本文是由美国国立卫生研究院拨款，以耶。我想感谢亚历戈特沙尔克博士提供胆碱能运动神经元在录音示威中所使用的转基因表达渠道的视紫红质的蠕虫。

A.准备清扫工具。 <ol><li>解剖/录音室：我们通常构造我们的录音室，一个1 / 16英寸磁板到大到足以容纳一个直径为22毫米的圆形玻璃盖的中心，这是钻了圆孔，。香港总商会的外部尺寸将取决于用来做录音显微镜阶段。在磁板的背面，我们重视一个48 × 60毫米盖玻片，我们坚持低熔体Paraplax组织包埋蜡颗粒放置在盖的每个角落室滑夹在玻璃和商会之间的。蜡，然后融化在热板，玻璃板朝上，直到它形成一个连续的层粘合盖玻片室。不要离开这一步无人值守的蜡融化迅速。被删除，冷却室应尽快蜡融化。使用刀片边缘，可以去除多余的蜡，其上有22毫米的圆形孔洞的内缘渗透。 </li><li> Sylgard涂层的盖玻片：22毫米，是放置在录音室的圆形空腔的Sylgard涂层盖玻片上进行剥离。 Sylgard大衣有助于坚持cyanoacrylic胶盖滑时胶合蠕虫，也作为一个气垫上打破胶枪头当它成为插入的行为。 Sylgard是由新鲜根据制造商的说明（10份有机硅基地，1份固化剂按重量计）。一个Sylgard小降，然后放置在每个盖玻片和整个使用刀片边缘，使表面均匀抹上。涂盖玻片放置在大型平板盖的容器中，并在65 °度的烤箱一夜之间治愈。 </li><li>移液器：通常情况下，我们使用相同的1毫米外直径长丝硼硅玻璃产生胶合蠕虫下降移液器，使肌肉从角质层的切口和录音，使用一个电极的车夫。明智的做法是拉，然后再尝试一个夹层，这个阶段可能需要几个快速交流的移液器一罐的移液器。 </li><li>胶机：胶是胶水吸管尖口压力，加载和应用。胶合吸管插入一块相匹配的吸管玻璃（通常为1毫米）外径与内径的聚乙烯管材的2脚的一端。油管的另一端持有的Eppendorf枪头，作为一个口片。 </li><li>提取移液器：必须从蠕虫腔移除蠕虫内脏，一旦角质层切口已取得。这是实现与油管连接到一个小指枪头类似胶机，但在这种情况下玻璃提取枪头被打破，足以让蛋和内脏，容易吸尘蠕虫腔。 </li></ol> B.剥离<ol><li>在录音室中绘制一个圆形的蜡笔线接近的一个Sylgard涂层的盖玻片压在坚决室（Sylgard涂层朝上）的周长。这蜡线无法移动盖玻片，防止渗下撞出解剖和录制时的盖玻片的录音解决方案。会议厅内充满了录音解决方案外，一些蠕虫在使用蠕虫挑中心放置。蠕虫会在溶液中游泳，但是与实践，这些蠕虫可以粘下来没有任何进一步的程序，如冷却，以防止运动。在训练阶段，它可以帮助实践胶合突变，其中游泳是受损的蠕虫（如UNC - 31突变体）。 </li><li>古板，蜡或黏土举行的PCR第一个胶容器是用来举行cyanoacrylic胶股票。胶类似的提示尺寸录音移液器吸液管（通常〜4兆欧电阻）用于申请胶水。使用胶机，少量的胶水的胶吸管吸，用解剖的范围，以形象化的枪头胶水是吸了。 </li><li> cyanoacrylic胶（HistoacrylBlue，Aesulap）聚合接触外录音解决方案，因此重要的是要保持正压上胶吸管，因为它进入会议厅的解决方案，以防止胶水硬化和堵塞吸管。只要在会议厅内的解决方案是胶吸管，小，但络绎不绝的胶水需要应用到Sylgard表面下口压力控制，以防止堵塞。如果插入吸管变成它有时可以轻轻拍打sylgard镀膜玻璃的一角畅通。建议你练习，然后再尝试粘合胶蠕虫。 Sylgard上的胶，如果你能经常写你的名字与线比蠕虫宽度薄的信件，你准备PROCEED到蠕虫角质层粘合。 </li><li>粘上一种蠕虫病毒，使用正压蠕虫的头部或尾部附着少量的胶水，并迅速Sylgard表面上绘制蠕虫。试图附加商会中心附近的蠕虫病毒，蠕虫粘太靠近边缘，将很难达到录音移液器。然后，应用胶沿背侧缘蠕虫的角质层开始附件络绎不绝，迫使与所面临的外来外阴（腹侧肌肉录音）的C位置的蠕虫病毒。在这种胶线的任何空隙，所以应填写该蠕虫强烈重视，在角质层切口步骤分离，以防止蠕虫。 </li><li>为了使角质层切口，切换到手持式清扫吸管。这种吸管需要比胶水/录音移液器更清晰，有一个短的，坚固的轴。解剖范围内使用的最高放大倍率，对准吸管蠕虫的纵轴平行，约沿切口在角质层/胶水界面（外阴）蠕虫的中点插入的提示。这个切口，释放蠕虫的静水压力，迫使通过切口点鸡蛋和肠。继续对蠕虫的头切割切片类似的议案，打开信的角质层，直到您到达咽。切换到提取吸管真空机关内部的蠕虫。打开角质层清除切口后，将保留它的圆柱形状。使用新鲜的粘合吸管点焊到Sylgard表面的角质层切边。重要的是，在这个阶段中使用最少的胶点，以避免损坏或模糊的NMJs。 腹侧神经线和体壁肌肉，现在暴露出来。 </li><li>要删除基底膜覆盖的NMJs，从腔吸外的录音解决方案，并申请胶原酶（外录音解决方案0.4mg/ml）约10-20秒;删除，并用新鲜外录音解决方案几次。