我们的工作是由神经科学（PAS），国家药物滥用研究所（R01 DA019022; PAS）的麦克奈特基金的财政支持和博士后NIH印度国家遥感局奖学金到PA（F31AA017042）从美国国家酒精滥用研究所和酗酒等。 GIRK2 - L257W突变提供了Prafulla阿亚尔博士。 荧光显微镜和赞助，请提供徕卡仪器。

1。我要定向诱变我们用真核表达质粒pcDNA3.1 GIRK2的cDNA编码，并引入点突变，使用安捷伦科技公司（Stratagene公司）Quikchange XL II站点定向诱变套件，根据制造商的指示。 引物序列，可以使用在线引物设计方案可在很容易产生： <a href="https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&amp;SubPageType=ToolQCPD&amp;PageID=15">https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&amp;SubPageType=ToolQCPD&amp;PageID=15</a> 请注意：我们使用标准的Eppendorf 1.5 mL管屋宇署14毫升管在协议中所描述的地方。然而，使用XL10金ultracompetent 大肠杆菌大肠杆菌与β-巯基乙醇的预处理，30多岁的热休克时间和 NZY +（ 或 NZYM）媒体是成功的诱变的关键。如果可能的话，工程，创建一个新的酶切位点的一个额外的沉默突变可以方便下面筛选突变菌落。 2。小量制备，DNA测序和Maxiprep 2.1 Qiagen公司的小量制备和测序。 个别细菌菌落采摘和氨苄青霉素（AMP）在LB培养基中生长。我们遵循制造商的指示，为净化的cDNA。一个简单的限制性消化，可用于确认成功引进的突变，如果在上述步骤1中的限制站点。 DNA自动测序完成场外，是必不可少的，以确认基因的突变在，以及评估是否有任何不必要的突变。这一步是非常重要的。我们整个GIRK编码序列使用T7启动子和反向BGH测序引物序列，侧翼的pcDNA3.1质粒的多克隆网站。 注：重要的是要仔细检查，以保证足够的测序数据质量的测序电泳。高品质的电泳锐利，背景噪声小非叠嶂。 2.2 Qiagen公司maxiprep。 我们遵循制造商的指示。 注意：这一步是在我们的实验中，以提高纯度的cDNA，我们发现，高效转染的重要。 3。 HEK293T细胞培养和转染所有这些措施应在无菌组织培养罩。 3.1细胞培养 HEK293T细胞，因为它们很容易文化（37 ° C，5％CO2培养箱）方便地使用，具有快速复制的时间，转染效率高，适合全细胞膜片钳电。 HEK293T细胞表达SV40大T抗原，确保染pcDNA3.1质粒的episomal复制。 HEK293T细胞培养在低糖DMEM辅以L -谷氨酰胺的媒体和10％胎牛血清（FBS）。传代细胞，加入0.25％胰蛋白酶/ EDTA融合（90-95％）HEK293T细胞，等待，直到细胞脱离组织的菜，然后停止含有胎牛血清/新媒体除了酶的活性。板〜1 × 10 5细胞/孔到12孔细胞培养皿，无抗生素的培养基，每孔用1毫升。 （1 × 10 5细胞是〜350μL的细胞悬液，从融合的T25烧瓶重悬于10毫升媒体获得）。 3.2转染 8-24小时后，HEK细胞会坚持到塑料和准备进行转染。与0.2微克通道的cDNA，0.4微克M2R受体cDNA和0.04微克YFP的cDNA的使用Invitrogen公司脂质体2000转染细胞。请注意：我们使用的YFP的少量基因，以确定哪些细胞是成功转染（绿色细胞可能会包含GIRK渠道和M2受体）。每个克隆的cDNA使用的浓度将需要调整。 3.3转染细胞的荧光可视化。 置于12孔板倒置荧光显微镜的舞台上，并检查YFP的表达的细胞。比较DIC的形象，并注意绿色细胞的大致数量。这提供了一个转染效率的估计。 3.4制备24菜和聚- D -赖氨酸涂布盖玻片 12毫米的玻璃盖玻片（华纳，CS - 12R）清洁摇晃他们Radiacwash过夜，用去离子水洗涤，并在一夜之间在95％的乙醇晃动。商店在70％，直到使用乙醇清洁盖玻片。 在培养皿中与乙醇，使用镊子和燃烧器的火焰去乙醇，将盖玻片D发布在一个无菌24孔板。 盖每孔250μL聚- D -赖氨酸（PDL）解决方案[0.2毫克/毫升，让盖玻片坐通宵沉浸在客运专线的解决方案，在无菌罩。在封口膜包裹的24孔板，以避免蒸发。 第二天早上，吸客运专线，并用无菌水冲洗2倍。离开板打开引擎盖下干，再盖。与涂层盖玻片以及24菜用无菌铝箔包裹，并存放于室温数周。 3.5封面上的电镀转染HEK293T细胞单 24小时后转成24孔盘（〜4 × 10 3细胞/孔），分裂细胞含玻璃盖单涂层与D -聚赖氨酸。注：转染的细胞，可用于高达24-72小时后转。细胞分裂提供个别盖玻片膜片钳电生理实验。 4。电 4.1准备解决方案外（浴）解决方案：20毫米140毫米氯化钠，氯化钾，0.5毫米氯化钙 ，氯化镁2 2毫米和10毫米HEPES（pH值7.4用NaOH）。 分装20K液，并添加适当的调制器<ul><li> 20K + BA 2 +溶液中含1毫米BaCl 2：向内整流电流的特异性抑制剂</li><li> 20K +卡巴胆碱：5微米卡巴胆碱的解决方案：一个特定的M2R激动剂; M2R夫妇G蛋白开放GIRK通道</li><li> 20K +乙醇液含100毫米乙醇直接激活GIRK通道</li></ul>注射器的快速交换灌注系统（溶液的容器）放入浴缸的解决方案。请注意：我们使用华纳夹管阀系统控制的沐浴解决方案。 PE管材建议。 内部/电极解决方案：氯化钾140毫米，20毫米氯化钠，EGTA 5毫米，5.4毫米MgCl 2的 ，10毫米与2.5毫米K 2 ATP和0.3微米的李二的GTP HEPES（pH值7.4）。 我们没有ATP和GTP的电极解决方案。我们分别准备ATP和GTP的分装，储存于-80 ° C，并添加到电极上一天的实验解决方案。我们一直在冰上的最后电极解决方案，以维持ATP / GTP的注射器。我们无菌过滤器（0.2微米）的内部/外部的解决方案，以抑制微生物的生长和减少堵塞管道和电极的可能性。 4.2牵引电极我们使用Narishige两步垂直拉拔和华纳仪器（外直径1.5毫米，0.86毫米内径和7.5厘米的长度）的高硼硅玻璃电极获得3-7电阻MΩ时，与细胞内的解决方案填充的电极。 注：这是必要的实验，以确定在你的实验室电极的玻璃电极拉马和类型参数。我们用我们的垂直拉拔，热定型的60.2拉头和第二个下拉〜43。 