威康信托基金资助下，该协议。我们感谢为维护飞股票和多年来的准备飞食品莫琳·辛克莱女士。我们还想感谢他的帮助和技术支持，在此协议的发展迈克尔·艾奇逊先生。

1。转基因果蝇和维护<ol><li> 转基因果蝇在此示范使用：-UBQ UBQ-GFP-CDC20（二），RFP-His2B（X）; UBQ-GFP-CDC20（二）UBQ-GFP-CDC20（二）; MAD2 安永 （三*）和UBQ-GFP-CDC20转基因果蝇先前在实验室中产生的，通过一个标准的P-元素介导的转基因方法10,11和UBQ RFP-His2B的是在UMR CNRS的144 /研究所居里从YohannsBelaïche样的礼物法国巴黎。他们介绍到Mad2的通过标准的果蝇基因突变的背景。 MAD2 安永原突变系从布卢明顿股票中心购买。我们将不讨论用于提高在本协议中的转化过程。 注：*表示的染色体数目。 </li><li> 维修：转基因果蝇保持在25°C间塑料瓶containin中Ğ飞食品与顶端额外的干酵母粉。小瓶经常更换每3-4周，根据生长条件（ 图1）。 </li></ol> 2。飞食品制备（实验室规模） <ol><li>苍蝇的食物混合适量，加热并不断搅拌，溶解的组成部分。 </li><li>作为浆分发到每个塑料瓶使用Jencons科技有限公司蠕动泵（直径为2.5厘米×8厘米长），约8-10毫升这一媒介。 </li><li>当食品浆料和冷却至室温，小瓶，然后用棉花泡沫插头插入。这些食物小瓶被放置在一个托盘，然后将其密封在一个塑料袋内，并保存在4℃，以后使用。 </li></ol> 3。小规模的蛋收集<ol><li>约50 2-3日龄成年果蝇对被转移到一个新的飞食品小瓶提供额外干酵母粉在其表面在25°C间裁员ING胚胎。 </li><li>苍蝇，然后转移到一个新的小瓶，每隔一小时30分钟离开的胚胎在小瓶，以确保收集的一些胚胎周围核的分裂周期8-10岁的细胞核逐渐迁移到皮质和组织作为单面单层。通常被丢弃，因为它往往包含被保留在女性的身体条件不适合铺设的中年胚胎的第一个小时集合。 </li></ol> 4。准备盖玻片和幻灯片<ol><li>取出一个50×22毫米的盖玻片，稍湿的水非常小的量使用湿润的细笔刷，放在显微镜幻灯片一侧的四个角落，使盖玻片不移动，因为毛细管表面张力薄液膜所造成的。 </li><li>适用的薄带钢跨中间盖玻片庚胶水，庚应在几秒钟内蒸发盖玻片上留下的胶水。 </li><li>剪下一个小方块（约1.5毫米2）金刚石笔的另一盖玻片，拿起一个放在胶条结束（这是用来打开需要显微注射时针头），保存以供将来的人使用。 </li><li>再举显微镜幻灯片和双面胶带粘片约2厘米长，它和封面纸剥离。 （参见图2A和B）。 </li></ol> 5。 dechorionate胚胎<ol><li>转移到一个新的食品小瓶苍蝇，并用适量的液体湿的细笔刷双面胶带将其转移到幻灯片上的约50鸡蛋，让鸡蛋散开。当液体蒸发的鸡蛋会坚持到磁带上。 </li><li>在解剖显微镜下触摸沿其长轴蛋多次使用到一半的镊子绒毛膜外侧轻轻被打破，开放，但留在楚胚胎离子外壳，以防止快速脱水。继续，直到鸡蛋10-20（在一个单一的实验所需的鸡蛋的数量而定）已被视为这一进程。 </li><li>胚胎，然后拿起并从绒毛膜壳和胶条轻轻地放在一个。鸡蛋可以放在与胚胎平行或反平行于长边胶条，取决于如果他们将被显微注射的长边对齐。 </li><li>胚胎可以立即被覆盖的Voltalef 10S油适量（西格玛700 Holocarbon油和27油的5:1混合，），以防止进一步的即时成像干燥或干燥室中放置3-8分钟他们进一步脱水取决于房间的湿度和实验的目的。 10S油脱水的胚胎应涵盖在适当的时候，以防止过度脱水，如果需要显微注射。 （参见图2C，丁。急症室） </li></ol> 6。成像胚胎<ol><li>时间推移或单个活胚胎图像采集用1 HCX PL亚洲生产力组织的政务司司长40X ​​1.25油浸物镜（60或100倍的目标太取决于实验的目的，可以使用的Leica TCS SP2倒置共聚焦显微镜系统放大倍率使用漂白会发生更多的照片）。 </li><li>当多个荧光成像，其中有重叠的光谱和共同表达在同一胚胎，每个荧光蛋白的发光波长连续收集。激发和发射波长的Leica TCS SP2系统是：绿色荧光蛋白，λ：488 nm和λEM：500-600纳米。利福平，λ：543 nm和λEM：555-700纳米。 </li><li>一个单一的形象普遍获得平均2扫描线与平均2帧扫描。这需要至少6.3秒，扫描512×512带够ixel同时扫描模式，15.44秒的顺序扫描模式和图像产生一个很好的信号信噪比。设立时间推移图像采集的时间框架时需要考虑这些时间。 </li><li>针孔一般设置1-2个AU（轻快单位），和激光功率调整，以可能的最低程度，以避免任何重大的漂白。 </li></ol> 7。通过适当的试剂微量注射胚胎挑起国资委<ol><li>实验前的准备针。我们喜欢拉siliconised玻璃毛细管（GC-100-10，哈佛）针头使用燃烧/棕色微量器（萨特仪器，型号：P-97）的参数为：热量= 600，拉= 90，VEL = 70 ，德尔= 150。 </li><li>回填罚款装载头（20μL的Eppendorf很多：W215818J）使用适当的浓度在注射液的试剂1-2毫升的针。 </li><li>安装在针针议员lder连续离心喷射控制系统相连的压力管道的显微镜。 </li><li>查找胚根据40X客观的和不改变针移动到视野的中心，并逐步降低它的权利焦平面焦平面针的影子。 </li><li>将胚胎轻轻找到破盖玻片预先安装胶条一端的小广场。很轻轻移动针头，直到它击中平方米的小盖玻片的边缘，轻轻地打破它打开。 </li><li>将胚胎放回视图，并选择胚胎注射合适的年龄。我通常在5-7注入，因为这是胚胎细胞核移植前向外皮质核分裂周期。核的分裂周期的各个阶段，可以决定由GFP-CDC20或核组蛋白的RFP-2B信号之前注射9荧光信号的快速扫描。 </li><li>小心移动了安莉芳Ø关闭和调整焦平面胚胎和针的针。移动胚胎进针，注入一滴试剂所需的位置和移动距离胚胎（或后续的显微注射系统的指示）。胚胎可以注射后或从侧面取决于实验的目的。 </li><li>一旦注入胚胎，准备在适当的时候成像。 （参见图2F＆G公司 ） </li></ol> 8。代表结果<img alt="图1" src="/files/ftp_upload/3763/3763fig1.jpg" /> 图1。所需材料：1。细笔刷，B。小方破眼镜的容器，C。 22×50毫米盖玻片，D。显微镜幻灯片，电子。双面胶带，F。孔上的盖子，G在试管中的干酵母粉。酵母粉煤灰维修，H用的颗粒。飞食品瓶， 我 。一个用于dechorionate胚胎1镊子的一半，J一对镊子，K。庚胶液容器（容量瓶）。 <img alt="图2" src="/files/ftp_upload/3763/3763fig2.jpg" /> 图2。图表显示在步骤4，步骤5和针注射在第7步准备在胚胎dechorionation盖玻片和幻灯片的准备。一个 。顶部的图片显示了一个跨的中间地带，庚胶水和小广场的一端粘在一个破碎的盖玻片盖玻片。底图为一个在其四个角的薄水层举行盖玻片显微镜幻灯片。使用幻灯片举行盖玻片的原因是要保持大致相同的焦距，发现当你在幻灯片上双面胶带，从而避免重新聚焦显微镜，而你ARË解剖和转移磁带和盖玻片之间的胚胎，B。用双面透明胶带的长度短的幻灯片应用前剥离其封面纸用于dechorionating胚胎，C。左边的图片显示有显着的前部和后部两端完整绒毛膜炮弹的胚胎;右侧图为在破碎的蛋壳壳的胚胎之前，他们被转移到盖玻片胶条，D，E图显示两个。胶水带dechorionated胚胎转移，安置和调整的方式。胚胎受10S油经过适当的干燥期。F＆G图显示如何打开针位置，使注入的尖端。 从上述描述的实验中获得的单个或延时的图像可直接分析使用莱卡软件或TIF作为一个开放的FO文件中保存rmat可为进一步量化，或使用其他常见的图像分析程序，如Photoshop和图像J，MetaMorph等两个例子来说明这个编辑分析讨论如下： 例1：一个时间推移电影（ 图3），显示着丝粒的GFP-CDC20和生活果蝇合胞胚胎染色体运动的动态招聘。