We thank Yixian Zheng (Carnegie Institution of Washington, Baltimore, MD) for the original protocol and assistance in initial setup and members of the Lei laboratory for critical reading of the manuscript. This work was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

1.启动循环:播种胚胎注:周期开始与来自前一个循环1.5克收集的材料的（一个胚胎和/或一些第一龄幼虫组成的混合物）。这种材料用活性酵母混合物直至蛹阶段被放置在一个塑料容器（见的具体材料/设备表）。容器将引入胚胎的生物混合物和幼虫与盖，以避免使幼虫逃跑之后被关闭。有必要使孔进入盖，以允许空气流通。为了避免通过孔幼虫逸出，使用泡沫塞。最后，建议戴上手套和白大褂，不仅在这一部分，而且在整个协议，以避免弄脏或染色的衣服用漂白剂。 <ol><li>设置塑料容器。 <ol><li>制作三个方孔用刀片在一个塑料容器盖pproximately2.2厘米。添加带（3/4英寸的标签带），以每个孔的四角，以确保紧配合用于泡沫塞（50×55毫米，DXL），然后将一个泡沫塞在每个孔中。 </li><li>用保鲜膜覆盖的塑料容器内。过该薄膜，添加棉层，以覆盖塑料容器的底部。撕裂用手指棉花。 </li></ol></li><li>准备飞食品。 <ol><li>加333毫升去离子水的500毫升烧杯中。在用磁性棒搅拌的烧杯中，加入丙酸167微升和1.08毫升磷酸，从储备溶液（分别为99.96％和85％）。 </li><li>慢慢加入77.5克活性干酵母，避免了大的团块。干酵母溶解后，加入38.8克蔗糖。 注:蔗糖应该加入的最后成分，因为添加此成分后立即，发酵将开始。 </li></ol></li><li>蔗糖溶解后，立即倒入食品在棉花，并确保均匀覆盖棉花。与盖合上的塑料容器内，避免污染实验室的食品中逃脱了苍蝇。 </li><li>悬浮1.5克从用5毫升70％的乙醇的前一循环的收获胚胎（未dechorionated）的。切成两半两个过滤纸，并且使用刮刀或具有宽尖端的移液管的4件均匀地分布在生物混合物（切如有必要）。躺在浸泡棉花顶部的滤纸，并盖上盖子。最后，孵育在RT（24ºC），直到蛹阶段塑料容器和湿度（35％）。 注意:所述的塑料容器不应该被放置在潮湿的腔室，否则这将鼓励细菌的生长。 </li></ol> 2.继续循环:从胚胎到苍蝇。 注:在循环的这一部分从放置在塑料容器的第1天，直到9天以后，当成蝇会出现胚胎进入从蛹。在这些8天什么也可以完成，但监视胚胎正确地进展到果蝇生命周期的下一阶段，直到羽化。如果幼虫开始死亡，并在此期间变黑，检查泡沫栓没有过紧，并提供足够的通风。这一时期是从上一个周期清洁苍蝇笼人口的好时机。 <ol><li>观察胚胎成为1 次龄幼虫约24小时后的成年雌淀积他们进入飞食品（参见步骤3）。