4.3全细胞膜片钳<ol><li>山盖在一个35毫米的菜的滑移，使用少量的凡士林，盖倒置荧光显微镜舞台上与外部的解决方案和地方菜。使用DIC的重点和发现细胞中，切换到YFP的荧光定位YFP的阳性细胞。 </li><li>灌注附近的绿色细胞的多方面的利息= 2-3个细胞的长度，除了从单元格（20-30微米）的位置提示。 </li><li>填充溶液中的电极，将它附加到Axopatch 200B中探头放大器（Axon仪器）以下的初始设置：1，增益配置：全细胞β= 1，模式V型钳。 </li><li>应用电极上的正压，以防止堵塞吸管，并使用机械手放置电极尖端的单元格上方。 </li><li>零吸管潜力，选择密封试验按钮提供一个测试电压一步。 Clampex密封测试窗口将出现在5 mV的矩形电压一步。调整放大器的移液器偏移电位带来0 nA的电流基准线。另外，零开关V型轨道模式和仪表旋钮Vtrack和使用移液器偏移电位放大器，放大器上的仪表显示“0.00”带来的电压放大器的模式选择吸管潜力。 </li><li>检查使用的Clampex 8.2软件（应为3-7MΩ）密封测试命令电极电阻。 </li><li>打开一个保存的协议文件或创建一个新的协议。开放的协议文件，从主菜单中选择收购然后打开协议，并选择一个保存的电压协议文件。在这个实验中，已创建的协议举行的差小于40mv膜电位，提供一个单一的50 ms的阶跃电压-100 mV和坡道40比为2 mV00毫秒。此协议是执行每2秒。斜坡协议允许转回的潜在的和外来的GIRK通道的整改观察。一个长方形的电压阶跃电压门控离子通道，可能更为合适。 </li><li>方法采用微调操纵细胞，释放出积极的压力，接触细胞表面后，采用负压，监测阻力增加，密封试验示波器窗口。一旦电阻GΩ的范围内，电极密封到膜（Gigaseal已形成）。 </li><li>应用负压脉冲，打破细胞膜，并获得进入细胞的访问。一旦在整个单元配置中，您将注册增加的容性电流。 </li><li>切换到膜Clampex测试，并写下访问电阻Ra，膜电阻Rm，膜电容Cm的验证眼指数拟合的理论曲线，真正的电容性电流。膜电容单元的大小成正比，稍后将用于计算电流密度。 </li><li>切换回密封测试，在10 kHz至-40 mV和过滤器设置的V -输出（膜电位） </li><li>放大器上的膜电容和串联电阻补偿以下步骤： <ol class="lalpha"><li>切换整个细胞上的第开关。调整全细胞第串联电阻拨号来回，直到你得到一个平坦的测试脉冲。您可能还需要调整吸管电容快速第旋钮。 </li><li>打开旋钮％预测60-80％左右，再次重新调整全细胞CAP和串联电阻拨号（您可能需要调整吸电容补偿以及快速第旋钮）。 </li><li>打开％COMP - 100％，无振荡的细胞，同时试图保持脉冲重新调整全细胞CAP和串联电阻（你可能需要调整吸电容补偿FAST第旋钮以及）单位。通过减少时间常数调整的滞后性。 </li><li>写下厘米（膜电容），RS（串联电阻），COMP％的值，％PRED和放大器在笔记本上设置的滞后时间。 CM和RS应约厘米，在上面的膜测试测量室相同。 </li></ol></li><li>打开外部解决方案阀来控制槽液。 8极Bessel滤波器放大器设置到2kHz，开放电压钳协议，并开始录制电流。 </li><li>我们记录GIRK使用一个坡道协议步骤-100 mV的为50毫秒，然后从100 mV上升到40毫伏超过200毫秒，并交付了每2秒的电流。斜坡协议允许的反转电位的观察和GIRK通道内向整流财产。在室温（22-25℃），目前GIRK应扭转近-50毫伏（用20毫米的浴缸氯化钾），这是为K（K = -50毫伏）计算出的平衡电位附近，然后保持在E K积极的潜力相当平坦。 </li><li>实验议定书“：记录一个稳定的基线后，切换到20K +卡巴胆碱的解决方案，并等待响应峰值，切换回20K液，然后申请20K +乙醇和等待峰值响应，然后切换到20K液，最后以20K + BA 2 +解决方案。在这些药物的应用，你会看到GIRK电流（在-100 mV的最明显）的幅度的变化。注意跟踪号码对应不同外解决方案应用在笔记本。一个单一Clampfit的文件将包含所有的扫描整个实验。 </li></ol> 5。数据分析我们使用Clampfit 9.2软件分析数据在-100 mV的GIRK电流。 <ol><li>打开录音文件，设置在-100 mV的光标。默认情况下，你会看到所有在屏幕上覆盖的痕迹。按“写游标”图标，您将创建类似Excel电子表格中的数值数据。 </li><li>分配X跟踪数列和Y光标列，按“创建图形”图标，你可以快速绘制的实验时间，当然情节。 </li><li>减去当前存在BA 2 +在外部仅灌注液（“20K目前”减“20K + BA 2 +电流”）录得的电流计算基底电流。感应电流计算减去基底电流激活电流在20K（20K +卡巴胆碱目前“减”20K目前“，”20K +乙醇“减”20K电流“）。膜电容（直接读取放大器）除以当前计算电流密度（PA / PF）。计算除以基底电流的感应电流，再乘以100％激活。 </li></ol> 6。代表性的成果您应该能够从转染细胞与野生型渠道电流记录。对于GIRK通道的措施d在包含20毫米的解决方案外钾离子（K +的细胞内和145毫米），他们应该表现出很强的内向整流和反转电位〜-50 mV时，这是一种钾离子通道的本质属性。野生型的电流将增加，强烈呼吁卡巴应用（超过基底电流的3-5倍）和100毫米乙醇作出同样反应。注：电签署公约，内向电流负和外向电流是积极的。对于酒精结合口袋GIRK2 L257W突变，你会看到减毒卡巴和乙醇。这表明，L257 GIRK2渠道的酒精和G蛋白激活。对于进一步的例子GIRK2诱变的结果，请参阅阿亚尔等最近出版的 1 。 <img src="/files/ftp_upload/2189/2189fig1.jpg" alt="图1" /> 图1。野生型GIRK2录音Clampfit 8.0显示数据文件的窗口 。左上面板显示叠加扫描记录在不同的外部BAH解决方案存在的斜坡电压协议。光标1定位在-100 mV的。游标值写入到电子表格（右图）。左下面板显示扫描的数量，与目前的情节。注意用乙醇（EtOH）和卡巴（CARB）和电流的抑制与Ba 2 +电流增加（向下偏转） 。 <img src="/files/ftp_upload/2189/2189fig2.jpg" alt="图2" /> 图2。 Clampfit 8.0显示数据为突变GIRK2 - L257W录音文件的窗口 。说明同图1 。注意卡巴（CARB）的活化和减少乙醇诱导电流突变渠道的损失。

