果蝇中估计的 450 个细胞簇的图谱。 重要的是，这些研究产生的分裂 Gal4 系将使我们能够解决长期存在的问题 果蝇领域，即现有 Gal4 系的特异性，正如我们和其他人观察到的 大多数所谓的“组织特异性”Gal4 驱动线也在额外的、不需要的细胞中表达 类型或组织。最后，由于当前的 split-Gal4 系统与时间控制不兼容，因此无法 使用 split-Gal4 系进行需要阶段特异性激活 UAS 驱动转基因的实验， 目标 3，我们将开发一种鲁棒的 split-Gal4 方法，该方法允许时间控制并生成 收集 200 个诱导型分裂 Gal4 系，可产生 100 个可控组织 特定的 Gal4 模式，覆盖大多数幼虫和成虫组织。这些资源共同将提供 拥有急需试剂的社区将尽快提供和分发 由布卢明顿果蝇库存中心生产。