从的帕迪拉实验室成员的意见和评论，我们想表达我们的感谢。线虫株在这项工作中所提供的秀丽隐杆线虫的遗传学中心，这是由美国国立卫生研究院国家研究资源中心（NCRR）。我们承认并感谢经度特恩布尔博士激光共聚焦显微镜技术援助。这项工作已经得到了美国国家科学基金会（NSF-IOS，职业）的资助PAP

1。样品制备<ol><li>生产或获得适当的转基因C.线虫应变计用转基因的方法，或从秀丽隐杆线虫基因联合中心（CGC），或同事。在这种情况下，我们使用的菌株TH32（ 馅饼1 :: TBG-1 :: GFP饼1 :: GFP :: H2B）5可视化标记细胞分裂染色体和中心体。 </li><li>生成一个同步的人口使用以下三种方法之一：1）瓦解妊娠成人次氯酸钠溶液，并使用剩余的胚胎，2）种子盘上挑妊娠的成年人，让他们打下的胚胎1-2小时，或3）挑L4幼虫从混合的人口和移动到一个新的种子板。 </li><li>成长线虫在20°C至妊娠年轻的成年，这将需要约96小时孵化，从L1幼虫的72小时或24小时后L4蜕皮。 在子宫内的胚胎（2-20芯）含有大量的卵裂球核是最佳的成像子细胞和亚核的变化，分别。这种方法提供了一种手段来提供的青壮年人口早期胚胎，含有丰富的。 </li></ol> 2。设置幻灯片的制备及缺氧商会<ol><li>一个的湿热试验箱持有预制玻片，放置在一个大培养皿或垃圾桶的盖子覆盖着相当规模的湿纸巾。 </li><li>准备熔融的2％琼脂糖在去离子H 2 O，通过加热在玻璃器皿的混合物通过微波或水浴。 </li><li>将1-3滴在干净的玻璃载玻片温暖的琼脂糖。立即放置一个第二滑动翻转顶部琼脂糖滴（）垂直，按略有，使所得焊盘是薄的和不含气泡。 </li><li>等待一段时间，冷却，慢慢分开的两个显微镜玻片上，留下一个完整的幻灯片的琼脂糖凝胶垫。重要的是要确保琼脂糖垫是足够薄，不interfe重新聚焦显微镜的能力。能力，可视化样本也是依赖于使用中的目标的特定的光学距离之间的显微镜，它可以改变。 </li><li>使用干净的刀片塑造成一个小正方形琼脂糖凝胶垫。储存在准备湿热试验箱，直到准备使用。 </li><li>仔细，使用相同的清洁刀片，采取琼脂糖垫的边缘，并将其转移到一个圆形的25 mm微型盖玻片从滑动，避免垫下方的气泡的积累。选择使用适合的圆形玻璃罩适当在microincubator。 </li><li>添增下拉到琼脂糖垫作为麻醉剂使用的麻醉剂（0.5％三卡因，在M9缓冲液中的0.05％四咪唑）。选择5-10蠕虫和地方麻醉剂的下降。允许1-3分钟蠕虫停止运动。 </li><li>成人的子宫内，观察胚胎代替解剖胚，的理由是，以减少潜在的胚胎desiccaTION和由于氮气流横跨胚胎运动。 </li><li>立即加一滴卤烃油在上面的蠕虫病毒，防止蠕虫脱水，气体流过腔。