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）设置或者37 ° C或28 ° C 。与细胞的所有工作是在层流罩。 </li><li>分裂的细胞通常双周刊，从〜90％汇合，在一个星期一1:8稀释，星期四1:12。 （参见图1在不同密度的HEK - 293T细胞镀图像） </li><li>分裂，媒体是从细胞培养瓶吸，细胞通过应用胰蛋白酶（Sigma公司Aldrich公司）加热到37 ° C至倒瓶的解决方案1ML分离。随后倒，轻轻摇晃烧瓶，手工等，胰蛋白酶溶液过几次贴壁细胞的移动。胰蛋白酶，然后迅速取出的愿望和250μL胰蛋白酶再次震撼移到细胞的胰蛋白酶溶液的烧瓶micropipetted。 </li><li>保温瓶，然后设置中的CO 2培养箱中孵育至37 ° C 3-5分钟，吸管是用来中止细胞在温暖的补充DMEM培养液4ml。不完整的胰蛋白酶和暂停会导致细胞的团块，并可能导致细胞生长向上，而不是预定的单层。过胰蛋白酶，可能会损坏细胞。 </li><li>对于1:8分割，添加悬浮细胞500μL被添加到一个新的烧瓶中含有DMEM培养液5.5mL;（见图1）为1:12的分裂，细胞333μL被添加到一个新的烧瓶中含有DMEM培养液5.7mL 。保温瓶是轻轻地混合，然后转移到37 ° C培养箱中培养。在3 - 4H的潜伏期在37 ° C后分裂，细胞将泥沙和坚持烧瓶。细胞最初环顾，但将扁平化，并更加坚定附件的扩展名（图1）（伪）。 细胞应该在一个在可预见的方式贴壁单层的增长。如果细胞的生长和外观非典型或不一致的，技术差或污染（细菌/支原体）应考虑。组织培养成功的关键是一致性。 </li></ol> 2。重组离子通道的cDNA转染的HEK - 293T <ol><li>在这里，各种离子通道的cDNA克隆入pI​​RES2 - EGFP质粒（BD Biosciences公司Clontech公司），允许正转染的细胞，通过增强型绿色荧光蛋白（EGFP）荧光识别。 EGFP是在相同的克隆插入基因插入基因和EGFP的编码序列（图2）上游下游内部核糖体进入网站发起，通过翻译，成绩单转染的细胞产生。积极与pIRES2 - EGFP结构中的钙离子通道亚基转染的HEK - 293T荧光图像，请参阅图3。 </li><li>转染前，是一个完全融合烧瓶一个新瓶分成1:4，和细胞可以附着在37 ° C为3-4小时（见图1）中的CO 2培养箱。 </li><li>细胞连接后，一个标准的磷酸钙转染战略（冷泉港协议）是用于引进包含到HEK细胞的离子通道和配套亚基的cDNA的结构。通常情况下，每一个12μg的DNA共有600μL转染的解决方案ISE瓶/转。 </li><li>转染后的解决方案是在细胞下降明智的吸管​​，烧瓶中放入37℃培养箱中过夜。 </li><li>以下的转染，细胞仔细洗净三次，用温水媒体或磷酸盐缓冲液（4-5毫升），通过洗涤液吹打成一个倒置的瓶中，然后缓缓转动烧瓶直立和摇晃烧瓶中，使洗涤液在移动细胞。如果不这样做仔细的细胞可以detatch很少会后三个步骤洗左。重复洗两次，然后是6毫升媒体加入到烧瓶中，它是在37 ° C为2小时，然后转移到28 ° C培养箱中培养。 </li><li>成功转染pIRES2 - EGFP的结构可以确认使用epifluorescence显微镜下转染后1〜3天。当暴露在紫外线下发出荧光绿色的细胞正转，应该表达异源的离子通道和EGFP蛋白。 </li></ol> 3。电镀转染HEK293T细胞电生理记录准备NT“&gt;不同的离子通道表达不同的HEK细胞的细胞膜的效率，正因为如此，本协议的其余部分需要一些优化，以最大限度地提高成功的电生理记录的通道表面表达。，在HEK细胞异种离子通道的表达，尤其是那些从非哺乳动物来源，可能会出现一些挑战，包括（1）大蛋白的大小，需要较长时间的翻译和本地化的质膜（2）缺乏或折叠和定位上的通道蛋白在哺乳动物图案必要的异同细胞系（3）在密码子使用频率的差异，从物种的物种。所有这些因素综合起来，可能会导致在一个长期的孵化时间要求在28 ° C，这确保没有有效的蛋白表达和细胞分裂的表面本地化（会冲淡异的cDNA和蛋白质）。不幸的是，孵化长时间在28 ° C，导致细胞脱落和膜的稳定性差，难以电生理记录，我们决定修改传统的转染战略（2005年托马斯和智能审查），以尽量减少孵化在28 ° C前的记录和最大限度地表面的表达。