薪金和研究的支持，提供由美国大豆委员会，由州和联邦基金拨美国俄亥俄州立大学/俄亥俄州农业研究和发展中心。这项研究部分由国家科学技术委员会，墨西哥，奖学金CMHG的支持。文中提到的商标或专利产品，并不构成了产品的担保或由俄勒冈州立大学/ OARDC保修，也并不意味着批准排斥其他产品，也可能是合适的。期刊论文的HCS 09-17。

以下方法概括为使用一个自动化的图像采集系统分析基因表达的形象的协议。为了便于说明，细分的总体思路是分为四个步骤：1）种子准备，2）基因导入用粒子轰击，3）机器人的图像采集，及4）图像分析。虽然这一般方法可用于范围广泛的其他应用程序使用，目前的协议集的基础上使用绿色荧光蛋白（GFP）的基因，这是一个有用的记者跟踪基因的表达基因在同一块随着时间的推移活组织。 一，种子的制备<ol><li>利马豆（ 菜豆lunatus CV恒基布什）的种子就可以买到，或生长的植物在生长室（相对湿度50％，16 / 8 h光照：黑暗，25/23 ° C昼/夜），理想收获。收获后的种子质量可保持在-20 ° C储存的种子</li><li>准备发芽的种子品红GA7容器。折叠滤纸或纸巾，以适应在洋红色箱底部。每个容器添加〜25毫升去离子水，滋润纸巾，并倒出未吸收的水。高压灭菌20分钟浸湿的纸的盒子。 </li><li>用50毫升的一次性离心管中，10％的商业漂白剂溶液在回旋筛晃动在60转20分钟（20种子在20-40毫升）的种子消毒。 注：所有的后续步骤，应在层流罩。 </li><li>每个冲洗过程中，轻轻摇动30秒，用无菌去离子水冲洗种子4-7次。 </li><li>将5-6位于每个无菌品红箱折叠纸层之间的无菌种子。 </li><li>在下列条件下孵育4天种子：25℃，16 / 8 h光照：黑暗和40μEm-2 s - 1时。 </li></ol>二。基因导入使用粒子轰击<ol><li>建立使用您最喜爱的表达载体可以在活体组织监测与报告基因的基因结构。我们已经设计了一个高拷贝数表达载体启动子和启动子元素分析的有用。这个载体含有绿色荧光蛋白基因，这是用来显示子/启动子元素转化的组织功能。 </li><li>大约1-2 h前的轰炸，消费利马豆萌发幼苗子叶和去除种皮。适合轰击对子叶应为黄色，浅绿色，平整和无任何损害，可能会干扰进一步的图像分析。 </li><li>广场上的OMS文化媒介包含的MS盐1，B5维生素2，3％蔗糖和0.2％Gelrite（pH值5.7）被切除后立即子叶。 </li><li>沉淀的DNA构造上M10钨粒子（西尔韦尼亚，托旺达，PA，USA）。在0.6 ml离心管中，加25μL钨颗粒（颗粒悬浮于权使用前，无菌水，100毫克毫升 -1），5μLDNA（1微克μL-1），25μL2.5米氯化钙（Sigma公司- Aldrich公司的猫。C3881 - 500G）和10μL100毫米亚精胺（西格玛猫S - 2626）。涡简要彻底混合所有的组件。 </li><li>冰浴5分钟的孵育DNA的制备。然后，删除和丢弃50μL上清液。 </li><li>由涡旋重悬DNA包覆颗粒，并立即删除2μL等份。重复此涡旋的步骤每一个等分是从管中删除的时间。在冰和包覆颗粒应保持在15分钟内使用。 </li><li>通过注射器过滤器的顶部，放置2μL包覆颗粒在滤网中。将包含在过滤器内的粒子枪室控股单位的包覆颗粒过滤装置。 </li><li>放置了利马豆子叶近轴侧挡板，并将其放置在粒子枪室挡板。挡板，融化到一个烧杯的底部屏幕组成，是用来作为一个平台，以支持组织在轰击。 </li><li>炮击使用粒子枪（在这种方法中，我们使用一个简单而廉价的粒子流入枪 3，猪，在我们的实验室设计）的组织如下：a）打开阀门导致的真空疏散室， b）在真空度达到（30）760毫米汞柱，启动电磁阀和释放氦气推动粒子，C）粒子轰击后，关闭真空阀门和使用排气阀释放真空。用来加速粒子的氦气压力是50psi。 </li><li>释放真空后，打开箱门，检索轰炸的子叶和子叶，近轴侧返回，OMS培养基。 注意：对于定量分析和表达分析，我们通常轰炸至少三个每个DNA构造子叶。阳性对照，这是通常的DNA构造，给出了一个研究基因的表达谱，作为参考。 </li></ol>三。自动图像采集<ol><li>打开汞灯，等待灯30分钟的热身。 </li><li>打开电源现货RT CCD相机（诊断仪器公司，斯特林高地，心肌梗死，美国）和机器人技术平台电机控制器，驱动机器人平台的运动。 </li><li>设置过滤器上设置一个MZFLIII解剖显微镜检测绿色荧光蛋白（GFP2过滤器设置;前四十分之四百八纳米，EM 510唱片。）（莱卡，Heerbrugg，瑞士）。 </li><li>用70％乙醇喷洒消毒增厚聚碳酸酯培养皿盖子。专门盖，用来盖培养皿基地防止凝结在图像采集4。 </li><li>载有关于机器人平台的自动图像采集系统（Arrick机器人），赫斯特，美国德克萨斯州的轰炸子叶板放置。设置由步进电机驱动的机器人平台和表包括8个培养皿中的附件。该平台是建聚碳酸酯，聚丙烯，铝。固定位置，固定螺丝拧紧外侧塑料板。 </li><li>打开自定义的应用软件，控制机器人平台和图像采集。此外，打开软件白光控制器开关。 </li><li>在“电机运动”标签，点击“电机”和“电机之家”。软件方向的起始位置的平台后，我们准备开始进入每子叶的位置。 </li><li>在“电机运动”标签，选择第一盘。该平台将显微镜的目标定位在培养皿的中心。使用移动距离按钮，精确定位图像采集的第一个子叶。随着白光控制器，可以观察到的目标区域，通过电脑显示器上的“直播模式”功能转向。 </li><li>对于第一个子叶，设置的重点和放大倍数（1.6倍的放大倍率是首选）使用解剖显微镜上的手动旋钮。