Name: xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments
Uploaded: 2016-12-31T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

动物实验已通过了伯尔尼州Kantonstierarzt的地方委员会，KantonalerVeterinärdienst伯尔尼（BE85 / 15）。 1. 爪蟾卵母的制备<ol><li>保持在12小时/ 12小时光照/黑暗中水的循环是严格保持在20℃，蛙（ 爪蟾 ）。 </li><li>从雌蛙9取出卵巢的叶。 注意:凸起的除去是用于再生的刺激，并经常在卵母细胞的质量与手术改善。卵母细胞被卵黄层，卵泡细胞的层，并且含有血管21结缔组织覆盖。整个结构被称为"毛囊"。 </li><li>放置被密集用含有无菌巴特的盐7（MBS）的卵母细胞补充有青霉素和链霉素（88毫摩尔NaCl，1mM的氯化钾，2.4毫毛囊卵巢的裂片的NaHCO 3，0.82毫硫酸镁 ，0.41毫氯化钙 ，0.33毫米的Ca（NO 3）2，10毫4-（2-羟乙基）-1-哌嗪乙磺酸; HEPES（pH值7.5，氢氧化钠），100微克/毫升青霉素和100微克/ mL链霉素）。 </li><li>单出阶段V-VI 21卵泡。握住钳子卵巢叶。降低铂环（ 图1）在卵泡并轻轻撤回循环扰乱含有从卵巢组织卵泡结缔组织。 注:第五阶段-Ⅵ21卵泡的特征在于它们的尺寸（1.0 - 1.2毫米直径），并通过暗着色动物半球和淡黄色植物性半球之间的良好的对比度。 </li><li>通过塑料巴斯德吸管（切回1.5mm的开口直径）的装置仅传送看起来健康的卵泡（ 即完美的球形，并用完整的色素沉着）到35毫米直径的培养皿。 </li></ol>注:此处描述的显微注射系统是从由Kressmann和Birnstiel 22日报道的。 <ol><li>准备从相应的cDNA 通过体外转录A受体，增加了一个多聚（A +）尾和RNA定量的用凝胶电泳23编码的α4β2δGABA的亚基的cRNA。 </li><li>从使用微量牵拉硼硅玻璃毛细管（1.0外径（OD），0.58内径（ID），100毫米长）制备显微注射移液器。断的玻璃毛细管的尖端使用显微和微丝创建12的顶端直径，在显微镜下 - 15微米与斜尖。 </li><li>回填用10毫升的注射器石蜡油显微注射吸管，然后装入显微注射吸管到一个自制的MICRoinjection装置（ 图2）。 注:自制液压油喷射装置包括一个脚踏开关控制烧烤电机。一个附加的开关改变马达的转动模式。该电机驱动的推进或缩回10μL玻璃注射器的柱塞螺旋测微计（0.5毫米/转）。注射是在约3 rpm的速度和0.5转弯对应于50 NL注射到毛囊。注射器的前端连接到本身由非常短的一块Tygon管的连接到注射针厚壁聚四氟乙烯管材。加样毛细管是由一个由显微控制的钻夹头保持。整个系统充满了石蜡油。任何气泡应该避免，因为它们会损害喷射系统。设置点亮了冷光源。立体显微镜需要光学控制。卵泡在冷光可视化与stereomicroscope需要（40X放大倍率）。注射设定的性能由注射放射性示踪剂进入爪蟾卵母测试。 45卷 - 55 NL应该每次注射交付。 </li><li>使用移液管，用无菌塑料尖端，通过将无菌水滴到耐湿热塑性（2×2厘米）的清洁内部测试设置。沉浸注射吸管到该液滴的前端，然后转动电机缩回柱塞（注射吸管内部负压力）。 注:水应该进入吸管和形成与油中的可视界面。 </li><li>退离液滴的吸管尖和施加正压到注射移液管的内部。水滴应该形成在注射吸管尖。 </li><li>粘到PE的底部:通过衬里它们与植物性磁极在尼龙网的空隙（0.8毫米G）的朝上准备毛囊的喷射三菜（60 MM）覆盖着MBS。 </li><li>使用移液管，用无菌塑料尖端到热塑性的清洁内部分配mRNA的2微升。通过施加负压到注射移液管的内部取的mRNA向上进入注射针。 </li><li>注射针在使用显微单个毛囊的位置。 </li><li>插入注射针进入植物极的中心与通过施加正压力，以移液管的内部注入mRNA的50 NL以0.6微升/分钟的流速。从毛囊取出注射枪头，以避免逃跑的mRNA前10秒 - 等待5。 注:基因应注入的植物（淡黄）极，以避免注射到细胞核中，它位于在动物半球。 </li><li>传输所注入的毛囊填充有2毫升的MBS的新的培养皿（35毫米）。放置盘成酒冷却器设定为18℃。孵育1注入的卵泡 -记录前7天，这取决于新表达的蛋白的身份。 </li></ol> 3.卵泡剥离（图3） 注:如上所述，由卵黄层，卵泡细胞，结缔组织，其中包含血管24所覆盖的卵母细胞，被称为"毛囊"。除了卵黄层，它提供机械稳定性不妨碍到细胞表面的解决方案的进入所有的层，必须电生理实验之前被移除。周围无细胞层卵泡先前已称为"洁净"卵母细胞25。此步骤通常在同一天的电生理实验进行。 <ol><li>转移10注入卵泡到含有0.5毫升的MBS，1毫克/毫升胶原酶和0.1mg / ml胰蛋白酶抑制剂的硼硅玻璃管。浸入管保持在36℃水浴。孵育20分钟卵泡和动摇管偶尔。 </li><li>通过将它们转移到含有1毫升的MBS在RT第二管冲洗卵泡。让他们在管约10秒。 </li><li>转移卵泡到包含含有4mM乙二醇双（2-氨基乙醚）加倍浓缩的MBS 0.5mL的第三管- N，N，N&#39;，N&#39;-四乙酸（EGTA），并孵育他们在4分钟RT。偶尔摇管。 注:高渗溶液（加倍浓缩MBS）用于诱导卵泡细胞层内的卵母细胞，从而分离从卵母细胞滤泡细胞层的收缩率加入EGTA以抑制胶原酶活性。 </li><li>通过将它们转移到含有1毫升的MBS在RT第二管冲洗卵泡。让他们在管约10秒。 </li><li>转移的卵母细胞，以含2毫升的MBS培养皿（35毫米直径）。在立体显微镜，通过分开毛囊外封套简单地推裸卵母细胞离开使用铂环。 注:卵母细胞的外信封牢牢粘在培养皿，并可以从卵母细胞容易地分离。有些卵母细胞会自发地失去他们的外封套，将被回收裸卵。此过程导致用于保存的卵母细胞的球形外观相对稳定的制剂。 </li></ol>周围的卵母细胞4.快速变化的解决方案<ol><li>进行电压钳实验中，如所描述9,26。 </li><li>通过根据需要由试验转动灌注开关改变重力供给灌注系统的解决方案。 <ol><li>用玻璃毛细管1.35毫米，口，其中放置约0.4mm从卵母细胞表面的内径施加在6毫升/分钟的灌注溶液，以允许围绕卵母细胞的激动剂浓度的快速变化。 注意:变化率一直此前估计ð在小于0.5秒27的70％。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这篇文章中描述的方法，从那些传统使用的7-9偏离。它是标准的对卵巢的裂片暴露于1至2小时胶原酶处理8;隔离损坏，裸卵;与使用商业注射装置的mRNA注入它们。此经典程序具有以下缺点:1）卵母细胞很可能是由长曝光于高浓度的胶原酶被损坏。 2）不稳定裸露的卵母细胞必须被存储，直到实验。 3）裸卵更有可能比注射过程中的卵泡挨。商业喷射设备使用大直径（约20微米）注射针，可能造成损坏的相对高的速率。这里描述的改进的过程的优点如下:暴露于胶原酶限于20分钟，裸露的卵母细胞不必被存储，并且所述移液器的小尖端直径 - 用于microinjecti（约12 15微米）上不需要事先毛囊为与mRNA注射的"defolliculation"。单个关键步骤是，在胶原酶溶液卵泡培养温度应仔细调整到36℃，并应不超过它。 在电生理实验溶液的变化，经常在测量室中的溶液被改变，这需要的大量时间，这取决于该腔室的尺寸和灌注速度。使用避免了这种灌注毛细管。 离子通道的爪蟾卵母中表达的主要缺点是它们的巨大小。电压控制的速度比约为2 ms是困难的，而且速度非常快（&lt;0.5秒）解决方案的改变需要复杂的程序。此外，强疏水性物质可能几乎是不可逆地结合到卵母细胞的蛋黄。 这里介绍的方法使我们能够调查在一个省时的方式的重组GABA A受体，通过合成的化合物和植物物质，其亚基排列其调制，并在不同的亚单位组成的受体的药物结合位点的位置的功能特性。此处所描述的方法适合于所选择的任何蛋白在非洲爪蟾卵母细胞中的表达。周围的卵母细胞的培养基中快速解的变化非常简单的方法可以应用于任何配体门控离子通道或载体的研究。