进入录制阶段。 </li></ol> C.膜片钳记录<ol><li>位置上升显微镜10倍的目标，中心蠕虫阶段的录音室。切换到一个40倍的水，再现与DIC的目标和检查，体壁的肌肉和神经线都完好无损。蠕虫的位置，使蠕虫的纵轴是与前边缘的范围平行，使电极的左，右两侧推出。 </li><li>使用标准的膜片钳技术来修补肌肉。约4兆欧电阻的记录电极工作。一旦电极接触的肌肉膜，增加负压，直到获得一个千兆欧密封。对于全细胞电压钳记录模式，适用于更多的吸力和ZAP膜破裂，直到千兆欧密封。在一个良好的记录，通常有野生型成虫体壁肌肉细胞膜的〜70 pF的电容和可稳定在-60以最小的控股电流（〜50 mV的控股潜力至少10分钟的记录PA）。 </li><li>外的录音解决方案，包括（毫米）：2 5 150氯化钠，氯化钾，氯化钙，氯化镁 2 4 5，蔗糖，葡萄糖10，HEPES 15（pH值7.3〜330mOsm ）。包含的修补程序吸管（毫米），氢氧化钾20 120氯化钾，氯化镁2 4 5（的N -三羟甲基] mthyl - 2 - aminoethane磺酸）工商业污水附加费，二水氯化钙 0.25，NA 2 ATP 4，蔗糖36 5，EGTA（pH值7.2〜312 mOsm）。注：这些解决方案人为负载氯化肌肉细胞，使GABA受体介导的电流向内由于氯在保持电位-60 mV的外排。这些解决方案的变化，已使用（详情请参阅出版物）。 </li><li>诱发释放。通常我们把第二个电极包含腹侧神经脊髓前修补肌肉细胞外液。这种松散的补丁配置需要大量的去极化刺激持续时间短（10S伏）（1毫秒），以达到最大的诱发反应（通常为2.0-2.5 NA）在野生型蠕虫。这种方法的局限性是：1）只有少数诱发反应可交付前的神经线造成不可挽回的损坏，原因不明2），只胆碱诱发的反应可以引起使用这种刺激的配置。 </li><li>光激活通道的视紫红质的刺激。最近，戈特沙尔克实验室已经产生了转基因蠕虫表达藻类衍生的通道视紫质，光刺激后的膜去极化神经元亚群，用于驱动表达4的启动子上。在的C.线虫 NMJ，表达的胆碱能运动神经元或GABA能运动神经元通道的视紫红质的综合线现在availablE.由于通道的视紫红质只响应光时，全反式视黄醛与视蛋白基团，用于此应用程序的蠕虫，必须对视网膜股价的细菌生长。一个绿色的光脉冲，然后可以用来反复激活通道的视紫红质，造成诱发ACH或GABA的释放，这取决于使用转基因蠕虫。这些转基因的蠕虫可以越过边界进入突变株。这比以前的电刺激技术的优点包括：1）光激活不引起神经元的损伤，使许多重复的刺激要执行和2）诱发释放要么GABA能和胆碱能运动神经元可独立研究。 </li><li>高渗诱导释放。要测量的很容易，可释放囊泡池（反映的催芽囊泡总结所有到的记录肌肉NMJ的池），一个压力，吸管含高渗外解决方案，通过添加额外的蔗糖〜850 mOsm使用的大小。 1-3秒的压力脉冲应用，产生突触后使用上述标准的解决方案的异步事件接二连三。 </li></ol><img src="/files/ftp_upload/old/1165_2009_1_22_3/figure1.png" alt="" width="600" height="449" /> 图1：原理概述了C.线虫解剖和神经肌肉记录配置 一个sylgard涂涂抹隔着玻璃和固化Sylgard下降盖玻片一夜之间被放置在圆形空腔预制录音室与录音解决方案涵盖。在中心放置了一个蠕虫病毒是从头部或尾部，蠕虫的背侧施加胶，用胶吸管，粘Sylgard表面。刷胶是完成后，毗邻外阴切口，在接口之间的角质层和胶水是用玻璃针和扩展向头部（见红穿孔线）。提取内脏和体腔的鸡蛋，切口切缘是现场焊接用胶水Syl​​gard露出腹侧神经（绿色）和体壁肌肉（红色）。外阴前腹内侧肌肉补丁钳位和刺激电极放置在上游的神经线。 代表性的成果<img src="/files/ftp_upload/1165/Reprensentative.png" alt="Represenative图" width="600" height="404" />全细胞电压钳位体壁肌肉录音显示每个刺激（电诱发，光诱发高渗反应）上文所述的三个协议。在所有情况下的腹内侧的体壁肌肉被钳制在-60 mV的。 A.野生型的肌细胞通常表现出内源性抵港的缩影，在我们的标准录音解决方案的突触电流的高利率。 B.一个2ms的去极化的前腹神经索的原因造成约2-2.5 nA的一个大诱发的反应，在野生型蠕虫的几个突触同步发行。野生型的痕迹权的代表在A和B的痕迹，显示在严重不协调的突变（UNC - 18）的突触释放。 C组显示了从野生型蠕虫，胆碱能神经元的表达通道的视紫红质的神经肌肉交界处的光诱发反应。 2ms的光脉冲，通常会产生1.5-1.8 nA的响应。 D.压力弹射到1秒的肌肉高渗盐水850 mOsm，生产的微型突触电流囊泡容易松解池的异步攻势。

