Name: electroporation of functional bacterial effectors into mammalian cells
Uploaded: 2015-01-19T13:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1.提前准备<ol><li>温暖的无菌磷酸盐缓冲盐水（PBS），以37℃。 </li><li>温暖的Dulbecco改性的Eagle培养基（DMEM）和极限必需培养基（MEM），补充有10％胎牛血清（FBS），100国际单位/毫升青霉素和100微克/毫升链霉素至37℃。注:分别为这些代表正常生长介质（NGM）为RAW和HeLa细胞。 </li></ol> 2.准备细胞<ol><li>生长的RAW 264.7细胞中的NGM 70-90％汇合。 <ol><li>保持在湿润的95％空气/ 5％CO 2的气氛中的细胞在37℃。 </li></ol></li><li>生长的HeLa细胞中的NGM 70-90％汇合。 <ol><li>保持在湿润的95％空气/ 5％CO 2的气氛中的细胞在37℃。 </li></ol></li><li>收集前，用无菌PBS洗涤细胞单层一次。 </li><li>收集在无菌锥形管中预汇合的细胞。 <ol><li>轻轻刮去RAW细胞用橡皮警察在PBS，反复吹打分散细胞聚集。 </li><li>分离HeLa细胞与0.25％胰蛋白酶溶液，直到视力检查显示了从培养表面解离。例如，使用2〜3毫升为T-75烧瓶中。相应地调整音量，保证胰蛋白酶溶液覆盖整个生长表面。 <ol><li>淬火用含有10％FBS的NGM离解反应。 </li><li>使用NGM的至少两倍的量，以胰蛋白酶，反复移液以分散细胞聚集。 </li></ol></li></ol></li><li>轻轻离心沉淀细胞，在900×g离心4分钟。 </li><li>重悬在相同体积用无菌PBS胰蛋白酶/骤冷溶液。 </li><li>计数使用血球或粒子计数器细胞。 </li><li>轻轻离心沉淀细胞，在900×g离心4分钟。 </li><li>重悬在PBS中的适当体积为5.5×10 6〜6.0×10 6个细胞/ ml。注:例如，一个T-75瓶将产生逼近的ately 7.5×10 6个细胞29。 </li><li>保持细胞悬液在冰上直到电。 </li></ol> 3.准备电穿孔<ol><li>预冷电试管（0.7厘米的缝隙）冰上。 </li><li>打开电设备和设置电压0.3千伏。 注:电容和电阻并不在我们的电穿孔（由制造商设定在10μF和600Ω）可调设置。 </li><li>填回收板的NGM，并在37℃下平衡的湿润的95％空气/ 5％CO 2的气氛中</li></ol> 4.电穿孔<ol><li>放置400微升细胞悬浮液中预先冷却的反应杯，并添加20微克选择蛋白与样本池（50微克/毫升）。 </li><li>弗里克试管轻轻〜10次混合而不损伤细胞。注意:反应杯也可以倒置几次以调匀，但不要吸管上下或涡流，以避免损坏电池。 </li><l我&gt;干外实验皿用纸巾或其它抽头的，以避免在所述电穿孔的电弧。 </li><li>在0.3千伏为1.5-1.7毫秒电​​穿孔的样品。注意:这是典型的用于本研究。 </li><li>紧接着电轻弹试管轻轻〜10次调匀。 </li></ol> 5.电镀细胞<ol><li>商店试管在冰上，直到准备电穿孔细胞放置在复苏板块。 </li><li>对于大多数下游分析，洗涤细胞1×预热的NGM去除多余的效应蛋白。 <ol><li>去除细胞进行分析，并暂停在3-5毫升NGM的。 </li><li>通过离心沉淀细胞，在900×g离心4分钟。 </li><li>悬浮在NGM足够的音量所需的板尺寸（ 如 2毫升35毫米盘）。 </li></ol></li><li>对于下游分析去除细胞的适当的量。 <ol><li>例1:板到玻璃底菜显微镜分析。 </li><li>例2:板INTØ细胞培养塑料蛋白质分析等亲和纯化。 </li></ol></li><li>允许细胞在平衡板在加湿的95％空气/ 5％CO 2的气氛中以回收在37℃下至少4小时。 </li></ol> 6.显微镜分析 6.1）固定/免疫染色<ol><li>洗涤细胞后4小时恢复期1×用无菌PBS。 </li><li>固定的细胞在100％甲醇中，在室温下2分钟。使用足够的甲醇，以完全覆盖细胞（ 如 2毫升35毫米盘）。 </li><li>洗3次，用无菌PBS洗涤。 </li><li>通透细胞用0.4％的Triton X-100的PBS 15分钟。例如，使用方法1毫升35毫米直径板。 <ol><li>根据表位与目标蛋白的位置调整通透的Triton X-100和实力的长度。注意:最好的结果将需要凭经验为每个目标确定的，但是在上述条件应足以大多数Cytosolic目标。 </li></ol></li><li>用5％的牛血清白蛋白（BSA）的PBS阻断1小时，在室温。例如，使用方法1毫升35毫米直径板。 </li><li>洗涤3次，用PBS。 </li><li>孵育在抗体结合解第一抗体（在PBS中0.1％的Triton X-100和1％BSA）中过夜，在4 &#160; °下与温和的摇摆。例如，使用0.5毫升为35毫米直径板。 <ol><li>按照制造商的建议抗体稀释。注意:例如，链霉亲和结合肽标签（SBP-标签）抗体被用于以1:1000稀释，而PKN1抗体被用于以1:200的稀释。 </li></ol></li><li>洗涤4X，用PBS。 </li><li>孵育在抗体结合解适当荧光标记的二抗1小时，在室温下，避光。 <ol><li>例如，使用的Alexa 488或Alexa 647进行1小时，在室温下进行。 <ol><li>按照制造商的建议蚂蚁ibody稀释。 （ 例如，约1:1000本研究）。 </li></ol></li><li>添加其它污渍根据需要，例如，5微米的小麦胚芽agglutinnin（WGA）缀合的Alexa 647或DAPI根据制造商的建议，进行1小时，在室温下进行。 </li></ol></li><li>洗涤5次，用PBS，并在4℃储存，避光，直到准备图像。 </li></ol> 6.2）共聚焦显微镜和图像分析<ol><li>在使用63X油浸物镜倒置共聚焦显微镜图像样本。 <ol><li>图像的绿色通道使用氩激光的488nm处线，具有492-542纳米之间的带宽发射。 </li><li>图像的红色通道用一个633纳米二极管激光器，具有640-718纳米之间的带宽发射。 </li><li>图像蓝色通道使用一个405nm的二极管激光器，具有407-453纳米之间的带宽发射。 </li><li>确保多通道的z堆叠覆盖细胞体积的全部。 </li></ol></li><li>处理图像与适当的图像处理软件。 </li></ol> 7.亲和纯化<ol><li>后4小时恢复期间用4℃的PBS洗两次电穿孔的细胞。 </li><li>裂解用〜加入1.0ml裂解缓冲液（1％的Triton X-100与蛋白酶抑制剂混合物和磷酸酶抑制剂的PBS）在冰上。根据制造商的说明，使用抑制剂。注意:例如，无论是在本研究中使用的磷酸酶和蛋白酶抑制剂在100倍供给的，但其他的制剂应该工作得同样好。 </li><li>及时刮掉细胞橡胶警察并收集到锥形管。 </li><li>裂解剧烈涡旋和超声。注:其它裂解方法可工作得同样为好，但将需要效率来凭经验确定作为对测定规定的适用性。 <ol><li>超声处理3×30秒，间歇漩涡。 </li></ol></li><li>离心机以10,000×g离心10分钟，在4℃以收集细胞碎片和不溶性聚集体;保存上清液。 </li><li>结合电穿孔细胞裂解物等体积（〜1.0ml）中，用50微升链霉亲和琼脂糖树脂悬浮液在4℃下过夜终过端旋转。注意:此树脂捕获经由其链霉结合肽的亲和标签的电穿孔蛋白（和相关的复合物）。 </li><li>离心在2500 xg离心2分钟，弃上清。 </li><li>用40柱床体积（约1毫升）的PBS洗涤两次。 </li><li>添加30微升4×LDS加样缓冲液（141毫摩尔Tris碱，2％的LDS，10％甘油，0.51毫摩尔EDTA，0.22毫SERVA蓝G，0.175毫酚红，pH值8.5），20微升DIH 2 O和1微升0.5M的三（2-羧乙基）膦（TCEP），允许一些量留在珠。 </li><li>热，在95℃下进行10分钟，并在冰上冷却。 </li><li>自旋&gt; 10,000 xg离心在4℃下5分钟。 </li><li>收集上清。 </li><li>使用合适的抗体注意进行免疫印迹:他重，抗PKN1初级抗体被使用。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., Galan, 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=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