所得幼虫将在4天从在步骤1中制备的飞食品饲料，种植和蜕皮两次成2 次和第3 龄幼虫。 </li><li>清洁苍蝇笼人口。 <ol><li>放置在4ºC笼为至少30分钟，以减慢蝇的活性。取下仪表外壳笼的一侧净，并用快速移动，拥有大量生物危害塑料盖打开的侧袋。倒入笼中的内容纳入囊中。 </li><li>与生物危害袋仍然覆盖所述保持架的一侧上，定位在笼上垂直与生物危害袋侧下一个接收器。小心取下塑料袋，以防止苍蝇逃跑，并在生物危险废物的容器将其丢弃。 </li><li>小心打开上部净不足以创造一个小洞，用清水冲洗笼子，将洗净的苍蝇到水槽内。慢慢增加孔的大小和消除在笼子里面所有的苍蝇。 </li><li>清洗笼和用水和肥皂两个网。虽然篮网还是湿的将其固定在干燥清洁笼子的两侧，最后把一个实验室透雨纸，这将阻止在步骤1.1.2粘到笼子里准备的塑料薄膜内。现在笼是准备进行下一个循环。 </li></ol></li><li>在初始安装后四天，观察幼虫化蛹。幼虫将保持在此阶段为4天。 注:During这个阶段的蛹将覆盖塑料容器内的整个棉的表面。 </li><li>第一苍蝇出现在蛹期和前，打开塑料容器，并将含棉与所有的蛹在干净的人口架内的实验室透雨纸塑料薄膜。用双结收网并清洗塑料容器并为下一个周期的盖子。 注:蛹固定到盖子应该被丢弃并通过高压灭菌或羽化之前冻结破坏。 </li></ol> 3.成蝇。 注:经过蛹的这4天上演的第一个苍蝇就会从蛹涌现。在24 - 48小时的所有苍蝇应该eclosed。在循环的这一部分，它向他们提供创建用于再现的合适的环境的目的的食品是重要的。 <ol><li>准备糖蜜托盘。 <ol><li>结合在一个1升烧瓶556.25毫升去离子H 2的O，90毫升糖蜜，和22克琼脂的。用高压灭菌30分钟，清凉搅拌棒，并添加9.25毫升10％Tegosept在95％乙醇。 </li><li>将混合物倒入的21×14.5厘米肉托盘。确保音量足以覆盖15 - 17盘。等待10 - 20分钟以固化并覆盖各托盘，用塑料薄膜，以避免干燥。的1升几个烧瓶可以在同一时间制得。在4ºC包裹在塑料存储托盘糖蜜直到需要。 </li></ol></li><li>添加覆盖着成蝇笼内湿酵母糖蜜托盘。 <ol><li>添加板之前，放置在4ºC笼在冷室中30分钟。 </li><li>加入200毫升去离子H 2的O到烧杯中，并慢慢倒入干酵母而用勺子搅拌。停止加入干酵母当该混合物变为约花生酱的防止蝇陷入到食物的一致性。 注意:不要让食物溶液固化否则这将是非常困难的覆盖是非常重要的糖蜜托盘和在收获过程（参见4.3节）提取鸡蛋。 </li><li>从一个糖蜜纸盒中取出塑料薄膜和用勺子或锅铲新鲜准备湿酵母一层覆盖。可以肯定的整个表面被覆盖。 </li><li>虽然笼仍然是在寒冷的房间，打开网和小心加入湿酵母糖蜜盘入笼。收网，并把笼子在室温。 注:可能是覆盖在插入一个空盘肉盘有用的，但一定不要阻止苍蝇进入糖蜜托盘。 </li></ol></li><li>改变食物板每24 - 48小时，以避免酵母太干燥。