我们的方法需要转染的细胞，培养3-7天的时间在28℃，然后由胰蛋白酶消化分离细胞盖玻片上replated和培养，在37 °彗星restabilizes膜和强烈的依恋到盖玻片的细胞允许细胞，然后能够马上被记录，或可用于额外的时间内培养28 ° C时我们发现在稍后阶段将盖玻片细胞replating在我们的方法是绝对必要的生产具有高的数字通道表达的，独立的贴壁细胞的盖玻片。 我们提出了两种可供选择的战略，我们宁愿使用的离子通道和离子通道的复合物，不含有大量的胞外域可能是脆弱的胰酶消化（如Cav3电压门控性钙通道; 2010年塞纳托雷和Spafford），我们使用的离子通道，含有大量的胞外域（如L -型电压门控性钙通道及其配套亚基 ;塞纳托雷，等2010） 。 <ol><li><ol><li>细胞培养在29 ° C为2-6天，以防止细胞分裂，并让翻译蛋白质的积累。此孵育时间必须凭经验确定每个离子通道，因为表面表达的是依赖于内在因素，决定如何交互渠道“新生的mRNA和多肽序列的平移机械和分泌途径，分别。 </li><li>电镀之前，圆形盖玻片（圆1号- 0.130.17毫米厚;尺寸：12毫米，费舍尔科学加拿大，渥太华，安大略省）都涂有聚- L -赖氨酸（Sigma公司）生产商的指示。 </li><li>从转染细胞，媒体被删除吸1毫升37 ° C胰蛋白酶溶液（Sigma公司）是一个倒置瓶（细胞的一面朝上）micropipetted。烧瓶，然后轻轻地倒到直立位置，使胰蛋白酶在细胞上运行，并烧瓶中，然后重新倒吸删除和胰蛋白酶。 250μL温暖的胰蛋白酶，然后直接添加在细胞和烧瓶中培养在37 ° C为3-5分钟，直至细胞detatch。 </li><li>同时，聚- L -赖氨酸涂层的盖玻片放入6毫米培养皿中（每盘3-8）和5毫升的温暖文化传媒添加到每一道菜。 </li><li>胰酶消化细胞悬浮与温暖的文化传媒4ML移液器向上和向下〜6倍，然后重悬细胞250 -500μLmicropipetted到文化含有盖玻片的菜肴。除了细胞之前，使用的微尖的盖玻片上推下来，以确保它们在底部的菜。 </li><li>细胞，然后在37 ° C为3小时，让他们坚持的盖玻片，然后将它们转移到28 ° C此时，细胞电生理记录，这是最好单独的贴壁细胞（图3说明转染细胞前后镀上盖玻片）做准备。离开太久的细胞在37 ° C会导致细胞分裂。 </li></ol></li><li><ol><li>细胞表达渠道/大外域通道复合物的制备是在大致相同的方式如上所述进行的异常，盖玻片，是在28 ° C是一个额外的电生理记录前2-4天进行孵育。这使得重新积累的通道/通道Complexes后胰蛋白酶的细胞膜。 </li></ol></li></ol> 4。重组渠道的HEK - 293T细胞的电生理记录几个综合资源描述的基本电生理记录（如奥格登和斯坦菲尔德1987年，Molleman 2003年）的一般概念。 4.1电钻机一个典型的电钻机（图4）包括一个放大器（如Axopatch 200B中或Multiclamp 700B放大器; Axon仪器，盟市，CA），pClamp10.1一起与Digidata 1440A模拟到数字转换接口配备一台PC计算机软件（Molecular Devices公司，位于加利福尼亚州森尼韦尔），机动，双吸液管造（MPC - 385 - 2，萨特仪器公司），epifluorescence显微镜（AXIOVERT 40紧凑型荧光灯，蔡司加拿大，多伦多，安大略省）和灌注系统（图4和5 ）。振动是有​​限的设备上安装一个TMC的63-500系列隔振表（技术制造公司，皮博迪。硕士）。杂散电噪声是有限的，使用40“高II型法​​拉第笼（TMC）的周围电气设备安装隔振表。，电子控制系统（如电脑及显示器，放大器，模拟到数字转换器，电动操纵器和Valvelink灌注控制系统）安装法拉第笼外的一个独立的五金电器架（哈蒙德制造，安大略省圭尔夫）所有电气设备的接地铜经销商和插入到（1800W的Tripp Lite的医疗隔离等级Tranformer UL60601 - 1），其中提供了隔离线，一以贯地面和浪涌抑制。 4.2补丁移液器硼硅玻璃毛细管长丝（BF150 - 86 - 15;外径1.5毫米，外径0.86毫米，15厘米长度萨特仪器公司）减少一半，并插入一个燃烧/布朗微管牵引器型号的P - 97（萨特仪器公司） 。吸管比赛项目优化方案是始终如一地生成25MΩ从电极到地（火抛光后;图6）之间的电阻移液器移液器个别火抛光允许对细胞膜紧密密封的枪头表面平滑（ MF - 830微锻造; Narishige，日本;图6）。