作者什么都没有透露。

从致病细菌分泌的效应已经发展到在宿主细胞内环境的作用，因此它是有帮助的主机内研究它们在原位 。引入感兴趣的特定效应物到宿主细胞中允许相关病原体 - 宿主相互作用进行研究孤立，而不与其他细菌蛋白质的干扰。的目标是探讨电穿孔以引入细菌效应蛋白进入真核宿主细胞，从而避免了与转染或转导相关的挑战的一种手段。绿色荧光蛋白作为对照和沙门氏菌效应蛋白进行了...

许多革兰氏阴性菌采用专门的分泌系统注入毒性相关蛋白（称为效应器）直接导入宿主细胞1-5。这些效应物具有广泛的生物学功能，包括:抑制宿主免疫的，细胞骨架的变化，细胞内运输和信号传导，转录变化的修饰，和宿主蛋白酶的改变6-9。某些效应的功能是已知的，但是在主机的目标和其他许多人的生化作用（多个）仍有待确定。而比较野生型和重组体细菌感染是研究细胞内效应器毒力机制的有效的方法，它通常是有利的引入个体效应入​​宿主细胞。因此，简单的方法，用于在宿主细胞中的上下文中引入和表征细菌效应蛋白是高度期望的。 简化了实验分析的Wi第一个效应是至关重要的其它效应可能有相反的或多余的功能。要做到这一点简化，研究人员先前推出的大分子物质进入细胞的许多不同的方法，包括病毒转导10，显微注射11，刮装载12,13，细胞融合与化学诱导注射14，专有蛋白"转"试剂15，磷酸钙沉淀16，和电穿孔17-20。所引入的分子范围从核酸包括DNA，RNA和RNA干扰物种的蛋白质，细胞不透染料和抗体的细胞内目标21,22。一些方法都有局限性，包括大分子的可以引入的类型，以及特定下游分析可由于高的细胞毒性，作用破坏机制，低功效，或导入效率的限制。转染，一个ofte正使用的表达方法在哺乳动物细胞中的细菌的基因，也受到限制，一些有关的宿主细胞类型，例如巨噬细胞和原代细胞，是朝着转染特别耐。除此之外，它难以控制导入外源DNA后产生的细菌蛋白的水平。 许多工作已建立的核酸引入细菌和哺乳动物细胞作为通用实验室技术电穿孔;然而，人们一直在研究转化为在生理条件下提供的蛋白质进入细胞的最佳方法。对蛋白质的转染报告是有希望的，但需要昂贵的试剂和优化。引入潜在的有毒细菌效应成各种各样的以最小的成本细胞靶的愿望促使我们考虑电穿孔作为用于体内研究这些蛋白质的方法。 蛋白质电23-25 ​​是一个满足HOD引入蛋白质成经由electropermeabilization活细胞，也被称为电-转染或电注入26。这种技术使用高强度的电脉冲以在细胞膜孔。这些可逆孔隙允许通常由细胞内的空间中排除大分子进入细胞。在去除外部电场，膜可以重新密封，使细胞能留存穿过孔27,28的分子。 一个标准的电穿孔被用于本研究中一致地引入细菌效应入两个小鼠巨噬细胞样细胞和人上皮细胞。该方法是快速的，有效的，廉价的，在细胞生存力没有明显的下降。引入的蛋白可通过免疫荧光显微镜进行可视化，或用于功能测定法。这已被证明使用绿色荧光蛋白（GFP），为无毒的标准，以及2 沙门氏菌效应蛋白，SspH1和GtgE。我们建议蛋白电作为剧目的细菌毒力的蛋白质及其功能的真核宿主细胞研究的一个额外的工具。

<table><tbody><tr><td>0.25% Trypsin-EDTA Solution</td><td>Cellgro</td><td>25-050-Cl</td><td></td></tr><tr><td>0.4 cm Gap-disposable electroporation cuvettes</td><td>Bio-Rad</td><td>165-2088</td><td></td></tr><tr><td>100% Methanol</td><td>Any</td><td>N/A</td><td>Flammable, Toxic</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4919</td><td></td></tr><tr><td>Cell Counting Apparatus - Hemocytometer or Coulter Counter</td><td>Beckman Coulter</td><td>Model Z1</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Any</td><td>N/A</td><td>Humidified 95% air/5% CO&lt;sub&gt;2 &lt;/sub&gt;atmosphere at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Cell Culture Plastic</td><td>Any</td><td>N/A</td><td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&amp;rsquo;s Medium (DMEM)</td><td>Cellgro</td><td>10-013</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Electroporator</td><td>Bio-Rad</td><td>E. coli Pulser</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Cellgro</td><td>35-016-CV</td><td></td></tr><tr><td>Fluorescent confocal microscope&amp;nbsp;</td><td>Ziess</td><td>Model&amp;nbsp; LSM 710&amp;nbsp;</td><td></td></tr><tr><td>Glass Bottom Dishes for Microscopy</td><td>Wilco Wells</td><td>HBSt-3522</td><td></td></tr><tr><td>HALT Protease Inhibitor Cocktail</td><td>Pierce</td><td>78430</td><td>Corrosive, Toxic</td></tr><tr><td>HeLa Cell Line</td><td>ATCC</td><td>ATCC CCL-2</td><td></td></tr><tr><td>LDS 4X Loading Buffer</td><td>Invitrogen</td><td>NP0007</td><td></td></tr><tr><td>Minimal Essential Medium (MEM)&amp;nbsp;</td><td>Cellgro</td><td>10-010</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-Cl</td><td></td></tr><tr><td>RAW 264.7 Cell line</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>Primary Antibody Against Target of Interest</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Secondary Antibody Conjugated to Fluorophore</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Any</td><td>N/A</td><td>Chill to 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Sterile Phosphate Buffered Saline</td><td>Any</td><td>N/A</td><td>Warm to 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Streptavidin Agarose Resin Suspension&amp;nbsp;</td><td>Pierce</td><td>20353</td><td></td></tr><tr><td>Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td><td>Any</td><td>N/A</td><td></td></tr><tr><td>TCEP</td><td>Sigma-Aldrich</td><td>646547</td><td>Corrosive, Toxic</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8585</td><td>Irritant, Toxic</td></tr></tbody></table>

