原设计的扭矩补偿的起源与卡尔戈茨。在此演示文稿中的特定的设置是对贷款在很大程度上，最初是由马丁海森堡和莱因哈德狼开发。我特别感谢他们的继续支持，鼓励和专业知识，这两种人。

飞介质飞食品的成分，是学习的关键（郭等，1996）。 <ul><li>水1000毫升</li><li>玉米面180克</li><li>大豆10克</li><li>酵母18.5克</li><li>琼脂7.5克</li><li>糖蜜40克</li><li>糖浆（甜菜）40克</li><li>尼泊金2.5克</li></ul>每一小瓶是提供一个新鲜的，活的酵母膏民建联和滤纸提供了一个额外的苍蝇和蛹表面。 苍蝇繁殖和分期下面的过程是每天执行，导致精确地上演在适当的密度种植的动物。所有新ecclosed的苍蝇，因为就在前一天的最后一个步骤是收集繁殖和实验。最古老的小瓶无任何遗留的生活蛹被丢弃。四天老苍蝇被添加到一个新鲜瓶过夜产卵。雌蝇密度应约20每个小瓶，小瓶的大小调整和使用的应变。理想的密度是一个足够高的飞中型液化在幼虫期和足够低，这样，所有的幼虫前第一苍蝇ecclose pupated。前一天的产蛋苍蝇拆除和丢弃。 飞行准备苍蝇标准玉米面/糖蜜介质保持在25以上所述° C和60％，一个12小时light/12hr的黑暗制度的湿度。在简要固定冷麻醉24 - 48H老苍蝇，苍蝇被粘一个三角形状的铜钩的头部和胸部（美国强力胶紫外线玻璃胶，505127A，步行者技术，Cucamonga，CA。）（直径0.05毫米实验）的前一天。然后单独保存的动物都含有蔗糖几粒小湿润商会过夜。 仪器设置的核心器件是转矩补偿（扭力​​计）（格茨，1964年）。它措施一只苍蝇绕其垂直体轴角动量，造成预定飞行演习。飞，粘在上面所述的钩，是连接通过一个夹具，扭力计，完成在一个圆柱形全景（舞台，直径58毫米），这是从背后均匀照明中心固定航班。光源是100W，12V的灯泡钨碘。对于绿色和蓝色的舞台上的照明，光线通过单色宽带柯达Wratten明胶滤镜（＃47和＃99，分别）。过滤器可以由0.1秒内快速电磁交换。另外，舞台上亮起了“日光”，通过一个蓝绿色过滤器（罗斯科“surfblue”号5433），或没有过滤器在所有。罗斯科的蓝绿色在这项研究中所使用的过滤器的透射谱相当于一个BG18过滤器（肖特，美因茨），构成柯达蓝色和绿色过滤器（Brembs和亨普尔 - 伊瓦拉，2006年之间的一个中间，刘等人，1999）。竞技场可旋转周围使用了计算机控制的电动机动态。在这样的“飞行模拟器”的局面，舞台上的角速度成正比，但对果蝇的偏航力矩（耦合系数K =- 11 ° / S•10 - 10NM）。这使飞行稳定的全景，并控制其角度方向。这虚拟的“飞行的方向”（即，舞台上的地位）是通过一个圆形的电位器（Novotechnik，A4102a306）连续记录。模拟到数字转换卡（PCL812;研华）送入计算机存储以供日后分析的痕迹（采样频率20Hz）到舞台上的地位和偏航力矩信号。申请可调红外激光（825纳米，150兆瓦），从后面和上面的指示，到果蝇的头部和胸部，的热量是通过处罚。激光束脉冲（约200ms的脉冲宽度〜4Hz的），其强度降低，以确保飞的生存。 实验模式学习对于传统的学习模式实验（Dill和海森堡，1995年，莳萝等，1993年，1995年;刘等，2006;刘等，1998;刘等，1999年，狼和海森堡，1991年） ，均匀分布在舞台上墙（模式宽度ψ= 40 °，高度θ= 40 °，酒吧宽度= 14 °，看到四个黑色，T形交替方向模式（即，两个一身正气，两个倒）从飞行的位置）。一种计算机程序，分为4个虚拟90 °象限的舞台上的360 °，其中中心表示的模式。控制苍蝇的角位置与它的偏航力矩（飞行模拟器情况）的模式。在培训过程中，热的惩罚是连续出现在正面的视野格局方向之一。强化每个模式总是扳平组内。在测试过程中，热量是永久关闭和记录飞行的模式偏好。 颜色学习如前所述，颜色学习（Brembs和海森堡，2000; Brembs和亨普尔 - 伊瓦拉，2006年; Brembs和维也纳，2006年，狼和海森堡，1997年）。舞台上是划分成四个虚拟90 °象限，其中心是由四个相同的竖条纹（宽ψ= 14 °，高度θ= 40 °）表示。苍蝇控制的四个相同的条纹其偏航力矩的T形图案，上面所述的角度位置。一个虚拟象限边界时通过一个点在前面飞，整个舞台照明的颜色改变。在培训过程中，热处罚是由两种颜色之一的队伍。在测试过程中，热量被永久关闭，并记录果蝇的色彩偏好。当然，颜色可以结合复合空调“（Brembs和海森堡，2001）的模式。 偏航扭矩学习如前所述（Brembs和海森堡，2000年海森堡和Wolf，1993），执行偏航扭矩学习。分为“左”和“右”的域，大约对应的左边或右边轮流的果蝇的自发偏航扭矩范围。有在舞台上墙没有模式。在训练期间，热应用每当飞的偏航力矩是在一个域和关闭时的扭矩传递到其他。在测试阶段，热是永久关闭和果蝇的偏航力矩域选择记录。 综合学习综合学习的偏航力矩学习的延伸，前面描述的“（Brembs和海森堡，2000）。基本上，偏航力矩学习和色彩的学习相结合，相当于操作性（偏航扭矩）和经典（颜色）的预测实验。在培训过程中，飞被加热时，飞的偏航力矩传递到处罚相关的域名。每当飞开关偏航扭矩域，不仅耐高温，而且舞台上的色彩改变（从绿色变为蓝色或反之亦然）。因此，偏航力矩域和色彩作为热量相当于预测。在测试阶段，热是永久关闭，只苍蝇的偏航力矩域/颜色的选择是记录。 讨论这个实验装置，结合先进的遗传模式生物实验的情况下，高超的控制。使用在此演示文稿中所描述的程序，各种行为特征的分子和神经生物学的基础，可以进行调查，包括但不限于，自发行为的产生机制，操作性和古典空调，模式识别，色觉或过程控制。