探头安装在一个专门的持有人（1 - HL - U;分子器件，桑尼维尔加州）微量移液器。 4.3接地电极硼硅玻璃，用来做补丁移液器管也用来做参比电极。 15cm长管切成3件。 2细尖钳，管子被关押在本生灯火焰，直到玻璃的延展性和管材可弯曲的“L”或“曲棍球棒”与一个90度角的形状。虽然玻璃管依然火爆，一端是立即沉浸在炎热的3M铯和1.5％琼脂糖的解决方案，这是绘制成管通过毛细作用。一旦参比电极是完全填补与解决方案，它被放置在一个烧杯中的冷水。所有的参比电极填充后，他们逐个消灭Kimwipes删除从外面额外琼脂糖和淹没在3M铯解决方案。参比电极的电生理记录，举行一个微电极架半电池（持有90 F颗粒1.5毫米;世界精密仪器公司，美国佛罗里达州萨拉索塔;＃MEH3RF15）充满3M铯解决方案。 （在沐浴液的接地电极的插图，请参阅图7） 4.4电录音解决方案电压门控离子通道的不同类型需要不同的录音解决方案，取决于离子通透性/选择性。电压门控性钙通道，细胞通常是沐浴在含有不同浓度或者钡或钙的外部解决方案。例如，无脊椎动物Cav3电压门控性钙通道（LCav3）的电生理记录可以使用外部5mm 的氯化钙，166mM四乙基胺（TEA - CL），10MM的HEPES溶液中含有与茶- OH调整pH值至7.4 。补丁移液器充满了1MM，氯化镁， 氯化钙 2MM 125MM CSCL，10MM EGTA 2，4MM MgATP，0.3MM的Tris - GTP与pH值7.2（塞纳托雷和Spafford，2010）10MM的HEPES的内部解决方案。无脊椎动物的L -型电压门控性钙通道（LCav1）可以使用外部的解决方案包含的电荷载体（15mm的氯化钡，1毫米氯化镁2，10毫米的HEPES，40MM茶- CL，72.5mM CSCL，10mm的葡萄糖，钡记录TEA - OH调整至7.2的pH值）和一个内部的解决方案包含108MM CS -磺酸，4mm的9MM EGTA 2，氯化镁，9MM HEPES，CsOH（ 塞纳托雷等调整pH至7.2，2010）。 4.5全细胞记录<ol><li>含有转染细胞（表达异源离子通道或离子通道复合物）的盖玻片被转移到一个35mm的塑料培养皿中含有〜3ML的外部录音解决方案。录音解决方案的体积必须精确测量时进行药物实验药物已知金额被添加到与微菜。电生理记录通常是在室温下进行，虽然研究者可能想改变这个温度取决于实验。 </li><li>细胞通过epifluorescence显微镜观察，一个孤立的，正转，贴壁绿色的细胞在视野中居中。 </li><li>补丁吸管放进洗澡使用的显微操纵器，完成补丁吸管电极和参比电极之间的电路解决方案。虽然在洗澡的模式，pClamp10.1软件使放大器产生小重复步骤吸管和参比电极之间的电压，并显示为一个方波跟踪（图8A）所产生的电流。这方波跟踪必须归零，使用前修补细胞放大器的偏移吸管。吸液管电阻测量的pClamp10.1软件，并应在2 -5MΩ。 </li><li>在浴缸模式时，吸管是到邻近的细胞，它可以作为在快速波动的方波跟踪可视化带来的，然后吸少量应用的修补吸管形式之间的吸管和gigaohm密封膜（导致方波跟踪一个完整的平面线图8B）。 </li><li>该程序，然后切换到补丁模式，吸管电容（电流的微小的光点，平当前跟踪的前缘和后缘可见;图8B）是吸电容补偿放大器上的补偿。 </li><li>一个吸量小，但尖锐的应用膜破裂的修补程序，并允许进入细胞内的电极。成功的膜破裂是由外观也大电容瞬态电流波形跟踪的边缘（图8C）的开头和结尾的表示。一旦突破已经实现，程序切换到细胞的模式和整个电池容量补偿调整放大器上消除电容瞬态。有几分钟的延迟，细胞内进入细胞之前，录音的录音解决方案，以便平衡。 </li><li>产生电压指令和数据是使用一个Digidata 1440A结合pClamp10.1软件模拟到数字转换器接口配备一台电脑收购。独特的电压钳协议被写入每个实验，这些都是用来记录转染细胞的离子通道电流。图9显示了记录的HEK - 293T细胞表达的无脊椎动物LCav3电压门控性钙通道钙电流。在- 110mV的电压，细胞被送往- 70mV和20 mV的增量之间的depolarisations引出内向钙电流。 </li><li>录制的电流是在10 kHz的低通贝塞尔滤波器过滤和采样频率为2 kHz数字化。