Name: high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)
Uploaded: 2012-09-13T13:00:00
Description: Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

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...

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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 ...

Research

JoVE Journal

Bioengineering

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

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)

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

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 ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

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

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

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 ...

Easy and Accurate Mechano-profiling on Micropost Arrays

在Micropost阵列方便，准确机械力分析

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

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

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

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

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 ...