成蝇对干燥非常敏感。 </li></ol> 4.结束循环的:收获的胚胎注意:最好的飞生育3 - 羽化后5天。因此，在这段时间收集会得到最好的胚胎产率。所需后馆藏完井泰德，周期结束。苍蝇应该牺牲和清洁的笼子步骤2.2指定。 <ol><li>通常情况下，苍蝇羽化后第2天，引入一个新的糖蜜托盘新鲜的湿粮入笼，如第3.2.5解释。以提高蛋类沉积的产率，用手指的帮助下，进行湿酵母的表面不规则越好。如果旧的食品托盘是目前在笼子里，添加新板之前，小心地取出，并将其丢弃到一个生物危害袋。用于增加产量的另一个末端是去除棉层因此所有鸡蛋会在食品敷设。维持RT笼。 </li><li>观察胚胎的小小白点。 注意:鸡蛋准备收集。对于飞笼的日常维护，一日2〜收集建议的时间。这个系列将包含多胚胎和几个1 日龄幼虫。请参阅用于各种科尔收获胚胎的典型代表单产结果ction时间点。 </li><li>收获的胚胎<ol><li>放置在冷室的笼约30分钟。在平均时间准备下一循环中的塑料容器和飞食品如在第1节说明。 </li><li>在8升高压釜盘加水至¼总容量，然后加入1 ml 10％的Triton X-100。而笼中的冷室小心地取出糖蜜板，并将其放在一个生物危害袋内。 </li><li>从包装袋中取出托盘，并迅速淹没它（防止苍蝇逃跑）在高压釜托盘中的洗涤液。关闭生物危害袋放在一个生物危害容器中。苍蝇不会淹没在水中不含有洗涤剂。 </li><li>使用大画笔和蒸馏水枪冲洗胚胎和酵母关糖蜜。弃板和糖蜜成生物危害框。 </li><li>通过三套筛（粗筛30之上，40在中间，100在底部）小心倒入解决方案。洗团块Øñ用蒸馏水枪顶层，直至无团块仍然存在。 注意:成蝇和3 龄幼虫将在筛30被保留。 <ol><li>用蒸馏水冲洗该高压釜盘，并通过筛倒漂洗水。卸下顶部筛和重复的过程，如果酵母团块仍然存在。大部分的1 次和所有的第二龄幼虫的将保留在筛40。 </li></ol></li><li>删除第二个筛子。在第三（底部）筛微黄材料是胚和小1 次龄幼虫的混合物。用蒸馏水枪的所有鸡蛋移动到​​筛的一侧。用抹刀收集并权衡它们。 </li><li>在这些周期胚胎收集产量/幼虫范围7至13克之间。必须使用1.5克该材料开始新的周期（第1节）。使用胚胎的其余部分作进一步处理，或者可以被丢弃。 </li></ol></li></ol> 5.进一步处理注:不用于人口的延续的胚胎可以立即用于实验或备选地可在-80ºC被冻结。对于这两个选项中，将胚可能需要首先被dechorionated。 <ol><li>对于dechorionation，洗的胚胎在50％的漂白粉溶液2分钟，并用蒸馏水彻底冲洗。 </li><li>通过用纸巾用力按压冷冻胚胎，第一干燥，然后称重并将它们放置，用刮铲的帮助下，到15或50毫升锥形管中。最后浸没在液体N 2锥形管几秒钟。 </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacol Rev. 63, 411-436 (2011).</li></ol>

