高效率，站点特定的贴壁细胞转染siRNA的微电极阵列（MEA）
Summary
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）。
Abstract
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“>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。
Protocol
1。 MEA制备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（http:/www.multichannelsystems.com/）和Alpha MED科学，公司从供应商直接购买（ http://www.MED64.com） 。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…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Cell media: Advanced MEM L-Glutamine 200 mM Penicillin/Streptomycin Fetal bovine serum |
Gibco/Invitrogen Himedia Laboratories/VWR Lonza group Ltd. Gibco/Invitrogen |
12492-013 95057-448 09-757F 16000-044 |
Cell media composition: 2% FBS, 2%L-glutamine and 2% Pennstrep in Advance MEM |
| Trypsin EDTA | Mediatech, Inc. | 25-053-CL | |
| PBS | Mediatech, Inc. | 21-040-CV | |
| Alexa 488 and rhodamine tagged scrambled sequence siRNA | Qiagen, Inc. | 1027292 | |
| Electroporation buffer | Biorad Laboratories | 165-2677 | |
| Waveform generator | Pragmatic | 2414A | Any waveform/pulse generator that can deliver the desired pulses can be used. |
Referências
- Fire, A. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391, 806-811 (1998).
- Caplen, N. J., Parrish, S., Imani, F., Fire, A., Morgan, R. A. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proceedings of the National Academy of Sciences. 98, 9742 (2001).
- Elbashir, S. M. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498 (2001).
- Brummelkamp, T. R., Bernards, R., Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 296, 550 (2002).
- Lee, N. S. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500-505 (2002).
- Miyagishi, M., Taira, K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497-500 (2002).
- Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505-508 (2002).
- Xia, H., Mao, Q., Paulson, H. L., Davidson, B. L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006-1010 (2002).
- Chu, G., Hayakawa, H., Berg, P. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 15, 1311 (1987).
- Felgner, P. L. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences. 84, 7413 (1987).
- Raptis, L. H. applications of electroporation of adherent cells in situ, on a partly conductive slide. Mol. Biotechnol. 4, 129-138 (1995).
- Jordan, M., Schallhorn, A., Wurm, F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 24, 596-601 (1996).
- Gao, Y., Liu, X. L., Li, X. R. Research progress on siRNA delivery with nonviral carriers. International Journal of Nanomedicine. 6, 1017 (2011).
- Ziauddin, J., Sabatini, D. M. Microarrays of cells expressing defined cDNAs. Nature. 411, 107-110 (2001).
- Yamauchi, F., Kato, K., Iwata, H. Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes. Nucleic Acids Res. 32, e187 (2004).
- Yamauchi, F., Kato, K., Iwata, H. Layer-by-Layer Assembly of Poly (ethyleneimine) and Plasmid DNA onto Transparent Indium? Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer. Langmuir. 21, 8360-8367 (2005).
- Jain, T., Muthuswamy, J. Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells. Biosensors and Bioelectronics. 22, 863-870 (2007).
- Jain, T., Muthuswamy, J. Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture. Lab on a Chip. 7, 1004-1011 (2007).
- Jain, T., Muthuswamy, J. Microelectrode array (MEA) platform for targeted neuronal transfection and recording. Biomedical Engineering, IEEE Transactions on. 55, 827-832 (2008).
- Jain, T., McBride, R., Head, S., Saez, E. Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays. Lab on a Chip. 9, 3557-3566 (2009).
- Huang, H. An efficient and high-throughput electroporation microchip applicable for siRNA delivery. Lab Chip. 11, 163-172 (2010).
- Li, Z., Wei, Z., Li, X., Du, Q., Liang, Z. Efficient and high-throughput electroporation chips. , (2011).
- Jain, T., Papas, A., Jadhav, A., McBride, R., Saez, E. In situ electroporation of surface-bound siRNAs in microwell arrays. Lab Chip. 12, 939-947 (2012).
- Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. Single-Cell Electroporationfor Gene Transfer In Vivo. Neuron. 29, 583-591 (2001).
- Akaneya, Y., Jiang, B., Tsumoto, T. RNAi-induced gene silencing by local electroporation in targeting brain region. J. Neurophysiol. 93, 594 (2005).
- Lee, W. G., Demirci, U., Khademhosseini, A. Microscale electroporation: challenges and perspectives for clinical applications. Integr. Biol. 1, 242-251 (2009).
- Boukany, P. E. Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nature Nanotechnology. 6, 747-754 (2011).
Citar este artigo
Patel, C., Muthuswamy, J. High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA). J. Vis. Exp. (67), e4415, doi:10.3791/4415 (2012).