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

购买Olympus SZX 7荧光体视显微镜用于后期解剖 果蝇胚胎及GFP/RFP标记基因筛选 该提案旨在收购Olympus SZX 7荧光立体显微镜， 替换了一个旧的和坏的荧光立体显微镜，这是用来选择绿色荧光蛋白， 阳性蝇胚，并在提供环形光时解剖蝇胚。美国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 在果蝇气管管成熟，这是一个首要的系统，以揭示基本的 管状器官形成的机制。 果蝇的气管是一个由上皮细胞构成的分支网络， 细胞围绕顶端腔。在管成熟过程中，顶端分泌物爆发沉积 大量的管腔基质成分进入顶腔。然后，清除该管腔 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“更广泛的协调者”。为了验证这个假设，我们 将完成以下三个具体目标：目标。1确定Osi基因在细胞中的功能。 管成熟过程中顶端腔基质的运输。瞄准2：确定的功能 Osi基因作为协调子控制内体介导的蛋白质运输。瞄准3：识别 与Osi蛋白直接结合的蛋白质。为了进行实验，我们将分析 蛋白质运输和结构变化在后期胚胎的免疫染色。此外，本发明还提供了一种方法， 我们将通过观察GFP标记的 囊泡标记和RFP标记的蛋白质标记，同时在Osi突变体背景。 Osi突变体的气管表型出现在晚期胚胎（中期17）。由于 角质层的形成，抗体不能有效地进入胚胎不解剖。所以我们 需要在免疫染色前解剖晚期胚胎。此外，我们将产生苍蝇 Osi中携带GFP标记的囊泡标记物和RFP标记的管腔蛋白标记物的菌株 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突变体的遗传背景。因此 携带GFP或RFP标记标记基因的潜在活重组苍蝇胚胎可以 使用立体荧光显微镜进行选择。不幸的是，我们的旧荧光立体声 一种显微镜，可以用来解剖后期的苍蝇胚胎，以及选择活的胚胎 携带荧光标记基因的细胞被破坏得无法修复。因此，这要求奥林巴斯 SZX 7荧光立体显微镜将允许我们进行所提出的实验（目的。 1、瞄准2)按时没有它，我们的项目进度将大大延迟。

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.