<ol>
	<li>Aryal, P., Dvir, H., Choe, S., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+alcohol+pocket+involved+in+GIRK+channel+activation.">A discrete alcohol pocket involved in GIRK channel activation.</a> Nature Neurosci. 12, 988-995 (2009).</li><li>Panchenko, V. A., Glasser, C. R., Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+between+glutamate+receptor+channels+and+K++channels+examined+by+scanning+mutagenesis.">Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.</a> J Gen Phys. 117, 345-360 (2001).</li><li>Doyle, D. A., Cabral, M. o. r. a. i. s., Pfuetzner, J., Kuo, R. A., Gulbis, A., Cohen, J. M., Chait, S. L., &amp;amp, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+potassium+channel:+molecular+basis+of+K++conduction+and+selectivity.">The structure of the potassium channel: molecular basis of K+ conduction and selectivity.</a> Science. 280, 69-77 (1998).</li><li>Nishida, M., Cadene, M., Chait, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Kir3.1-prokaryotic+Kir+channel+chimera.">Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.</a> EMBO J. 26, 4005-4015 (2007).</li><li>Tao, X., Avalos, J. L., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+eukaryotic+strong+inward-rectifier+K++channel+Kir2.2+at+3.1+&amp;Aring;+resolution.">Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 &amp;Aring; resolution.</a> Science. 326, 1668-1674 (2009).</li></ol>

