这项工作是由国家卫生赠款R01 - HL70393和R01 - NS060706到JCJC研究院支持，斯宾塞吨奥林基金会是一个生物医学工程教授。

1。注射到卵母细胞的mRNA <ol><li>注入0.05 - 50吴爪蟾卵母细胞，使用Nanoject II自动纳升进样器（德拉蒙德科学公司，模型3-000-204）（第五和第六阶段 ），在体外转录成信使RNA。重复上的每一个基因12个卵母细胞的注射。 </li><li>冲洗两次使用注入卵母细胞ND - 96解决方案（96毫米氯化钠（NaCl，议员58.44克/摩尔），2毫米氯化钾（KCl，议员，74.56克/摩尔），1.8毫米氯化钙（ 氯化钙 2•2H 2 O议员147.02），1毫米氯化镁（氯化镁2•6H 2 O先生203.31克/摩尔），5毫米4 - （2 -羟乙基）- 1 - piperazineethanesulfonic磺酸（HEPES，议员238.31克/摩尔），2.5毫米丙酮酸钠（主席110.04克/摩尔）和1x青霉素，链霉素，pH值7.6），然后放置在18 °在培养板中，孵育2天的彗星。 </li></ol> 2。准备灌注系统<img src="/files/ftp_upload/2269/2269fig1.jpg" alt="图1" /> 自动化ValveLink 16灌注系统的插图。灌注系统采用加压氮气推动解决方案的水库灌注的解决方案，通过灌注导管灌注铅笔和小费。每灌注液的流的流动是一个水库阀和电子膨胀阀控制。在这个协议中，水库之一，是不加压和作为废物收集，以及一个压力释放机制的职能。 （从供应商的网站http://www.autom8.com适应的图片） <ol><li>连接导管灌注铅笔。打开电子膨胀阀控制器，电子阀打开。 </li><li>为了保证管路及灌注铅笔无堵塞和空气气泡，通过每个使用去离子水填补了注射器管推去离子（DI）水。 </li><li>的油管连接的解决方案，水库并打开煤气阀，气缸申请加压氮。 </li><li>打开水库阀门，水库的解决方案，以填补油管。弗里克水库阀在解决方案中删除，如果需要的气泡。关闭阀后的管充满与解决方案。 </li><li>填写灌注水提示，并拧到灌注铅笔。的提示连接时要小心，不要捕获任何气泡。 </li><li>修正灌注铅笔使用橡皮泥浴阶段。确保灌注尖端的卫浴空间。现在成立灌注系统。 </li><li>在洗澡添加去离子水。确保淹没在水中灌注提示。 </li><li>测试每个灌注液灌注提示正确。一次灌注液（140毫米的氢氧化钾（KOH，关闭电子废物收集管相连的阀门，并打开阀门 ř 56.1克/摩尔，中号20毫米4） - （2 -羟乙基）-1 - piperazineethanesulfonic磺酸（HEPES，男ř 238.31克/摩尔），2毫米氯化钾（KCL米ř 74.56克/摩尔），1毫米乙二醇双（2 - aminoethylether）- N，N，N&#39;，N&#39; -四乙酸（EGTA米ř 380.35克/摩尔），pH值调整甲磺酸（中间3米ř 96.1克/摩尔）， 钙 7.1-7.2 +和Mg2 +添加到所需浓度，在需要时） 。在显微镜下，观察灌注液灌注尖端的喷气机。关闭灌注液阀，并打开阀门废物收集。灌注飞机几乎消失。重复相同的测试的所有灌注解决方案。 </li><li>当您已经验证所有灌注流量的解决方案正确，更换槽液浴（140毫米的氢氧化钾（KOH，DI水ř 56.1克/摩尔，中号20毫米4） - （2 -羟乙基）- 1 - piperazineethanesulfonic磺酸（HEPES，男ř 238.31克/摩尔），2毫米氯化钾（KCL米ř 74.56克/摩尔），pH值调整到7.1-7.2甲磺酸（MESO 3，M ř 96.1克/摩尔））。 </li></ol> 3。膜片钳<ol><li>重涂与氯化银银记录电极膜片钳在每个会议。首先，从吸管持有人，淹没在一个小瓶新鲜的漂白剂含有至少15分钟的电极丝的尖端一半的电极丝。这存款一层AgCl的电线上。 </li><li>用去离子水冲洗电极丝和吸干。 Ag / AgCl电极，然后安装在吸管持有人。 </li><li>洗澡（参考）电极连接的探头，它在洗澡的地方。这准备记录电极。 </li><li>现在准备进行数据采集。打开您的计算机上，插入您的计算机的打印机端口的硬件密钥。必须插入硬件密钥，供您使用的数据采集软件，这是HEKA脉冲。 </li><li>开关放大器（Axon仪器，AXO补丁200B）并开始HEKA脉冲。负载的协议文件，并调整配置设置，如经济刺激规模。调整配置设置的目标，以保证测试的潜力是完全平等的命令的潜在。这准备的数据采集设备。 </li><li>准备卵母细胞。淹没卵母细胞中剥离液（N -甲基- D -葡萄糖胺（NMG，男ř 195.22克/摩尔），200毫米的天门冬氨酸（没有K +）（M ř 133.1克/摩尔），2毫米氯化钾（氯化钾200毫米中号ř 74.56克/摩尔），10毫米乙二醇-双（2 - aminoethylether）- N，N，N&#39;，N&#39; -四乙酸（EGTA，M ř 380.35克/摩尔），1毫米氯化镁（氯化镁2•6H 2 O，中号，10毫米4 ř 203.31克/摩尔） - （2 -羟乙基）- 1 - piperazineethanesulfonic磺酸（HEPES，男ř 238.31克/摩尔），pH值是由氢氧化钠调整至7.4（氢氧化钠，中号ř 40.0克/摩尔）5 - 10分钟剥离的解决方案从质膜，这使得它可以地带卵黄膜分离卵黄膜。 </li><li>轻轻地带使用两个双钳卵母细胞的卵黄膜。一个devitellinized卵母细胞，是极其脆弱和必要的少，因此应移到。 </li><li>同时注意防止devitellinized卵母细胞暴露到空气或气泡，充满了足够的解决方案的卵母细胞转移到洗澡使用的玻璃吸管。这准备录制的卵母细胞。 </li><li>准备补丁移液器。拉后插入一个萨特的P - 97火焰山/布朗微管牵引器的玻璃毛细管（VWR International的猫＃53432-921）玻璃吸液管。在显微镜下观察移液器，以确定针尖的形状和开口直径。开口直径为2-4微米。 </li><li>在录制过程中为了减少电容电流，以确保一个平稳的提示，大衣用蜡的一角，然后火抛光。 </li><li>填充液（140毫米的氢氧化钾（KOH米ř 56.1克/摩尔），20毫米4放置吸管尖枪头- （2 -羟乙基）- 1 - piperazineethanesulfonic磺酸（HEPES，中号ř 238.31克/摩尔），2毫米氯化钾（KCL米ř 74.56克/摩尔），2毫米氯化镁（氯化镁2•6H，男ř 203.31克/摩尔），pH值调整到7.1-7.2甲基酸（MESO 3，M ř 96.1克/摩尔））和绘图柱塞。这弄湿技巧。 </li><li>填充吸管的解决方案中使用的注射器吸液管的1 / 3满，并将其放置在吸管吸管持有人的Ag / AgCl电极内部和与电极内液接触。这准备修补吸管。 </li><li>要准备的卵母细胞的消费税从内而外的补丁，在显微镜下找到明确的卵母细胞的边缘。移动吸管接近使用机械臂的卵母细胞的修补程序。记录的吸液管的串联电阻。补丁吸管理想的串联电阻时充满电极内液和沐浴液淹没的1-1.5MΩ。 </li><li>慢慢推对卵母细胞的吸管，直到阻力大约增加一倍。 </li><li>申请经口轻柔吸膜通过吸痰管，它是连接到吸管持有人，直到获得一个千兆欧姆密封。稳定在-30 mV的电压为几分钟的修补程序。 </li><li>要获得一个由内向外的补丁，消费迅速推进吸管进一步进入卵母细胞，然后轻轻拉出补丁。 </li><li>内而外的补丁现在是准备用于夹紧。移动吸管内而外的补丁约100微米，从开放灌注提示。 </li><li>关闭电子废物收集阀，并打开所需的灌注液水库阀</li><li>开始记录电流穿过补丁。更改的解决方案所需的电压和灌注。 </li><li>当您完成录制，推动通过去离子水，以避免堵塞清洁灌注导管，铅笔和小费。 </li></ol> 4。代表性的成果在膜片钳的卵母细胞的准备，剥离液5-10分钟治疗，脱离质膜，这使得有可能剥离卵黄膜卵黄膜。 补丁吸管理想的串联电阻时充满电极内液和沐浴液淹没的1-1.5MΩ。 以下是BK一个卵母细胞质膜补丁上的野生型渠道的代表性录音。在左上角，方波表示适用于修补程序的电压 - 从50 mV到200 mV的增量为50 mV的电压升高的第二个步骤。下面是相应的电流的痕迹时，灌注液公称0的Ca 2 +（免费的[Ca 2 +]约为0.5纳米）。在电流幅值的折痕表明，BK通道的开放概率增加电压。在右侧，相同的修补程序灌注1.0μM的Ca 2 +具有相同的电压协议。相同的电压下电流的幅度增加，表明开放概率增加的Ca 2 +]。 <img src="/files/ftp_upload/2269/2269fig2.jpg" alt="图2" />

