1。 MEA制备<ol><li>多边环境协定电实验中使用的，可以使用标准光刻技术制作如前所述，18岁或MEA制造商，如多通道系统<a href="http://www.multichannelsystems.com" target="_blank">（http:/www.multichannelsystems.com/）</a>和Alpha MED科学，公司从供应商直接购买<a href="http://www.med64.com/" target="_blank">（ http://www.MED64.com） 。</a>在我们的实验中所用的多边环境协定是捏造的房子在洁净室的设施，这是由固体电子学研究（CSSER）中心在亚利桑那州立大学。在我们的实验中所用的多边环境协定有32个铟锡氧化物电极在8 4与电极直径在1厘米内径的玻璃以及从50-200微米大小的阵列。玻璃以及聚二甲基硅氧烷（PDMS）的MEA使用是否键合。 </li><li>在高压釜中灭菌的MEA细胞前eeding。如浸泡在70％乙醇中的灭菌，如其他地方描述的UV处理和热水处理的其它方法也可以被使用，只要它们不会危及在MEA的完整性。 </li><li>转移蒸压MEA层流罩，并将其放置在一个标准的无菌聚苯乙烯培养皿中。在种子细胞上的MEA，消毒层流罩接通20分钟的UV光。如果MEA UV光覆盖的MEA的培养皿中，用无菌铝箔流罩是在紫外光照射下是敏感的。 </li></ol> 2。种子细胞在MEA <ol><li>从一个正在运行的细胞粘附的哺乳动物细胞，用胰蛋白酶/ EDTA，并提高它们的浓度为100-200个细胞/微升用于电镀在1ml细胞介质中收获细胞。在我们的实验中，我们已经成功地培养NIH 3T3成纤维细胞的细胞系，HeLa细胞和初级皮层和海马神经元的MEA。 </li><li&gt;将媒体与细胞在MEA 20-30微升下降。把MEA培养皿中的孵化器。这是重要的，以确保该单元格的液滴尺寸是大到足以覆盖所有的电极上的MEA。 </li><li> 2小时后，播种的细胞从培养箱中与MEA的陪替氏培养皿转移到层流罩，并轻轻地添加400-500微升预温热媒体（37℃）的MEA以及。在显微镜下检查细胞仍然连接和MEA表面均匀地涂在。孵化器转移MEA回。 2小时媒体除了之前的等待期间，确保足够的时间进行的与MEA表面细胞附着。添加太早接种细胞后，细胞培养基可以洗去细胞。如果细胞不能很好地附着在MEA，其表面可以处理与细胞附着的等因素aspolylysine，纤连蛋白和层粘连蛋白，改善细胞的粘附。在我们的HeLa细胞的实验中，我们一般将MEA表面fibronectiN（10微克/毫升）1-2小时。 </li><li> 8-12小时后接种细胞，进行1-2与PBS清洗，以消除任何的死细胞。细胞培养基可以根据需要24-48小时更换一次。在一个典型的实验中，细胞是通常准备用于转染24-48小时后播种。为了保持电效率的一致性，建议，等到文化是至少80％汇合前电。 </li></ol> 3。