只有以最小的泄漏（&lt;10％）录音是用于分析和离线泄漏减法是使用Clampfit 10.1软件进行。 </li></ol> 4.6记录细胞的药物研究药物可直接到正在录制的菜吸管。如果药量增加和沐浴液量是已知的，可以计算药物浓度。聚酰亚胺灌注铅笔，一个250微米的可移动提示使用八通道Smartsquirt压力微灌注系统（自动化科学）或重力流系统通过聚四氟乙烯阀门操作昂贵​​的药物或数量有限，也可能是添加从一个多管歧管microperfusion Valvelink8阀控制器（自动化，科学化;灌注系统设置见图5）。 microperfusion系统的流速在每分钟0.1ml的一个退出灌注铅笔记录细胞从400微米的流。灌注笔尖记录电极尖端的距离应该是一致的补丁修补的;图10说明了建议的距离在40倍放大倍率可见。足够的流速提供饱和的记录细胞的灌注。使用microperfusion前进行药物实验，细胞是在一个连续的数据流与控制槽液microperfusion的平衡。流最小的中断是避免在快速切换控制浴，药物或洗涤液的实验。在录音碟的边缘上的吸力可以采用tØ删除溢出的解决方案，通过手动吸用注射器或使用蠕动泵驱动装置（例如MINISTAR微型直流蠕动泵，世界精密仪器）。必须小心，因为极为迅速灌注诱导的剪切力，可能会改变钙电流（Peng等，2005），可能会洗去被记录下来的细胞，并耗尽药物水库太快。 直接pipeting入菜与灌注系统 <table border="1"><tbody><tr><td></td><td>直接吹打</td><td>微型灌注系统</td><td>重力流灌注系统</td></tr><tr><td>需要卷药物</td><td>中度</td><td>低</td><td>高</td></tr><tr><td>技术技能</td><td>低</td><td>中等</td><td>中等</td></tr><tr><td>设备成本</td><td>低</td><td>显着</td><td>显着</td></tr><tr><td>每天的时间设置和清理</td><td>没有</td><td> 20 +分钟</td><td> 20 +分钟</td></tr><tr><td>污染设备</td><td>没有</td><td>是</td><td>是</td></tr><tr><td>故障排除</td><td>轻松</td><td>综合</td><td>综合</td></tr><tr><td>保证药物在菜</td><td>是</td><td>无</td><td>无</td></tr><tr><td>失去细胞与药品加成</td><td>是</td><td>是</td><td>是</td></tr><tr><td>大大降低药物浓度。通过洗涤</td><td>很难</td><td>简单的</td><td>简单的</td></tr><tr><td>易于做多的剂量反应曲线</td><td>不是最佳</td><td>简单的</td><td>简单的</td></tr><tr><td>结果的重复性</td><td>确定 - 依赖于位置的pippeting </td><td>好</td><td>好</td></tr></tbody></table>

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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) ...

optimized-transfection-strategy-for-expression-electrophysiological

Research

JoVE Journal

Biology

32.2K 次观看.  University of Waterloo. 高效可靠的方法在体外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。

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

科研

生物学

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

多功能，微管为基础的法团细菌机械敏感离子通道与刺激的液滴界面双层膜

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...