定点突变是一个经典的方法来研究离子通道的结构与功能的关系。它是基于的前提下，通过改变离子通道的关键残留之一，应该能够观察到的通道功能的明显变化。与此相反，在许多非关键位置的突变，可以引进无通道功能的主要扰动。通常情况下，残留问题首先是突变为丙氨酸，一个小型的非极性氨基酸，色氨酸，氨基酸最大的2突变。如果残留物是很重要的通道功能，这两个基因突变会改变通道活动。如果一个较次要的残基突变，突变，特别是丙氨酸的影响往往是不值一提。色氨酸突变更容易引起的变化，由于色氨酸大小的立体障碍。通常情况下，当结构信息缺乏，系统内的通道（称为丙氨酸或色氨酸扫描）片段突变为丙氨酸或色氨酸可用于定位的关键残留。重要的是要注意，不同的点突变的多个克隆可以并行测试多个残留在相对较短的时间内准备。最近，多个渠道的晶体结构已成为可用3,4,5。它们可以用来使用在本议定书中所描述的方法进行测试，提出潜在的关键残基。 野生型通道的测量值应始终伴随着突变的渠道，作为阳性对照，以确保转染和电生理实验做正确的测量。点突变可能会导致缺乏功能电流，电流类似野生型或改变通道活动调控。可能是由于不当的折叠，变造贩运缺乏可测量的电流。在这种情况下，它可能是有用的的，找出通道流量是否正确地使用生物素法或免疫荧光染色对细胞外标签（无细胞通透性）的细胞膜和细胞内保留的通道上区分非功能之间的渠道表面。最后，这是最好的渠道，但也航道整治突变的后果进行调查，不仅基底活动。不同的通道功能的调节机制，可以研究离子通道的利益，包括调控，PIP2的G蛋白，离子，ATP浓度，磷酸（使用酪氨酸，丝氨酸或苏氨酸突变），泛素化或SUMO化（使用赖氨酸突变）直接通道开放剂或阻滞剂。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> 
 