时间推移从原来的序列图像感兴趣区域确定/编辑和使用Photoshop软件自动批量转换。电影组装使用QuickTime软件。 <img alt="图3" src="/files/ftp_upload/3763/3763fig3.jpg" /> 图3。一时间推移电影显示着丝粒的GFP-CDC20和生活果蝇合胞胚胎染色体运动的动态招聘。 （一）时间推移图像合胞从转基因胚胎，共同表达GFP-CDC20（绿色）和RFP-2B组蛋白（红色）融合蛋白，并记录在18°C在细胞核分裂周期7​​-8使用了Leica TCS SP2共聚焦系统。帧被每10秒钟。细胞已经在前期的帧被视为零时间10点， <a href="http://www.jove.com/files/ftp_upload/3763/3763movie3A.mov" target="_blank">点击这里查看图3A的电影</a> 。（二）绿色荧光蛋白CDC20可以随时观察前期和早中期，着丝粒（B3＆4，白色箭头）和坚持在中期和后期着丝粒（B5＆6，白色箭头）。 GFP-CDC20被排除从相间细胞核（白色箭头），早前期进入细胞核。 （B8-14）作为标记共同表达His2BmRFP的使用染色质形态。酒吧为5mm。 例2：国资委的功能可以通过操纵胚胎显微注射荧光标记的抗体，蛋白质或chemica研究L化合物的利益，可能引发国资委。例如注射秋水仙碱，使微管解聚挑起国资委表示在图4 10。 <img alt="图4" src="/files/ftp_upload/3763/3763fig4.jpg" /> 图4。 MAD2是必不可少的秋水仙碱援引国资委功能 10。 GFP-CDC20; MAD2 + / + GFP-CDC20; MAD2 安永 （MAD2 - / -空突变）和“绿色荧光蛋白MAD2; MAD2 安永胚胎内注射秋水仙碱治疗。时间推移聚焦图像被前（01，07和13）或注射后（02-06，前面板; 08-12，中板; 14-18，底部面板）。绿色荧光蛋白CDC20（顶部和中间板）或GFP-MAD2（底部面板）着丝粒信号被用来作为细胞周期进展的标志。顶部面板，箭头表示在02-06被捕的着丝粒。在01箭头标记的区域表明，秋水仙碱治疗前，GFP-DC20被排除后期相间核。中间面板，在缺乏内源性MAD2，GFP的-CDC20信号，继续振荡和细胞核中，开启和关闭的着丝粒，虽然胞质出现有缺陷，将预计在缺乏微管的胚胎，在存在秋水仙碱（在07-12箭头表示）建议在逮捕细胞秋水仙碱治疗失败的国资委功能。女儿细胞核分离失败图片10。底部面板，在14-18的箭头表明被捕的着丝粒积累的GFP-MAD2融合蛋白MAD2突变胚胎中的国资委缺陷型救援功能的GFP-MAD2建议。 13箭头表明在晚间期核中细胞MAD2积累。禁止为5mm。胚胎显微注射〜1％的100mg/ml秋水仙碱在1×PBS原液的蛋量。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+Techniques+for+Studying+Mitosis+in+the+Drosophila+melanogaster+Syncytial+Embryo.">Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo.</a> J. Vis. Exp. (31), e1382 (2009).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Cell+Shape+Change+in+Living+Drosophila+Embryos.">Imaging Cell Shape Change in Living Drosophila Embryos.</a> J. Vis. Exp. (49), e2503 (2011).</li></ol>