The authors have nothing to disclose.

用1.5g材料之一，开始可以获得每个周期7和13 g的收集胚胎的产量。向得到的材料的这样的量是维持飞行周期的所有阶段的权利培养条件是至关重要的。 最重要的参数是温度和湿度，这应该是分别为24ºC和35％。如果这两个参数不能在实验室的正常环境中保持恒定，一种可能性是将飞笼子放置到培养箱或环境室。其他协议建议70％的湿度，并且也是恒定24小时的光-暗循环，以增加所产生的蛋9,10的产率。但保持35％左右的湿度避免了细菌污染，而且由于该协议的目的只有一个人口笼的维修，苍蝇被关在实验室的正常光线环境。 另外重要的一点是要保持迪sturbances的成蝇尽可能低。它可能是可取的保持笼中从飞室分开的位置，以避免来自其它苍蝇交叉污染。 不推荐大种群的转基因和突变蝇的培养中，因为它是非常困难的，以保持它们的纯度，它们可以在较大的人口笼14表现出明显不正常的交配行为。 一个可能的问题，同时在大批量培养果蝇是其他生物一样螨和/或霉菌，这将用于食品的竞争，从而降低生产的蛋的产量的存在。为了避免这种情况，以保持所有设备清洁，洗涤该笼，蚊帐和用水和肥皂，以及每个周期后丢弃的一次性材料（泡沫塞）飞盒这是非常重要的。为了降低模具增长，丙酸和磷酸步骤准备飞食品时加入湿酵母1.2和Tegosept准备糖蜜托盘时，在步骤3.1。它有时是有帮助的地方材料，如在-20℃的塑料盒或筛子，当时间短，材料不能马上清洗。 整整一个回收周期发生在14 - 15天，当胚胎接种成飞盒，与上次收集日结束的开始。在此期间的时间，建议以记住所有的维持人口蝇笼（ 图2）的必要的步骤，在该协议中有详细说明，以组织的时间表。从一天，蛋是种子，直到成年果蝇出现，它仅需要检查塑料盒，并且当化蛹发生时，将它们放置在笼子内。在这之后，苍蝇具有每2至供给 - 1天直到胚胎接种为一个新的周期。在整个协议，最长的一天是胚胎用于开始一个新的周期收集和播种期间。一个■在该协议的评论，最好的鸡蛋产量为3 - 成虫羽化后第5天，终于下降在2天后。这为我们提供了一定的灵活性，以便选择在该卵的收获将执行新的一天。 如果由于任何原因收集胚胎的产率小于所需起始量（1.5克），一个总是可以添加新的糖蜜托盘和收集更多的蛋的第二天。对于恒定的集合，它建议保持平行2笼，和如果需要的胚胎的更高数量时，它也有可能使用更大的笼中。在这样做的很短的时间集合的情况下，以提高产率的一种方法是采取在上午产蛋突发的优势。 目前正在收集大量不同发育阶段的许多优点。例如，从人口笼收集胚胎已在免疫沉淀测定法6-8，质谱收藏品非常成功从分离的幼虫幼体组织中N有证明是一个很好的来源，3C实验4和RNA制剂15，并从成蝇头已被用于芯片实验16。此外，通常需要成人使蝇提取物用于组织培养17。 一个保持架的最有前途的应用是用于高通量测定法，允许基因，转录，蛋白质和代谢物的分析和筛选响应于病原体，生物分子，化学物质和电离辐射曝光提供材料。在这些大型试验大量个人是必需的，这里所描述的人口动态笼可为了在果蝇的生命周期，为他们的分析和筛选18的不同阶段来获得材料的大量非常有益的。

遗传和生化方法结合起来的能力已取得果蝇对生物化学和分子生物学的研究1-3特别合适的生物体。这些研究通常需要大量的生物材料，不仅从成蝇，而且从幼虫4，蛹5和胚胎6-8。为了获得大量的材料，研究人员使用一种称为飞"人口笼"大型集装箱养殖苍蝇。这些笼子包括由塑料制成的由两侧净覆盖，以允许保持器内引入的食物没有苍蝇逸出的圆柱体。这些笼可以通过自制9-11或者从公司购买（见具体的材料/设备表）。 使用该系统成长大量苍蝇的一个主要优点是，水果的周期飞12可以在所有的苍蝇在重新开发的方式来控制latively同步的方式。这种同步由播种新的胚胎，喂养幼虫/苍蝇和在精确的时间牺牲成蝇实现。使用同步飞的人口是发育研究13特别有用。 从几只苍蝇一个新的人口笼的开始是一个耗时的过程，需要放大9-11的许多周期。即使使用的苍蝇，培养瓶或minicages更大的容器，整个过程可以持续数月。为了避免这种耗时的一步，许多果蝇实验室经常保持这样的笼子。它是最方便的，开始从胚胎收集从已经建立的人口笼开始一个新的笼子。一般情况下，大多数实验室保持野生种群笼，比如俄勒冈R或广S.这手稿提出了一个详细的协议，以保持飞行人口笼。