 <div>
 <table border="1">
 <thead>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 </thead>
 <tbody>
 
<tr>
<td>Chemicals</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Varies</td>
<td>&nbsp;</td>
<tr>
<td>Mini Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>Radiacwash</td>
<td>&nbsp;</td>
<td>Biodex</td>
<td>005-100</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>CB40210</td>
<td>Poly-L-lysine can also be used</td>
</tr>
<tr>
<td>Maxi Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12263</td>
<td>&nbsp;</td>
<tr>
<td>QuickChange XL II Site Directed Mutagenesis Kit</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>200512-5</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-027</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips &#x02C6;&#x2026; 12 mm</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>CS-12R</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Glass Tubing Borosilicate-standard wall</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>G150-3</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="_blank">Inverted fluorescence microscope</a></td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMI3000 B</td>
<td>&nbsp;</td>
<tr>
<td>Electrophysiology</td>
<td>&nbsp;</td>
<td>Nikon/ Leica/ Zeiss</td>
<td>Any inverted DIC (Hoffmann) microscope with fluorescence</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Axon Axopatch 200B amplifier</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion system</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>VC-6 Perfusion MM-6</td>
<td>&nbsp;</td>
<tr>
<td>Digitizer</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Digidata 1320 digitizer</td>
<td>&nbsp;</td>
<tr>
<td>Manipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Manipulator: MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC10 vertical electrode puller</td>
<td>&nbsp;</td>
<tr>
<td>Isolation Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing Corporation</td>
<td>Air table</td>
<td>&nbsp;</td> </tbody>
 </table>
 </div>

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

我们将演示如何研究引入点突变在离子通道的功能效应。我们研究G蛋白门控内向整流钾（简称为GIRK）通道，这是调节神经元的兴奋性的重要。有四个不同的哺乳动物GIRK通道亚基（GIRK1 GIRK4） - 我们GIRK2重点，因为它形成一个homotetramer。 G蛋白偶联受体（GPCRs），如毒蕈碱受体（M2R），不同类型的刺激导致激活GIRK通道。酒精也可直接激活GIRK通道。我们将展示如何具体改变的cDNA为GIRK通道的一个或多个核苷酸变异的一种氨基酸。这种突变的cDNA序列，将扩增细菌，净化，和点突变的存在将通过DNA测序证实。将突变和野生型GIRK通道的cDNA转染入人胚肾HEK293T细胞在体外培养。最后，将用于全细胞膜片钳电生理研究宏观钾电流通过异位表达野生型或突变GIRK渠道。在这个实验中，我们将研究在GIRK2 M2R依赖和酒精依赖的活化渠道L257W突变的效果。

Watch this Scientific Journal Video about Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells at JoVE.com

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

我们将演示如何研究的单点突变对离子通道的功能的影响。

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 ...

mutagenesis-functional-analysis-ion-channels-heterologously-expressed

Research

JoVE Journal

Biology

17.3K 次观看.  Salk Institute for Biological Studies. 我们将演示如何研究的单点突变对离子通道的功能的影响。

视频: Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian 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

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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 ...

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

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

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) ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...