在同一板块其余子叶的重点作出调整，三个找平每盘下方的螺丝，使用机器人平台上。 </li><li>一旦已设置感兴趣的区域，单击“添加这个位置”按钮。该软件将增加本地区的坐标位置的时间表文件，该平台将返回到该板块的中心。 </li><li>设置岗位和重点上的所有使用移动距离按钮和手动调平螺丝板剩余子叶。子叶的坐标已经输入后，保存的位置的时间表坐标。 </li><li>输入图像采集参数。在“图像设置”选项卡，选择所需的类型;白色或蓝色光。绿色荧光蛋白检测，只有蓝色的光芒，应使用。此外，进入曝光设置。虽然我们可以修改为红色，蓝色和绿色的色彩独立的曝光时间，我们通常使用相同的设置，使我们能够直接和GFP照片文档（20，14和红色，绿色和蓝色通道，分别为14秒）不同的DNA结构相一致的比较。 </li><li>在相同的“图像设置”选项卡，指定一个空的文件夹中的图像将被存储。每个一系列图像将被保存在这个独立的文件夹中的主文件夹，根据子叶进入阵地的命令按顺序编号。 </li><li>转到“捕获时间控制”选项卡，并设置图像采集的时间间隔和图像采集周期的总数。图片通常是收集每个小时为100小时，产生良好定义的基因表达谱。 </li><li>图像采集开始通过点击“运行时间表！”在位于“位置的时间表”标签的按钮。 </li><li>在100小时的图像采集，传输从进一步的图像分析任何大容量硬盘驱动器或计算机控制的机器人的计算机中的所有图像。高清晰度图像（1600 × 1200像素）收集为TIF文件测量〜5 MB每个。 </li></ol>四。图像分析<ol><li>组装100个连续的影像，从每个使用Adobe ImageReady中的文件夹。 <ol><li>打开ImageReady中导入所有的撰写一系列作为帧的序列图像。 </li><li>调整原始图像的大小为800 × 600像素。收集到的图像分辨率高，产生大量的文件，它可以使图像处理速度慢。 </li><li>滚动显示的图像，并找到一个点（S）在大部分图像中可见。 </li><li>选定的现场（S）变焦高达300％。使用高倍率图像的对齐方式可以更精确的登记。 </li><li>插入一个层上的所有图像上，并确保在所有帧中可见。标记使用“画笔”工具，在现场（S）的位置。 </li><li>开始调整所有帧，使用“移动”工具，并到上一层作为参考标记。 </li><li>当校准完成后，删除层用作参考，并保存为一个单一的“PSD”文件的影像。此外，出口作为一个单一的“MOV”文件中的所有帧，使用的最高分辨率。每个图像系列的手动对齐约需10分钟。 </li></ol></li><li>使用ImageJ的基因表达进行量化。 <ol><li>在ImageReady中打开ImageJ软件和开放的“MOV”文件以前保存。 </li><li>使用选择工具，选择一个400 × 300像素的面积和作物的形象。这个新的文件应保存为一个“AVI”文件。 </li><li>连续的影像，在位于新的“AVI”文件分为红色，蓝色和绿色通道，点击“图像”，“色”和“RGB分裂”在ImageJ分开。 </li><li>在绿色和红色通道的所有图像中减去背景荧光。使用绿色通道的图像系列，选择与非表达细胞从一个地区一个20 × 20像素的面积，并记录在“投资回报率管理器”工具，它的位置，所以，同样的面积将在选定的两个通道。 20 × 20像素的正方形活跃在绿色通道，位于ImageJ任务栏“插件”命令，然后点击“减去测得的投资回报率，为每个切片”工具插件。 </li><li> ImageJ，点击“图像”，“调整”，然后“门槛”来定义或段绿色荧光蛋白表达的像素。调整门槛窗口拖动栏获得的平均现货20-30像素大小的阈值水平。 </li><li>确定GFP的表达，用我们设计了一个插件，来衡量每个像素的平均灰度值总数表达GFP的像素。 </li><li>复制的平均输出ImageJ并粘贴到Microsoft Office Excel中的灰度值。每个通道（红色和绿色）乘以平均计算GFP表达灰度总数的每个像素值表达GFP的每个通道的像素。两个通道的总和表达式的值，给出了GFP的表达。值可以被用来说明基因表达谱，并进行直接的比较。定时动画也可以使用“MOV”或“AVI”文件，提供了一个独特的基因表达谱和清晰的可视化生成。 </li></ol></li></ol>五，代表性的成果该机器人的图像采集和分析过程（图1）这里允许的基因表达的定量数据，在很短的时间内大量收购。对于植物推广使用绿色荧光蛋白基因的特性，这种方法不仅是有用的创建瞬时表达谱（图2），而且还细致地表达GFP在瞬时和稳定的转化植物组织 5,6跟踪。短的时间推移与收集到的序列图像生成的动画是基因表达的深入分析，在植物组织随时间的宝贵工具。这种方法也有很大的应用，以评估因素，直接影响基因的表达。例如，瞬时表达GFP沉默病毒的起源存在不同的抑制器下已经成功地使用我们的自动图像采集和分析系统 7,8研究。 <img src="/files/ftp_upload/1733/1733fig1.jpg" alt="图1" /> 图1。图像分析过程包括四个主要步骤。 （a）收购的形象系列使用机器人的图像采集系统，（二）图像分离成红色，蓝色和绿色通道，（三）通过调整阈值水平的表达像素分割，和（d）获得的输出结果包含灰度值和绿色荧光蛋白表达的重点计数。 <img src="/files/ftp_upload/1733/1733fig2.jpg" alt="图2" /> 图2。图表显示不同的瞬时表达融合到 GFP的植物促销员驱动的配置文件。使用我们的机器人的图像采集系统收集的数据和ImageReady中ImageJ软件进行分析。

