Name: optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in hek-293t cells
Uploaded: 2011-01-19T18:00:00
Description: Watch this Scientific Journal Video about Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells at JoVE.com

这项工作得到了发现工作津贴，从自然科学和捷工程研究理事会（NSERC），加拿大，NSERC亚历山大格雷厄姆贝尔加拿大研究生奖学金（博士）（A.塞纳托雷）和心脏与中风基金会，格兰特在援助NA＃6284。

<html> 1。细胞培养<ol><li>人类胚胎肾293T细胞（HEK - 293T，Invitrogen公司）是生长在通风25厘米2烧瓶（组织培养治疗格雷纳生物的之一;;蜂星）贴壁单层含贝科的改性老鹰中等（DMEM培养液;西格玛爱秩序）6ML辅以用10 ％V / V小牛血清（FBS;西格玛Aldrich公司），1％丙酮酸钠，在37和250微克/毫升青霉素/链霉素℃，5％的CO 2）。 细胞培养中的CO 2孵化器（5％的CO 2）设?...

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	<li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> J. Pharmacological and Toxicological Methods. 51, 187-200 (2005).</li><li>Senatore, A., Spafford, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+big+are+key+features+of+an+invertebrate+T-type+channel+(LCav3)+from+the+central+nervous+system+of+Lymnaea+stagnalis.">Transient and big are key features of an invertebrate T-type channel (LCav3) from the central nervous system of Lymnaea stagnalis.</a> Journal of Biological Chemistry. 285, 7447-7458 (2010).</li><li>Ogden, D., Stanfield, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+single+channel+and+whole-cell+recording.">Patch clamp techniques for single channel and whole-cell recording.</a> Microelectrode techniques: the Plymouth workshop handbook. , (1987).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamping:+an+introductory+guide+to+patch+clamp+electrophysiology.">Patch clamping: an introductory guide to patch clamp electrophysiology.</a> , (2003).</li><li>Peng, S. -. Q., Hajela, R. K., Atchison, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow-induced+increase+in+inward+Ba2++current+expressed+in+HEK293+cells+transiently+transfected+with+human+neuronal+L-type+Ca2+channels.">Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+channels.</a> Brain Research. 1045, 116-123 (2005).</li></ol>

<html>能够记录使用所描述的协议和解决方案的全细胞电流显示，转染成功，所需的电压门控离子通道或离子通道复合物在细胞膜的大量。细胞应该积极转（即绿色荧光），但没有录制的电流，更多的时间在28 ° C可要求允许必须包装妥当，并插入到细胞膜上的通道蛋白。注意，长期孵化28 ° C时，将会导致细胞分离从盖玻片，膜的破坏和堆积死细胞碎片，使膜片钳记录更具挑战性的。所有在本议定?...

<table border="1"><tr><td>材料</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td> pIRES2 - EGFP的质粒</td><td> Clontech公司</td><td> 6029-1 </td><td></td></tr><tr><td>圆盖玻片</td><td> Fisher Scientific则</td><td> 12-545-80 </td><td>圈第1号 - 0.130.17毫米厚，尺寸：12mm </td></tr><tr><td>放大器</td><td> Axon仪器</td><td> Axopatch 200B中或Multiclamp 700B </td><td></td></tr><tr><td>数据采集​​系统</td><td> Axon仪器</td><td> Digidata 1440A </td><td></td></tr><tr><td>电软件</td><td> Axon仪器</td><td> pClamp10.1 </td><td></td></tr><tr><td>移液器造</td><td>萨特仪器有限公司</td><td> MPC - 385 - 2 </td><td></td></tr><tr><td> Epifluorescence倒置显微镜</td><td>蔡司加拿大</td><td> AXIOVERT 40紧凑型荧光灯</td><td></td></tr><tr><td>探头</td><td> Axon仪器</td><td> CV - 7B </td><td></td></tr><tr><td>探头电极架</td><td> Axon仪器</td><td> 1，HL - U </td><td></td></tr><tr><td>接地电极的微电极持有</td><td>世界精密仪器</td><td> MEH3RF 15 </td><td></td></tr><tr><td>电毛细管</td><td>萨特仪器有限公司</td><td> BF - 150 - 86 - 15 </td><td>硼硅玻璃长丝，外径1.5毫米，OD0.86​​毫米，15cm长</td></tr><tr><td>火焰山/布朗微管拖轮</td><td>萨特仪器有限公司</td><td> P - 97 </td><td></td></tr><tr><td>微管火抛光</td><td> Narishige </td><td>微锻造MF - 830 </td><td></td></tr><tr><td>微型药物灌注系统</td><td>自动化科学</td><td> SmartSquirt8 Valvelink8.2 </td><td></td></tr><tr><td>药物灌注系统</td><td>自动化科学</td><td> Valvelink8.2与聚四氟乙烯阀</td><td></td></tr></table>

optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in hek-293t cells

在体外表达和重组在体外培养的人类胚胎肾细胞的电压门控离子通道的电生理记录（HEK - 293T）是一个无处不在的研究策略。 HEK - 293T细胞必须镀在足够低的密度，使他们在相互接触不是为了让电生理记录没有因接触与相邻的细胞混杂影响到盖玻片。转染的渠道也必须在全细胞膜片钳记录检测到的电流噪音水平以上的质膜与高效率的表达。异种离子通道往往需要长的潜伏期，在28 ° C转染后，以达到足够的细胞膜表达，但有越来越多的盖玻片细胞粘附和膜的稳定性，在此温度下的损失。要绕过这个问题，我们开发了一种优化策略，以转染和板的HEK - 293T细胞。这种方法需要在一个相对较高的汇合，被转染细胞，并培养28 °不同的潜伏期后转染，以便有足够的离子通道蛋白的表达转染细胞，然后接种到盖玻片培养在37℃为好几个小时，这对于刚性细胞附着到盖玻片和膜restabilization允许。细胞可记录后不久，电镀，或可转移至28 ° C的进一步孵化。我们发现，最初的潜伏期在28 ° C转染后，但在此之前，电镀，高效表达异源离子通道，一般不会在细胞膜表达的关键。正转，培养的细胞共同表达的eGFP或的eGFP确定重组离子通道的cDNA内部核糖体进入位和EGFP编码序列的上游，从双顺反子载体（如pIRES2 - EGFP）表达。全细胞膜片钳记录需要专门的设备，加上抛光的记录电极和“L”形的接地电极，高硼硅玻璃制作。药物输送到离子通道药理学研究的，可以实现直接micropipetting进录音碟药物，或使用microperfusion或重力流系统记录细胞产生不间断的药物溶液流。

Watch this Scientific Journal Video about Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells at JoVE.com

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

高效可靠的方法<em&gt;在体外</em&gt;表达和随后的重组中培养的人类胚胎肾细​​胞电压门控离子通道（HEK - 293T）的电生理记录。

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

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