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	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Richmond, J. E., Davis, W. S., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Neurosci. 2 (11), 959-959 (1999).</li><li>Richmond, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. 1, (2006).</li><li>Liewald, J. F., Brauner, M., Stephens, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature methods. 5 (10), 895-895 (2008).</li><li>Lockery, S. R., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in enzymology. 293, 201-201 (1998).</li><li>Brockie, P. J., Mellem, J. E., Hills, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 31 (4), 617-617 (2001).</li><li>Richmond, J. E., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Neurosci. 2 (9), 791-791 (1999).</li></ol>

遗传模式生物，线虫是非常适合的突触传递的研究，通过突触蛋白编码基因的突变在功能分析。在这里，我们描述了一个夹层技术，呈现了C.线虫 NMJs电生理分析访问。影随行描绘了C.的关键步骤线虫的解剖和典型的录音配置，用于测量突触活动。在学习这一技术带来的最大挑战的三个步骤; 1）蠕虫胶合一步，2）执行一个成功的切口，3）体壁肌肉的修补。一旦这些已经?...

dissecting and recording from the c. elegans neuromuscular junction

神经递质是其中神经元突触后的目标，快速的时间尺度上，通过化学信号传输的信息的过程。这一复杂的过程，需要许多前和突触后蛋白的协调活动，以确保合适的突触连接，电信号的传导，目标和分泌泡，钙传感，囊泡融合，本地化和突触后受体的功能的吸，最后，​​回收机制。作为神经学家，它是我们的目标是澄清在其中的每个步骤功能的蛋白质，并了解他们的行动机制。从突触的电生理记录提供一个量化的基础突触传递过程中发生的电气事件读出。通过结合这项技术可用于处理C.突触蛋白的分子和遗传工具，强大的阵列线虫 ，我们可以分析导致突触传递功能的变化。 c. 在线虫 NMJs之间形成运动神经元和体壁的肌肉控制运动，因此，不协调locomotory表型（UNC小号）的突变体，往往扰乱这些突触1突触传递。由于UNC突变体的一种细菌的食物来源丰富的供应维持，他们依然是可行的，只要保留一些咽抽水能力摄取食物。这一点，连同事实，C 。 线虫为雌雄同体的存在，使他们能够通过突变后代，而不需要精心设计的交配行为。这些属性，再加上我们最近从蠕虫记录NMJs 2,3,7这是一个很好的模式生物来解决如何准确 UNC突变影响神经递质的能力。 解剖的方法包括使用cyanoacrylic胶，以使切口暴露在蠕虫的NMJs角质层固定成虫。由于C。线虫成年人只有1毫米的长度进行解剖与解剖显微镜的使用，需要良好的手眼协调。 NMJ录音是由全细胞电压钳位个人体壁肌肉细胞和神经递质的释放，可以使用各种刺激的协议，包括电疗，光激活通道的视紫红质介导的去极化4和高渗盐水，所有这些都将是诱发简要描述。

Watch this Scientific Journal Video about Dissecting and Recording from The C. Elegans Neuromuscular Junction at JoVE.com

Dissecting and Recording from The C. Elegans Neuromuscular Junction

电的应用访问突触的突触活动提供量化的测量，分析突触的突变体。本文介绍了一种夹层的方法，用来揭露神经肌肉接头（NMJ）<em&gt;线虫（线虫）</em并简要讨论了一些可用于本制剂的使用。

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

dissecting-and-recording-from-the-c-elegans-neuromuscular-junction

Research

JoVE Journal

Biology

15.5K 次观看.  University of Illinois, Chicago. 电的应用访问突触的突触活动提供量化的测量，分析突触的突变体。本文介绍了一种夹层的方法，用来揭露神经肌肉接头（NMJ）线虫（线虫）

视频: Dissecting and Recording from The C. Elegans Neuromuscular Junction

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...