<ol>
	<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol Plant. 15, 473-497 (1962).</li><li>Gamborg, O. L., Miller, R. A., Ojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+requirements+of+suspension+cultures+of+soybean+root+cells.">Nutrient requirements of suspension cultures of soybean root cells.</a> Exp Cell Res. 50, 150-158 (1968).</li><li>Finer, J. J., Vain, P., Jones, M. W., McMullen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+particle+inflow+gun+for+DNA+delivery+to+plant+cells.">Development of the particle inflow gun for DNA delivery to plant cells.</a> Plant Cell Rep. 11, 232-238 (1992).</li><li>Finer, J. E., Finer, J. 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+simple+method+for+reducing+moisture+condensation+on+Petri+dish+lids.">A simple method for reducing moisture condensation on Petri dish lids.</a> Plant Cell Tiss Org. 91, 299-304 (2007).</li><li>Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+35S+and+Lectin+promoters+in+transgenic+soybean+tissue+using+an+automated+image+acquisition+system+and+image+analysis.">Comparative analysis of 35S and Lectin promoters in transgenic soybean tissue using an automated image acquisition system and image analysis.</a> Plant Cell Rep. 25, 290-296 (2006).</li><li>Chiera, J. M., Bouchard, R. A., Dorsey, S. L., Park, E. H., Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+two+highly+active+soybean+(Glycine+max+(L.)+Merr.)+promoters+and+their+characterization+using+a+new+automated+image+collection+and+analysis+system.">Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system.</a> Plant Cell Rep. 26, 1501-1509 (2007).</li><li>Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+extension+of+transient+GFP+expression+by+the+co-introduction+of+a+suppressor+of+silencing.">Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing.</a> Transgenic Res. 17, 1143-1154 (2008).</li><li>Dhillon, T., Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+six+different+viral+suppressors+of+silencing+using+image+analysis+of+transient+GFP+expression.">Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression.</a> Plant Cell Rep. 28, 639-647 (2009).</li></ol>

