We thank Yumeng (Helen) Xia (Rutgers University) for manuscript editing. The work on BASL polarity formation is supported by grants from the U.S. National Institute of General Medical Sciences to J.D. (R01GM109080) and Rutgers University.

1.质粒构建<ol><li>产生由如前所述1,6-克隆技术的构建体。 <ol><li>先用适当的引物高保真DNA聚合酶扩增BASL的编码序列，YDA和MPK 6和它们子克隆到条目的载体。查找7的详细协议。 </li><li>以生成（分别DNmpk6 8和DNyda 4）MPK 6和YDA的显性负版本，使用MPK 6和YDA的条目的质粒为模板，通过定点诱变方法9引入点突变。 </li></ol></li><li>要建立烟草瞬时测定的结构，进行标准的LR（attL位点¯x重组的attR），反应（7详细过程），以BASL融入pH35CG 10，DNyda和DNmpk6到pXNGW和pXCGW分别载体11。 <ol><li>确认通过酶切和测序产生的质粒。一旦ŝuccessful，变换二进制构建入根癌农杆菌菌株GV3101 12和在28℃生长他们选择平板2 - 3天。 </li></ol></li></ol> 2. 农杆菌渗透溶液的制备<ol><li>接种几新鲜转化的土壤杆菌的菌落到10ml的Luria-BERTANI（LB）具有适当的抗生素培养基（庆大霉素25微克/毫升，利福霉素25微克/毫升，壮观霉素100μg/ ml的对pH35CG，pXNGW和pXCGW构造;同样的浓度的庆大霉素和利福霉素与卡那霉素50微克/毫升为P19（表达抗转基因的沉默蛋白质）13。 </li><li>生长在28°C摇床农杆菌培养在200转的速度约16小时，然后测量外径600（估计达到1.5 - 1.8）（ 图2A-B）。降速文化在2500 XG 10分钟。重悬和WAsh的含MgCl 2的10mM的（渗透液）的细胞沉淀。 </li><li>再次降速培养并重新暂停与浸润溶液适量粒料达到最终OD 600 = 0.5（ 图 2C-D）。浸润植物之前，离开在室温下将细胞混合物为1 - 3小时。此步骤有助于维持细胞稳态。 </li></ol> 3.卷烟厂和叶注射液<ol><li>使用烟草（ 本生烟），增长4 -第5周龄在一个标准的植物生长室（23℃，16小时光照/ 8小时黑暗的循环）14叶注射液。 注意：在此阶段，烟草植物通常具有6叶（2大，2培养基，和2小）。这两个中等大小的叶子是最优化的叶注射液（这两个路数太旧了，而两个小的往往是太年轻）。 </li><li>浸润前一天，浇灌tobac合植物饱和的土壤中。 </li><li>在浸润天，预孵育在黑暗中的植物为1 - 3小时。此步骤允许气孔在表皮广开，渗透到叶表皮细胞时，极大地方便了根 。 </li><li>使用标有日期的颜色条，旗叶进行渗透。标记区域用黑色粗Sharpie笔渗入（可选的;渗入领域是被感染后可见）。 </li><li>取农杆菌再悬浮等体积并在通过温和涡旋（ 图2E）100×15毫米的培养皿混合。注：在我们的例子中，我们混合1毫升P19的1毫升CFP-BASL的1毫升DNyda-nYFP和1ml DNmpk-cYFP的。 </li><li>在浸润的过程中，填充渗透混合物3mL的无针注射器（1 - 2毫升）中，推入烟草叶（ 图2F）的底侧（背面）。而渗透，它是有帮助用一只手推，另一只手轻轻支撑上侧（正面，对抗推压力）。 注意：一旦渗透混合物被成功注入，受感染的区域将成为在表皮容易辨认。然而，渗透可能会失败，由于恶劣的注射液（组织损伤）或推不足（没有液体渗透）。对于每个测试，我们建议在两个独立的叶子至少两个注射（来自不同的植物）。 </li><li>放置感染植物放回生长室。一般来说，蛋白质的表达都是经过48小时以下的渗透检测。 </li></ol> 4.共焦成像<ol><li>在48小时后浸润，用打孔器从注射部位切除叶盘（通常为3 - 4个磁盘/可以生产区）（ 图2G）。使用镊子轻轻的叶盘水珠转移到幻灯片（远轴朝上），并安装它们。避免过压哈日在盖滑移ð因为挤压/受损细胞不会在共聚焦显微镜（ 图2H）下显示良好。 </li><li>共焦成像之前，扫描于外延荧光化合物显微镜安装样本，以检查的表达水平。注：根据我们的经验，这表明中间表达水平的细胞共聚焦显微镜下最好代表。 </li><li>开始对共聚焦显微镜的40X（NA 1.3）镜头。调节滑动到所需的位置，从而使表达荧光蛋白的中等水平的细胞保持在中心。对于CFP和YFP激发/发射光谱是458海里/ 480 - 500纳米和514纳米/ 520 - 540分别纳米。 </li><li>激活“实时”按钮来预览图像，然后调整激光强度和智能增益最均衡的荧光强度/噪音比。以改善成像质量，增加扫描像素和/或帧/线平均。最后，调整调焦旋钮，REACH中间焦平面并点击“捕获”采取的形象。 注意：CFP和YFP的图片可通过打开或关闭的“连续”扫描分别模式，选项检查被顺序地或同时捕获。 </li><li>当Z-投影期望的细胞的深入视图中选择“XYZ”摄像模式时，限定的扫描范围的深度（10 - 15微米），并收集从顶部图像到目标小区的底。编译通过在图像文件右键点击徕卡软件获得的图像，选择“重命名”，并导出图像为.tiff文件。 </li><li>图片30 - 40细胞定量分析。 </li></ol> 5.图像处理与定量分析<ol><li>使用斐济软件（http://fiji.sc/Fiji）来处理图像并进行量化。 <ol><li>绘制荧光强度图。 <ol><li>为了更好地可视化CFP / YFP表达是否均匀分布的，使用斐济证明CFP和YFP的信号强度沿着电池周边。要做到这一点，首先启动斐济，然后选择“文件”和“打开”开始图像。单击工具菜单中的“行”，并通过右键单击选择“分段线”。决定的感兴趣的区域，并绘制沿着电池周边的线迹在CFP或YFP的信号（ 图3A-C）。 注意：斐济测量沿着分段线的绝对荧光强度值。 </li><li>选择下拉菜单中的“分析”和“剧情资料”来生成与代表CFP / YFP沿分割线的水平位置和强度值的曲线图。如果需要的话，重复的强度值（通过选择菜单中的“复制”）到Excel或其它图形软件生成可比强度的曲线图。 </li></ol></li><li>量化极性程度。 <ol><li>收集大约20 represen从3重复实验对准焦点细胞，以评估CFP和YFP的烟草细胞的极性程度。计算在低荧光强度（定义为L）的基础上的高荧光强度（定义为H）的比率的极性程度。注意：定量方法在图3中说明。 <ol><li>为了测量H和L值，得出沿小区外围（如上所述）感兴趣区域分割的线，请从下拉菜单中选择“分析”，然后选择“测量”。当一个“结果”窗口弹出时，用“中庸”的价值观进行量化。 </li><li>从显示CFP和YFP， 例如相对均匀表达的细胞，CFP-BASL（ 图3A）或YFP的从DNyda-nYFP和DNmpk6-cYFP（ 图3B）的共表达补充，通过测量得到的H和L的值两个随机选择的圆周段具有相等的长度。 </li> &lt;li&gt;从呈现荧光蛋白的明显极性积累，在这种情况下，细胞，CFP-BASL，DNyda-nYFP和DNmpk6-cYFP（ 图3C），的共表达收集来自周边区域轰值与富集的荧光信号从低/无荧光积累区域LS。 </li><li>池H / L的所得比率从20个细胞一起并测试正态分布。通过计算学生的t检验的p值（正态分布）或柯尔莫哥洛夫-斯米尔诺夫（KS）测试（非正态分布）。 </li></ol></li></ol></li></ol></li></ol>