我们什么都没有透露。

这里描述的协议是一个成像飞合胞胚胎及使用的Leica TCS SP2激光共聚焦扫描显微镜，可以修改，以适应其他显微镜系统，也可以适应其他基因的功能，利用果蝇合胞胚胎研究的一般方法。我们已经用此协议纺锤体组装检查点，蛋白质动力学和蛋白质水解，使用转基因果蝇或多克隆抗体，以可视化的蛋白定位在生活或固定样本4,6,12-14研究的许多方面。 这是最简单的收集鸡蛋的小号码（约10 - 100）时，以保持新鲜飞食品瓶苍蝇。这样可以减少决策和准备额外的苹果汁琼脂平板和鸡蛋收集室在15,16其他出版物中所述的麻烦。盖玻片玻璃破碎的小广场一个在另外一端的盖玻片上的胶条使得它容易得多打开针注射量，并确定通过调整显微注射系统设置，而针浸泡在10S石油。重要的是，为避免泄漏和维持胚胎的可行性，特别是对于那些需要很长一段时间，或提高注射后转化时，实验成功的注射胚胎的干燥程度。然而，有没有需要究竟有多长干燥的金科玉律。这种变化从实验，实验和依赖注入的解决方案和工作环境的湿度量。 利益的特定蛋白质的功能，可以操纵特定的蛋白质抑制剂，通过显微注射单或多克隆抗体对一个特定的蛋白质，信使RNA或野生型或基因突变的背景下荧光标记的肽。这些突变线或GFP标记的转基因株系的许多公开AV从果蝇库存中心，其中大部分是上市ailable果蝇（ <a href="http://flybase.org/" target="_blank">http://flybase.org/~~V</a> ），可在线搜索。

必要的设备和试剂： 共聚焦成像系统：在本协议中所述的成像系统是在Leica TCS SP2激光扫描共聚焦倒置显微镜系统。介绍的方法也适用于可能与其他成像系统的一些小的修改。 解剖显微镜：我们使用的是奥林巴斯SZX7与东风PLAPO 1X-4镜头。 针拔：火焰山/棕色微量车夫（萨特仪器，型号：P-97）。 显微注射系统的Eppendorf显微注射系统的描述，但可用于任何其他适当的系统。 飞食品经销商：Jencons科技有限公司蠕动泵。 试剂： <table border="1"><tbody><tr><td> 成分 </td><td> 重量小号</td><td> 资源 </td><td> 地段编号 </td></tr><tr><td>玉米粉</td><td> 100.0克</td><td>英国的SUMA， </td><td></td></tr><tr><td>红糖</td><td> 50.0克</td><td>林顿的，英国</td><td></td></tr><tr><td>干酵母</td><td> 25.0克</td><td> DCL的酵母有限公司英国</td><td></td></tr><tr><td>洋菜</td><td> 12.5克</td><td> Fisher Scientific则</td><td> 106556 </td></tr><tr><td>山梨酸</td><td> 0.4克</td><td> BDH问题，英国VWR国际有限公司</td><td> 8829310 </td></tr><tr><td>苯甲酸</td><td> 2.9克</td><td> Fisher Scientific则</td><td> 1019599 </td></tr><tr><td>尼泊金 （甲基-4 - 海德鲁 xybenzoate） </td><td> 0.9克</td><td> BDH问题，英国VWR国际有限公司</td><td> K35969015 </td></tr><tr><td colspan="3"> H 2 O上升至1L </td><td></td></tr></tbody></table><p类=“jove_content”&gt; 表1。飞食品配料。 holocarbon 700和27油购自Sigma。 干酵母：托马斯·阿利森，英国。 准备庚胶：以约1.5米的双面透明胶带，把成猎鹰15毫升管庚烷5毫升，和旋转为3-5小时或过夜。时间后，分成4等分等分1.5毫升的Eppendorf离心管中，并在台式离心机在旋转速度为5分钟，以消除任何碎片。存储在10毫升容量瓶庚胶液，并继续使用。胶水的强度会有所不同，根据所使用的胶水的浓度和金额。通常情况下，更薄的胶水产生更低的背景噪音时，由共聚焦显微镜扫描。 <ul><li> 盖玻片：BDH问题，英国VWR国际有限公司</li><li> 显微镜幻灯片：BDH问题，英国VWR国际有限公司&lt;/ LI&gt; <li> siliconised玻璃毛细管 GC-100-10，哈佛</li><li> 罚款加载提示：20μL的Eppendorf很多：W215818J </li><li>镊子</li><li>细笔刷</li></ul>

studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living drosophila syncytial embryos

纺锤体装配检验点（SAC）的机制是一个积极的信号，它监视染色体着丝粒和纺锤体微管之间的相互作用，以防止后期发病，直到染色体是否正确连接。细胞利用这种机制，以防止非整倍体或基因组不稳定，因此，癌症和其他人类疾病，如出生缺陷和阿尔茨海默氏症1。 Mad1的，如国资委组件的数量，MAD2，BUB1，BUBR1，BUB3，Mps1，ZW10，罗德和Aurora B激酶已被确定，他们是所有着丝粒的动态蛋白质2。有证据表明，着丝粒是SAC信号启动。 CDC20的是国资委的首要监管目标。 CDC20是必要的APC /℃（， 一个 naphase 带够romotingÇomplex或Çyclosome）激活3之一，也是一个着丝粒的动态蛋白质4-6。当被激活时，国资委抑制活性的APC /防止两个关键基材，细胞周期蛋白B及securin的破坏，从而防止中期向后期过渡7,8。国资委信号究竟是如何启动和组装上的着丝粒和传达到APC / C的抑制其功能仍然难以捉摸。 果蝇是一个非常听话的实验系统;一个更简单和更易于理解的有机体相比，人类只有一个，普通股的基本过程。也许，这是一个最好的生物体活细胞生物成像研究中使用，特别是对有丝分裂活动在时间和空间可视化，为早期胚胎通过13快速核分裂周期同步（8-10分钟每个周期在25°C），并逐步组织在刚刚下皮层9单面单层的细胞核。 在这里，我提出了一种生物成像的方法，使用转基因果蝇表达星GFP（绿色荧光蛋白）或其变种针对性利息的Leica TCS SP2激光共聚焦扫描显微镜系统研究苍蝇国资委的功能，图像显示一些国资委组件的GFP融合蛋白，CDC20和Mad2蛋白作为例子。

Watch this Scientific Journal Video about Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos at JoVE.com

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

着丝粒是地方国资委启动其信号监测的有丝分裂的姐妹染色单体隔离。一种方法是描述与染色体运动的可视化招聘和营业额的着丝粒蛋白及其协调<em&gt;果蝇</em&gt;胚胎使用徕卡激光扫描共聚焦系统。

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

studying-mitotic-checkpoint-illustrating-dynamic-kinetochore-protein

Research

JoVE Journal

Biology

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

11.3K 次观看.  University of Newcastle, United Kingdom. 着丝粒是地方国资委启动其信号监测的有丝分裂的姐妹染色单体隔离。一种方法是描述与染色体运动的可视化招聘和营业额的着丝粒蛋白及其协调果蝇胚胎使用徕卡激光扫描共聚焦系统。

视频: Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

科研

生物学

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

研究线粒体结构与功能<em&gt;果蝇</em&gt;卵巢

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...