网站特定的siRNA转染<ol><li>准备核酸溶液。对于siRNA转染，准备一个1-2μM的siRNA电穿孔溶液冰冷的电穿孔缓冲液中，以达到最终体积为200-400微升。在这个视频文章中，我们展示了Alexa 488标记的扰码序列的siRNA转染HeLa细胞。 </li><li>从孵化器的无菌层流罩与细胞转移MEA。从MEA中取出的细胞培养基，并用PBS进行洗涤。此后，添加200-400微升的iCE冷1μM的siRNA电的很好的解决方案。 </li><li>应用阳极方形电脉冲的目​​标电极（参照到步骤4，用于确定最优参数的电脉冲）。在我们的实验中，我们使用的语用仪器2414A波形发生器产生电脉冲和双列直插式封装（DIP）16引脚剪辑的接触的MEA焊盘的。通过一个多路分路器，印刷电路板，以提供单个电极的选择性，可以被连接到的波形发生器的DIP剪辑。对于参比电极，在细胞培养基中放置一个钢阴极。同时降低钢在媒体上的参考电极，它是极为重要的，以确保它不会使与MEA的表面接触。接触时，它可能会导致损坏的细胞和MEA。它是建议降低参比电极的位置的1毫米以上的细胞。在放置了ref，高度为1mm的隔离物具有可放置在阱，以协助erence电极。使用外部基准电极的另一种方法是使用阴极 - 阳极对相邻的电极。 </li><li>的电脉冲施加到目标上的MEA的电极后，立即转移的MEA回到培养箱。 5分钟的潜伏期后，轻轻地取代75％的电穿孔缓冲液预温（37°C）的细胞培养基。购买，荧光成像可以做，以荧光标记的siRNA的细胞中确认交货。 </li></ol> 4。电穿孔参数优化<ol><li>电穿孔法，的设计电穿孔实验在脉冲幅度，脉冲宽度，和脉冲数的值的范围内，以确定最佳的电脉冲参数。在我们的经验与HeLa细胞中，我们观察到一个电压脉冲的幅值从3 V到9 V，和持续时间1毫秒至10毫秒的一个电极的大小为100μm与英里导致成功的电穿孔nimal细胞死亡。一个参数而变化的时间和其他被保持恒定，这是我们的建议。为了定量的电穿孔效率为每个脉冲参数中，我们使用碘化丙啶（PI），细胞浸透染料污渍核酸材料，和现场检测为基础的方法，描述在步骤4.2-4.5。 </li><li>按照在步骤1和2中列出的指示，建立的细胞培养上的MEA。 </li><li>电穿孔前制备300-500微升30μg/ ml的PI溶液在冰冷的电穿孔缓冲液和400微升4的μMcalcien AM的PBS溶液。 </li><li>转移MEA从培养箱中的层流罩。从MEA中取出的细胞培养基，并用PBS进行洗涤。此后，与PI冰冷的电穿孔缓冲液中添加300〜500μl的。 </li><li>应用电脉冲与所需的脉冲的幅度和持续时间的有针对性的电极（在我们的实验中所使用的设备，用于产生和施加电TRIC脉冲的MEA的步骤3.3）中描述的。对于参比电极的钢降低电极到在MEA的电穿孔缓冲液中，或使用相邻的电极作为参考。通过选择性地施加脉冲的变化的幅度或持续时间，以不同的电极，多个实验，可以在同一装置上进行。