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.

项目摘要 /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 可以帮助开发新试剂对功能研究有用。 SCRNASEQ研究的主要挑战之一 要解决的是将scrnaseq簇映射到解剖结构。虽然如果群集可以相对容易地完成此操作 由单个不同表达基因（deg）的唯一表达来定义，任务更多 复杂的如果只能使用DEG组合来定义一组细胞。确定组合 表达定义细胞簇的基因，我们在AIM 1中提出，以开发确定的算法 定义集群的最小独特标记基因集。许多方法和资源可以 被用来将标记基因的表达与解剖结合在一起；但是，在大多数情况下 需要多个基因将簇正确地映射到解剖结构，并且现有试剂受到限制。作为 拆分gal4方法，通过该方法，GAL4和转录激活域的DNA结合域是 在两个不同的增强子的控制下独立表示，可扩展以检测多个 基因，在AIM 2中，我们建议在我们既定的Trip/DRSC平台上构建并产生一个集合 将用于绘制估计的450个细胞簇的1012分式GAL4线。 重要的是，这些研究产生的分式gal4线将使我们能够解决一个长期存在的问题 果蝇领域，即现有GAL4线的特异性，因为我们和其他人都观察到 绝大多数所谓的“组织特异性” gal4驱动线也以其他不需要的细胞表示 类型或组织。最后，由于当前的拆分gal4系统与临时控制不兼容，因此 使用拆分gal4线用于实验，需要特定阶段的UAS驱动器转换， AIM 3，我们将开发一种强大的分式GAL4方法，该方法允许临时控制并生成一个 收集了200个诱导型分裂4系，这将允许产生100个控制器组织 特定的GAL4模式，覆盖大多数幼虫和成人组织。这些资源将共同提供 具有急需试剂的社区将尽快提供和分发 由布卢明顿果蝇股票中心生产。