由于卤烃油是粘性的，可以用一个的枪头或尖端的蠕虫挑到的蠕虫收集石油和下降的。 </li><li>含有蠕虫的圆形玻璃罩一旦准备就绪后，莱顿的两半封闭的灌注microincubator是分离的，盖玻片，然后小心地直立放置到底环。双方室紧紧地握在一起，然后关闭。 </li><li>然后将腔室连接到氮气罐经由柔性塑料管，并放置到适当的位置上的显微镜载物台（ 图1C）。在这个时候管和microincubator的应仔细检查是否有泄漏的油管和会议厅一侧的端口插头沿确保安全的依恋。人们可以AL如此轻率地散布少量的肥皂水，看是否有气泡，如果存在泄漏，这将形成管。 </li></ol> 3。显微镜在缺氧<ol><li>定位在低倍率的动物，然后，移动到合适的更高的放大倍率（此应用程序的64倍），图像的兴趣，如在成人的胚胎卵裂球或卵母细胞的组织或细胞。打开的488 nm的激光，以查看GFP信号和定位GFP融合蛋白在细胞中（s）的权益。 </li><li>阻滞细胞周期阻滞在特定阶段的可视化（ 如下旬前期）确定一个卵裂球在前一阶段的细胞分裂（ 例如，后期相间或早期前期）。如中心粒的位置，染色体缩合开始，染色体上的位置和核膜的存在或不存在的细胞标记将帮助确定细胞周期阶段（ 图2A）。 </li><li>用氮气（99.999％的腔室，然后灌注N 2 &lt;2 ppm的O 2）。在这种情况下，我们使用了一个6 psi的压力，但是压力可能需要根据每个microincubator大小和在使用的管子的直径进行调整和优化。继续监测卵裂球（S）的氮充满室，调整焦平面必要的。 </li><li>在这一点上，已经开始的时间推移成像。成像时，只要有必要进行捕获感兴趣的现象，在这种情况下前期逮捕和对接核膜内的染色体。图像不超过30分钟，每10秒一次。的图像捕获和设置，例如增益和激光功率的频率应根据需要调整。 </li><li>取决于缺氧曝光后对细胞周期的阶段，在该时间内的胚胎回应缺氧环境可以改变。通常情况下，缺氧诱导前期逮捕发生在20-30分钟开始氮气流。我们观察到缺氧诱导细胞周期阻滞occuRRING于&lt;20分钟。的图像可以在这段时间帧的方式获得，或可以分离出特定的图象，从时间推移系列购买。另外一个选择是使用视频显微镜胚胎中的亚细胞结构可视化。 </li><li>要记录从缺氧诱导被捕的状态恢复，气体被关闭和室被允许返回到常氧的扩散，同时记录时间的推移系列。恢复出坞染色体的细胞周期进程，通常发生在5-20分钟的氮气。 </li><li>请注意，在另外的实验中，一个缺氧指示器，刃天青，被安置在流体可通过的微容器，它被确定腔室变得缺氧氮气气流已经开始后约19分钟内的平均。 </li><li> Imaris，图像J或Photoshop处理图片和视频导入QuickTime显示。 </li></ol>