例如，在一个32电极的MEA，8的实验可以通过拆分进行的32个电极中的8个组，每个组由4个电极组成的。的8个组中的每一个，然后，可以在3 V-6 V的范围内变化的振幅在0.5 V增量和一组由一个单一的矩形电压脉冲的持续时间5毫秒的刺激，可以用作对照。 </li><li>后立即施加的电压脉冲的MEA转移到孵化5分钟。之后，轻轻地去除75％的电穿孔缓冲液的MEA，取而代之的是温暖的细胞培养基。孵化器转移MEA回。 </li><li>经过2-4小时的潜伏期以及取代的MEA中的细胞培养基，用300-500微升4μM钙黄绿​​素AM在PBS中的溶液。 MEA回转移到另一个30分钟的孵化器。 </li><li>钙黄绿素AM的细胞孵育之后，取代的钙黄绿素AM溶液中的MEA以及用PBS洗涤两次后，用PBS。获得使用与PI（激发/发射 - 535 nm/617纳米）和钙黄绿素AM（激发/发射 - 490 nm/520纳米）的过滤器，用光学显微镜的细胞的荧光图像。 PI的吸收，由于电，使细胞发荧光红和钙黄绿素AM导致细胞是活着的绿色荧光。有三种可能的情况下，可以观察到：1）细胞电穿孔和活着（会发出荧光红色和绿色），2）细胞存活，但没有电穿孔（荧光只是绿色）和3）细胞死亡电（荧光红刚） 。计算细胞存活率，在电极上的细胞存活的总数的百分比和电穿孔EFFiciency加载带有PI /的siRNA的细胞总数的百分数计算。 </li></ol> 5。代表性的成果 图1显示了特定网站的加载Hela细胞PI。可以观察到的，只在电极上的细胞表现出的PI（ 图1B）中的摄取。进行的现场试验后的电用于评估的可行性细胞（ 图1A）。电穿孔效率和在图1中所示的例子中的细胞存活率分别为81.8％和96.1％。高的细胞存活率和电穿孔效率，可以实现优化的电穿孔脉冲参数。特定于站点的转染的HeLa细胞用荧光标记的siRNA为一个优化的电穿孔脉冲（8 V，1毫秒）在图2中示出。电穿孔效率为8 V，1毫秒的siRNA被发现是74.4±16.0％，而细胞活力被发现是87.9±8.4％（所有数据表示为平均值±标准偏差，N = 5）。 <img alt="图1" src="/files/ftp_upload/4415/4415fig1.jpg" /> 图1特定站点200μm的电极（6 V，5毫秒）在HeLa细胞中对交付PI。 A）Calcien根据现场试验进行了2小时后电。活细胞显示绿色荧光。 B）红色荧光表明在电极上的细胞吸收PI。 C）的叠加后的图像A和B的白色箭头表示的电穿孔和活的细胞。黑色箭头表示的细胞都死了。在A，B和C的白圈表示在电极的轮廓。 <img alt="图2" src="/files/ftp_upload/4415/4415fig2.jpg" /> 图2转染的HeLa细胞用荧光标记的scramblË序列的siRNA。 A）用Alexa 488标记的siRNA的（8 V，1毫秒）转染的HeLa细胞的图像上的100微米的电极。 B）的细胞核用DAPI染色的图片。的白色圆圈标记的边缘的电极。