electroporation of functional bacterial effectors into mammalian cells

蛋白质相互作用的活细胞中的上下文中的研究可以产生大约本地化，动力学和相互作用伙伴的关键信息。这些信息是在宿主 - 病原体相互作用的背景下尤为可贵。许多病原体的蛋白质的宿主细胞内的多种方式，如功能，从而使宿主的免疫系统和存活的逃避细胞内环境中。研究这些病原体蛋白宿主-细胞相互作用，几种方法是常用的，其中包括: 在体内感染用表达一个标记或突变蛋白，或引入病原体的基因通过转染或转导的菌株。每一种方法都有优点和缺点。我们试图直接引入外源蛋白进入细胞的一种手段。电穿孔通常用于将核酸引入细胞中，但已被更很少应用于蛋白质虽然生物物理基础是完全一样的。一个标准的电穿孔被用于引入亲和标签效应细菌到哺乳动物细胞。人类上皮细胞和小鼠巨噬细胞用传统方法进行培养，分离，并置于0.4厘米的空隙电试管与感兴趣的外源细菌病原体蛋白（ 例如鼠伤寒沙门氏菌 GtgE）。电穿孔（0.3千伏）和短（4小时）的恢复期后，细胞内蛋白质是由通过其亲和标记物的荧光标记的蛋白，并检查空间和时间分布通过共聚焦显微镜证实。电穿孔的蛋白质也被证明是功能性细胞内，并能正确的亚细胞贩卖和蛋白质 - 蛋白质相互作用的。而该外源蛋白倾向于积聚在细胞的表面上，将电穿孔样品具有大量增加的细胞内效应器浓度相对单独孵育。该协议是简单，速度不够快，在做AParallel时尚，允许病原体蛋白在宿主细胞中的高通量特征包括亚细胞靶向和毒性蛋白的功能。

作为概念的初步证明，纯化的绿色荧光蛋白被成功地引入到使用电穿孔哺乳动物细胞。绿色荧光蛋白，近似27 kD的分子量蛋白质通常引入哺乳动物细胞（通常为质粒DNA表​​达），为无显著细胞毒性的分子生物学工具。 HeLa细胞孵育（ 图1A）或电穿孔（ 图1B）与25微克/毫升的GFP，随后通过免疫荧光共聚焦显微镜检查荧光GFP信号。以证明GFP是蜂窝胞质溶胶内，将细胞也沾上?...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K 次观看.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

视频: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

科研

生物学

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

神经科学

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

使用睡美人转座子系统对原代人色素上皮细胞进行基于电穿孔的遗传修饰

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

医学

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

将质粒DNA电穿孔到小鼠骨骼肌中

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

Bacterial Transformation: Electroporation

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA. In nature, transformation can occur in certain types of bacteria. In molecular biology, however, transformation is artificially induced through the creation of pores in the bacterial cell walls. Bacterial cells that are able to take up DNA from the environment are called competent cells. Electrocompetent cells can be produced in the laboratory and transformation of these cells can be achieve via the application of an electrical field that creates pores in the cell wall through which DNA can pass.
The video explains the equipment used in electroporation such as an electroporator and electroporation cuvette. The video also goes through a step-by-step procedure about how to create electrocompetent cells and electroporate cells of interest. 
Prediction of the success of a transformation of an experiment, by observing the time constant, as well as the importance of removing salt from the solutions when electroporating, are also mentioned.

细菌转化:电穿孔法

The term &#x201C;transformation&#x201D; refers cellular ingestion of foreign DNA.  In nature, transformation can occur in certain types of bacteria.  In ...