<ol>
	<li>Guo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila;+Dependence+on+age,+practice+and+diet.">Conditioned visual flight orientation in Drosophila; Dependence on age, practice and diet.</a> Learn. Mem.. 3, 49-59 (1996).</li><li>Goetz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optomotorische+Untersuchung+des+visuellen+Systems+einiger+Augenmutanten+der+Fruchtfliege+Drosophila.">Optomotorische Untersuchung des visuellen Systems einiger Augenmutanten der Fruchtfliege Drosophila.</a> Kybernetik. 2, 77-92 (1964).</li><li>Liu, L., Wolf, R., Ernst, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+in+Drosophila+visual+learning+requires+the+mushroom+bodies.">Context generalization in Drosophila visual learning requires the mushroom bodies.</a> Nature. 400, 753-756 (1999).</li><li>Brembs, B., Hempel de Ibarra, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+parameters+support+generalization+and+discrimination+learning+in+Drosophila+at+the+flight+simulator.">Different parameters support generalization and discrimination learning in Drosophila at the flight simulator.</a> Learn. Mem. 13, 629-637 (2006).</li><li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+memory+traces+for+two+visual+features+in+the+Drosophila+brain.">Distinct memory traces for two visual features in the Drosophila brain.</a> Nature. 439, 551-556 (2006).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+organization+of+operant+behavior+as+revealed+in+Drosophila+flight+orientation.">Basic organization of operant behavior as revealed in Drosophila flight orientation.</a> J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol. 169, 699-705 (1991).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+visual+flight+orientation+in+Drosophila+melanogaster+abolished+by+benzaldehyde.">Conditioned visual flight orientation in Drosophila melanogaster abolished by benzaldehyde.</a> Pharmacol Biochem Behav. 61, 349-355 (1998).</li><li>Dill, M., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+memory+without+shape+recognition.">Visual pattern memory without shape recognition.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 349, 143-152 (1995).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+pattern+recognition+in+Drosophila+involves+retinotopic+matching.">Visual pattern recognition in Drosophila involves retinotopic matching.</a> Nature. 365, 751-753 (1993).</li><li>Dill, M., Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+analysis+of+Drosophila+landmark+learning+in+the+flight+simulator.">Behavioral analysis of Drosophila landmark learning in the flight simulator.</a> Learn. Mem. 2, 152-160 (1995).</li><li>Wolf, R., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Space+from+Visual+Motion:+Turn+Integration+in+Tethered+Flying+Drosophila.">Visual Space from Visual Motion: Turn Integration in Tethered Flying Drosophila.</a> Learn. Mem. 4, 318-327 (1997).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+operant+and+the+classical+in+conditioned+orientation+in+Drosophila+melanogaster+at+the+flight+simulator.">The operant and the classical in conditioned orientation in Drosophila melanogaster at the flight simulator.</a> Learn. Mem. 7, 104-115 (2000).</li><li>Brembs, B., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+generalization+and+occasion+setting+in+Drosophila+visual+learning.">Context generalization and occasion setting in Drosophila visual learning.</a> Learn. Mem. 13, 618-628 (2006).</li><li>Brembs, B., Heisenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+with+compound+stimuli+in+Drosophila+melanogaster+in+the+flight+simulator.">Conditioning with compound stimuli in Drosophila melanogaster in the flight simulator.</a> J Exp Biol. 204, 2849-2859 (2001).</li><li>Heisenberg, M., Wolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensory-motor+link+in+motion-dependent+flight+control+of+flies.">The sensory-motor link in motion-dependent flight control of flies.</a> Rev. Oculomot. Res. 5, 265-283 (1993).</li></ol>

