Acquisition of an Olympus SZX7 fluorescent stereo microscope for dissecting late-stage Drosophila embryos and selecting Drosophila embryos with GFP/RFP tagged genes

获取奥林巴斯 SZX7 荧光体视显微镜，用于解剖晚期果蝇胚胎并选择带有 GFP/RFP 标记基因的果蝇胚胎

• 批准号: 10795289
• 负责人: Lan Jiang
• 金额: $ 2.35万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10795289
• 关键词: Air; Antibodies; Apical; Autophagosome; Award; Binding; Binding Proteins; Biological Assay; Biological Models; Blood Vessels; Cessation of life; Chromosomes; Co-Immunoprecipitations; Cytoplasm; Cytoplasmic Vesicles; Defect; Deposition; Disease; Dissection; Drosophila eye; Drosophila genus; Drug Metabolic Detoxication; Embryo; Endosomes; Epithelial Cells; Epithelium; Extracellular Matrix; Fluorescence; Gases; Gene Family; Genes; Genetic; Glycocalyx; Glycolysis; Goals; Golgi Apparatus; Human; Image; Immunohistochemistry; Individual; Insecta; Invertebrates; Kidney; Knowledge; Lactoylglutathione Lyase; Life; Light; Liquid substance; Lung; Lysosomes; Mediating; Metabolic; Microscope; Morphogenesis; Organ; Organism; Pathway interactions; Phenotype; Polycystic Kidney Diseases; Property; Proteins; Pyruvaldehyde; Recombinants; Research Project Grants; Role; Shapes; Structure; System; Testing; Time; Trachea; Tube; Tubular formation; United States National Institutes of Health; Vertebrates; Vesicle; Yeasts; body system; exosome; experimental study; fluorescence microscope; fly; gene function; in vivo; malformation; member; monolayer; mutant; protein biomarkers; protein transport; repaired; secretion process; spine bone structure; trafficking

Acquisition of an Olympus SZX7 fluorescent stereo microscope for dissecting late-stage Drosophila embryos and selecting Drosophila embryos with GFP/RFP tagged genes This proposal is for the acquisition of an Olympus SZX7 fluorescent stereo microscope to replace an old and broken fluorescence stereo microscope, which was used to select GFP- positive fly embryos and dissect fly embryos when ring light is provided. The awarded NIH R15 research project is to study the role of a poorly understood Osiris (Osi) gene family in regulating tube maturation in Drosophila trachea, which is a premier system to reveal fundamental mechanisms of tubular organ formation. The Drosophila trachea is a ramifying network of epithelial tubes with a monolayer of epithelial cells surrounding an apical lumen. During tube maturation, the apical secretion burst deposits large amounts of luminal matrix components to the apical lumen. Then, this lumen is cleared before air fills the lumen. The goal of the awarded project is to reveal the functions of Osi proteins as “broader coordinators” of protein trafficking during tube maturation. To test this hypothesis, we will complete the following three specific aims: Aim. 1 Determine the function of Osi genes in the trafficking of the apical luminal matrix during tube maturation. Aim. 2: Determine the function of Osi genes as coordinators to control endosome-mediated protein trafficking. Aim. 3: Identify proteins that directly bind to Osi proteins. To carry out the proposed experiments, we will analyze protein trafficking and structural changes in late-stage embryos by immunostaining. In addition, we will analyze the dynamic coordination of vesicular trafficking in vivo by observing GFP-tagged vesicle markers and RFP-tagged protein markers simultaneously in Osi mutant background. The tracheal phenotypes in Osi mutants appear in late-stage embryos (mid-stage 17). Due to cuticle formation, antibodies cannot enter embryos effectively without dissection. Therefore, we need to dissect the late-stage embryos before immunostaining. In addition, we will generate fly strains that carry GFP-tagged vesicle markers and RFP-tagged luminal protein markers in Osi mutant background. Since some marker genes are located on the same chromosome as Osi genes, we will recombine these marker genes to Osi mutant background genetically. Therefore the potential live recombinant fly embryos that carry GFP- or RFP- tagged marker genes can be selected using a stereo fluorescent microscope. Unfortunately, our old fluorescence stereo microscope that can be used to dissect late-stage fly embryos as well as to select live embryos that carry fluorescently tagged genes is broken beyond repair. Thus, this requested Olympus SZX7 fluorescent stereo microscope will allow us to carry out the proposed experiments (Aim. 1 and Aim. 2) on time. Without it, our progress on the project will be significantly delayed.

购买奥林巴斯 SZX7 荧光体视显微镜用于后期解剖 果蝇胚胎和选择带有 GFP/RFP 标记基因的果蝇胚胎 本提案旨在购买一台奥林巴斯 SZX7 荧光体视显微镜 更换旧且破损的荧光体视显微镜，该显微镜用于选择 GFP- 当提供环形光时，检测果蝇胚胎呈阳性并解剖果蝇胚胎。荣获NIH R15 研究项目是研究一个鲜为人知的奥西里斯（Osi）基因家族在调节中的作用 果蝇气管的管成熟，这是揭示基本原理的首要系统 管状器官形成的机制。 果蝇气管是由单层上皮细胞组成的上皮管的分枝网络。 围绕顶腔的细胞。在管成熟过程中，顶端分泌物突然沉积 大量的管腔基质成分到达顶端管腔。然后，该管腔被清除 在空气充满管腔之前。获奖项目的目标是揭示 Osi 蛋白的功能 作为管成熟过程中蛋白质运输的“更广泛的协调员”。为了检验这个假设，我们 将完成以下三个具体目标： 目标。 1 确定Osi基因的功能 管成熟期间顶端管腔基质的运输。目的。 2：确定功能 Osi 基因作为协调员控制内体介导的蛋白质运输。目的。 3：识别 直接与 Osi 蛋白结合的蛋白质。为了进行所提出的实验，我们将分析 通过免疫染色观察晚期胚胎中的蛋白质运输和结构变化。此外， 我们将通过观察 GFP 标记来分析体内囊泡运输的动态协调 在 Osi 突变背景中同时检测囊泡标记和 RFP 标记的蛋白质标记。 Osi 突变体的气管表型出现在胚胎晚期（17 中期）。由于 角质层形成后，抗体在不解剖的情况下无法有效进入胚胎。因此，我们 需要在免疫染色之前解剖晚期胚胎。此外，我们将生成苍蝇 Osi 中携带 GFP 标记的囊泡标记和 RFP 标记的管腔蛋白标记的菌株 突变背景。由于一些标记基因与 Osi 位于同一染色体上 基因，我们将这些标记基因重组到Osi突变背景中。所以 携带 GFP 或 RFP 标记基因的潜在活重组果蝇胚胎可以是 使用立体荧光显微镜选择。不幸的是，我们的旧荧光立体声音响 可用于解剖晚期果蝇胚胎以及选择活胚胎的显微镜 携带荧光标记基因的细胞已被破坏且无法修复。因此，这要求奥林巴斯 SZX7 荧光立体显微镜将使我们能够进行所提出的实验（目标。 1、目标。 2）准时。如果没有它，我们的项目进展将大大延迟。

项目成果

The novel gene apnoia regulates Drosophila tracheal tube size.

新基因 apnoia 调节果蝇气管的大小。

• DOI: 10.1002/dvdy.29
• 发表时间: 2019
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists
• 作者: Scholl,Aaron;O'Brien,MichaelJ;Chandran,RachanaR;Jiang,Lan
• 通讯作者: Jiang,Lan

The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea.

• DOI: 10.1371/journal.pgen.1010571
• 发表时间: 2023-01
• 期刊: PLoS genetics
• 影响因子: 4.5