使用的机器人在人类生活的不同方面的巨大应用，具体而言，机器人已经被有效地使用在危险的环境中执行的活动，自动化冗长而复杂的活动，并在一个更精确的的方式来进行任务。在分子生物学，特别是基因表达分析，机器人可以帮助跟踪不仅功能的基因，但也是组织的生长和发展随着时间的推移。许多生物的现象时有发生动态，这可能是难以遵循，使用单一的时间点观测。 使用绿色荧光蛋白基因表达的研究观测组织的响应和增长带来额外的好处。对于我们的实验程序，GFP让我们按照组织随着时间的推移同一块绿色荧光蛋白检测是一种非破坏性的基因表达。此外，我们的GFP蛋白的版本不够稳定，让检测，但也显示了一些营业额，以尽量减少在植物组织中的积累，使我们能够遵循的兴起和基因表达下降。 我们已经利用我们的机器人的图像采集和分析系统的广泛应用。我们预计所需的其中一个动态的理解的一种现象是，许多生物学应用潜力高。例如，植物组织的生长和发展，可以使用我们的系统提供了宝贵的见解/对这些过程的信息的跟踪。此外，使用GFP报告基因的蛋白质运输的动力，可以轻松地可视化，使用时间推移动画。在这份报告中所描述的方法在技术上是复杂的，但概念是很简单的。我们的结果是强大的和新的应用正在不断被发现。

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<td>ImageJ</td>
<td>Software</td>
<td>U. S. National Institutes of Health</td>
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<td><a href="http://www.rsb.info.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a></td>
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robotics and dynamic image analysis for studies of gene expression in plant tissues

通常在植物组织中的基因表达研究化合物从植物组织的破坏性开采<em&gt;在体外</em&gt;分析。这里介绍的方法，利用绿色荧光蛋白（<em&gt; GFP</em&gt;）基因对基因表达的连续监测，在相同的组织，随着时间的推移。 “<em&gt; GFP</em&gt;基因划归不同的促销员的监管控制，并通过粒子轰击到利马豆子叶组织实施。然后放置在一个机器人的图像采集系统，该系统包括一个荧光解剖显微镜用数码相机和一个2维的机器人平台，定制设计，让安全依恋培养皿子叶。从子叶组织每隔一小时为100小时，图像采集，生成每个发起人的表达谱。每个收集的100幅图像系列是第一个遭受手动图像对齐，使用ImageReady中做出一定的绿色荧光蛋白表达灶一直保留内选定的分析领域。测量系列300 × 400像素的特定区域，然后选择提供GFP强度测量使用ImageJ软件作进一步分析。批量图像被分成红色，绿色和蓝色通道和绿色荧光蛋白表达领域被确定使用ImageJ的阈值功能。减去背景荧光（灰度值加减后的非表达每一个像​​素像素），在各自的红色和绿色通道，绿色荧光强度计算，乘以平均灰度在每个总数的表达GFP的像素，每个像素值通道，然后添加的红色和绿色通道的值。绿色荧光强度值分别收集所有100个时间点，收益率的表达谱。 GFP的表达谱的变化导致启动子的实力，一个沉默抑制的存在，或发起人的性质等因素的差异。除了绿色荧光强度的定量，图像系列也被用来使用ImageReady中生成的时间推移动画。定时动画显示，明显多数的细胞显示在GFP的表达，随后缓慢下降的较快增长。偶尔有些细胞的荧光显示突然丧失，这可能与细胞快速死亡。也观察到绿色荧光蛋白的表观运输穿过细胞膜和细胞壁，相邻的细胞。时间的推移动画提供了更多的信息，否则不能获得使用绿色荧光强度分布或单一时间点的图像集合。

Watch this Scientific Journal Video about Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues at JoVE.com

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

我们报告的引进，跟踪和绿色荧光蛋白基因在植物细胞中表达的定量分析方法。这种方法是利用一个半连续图像采集大量样品的定制设计的机器人系统，随着时间的推移。我们也证明了ImageJ和ImageReady中使用图像序列的分析。

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

robotics-dynamic-image-analysis-for-studies-gene-expression-plant

Research

JoVE Journal

Biology

12.4K 次观看.  The Ohio State University. 我们报告的引进，跟踪和绿色荧光蛋白基因在植物细胞中表达的定量分析方法。这种方法是利用一个半连续图像采集大量样品的定制设计的机器人系统，随着时间的推移。我们也证明了ImageJ和ImageReady中使用图像序列的分析。

视频: Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...