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没有利益冲突的声明。

一般使用和信号转导的烟草瞬时表达系统的可能的修改：荧光蛋白的过表达（FP）-tagged在烟草表皮蛋白已成功用于在植物细胞中蛋白的亚细胞定位的快速检测。然而，在植物中研究蛋白质复合物的动态信令分配是一项具有挑战性的主题，由于复杂的亚细胞环境中，瞬时蛋白质-蛋白质相互作用和信号转导事件。为了应对这一挑战，我们采取了附设系统的优势，研究MAPK信号的事?...

蛋白质错综复杂的分层网络，并形成络合物的发挥，在一个不可预知的细胞环境发生的几乎所有的生物过程的关键角色中进行互动。然而，从植物发育生长的响应，所以一直缺乏方便的工具，不仅可以快速而且有效地在亚细胞水平识别和监控这些动态信号事件。 烟草叶表皮瞬时表达系统已经呼吁优势，为在活细胞中可视化的荧光蛋白质。这个系统提供了半体内条件允许翻译后蛋白质修饰和蛋白质定位快速检查。通过检查补充YFP信号时，双分子荧光互补（附设）测定通知蛋白质 - 蛋白质相互作用的在植物细胞中的可能性。相比其他方法， 如酵母双杂交（Y2H）和Co-immunoprecipitation（联合IP）附设还提供了可视化，其中在亚细胞水平，可能会发生蛋白 - 蛋白相互作用室的有力手段。 因为不对称细胞分裂（ACD）保存干细胞群的同时用于组织/器官形成产生新的细胞类型，它对于促进真核多细胞不可或缺的机制。 拟南芥气孔发展已被用作模型系统，用于在植物中学习的ACD。的前体细胞，拟分生组织母细胞，除以不对称产生两种不同的子细胞中，拟分生组织（经历终止于一对保卫细胞之前干细胞样师）和一个气孔谱系接地单元（SLGC）（可分裂并分化成路面细胞），分别为（ 图1）。在气孔ACD，在天堂气孔不对称的新型蛋白质断裂（BASL）被premitotically极化带动分裂不对称，这INC路得身体不对称和细胞命运的不对称1。该MAPKKK YODA和的MAPKs，MPK3和6组成的MAPK级联是中部的气孔师图案和命运采用2,3，4,5。 近日，张某等人 。联极性蛋白BASL在拟南芥的气孔ACD 6 YDA-MAPK信号通路。规范YODA-MAPK通路，通MPK3 / 6，磷酸BASL并激活它的两极分化。磷酸BASL用作脚手架和募集YODA（YDA）和MPK3 / 6，以形成蛋白复合物，并在细胞皮质6集中的信令。 MAPK信号分量和BASL和YDA-MAPK途径之间的正反馈环的极化表示用于在植物细胞中的蛋白质偏振一种新的机制。当地丰富的MAPK信号是假设紧扣细胞命运分化气孔ACD 6（ 图1）。一项所述的k的一个支持这种模式安永实验数据从表现在BASL 6的诱导表达的的MAPKs的空间再分配烟草检测出来。 在一般情况下，这是不容易来监控发生MAPK信号因为MAPK分子经常发现到处一个细胞的内部。在这项研究中，我们所用的拆分YFP系统可视化的上游激酶和下游的人之间的相互作用暗示其中信令中继发生。我们通过进一步延长使用附设系统的共表达的第三蛋白（CFP-标签）与分割YFP对（暗示蛋白质 - 蛋白质相互作用的）来可视化是否以及如何补充YFP可以由共进行空间调制CFP表达的蛋白质。通过这样做，我们证明在烟草表皮的细胞皮层诱导YDA和MPK 6，之间的相互作用空间重组从均匀分布到极化图案CFP-BASL的共表达人的细胞。因此，这种系统具有用于当细胞被内部或外部刺激挑战监控的条件下在植物细胞中动态信令事件待开发的潜力（ 例如，其它蛋白质，化学应用，病原体攻击，或者环境变化等的共表达。 ）。

<table>
	<tbody>
		<tr>
			<td>Phusion DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D/TOPO</td>
			<td>Life Technologies</td>
			<td>K2400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickChange II XL Site-directed Mutagenesis Kit</td>
			<td>Agilent Technology</td>
			<td>200521</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>AMRESCO</td>
			<td>J106-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>AMRESCO</td>
			<td>J673-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G3632-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>R3501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>K4000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S4014-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>25x75mm Slide</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
		</tr>
		<tr>
			<td>24x50 mm Cover glass</td>
			<td>VWR</td>
			<td>48393-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica&#160;</td>
			<td>TCS SP5 II</td>
			<td>&#160;LAS AF Lite software</td>
		</tr>
		<tr>
			<td>40 x objective lens</td>
			<td>Leica</td>
			<td>HCX&#160;Plan APO&#160;</td>
			<td>HCX&#160;Plan APO&#160;, NA 1.30</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>SORVALL 6+</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 5 Forceps</td>
			<td>Canemco &#38; Marivac</td>
			<td>205EQA-Spec</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging spatial reorganization of a mapk signaling pathway using the tobacco transient expression system

在活细胞中的动态信号事件的可视化一直是个挑战。我们扩大建立瞬时表达系统，在烟草表皮细胞的生物分子荧光互补（附设）测定中，从检测蛋白质 - 蛋白质相互作用，以监测在植物细胞中的信号转导的空间分布。在这个协议中，我们所用的附设测定以表明相互作用和拟南芥 MAPKKK YODA和MAPK6之间的信令发生在质膜。当支架蛋白BASL被共表达时，YODA-MAPK相互作用引入并与CFP-BASL空间共极化。此改性烟草表达系统允许信令本地化和动态变化（小于4天），可容纳的荧光蛋白的颜色（至少三个）多个快速检查。我们还提出了具体的方法来定量蛋白质分布（非对称的空间定位，或“两极分化”;）在烟草细胞。这种先进的烟草表达系统具有用于在活的植物细胞动态信令事件的快速测试被广泛使用的潜力。

为了防止由过度活跃的MAPK信号诱导的细胞死亡，YDA（DNyda）和MPK 6（DNmpk6）的激酶非现行版本在烟草细胞共表达测定中使用。既不通过附设也不CFP-BASL本身产生不均匀分布图案（ 图3A-B）揭示DNyda和DNmpk6之间的相互作用。然而，当CFP-BASL引入DNyda-DNmpk6相互作用对，两者CFP和YFP信号被重新分配给一个高度偏振的方式（ 图3C）。这种重新分布表明BASL与YDA?...

Watch this Scientific Journal Video about Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System at JoVE.com

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

The overall goal of this procedure is to visualize dynamic spatial distribution of protein

imaging-spatial-reorganization-mapk-signaling-pathway-using-tobacco

Research

JoVE Journal

Biology

9.7K 次观看.  Rutgers, The State University of New Jersey. The overall goal of this procedure is to visualize dynamic spatial distribution of protein

视频: Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

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

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

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 ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

在单酵母细胞MAPK诱导表达的时空定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.