<ol>
	<li>Methfessel, C. <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+measurements+on+Xenopus+laevis+oocytes:+currents+through+endogenous+channels+and+implanted+acetylcholine+receptor+and+sodium+channels.">Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.</a> Pfl&#252;gers Arch. 407, 577-588 (1986).</li><li>Toro, L., Wallner, M., Meera, P., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maxi-K+(Ca),+Unique+Member+of+the+Voltage-Gated+K+Channel+Superfamily.">Maxi-K (Ca), Unique Member of the Voltage-Gated K Channel Superfamily.</a> News Physiol. Sci. 13, 112-117 (1998).</li><li>Fettiplace, R., Fuchs, 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=Mechanisms+of+hair+cell+tuning.">Mechanisms of hair cell tuning.</a> Annu. Rev. Physiol. 61, 809-834 (1999).</li><li>Du, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-sensitive+potassium+channelopathy+in+human+epilepsy+and+paroxysmal+movement+disorder.">Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.</a> Nat. Genet. 37, 733-738 (2005).</li><li>Ledoux, J., Werner, M. E., Brayden, J. E., Nelson, M. T., T, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-activated+potassium+channels+and+the+regulation+of+vascular+tone.">Calcium-activated potassium channels and the regulation of vascular tone.</a> Physiology (Bethesda). 21, 69-78 (2006).</li><li>Salkoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-conductance+potassium+channels+of+the+SLO+family.">High-conductance potassium channels of the SLO family.</a> Nat. Rev. Neurosci. 7, 921-931 (2006).</li><li>Shi, J., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Mg2++enhances+the+function+of+BK-type+Ca2+-activated+K++channels.">Intracellular Mg2+ enhances the function of BK-type Ca2+-activated K+ channels.</a> J. Gen. Physiol. 118, 589-606 (2001).</li><li>Magleby, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+mechanism+of+BK+(Slo1)+channels:+so+near,+yet+so+far.">Gating mechanism of BK (Slo1) channels: so near, yet so far.</a> J. Gen. Physiol. 121, 81-96 (2003).</li><li>Latorre, R., Brauchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+conductance+Ca2+-activated+K++(BK)+channel:+activation+by+Ca2++and+voltage.">Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage.</a> Biol. Res. 39, 385-401 (2006).</li><li>Cox, D. H., Cui, J., Aldrich, R. W., W, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+gating+of+a+large+conductance+Ca-activated+K++channel.">Allosteric gating of a large conductance Ca-activated K+ channel.</a> J. Gen. Physiol. 110, 257-281 (1997).</li><li>Cui, J., Aldrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+linkage+between+voltage+and+Ca2+-dependent+activation+of+BK-type+mslo1.">Allosteric linkage between voltage and Ca2+-dependent activation of BK-type mslo1.</a> K+ channels. Biochemistry. 39, 15612-15619 (2000).</li><li>Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Slo1+BK+channels+by+Mg2++coordinated+between+the+voltage+sensor+and+RCK1.">Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1.</a> 15, 1152-1159 (2008).</li><li>Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term={beta}+subunit-specific+modulations+of+BK+channel+function+by+a+mutation+associated+with+epilepsy+and+dyskinesia.">{beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia.</a> J. Physiol. , 587-1481 (2009).</li></ol>