<ol>
	<li>Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+stress+in+Caenorhabditis+elegans.">Longevity and stress in Caenorhabditis elegans.</a> Aging (Albany NY). 3, 733-753 (2011).</li><li>Schmidt, D. J., Rose, D. J., Saxton, W. M., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cytoplasmic+dynein+heavy+chain+in+Caenorhabditis+elegans+with+fast-acting+temperature-sensitive+mutations.">Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations.</a> Mol. Biol. Cell. 16, 1200-1212 (2005).</li><li>Sulston, J., Hodgkin, J., Wood, W. 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> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, 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=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. 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没有利益冲突的声明。

缺氧和假死虽然暴露于严重缺氧可能是致命的一些生物，有些生物能够生存暴露于缺氧。在的情况下的 C 线虫 ，缺氧的生存依赖于发育阶段，对缺氧的反应是一个可逆的假死状态进入观察到的生物过程中，一些被逮捕。细胞分裂，发展，运动，饮食和生殖过程停止，直到的氧气重新引入环境7,9。缺氧暴露的胚胎，细胞周期进程，可逆逮捕相间，下旬前期或...

<table border="1"><tbody><tr><td> 试剂/设备 </td><td></td><td></td><td> 组成 </td></tr><tr><td>次氯酸钠溶液</td><td></td><td></td><td> 0.7克KOH，12毫升5％的次氯酸钠，把50毫升DDH 2 0 </td></tr><tr><td> M9缓冲</td><td></td><td></td><td> 3克KH 2 PO 4，11克Na 2 HPO 4，5克NaCl，1毫升（1 M）每1L DDH 2 O MgSO 4干燥 </td></tr><tr><td>显微镜幻灯片</td><td> Fisher Scientific则</td><td> 12-550-343 </td><td> 3“×1”×1.0毫米</td></tr><tr><td>圆形微盖玻片</td><td>电子显微镜科学</td><td> 72223-01 </td><td> 25毫米直径</td></tr><tr><td>卤烃油700 </td><td>西格玛</td><td> H8898-100ML </td><td></td></tr><tr><td>麻醉剂</td><td></td><td></td><td> 0.5％TRICAINE，0.05％四咪唑</td></tr><tr><td>莱顿关闭灌注Microincubator </td><td>哈佛设备</td><td> 650041 </td><td></td></tr><tr><td> UHP氮气</td><td> Calgaz（法液空） </td><td></td><td> &gt; 99.9990％N 2 &lt;2 ppm的O 2 </td></tr><tr><td>塑料管</td><td> VWR </td><td> 89068-468 </td><td> 0.062“（内径）×0.125”OD </td></tr><tr><td>旋转盘共聚焦显微镜</td><td>麦克贝恩系统</td><td></td><td>蔡司倒置光学显微镜，荧光显微镜照明系统，CSU-10横河电机株式会社滨松共焦扫描仪，电子倍增CCD相机。 </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

已caenorhabdits线虫广泛使用在研究的胁迫抗性的成人和胚胎阶段的透明度，以及由遗传突变体和转基因株表达融合蛋白1-4无数的可用性，这是便利的。此外，可以被视为使用荧光标记的记者蛋白的动态过程，如细胞分裂。有丝分裂的研究，可以促进通过使用时间推移实验在各种系统中，包括完整的生物体，因此早期C.线虫的胚胎非常适合这项研究。这里介绍的是一种技术，其中在响应于缺氧（纯度为99.999％的N 2的 &lt;2 ppm O 2） 在体内成像的亚细胞结构应力是可能使用一个简单的气体流通过安装在一个高功率的显微镜。 ，甲microincubation室中使用结合用氮气流过和纺丝盘共聚焦显微镜创建一个受控的环境，在该环境中的动物可以在体内成像。用GFP标记的伽玛微管蛋白和组蛋白的动力学和逮捕的细胞分裂，可监视之前，期间和之后暴露于缺氧的环境。这种技术的高分辨率，详细的视频和图像的细胞结构内暴露于缺氧的胚胎卵裂球的结果。

线虫胚胎暴露于严重缺氧（缺氧）是能够生存下去，逮捕生物过程，包括发展和细胞分裂的7。细胞分裂的缺氧诱导逮捕可以被监测，在亚细胞水平，通过用氮气流过和纺丝盘共聚焦显微镜结合使用microincubation室创建一个受控的环境，在该环境中的动物可以在体内成像（ 图1）。蜂窝结构可以监视在胚胎暴露于各种气体的环境中，在这种情况下，我们用GFP标记的?...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

这里描述的是一个<em在体内</em&gt;技术图像亚细胞结构在动物中使用的气体流通过在纺丝盘共聚焦显微镜连同microincubation室暴露于缺氧。这种方法非常简单，具有足够的灵活性，以适应各种实验参数和模型系统。

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

use-time-lapse-microscopy-to-visualize-anoxia-induced-suspended

Research

JoVE Journal

Biology

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

11.5K 次观看.  University of North Texas. 这里描述的是一个

视频: Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Monitoring Actin Disassembly with Time-lapse Microscopy

拆装时间推移显微镜监测肌动蛋白

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...