<ol>
	<li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Caplen, N. J., Parrish, S., Imani, F., Fire, A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+gene+expression+by+small+double-stranded+RNAs+in+invertebrate+and+vertebrate+systems.">Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.</a> Proceedings of the National Academy of Sciences. 98, 9742 (2001).</li><li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550 (2002).</li><li>Lee, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+small+interfering+RNAs+targeted+against+HIV-1+rev+transcripts+in+human+cells.">Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.</a> Nat. Biotechnol. 20, 500-505 (2002).</li><li>Miyagishi, M., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U6+promoter-driven+siRNAs+with+four+uridine+3'+overhangs+efficiently+suppress+targeted+gene+expression+in+mammalian+cells.">U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.</a> Nat. Biotechnol. 20, 497-500 (2002).</li><li>Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+expression+of+small+interfering+RNA+in+human+cells.">Effective expression of small interfering RNA in human cells.</a> Nat. Biotechnol. 20, 505-508 (2002).</li><li>Xia, H., Mao, Q., Paulson, H. L., Davidson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+gene+silencing+in+vitro+and+in+vivo.">siRNA-mediated gene silencing in vitro and in vivo.</a> Nat. Biotechnol. 20, 1006-1010 (2002).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Res. 15, 1311 (1987).</li><li>Felgner, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proceedings of the National Academy of Sciences. 84, 7413 (1987).</li><li>Raptis, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=applications+of+electroporation+of+adherent+cells+in+situ,+on+a+partly+conductive+slide.">applications of electroporation of adherent cells in situ, on a partly conductive slide.</a> Mol. Biotechnol. 4, 129-138 (1995).</li><li>Jordan, M., Schallhorn, A., Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+mammalian+cells:+optimization+of+critical+parameters+affecting+calcium-phosphate+precipitate+formation.">Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.</a> Nucleic Acids Res. 24, 596-601 (1996).</li><li>Gao, Y., Liu, X. L., Li, X. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+on+siRNA+delivery+with+nonviral+carriers.">Research progress on siRNA delivery with nonviral carriers.</a> International Journal of Nanomedicine. 6, 1017 (2011).</li><li>Ziauddin, J., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays+of+cells+expressing+defined+cDNAs.">Microarrays of cells expressing defined cDNAs.</a> Nature. 411, 107-110 (2001).</li><li>Yamauchi, F., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+gene+transfer+by+electroporation+into+adherent+cells+on+plasmid+DNA-loaded+electrodes.">Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.</a> Nucleic Acids Res. 32, e187 (2004).</li><li>Yamauchi, F., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-Layer+Assembly+of+Poly+(ethyleneimine)+and+Plasmid+DNA+onto+Transparent+Indium?+Tin+Oxide+Electrodes+for+Temporally+and+Spatially+Specific+Gene+Transfer.">Layer-by-Layer Assembly of Poly (ethyleneimine) and Plasmid DNA onto Transparent Indium? Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer.</a> Langmuir. 21, 8360-8367 (2005).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsystem+for+transfection+of+exogenous+molecules+with+spatio-temporal+control+into+adherent+cells.">Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells.</a> Biosensors and Bioelectronics. 22, 863-870 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-chip+for+spatially+controlled+transfection+of+nucleic+acid+payloads+into+cells+in+a+culture.">Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture.</a> Lab on a Chip. 7, 1004-1011 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+array+(MEA)+platform+for+targeted+neuronal+transfection+and+recording.">Microelectrode array (MEA) platform for targeted neuronal transfection and recording.</a> Biomedical Engineering, IEEE Transactions on. 55, 827-832 (2008).</li><li>Jain, T., McBride, R., Head, S., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+introduction+of+nucleic+acids+into+mammalian+cells+grown+in+microwell+arrays.">Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays.</a> Lab on a Chip. 9, 3557-3566 (2009).</li><li>Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+high-throughput+electroporation+microchip+applicable+for+siRNA+delivery.">An efficient and high-throughput electroporation microchip applicable for siRNA delivery.</a> Lab Chip. 11, 163-172 (2010).</li><li>Li, Z., Wei, Z., Li, X., Du, Q., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+high-throughput+electroporation+chips.">Efficient and high-throughput electroporation chips.</a> , (2011).</li><li>Jain, T., Papas, A., Jadhav, A., McBride, R., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+surface-bound+siRNAs+in+microwell+arrays.">In situ electroporation of surface-bound siRNAs in microwell arrays.</a> Lab Chip. 12, 939-947 (2012).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporationfor+Gene+Transfer+In+Vivo.">Single-Cell Electroporationfor Gene Transfer In Vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Akaneya, Y., Jiang, B., Tsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+gene+silencing+by+local+electroporation+in+targeting+brain+region.">RNAi-induced gene silencing by local electroporation in targeting brain region.</a> J. Neurophysiol. 93, 594 (2005).</li><li>Lee, W. G., Demirci, U., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+electroporation:+challenges+and+perspectives+for+clinical+applications.">Microscale electroporation: challenges and perspectives for clinical applications.</a> Integr. Biol. 1, 242-251 (2009).</li><li>Boukany, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nature Nanotechnology. 6, 747-754 (2011).</li></ol>