The authors have nothing to disclose.

这个实验装置，结合先进的遗传模式生物实验的情况下，高超的控制。使用在此演示文稿中所描述的程序，各种行为特征的分子和神经生物学的基础，可以进行调查，包括但不限于，自发行为的产生机制，操作性和古典空调，模式识别，色觉或过程控制。

operant learning of drosophila at the torque meter

在扭力计的实验，苍蝇在飞中等标准保持在25 ° C和60％，一个12小时light/12hr的黑暗制度的湿度。一个标准化养殖制度，确保正确的幼虫密度和年龄相匹配的同伙。冷麻醉苍蝇粘一个三角形形钩实验前一天的头部和胸部。苍蝇的飞行演习，通过钳扭力计，测量绕其垂直体轴的角动量。苍蝇是摆在中心的圆柱形全景完成固定航班。模拟数字转换卡饲料偏航扭矩信号进入计算机存储以供日后分析跟踪。计算机控制的各种苍蝇的控制下，可通过关闭这些刺激和偏航力矩跟踪反馈回路带来的刺激。申请可调红外激光的热量是通过处罚。

Watch this Scientific Journal Video about Operant Learning of Drosophila at the Torque Meter at JoVE.com

Operant Learning of Drosophila at the Torque Meter

拴果蝇与扭力计测量偏航力矩，使得神经学家精致控制的实验动物的刺激情况。在果蝇中具有独特的遗传工具，用于各种各样的神经生物学研究范式。

果蝇在操作性学习扭力计

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

operant-learning-of-drosophila-at-the-torque-meter

Research

JoVE Journal

Biology

13.4K 次观看.  Free University of Berlin. 拴果蝇与扭力计测量偏航力矩，使得神经学家精致控制的实验动物的刺激情况。在果蝇中具有独特的遗传工具，用于各种各样的神经生物学研究范式。

视频: Operant Learning of Drosophila at the Torque Meter

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

重组工程同源重组构建<em&gt;果蝇</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...