<table>
	<tbody>
		<tr>
			<td>Bacto-Agar</td>
			<td>Beckton Dickinson</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Curity practical cotton roll</td>
			<td>Kendall</td>
			<td>2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry yeast</td>
			<td>Affymetrix</td>
			<td>23540</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>GE Healthcare Life Science</td>
			<td>1001-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam tube plugs</td>
			<td>Jaece</td>
			<td>L800-D2</td>
			<td>50 mm Diameter x 55 mm Length</td>
		</tr>
		<tr>
			<td>Fly population cage</td>
			<td>Flystuff</td>
			<td>59-116</td>
			<td>9&#8243; Diameter x 14.4&#8243; Length. Includes the nets for the cage.</td>
		</tr>
		<tr>
			<td>Meat tray</td>
			<td>Genpak</td>
			<td>1002S (#2S)</td>
			<td>8.25 x 5.75 x 0.5 inches</td>
		</tr>
		<tr>
			<td>Molasses</td>
			<td>Grandma&#180;s</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container</td>
			<td>Rubbermaid</td>
			<td>4022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic film</td>
			<td>Glad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Fisher Scientific</td>
			<td>S 93326</td>
			<td>Toxic. Handle in Chemistry Hood</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Fisher Scientific</td>
			<td>A258-500</td>
			<td>Toxic. Handle in Chemistry Hood</td>
		</tr>
		<tr>
			<td>Stainless steel&#160; sieve #100</td>
			<td>VWR</td>
			<td>57324-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel&#160; sieve #40</td>
			<td>VWR</td>
			<td>57324-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel&#160; sieve #30</td>
			<td>VWR</td>
			<td>57324-240</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>MP</td>
			<td>152584</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegasept</td>
			<td>LabScientific</td>
			<td>FLY5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

maintenance of a drosophila melanogaster population cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