卵母细胞表达系统是由于相对较低的内源性途径背景电压依赖性离子通道的电生理特性的理想选择。此外，因为它是一个瞬时表达系统，它提供了一个有效的方法执行这些渠道的诱变研究。然而，应该指出的是，卵母细胞表达系统在哺乳动物细胞中是不同的，从而有可能在翻译后修饰和与不同的亚基协会的差异。此外，血脂浓度可能会有所不同，两者之间的细胞这可能会影响通道的功能特性。 </p...

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		<th>Catalogue Number</th>
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<td>Nanoject II Auto-Nanoliter Injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>3-000-204</td>
<td>&nbsp;</td>
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<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Perfusion System and Electronic Controller</td>
<td>&nbsp;</td>
<td>AutoMate Scientific, Inc.</td>
<td>ValveLink 16</td>
<td>Inner diameter of perfusion tip: 100 microns</td>
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<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>CKX31</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>VWR International</td>
<td>53432-921</td>
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<td>Sutter Instrument Co.</td>
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<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
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<td>Computer Interface</td>
<td>&nbsp;</td>
<td>INSTRUTECH Corporation</td>
<td>ITC-18</td>
<td>&nbsp;</td>
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<td>Headstage</td>
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<td>Axon Instruments</td>
<td>CV 203BU</td>
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patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes

这里介绍的协议，旨在研究激活大电导，电压和通道的Ca 2 +激活的K +（BK）。该协议也可以用于研究等离子通道和神经递质受体1的结构与功能的关系。 BK通道广泛表达在不同组织中，并已在许多生理功能，包括调节平滑肌收缩，内毛细胞的频率调整和调节神经递质的释放 2-6牵连。 BK通道被激活细胞膜去极化和细胞内Ca 2 +，Mg 2 +的6-9。因此，该协议的目的是控制膜电压和细胞内的解决方案。在这个协议中，BK通道的信使RNA是注入爪蟾卵母细胞由2-5天的潜伏期，在18 ° C 10-13（第五和第六阶段）。膜补丁包含单个或多个BK渠道切除与内配置使用膜片钳技术10-13。灌注所需的解决方案，以便在录制过程中，可以研究不同条件下的通道激活细胞内的补丁。总结，BK通道的mRNA 注入非洲爪蟾卵母细胞表达的卵母细胞质膜上的通道蛋白;膜片钳技术是用来记录电流流经控制电压和细胞内的解决方案下的渠道。

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

BK通道的离子电流是利用膜片钳技术记录。 BK通道均以<em&gt;爪蟾卵母细胞</em注射信使RNA。在膜片钳记录细胞内的解决方案是由灌注系统控制​​。

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 ...

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

20.9K 次观看.  Washington University in St. Louis. BK通道的离子电流是利用膜片钳技术记录。 BK通道均以爪蟾卵母细胞

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

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

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 ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

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 ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...