没有利益冲突的声明。

在这个视频文章中，我们演示了如何使用MEA站点特定的扰码序列的siRNA转染HeLa细胞。这种技术的优点之一是其适用于不同的细胞系，包括原代细胞系。我们实验室先前已经展示了使用这种技术的位点特异性转染的原代海马神经元培养E18日龄大鼠和NIH-3T3细胞，加扰的siRNA序列和GFP质粒18，19。我们也有过成功提供功能性siRNA对HeLa细胞中内源性的目标（未发表的数据）。典型的市售MEA是兼容与?...

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>手机媒体： 高级MEM L-谷氨酰胺200毫米 青霉素/链霉素 胎牛血清</td><td> Gibco / Invitrogen公司 HIMEDIA实验室/ VWR 龙沙集团有限公司 Gibco / Invitrogen公司</td><td> 12492-013 95057-448 09-757F 16000-044 </td><td>细胞培养基组成为： 2％胎牛血清，L-谷氨酰胺和2％，2％Pennstrep提前MEM </td></tr><tr><td>胰蛋白酶EDTA </td><td>敏达公司</td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td>敏达公司</td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa的488和若丹明标记炒SEQUENCE的siRNA </td><td> Qiagen公司</td><td> 1027292 </td><td></td></tr><tr><td>电穿孔缓冲液</td><td> BioRad实验室</td><td> 165-2677 </td><td></td></tr><tr><td>波形发生器</td><td>务实</td><td> 2414A </td><td>可以使用任何波形/脉冲发生器，它可提供所需的脉冲。 </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

发现RNAi途径中真核生物的RNA干扰剂，如siRNA和shRNA的后续发展，都取得了沉默特定基因的功能基因组学和治疗学1-8的一种有效的方法。 RNAi技术为基础的研究中所涉及的一个主要挑战是交付的RNAi药物的靶细胞。传统的非病毒载体技术，如大容量电和化学品的转染方法往往缺乏必要的空间控制交付和9-12负担转染效率差。化学转染方法，例如阳离子性脂质，阳离子性聚合物和纳米粒子中的最新进展已经导致在高度增强的转染效率13。然而，这些技术仍无法提供精确的空间控制权交付，可以极大地有利于小型化，高通量的技术，单细胞的细胞 - 细胞相互作用的研究和调查。 NT“&gt;基因传递的最近的技术进步已经使高通量的贴壁细胞转染14日至23日 ，其中大部分使用微型电。微型电，提供了精确的时空交付控制（单细胞），并已被证明此外，实现高效率，19，24-26。电穿孔基础的方法不要求长时间的潜伏期与siRNA和DNA复合物作为必要的化学基的转染方法（通常为4小时），并导致裸siRNA和DNA直接进入因此基因表达的分子进入细胞的细胞质中。可以实现早六个小时后，转染27。我们实验室先前已经展示了采用微电极阵列（MEA）的位点特异性转染贴壁哺乳动物细胞培养17-19。在MEA为基础的方法，实现基因的有效载荷交付通过局部的微观尺度electroporati上的细胞。到选定的电极产生的电脉冲的应用程序，导致刺激电极的区域中存在的细胞电穿孔的局部电场。微电极的独立的控制提供了空间和时间的控制权转染，并还可以使多个转染实验吞吐量增加和减少培养至培养变异上要执行的相同的培养的基础的实验。 在这里，我们描述的实验装置和有针对性的粘附用荧光标记的使用电穿孔的扰频序列的siRNA的HeLa细胞转染的协议。相同的协议也可以用于转染的质粒载体。此外，这里所描述的协议可以很容易地扩展到轻微的修改的各种哺乳动物细胞系。使该技术的商业可用性多边环境协定与预定义的和自定义的电极图案访问吨大多数研究实验室基本的细胞培养设备。

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

文章详细介绍了协议的站点特定的扰码序列的siRNA转染贴壁哺乳动物细胞培养用微电极阵列（MEA）。

高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

13.4K 次观看.  Arizona State University. 文章详细介绍了协议的站点特定的扰码序列的siRNA转染贴壁哺乳动物细胞培养用微电极阵列（MEA）。

视频: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

科研

生物工程

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

网站特定的细菌染色体工程：ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

用于Vivo神经记录的 Ti3C2 MXene 微电极阵列的制造

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

使用人iPSC衍生的心肌细胞，心脏成纤维细胞和内皮细胞制造3D心脏微组织阵列

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...