蝇笼人口的维护是根据动态的生命周期。因此，（ 图1A）将胚胎和幼虫的初始生物学混合物在塑料容器后受精卵将成为幼虫在不超过2天，幼虫将4天生长，通过不同的龄幼虫阶段相对同步（参见图1B）。 幼虫已完成第三龄幼虫期之后，他们将化蛹并覆盖与一些盖子（ 图1C）的内表面上的塑料容器内的棉的表面上。这化蛹期会在接下来的4天，直到苍蝇完成变态过程。在此期间，强烈建议苍蝇转移到笼子里。第一个成蝇将开始9天至E关闭 - 10后到第11天所有的周期（ 图1D），并开始将已经从蛹涌现。 羽化（13天至15）后5天，最后在17个日跌幅，（羽化后7天） - 3中得到的胚胎集合的最佳产率。因此，建议将第13天补充的新鲜食品的最后托盘和收集在第15天的鸡蛋（ 图1E和F）。这将允许胚胎的最大数量的汇编。增加第三额外前一天收集可以导致食品过于干燥，因此会减弱收集胚胎的产率如表1所示的6个连续循环集合收率，与在每个循环为1.5g起始原料，与图2是一整个周期收集建议的时间表。 该材料收获第2天60，时间大多是由胚胎。然而幼虫少数也存在。笼维护的目的，这些幼虫都没有问题，因为某种程度的异步性的预期。然而对于某些生物化学实验，这些胚胎的纯度是非常重要的，但在同一时间，还需要收集大量的蛋进行这些实验。为此物在1小时和3小时进行几个短的连续集合，之后在RT快速添加与食物一新板，提供一种可以用这种方法得到的产率和纯度的估计（ 表2和图3） 。在1小时的连续集合开始以非常低的产率（14.5毫克），但是随后的胚胎增加收集每次的量，但在最后一次。以改变食物但后来驯化过程中被强调，苍蝇在第一低产量可能是由于。另一方面，没有拉尔VAE在五个连续的集合（ 图3A）进行观察，显示出较短的集合产生较高的纯度。如果棉花不从笼取出，从较早的时间点幼虫的可能性将保持并进入新鲜板增加。即使当棉被删除，在时间幼虫将徘徊在笼的侧面和收集过程中输入的食物。 为3小时的连续集合，产量介于840至1250毫克，是绕比在1小时的集合获得的产率的10倍以上。这些胚胎的纯度为接近100％（ 图3B）。偶尔幼虫进行观察。有些发育研究需要收集的胚胎非常严格同步。为了增加同步纯度，建议丢弃第一收集板，因为如果条件不理想，女性可以留住更多的成熟卵子和沉积m如果条件的改善，如引入新的板块。同样重要的是要知道，旧的成蝇（&gt; 6天）产生更少的同步胚胎。为了验证以高精度同步的程度，建议收集胚胎的DAPI染色。 <img alt="图1" src="/files/ftp_upload/53756/53756fig1.jpg" /> 图1. 果蝇的 生命周期在飞行人口凯奇（A）的循环开始播种1.5克胚胎放入塑料盒容器。作为参考，在下部面板，滤纸的长度为8.5厘米（B）的幼虫生长周期的开始后第5天相对同步。 （C）在第8天，大部分的苍蝇都在蛹阶段。在B（B&#39;）和C的下面板<html> （C&#39;）是在上面板所指示的区域的放大倍数。 （D）的成人蝇从循环开始后10天蛹出现。 （E）成人拥有人口笼内开始收集胚胎的过程中，在15天的淡黄色材料对飞食品之前是受精卵。 （F）胚胎（和一些幼虫）在100粗筛的底部收集，整笼周期之后， <a href="https://www.jove.com/files/ftp_upload/53756/53756fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53756/53756fig2.jpg" /> 图2.整个周期收集日程安排。图2示出了一个完整的循环集合建议的时间表。<html> T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53756/53756fig3.jpg" /> 图3. 1小时和3小时连续集合纯度。在1小时（A）和增加食品的新盘3小时后（B）收集胚胎的形象代表。 <a href="https://www.jove.com/files/ftp_upload/53756/53756fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table><tbody><tr><td> 循环次数 </td><td> 产量（g） </td></tr><tr><td> 1 </td><td> 10.3 </td></tr><tr><td> 2 </td><td> 9.5 </td></tr><tr><td> 3 </td><td> 10.3 </td></tr><tr><td> 4 </td><td> 12.7 </td></tr><tr><td>五</td><td> 10 </td></tr><tr><td> 6 </td><td> 7.1 </td></tr></tbody></table>ntent"FO:保持-together.within页="1"&gt; 表1.在 一些周期类别的产率 如表1所示的6个连续循环集合的产量，与1.5克的起始材料。在每个循环中，最后一个。 13天加入食品的托盘和收获物在第15天进行。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 集合＃ </td><td> 收集的长度（小时） </td><td> 产量（毫克） </td></tr><tr><td> 1 </td><td> 1 </td><td> 14.5 </td></tr><tr><td> 2 </td><td> 1 </td><td> 21.6 </td></tr><tr><td> 3 </td><td> 1 </td><td> 56.1 </td></tr><tr><td> 4 </td><td> 1 </td><td> 160.1 </td></tr><tr><td>五</td><td> 1 </td><td> 106.1 </td></tr><tr><td> 1 </td><td> 3 </td><td> 960 </td></tr><tr><td> 2 </td><td> 3 </td><td> 840 </tD&gt; </tr><tr><td> 3 </td><td> 3 </td><td> 1250 </td></tr></tbody></table> 表2的1小时和3小时连续类别产量的影响 。 表2示出的连续5集合1小时放置一个新的糖蜜板之后加入新的食品和连续3集合3小时之后的产率。

Watch this Scientific Journal Video about Maintenance of a Drosophila melanogaster Population Cage at JoVE.com

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

maintenance-of-a-drosophila-melanogaster-population-cage

Research

JoVE Journal

Biology

Maintenance of a Drosophila melanogaster Population Cage

13.1K 次观看.  National Institutes of Health. This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

视频: Maintenance of a Drosophila melanogaster Population Cage

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...