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

奥林巴斯SZX7型晚期解剖荧光立体显微镜的购置 果蝇胚胎及GFP/RFP标记基因果蝇胚胎的筛选 这项提议是为了购买奥林巴斯SZX7荧光立体显微镜，以 取代旧的和破旧的荧光立体显微镜，它被用来选择GFP- 当提供环形光时，阳性果蝇胚胎和解剖果蝇胚胎。获奖的NIH R15 研究项目是研究一个知之甚少的Osiris(OSI)基因家族在调节 果蝇气管中的管子成熟是揭示基本原理的首要系统 管状器官形成的机制。 果蝇的气管是由上皮管和单层上皮层组成的分支网络。 根尖管腔周围的细胞。在输卵管成熟过程中，顶端分泌物破裂沉积。 大量管腔基质成分进入根尖管腔。然后，这个管腔被清除 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。 作为输卵管成熟过程中蛋白质运输的“更广泛的协调者”。为了检验这一假设，我们 将完成以下三个具体目标：目标。1确定OSI基因在骨肉瘤中的功能 输卵管成熟过程中顶端管腔基质的运输。瞄准。2：确定的功能 OSI基因作为协调者控制内体介导的蛋白质运输。瞄准。3：识别 直接与Osi蛋白结合的蛋白质。为了执行拟议的实验，我们将分析 免疫染色观察晚期胚胎的蛋白质转运和结构变化。此外, 我们将通过观察GFP标记来分析体内囊泡转运的动态协调 在Osi突变背景中，囊泡标记和RFP标记的蛋白标记同时存在。 Osi突变体的气管表型出现在晚期胚胎(中期17)。由于 在角质层形成过程中，如果不解剖，抗体就不能有效地进入胚胎。因此，我们 在进行免疫染色之前，需要对晚期胚胎进行解剖。此外，我们还将产生苍蝇 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 背景突变。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从基因上重组到OSI突变背景中。因此 携带GFP或RFP标记基因的潜在活重组果蝇胚胎可以 用立体荧光显微镜选择的。不幸的是，我们的旧荧光立体音响 可用于解剖晚期苍蝇胚胎以及选择活胚胎的显微镜 携带荧光标记基因的基因已经损坏，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拟议的实验(目的。 1和Aim。2)准时。如果没有它，我们在该项目上的进度将被显著推迟。

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.