爪蟾卵母被广泛用作表达系统（参考文献2 - 3，和其中的参考文献）。它们能够正确地组装和整合功能活性多亚基蛋白到其质膜。使用该系统，有可能在功能调查膜蛋白单独使用或与其它蛋白质组合，为了研究突变，嵌合的，或连接在一起的蛋白质的性质，以及筛选潜在药物。 使用的卵母细胞比其它异源表达系统的优点包括巨细胞的简单处理，表达外源遗传信息，卵母细胞的环境的通过浴灌注装置的简单的控制的细胞的比例高，和膜电位的控制。 该表达系统的缺点是在许多实验室17-20中观察到的季节变化。这样做的原因变异目前尚不清楚。此外，卵母细胞的质量经常观察到强烈变化。传统方法7-9包括卵巢瓣的隔离，卵巢瓣的暴露于胶原酶一些小时，裸露的卵母细胞的选择，以及卵母细胞注射。在这里，一些替代方案，快速程序报道，使我们能够用这个表达系统工作超过30年，在卵母细胞质量无季节变化和变化不大。 此处描述了用于卵母细胞的分离，微注射用的cRNA，并除去滤泡细胞层的改性，改进的方法，可用于选择的任何蛋白在非洲爪蟾卵母细胞中的表达。周围的卵母细胞的培养基中快速解的变化非常简单的方法可以应用于任何配体门控离子通道和载流子的的研究。

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

非洲爪蟾卵母细胞作为异源表达系统用于蛋白质，首先由Gurdon 等人所述。 1并已发现以来被广泛使用（参考文献2 - 3，和其中的参考文献）。这使得外国通道表达卵母细胞有吸引力的一个特点是内生的离子通道4的丰度差。这种表达系统已被证明对于许多蛋白质，其中配体门控离子通道的特征是有用的。 GABA A受体在爪蟾卵母细胞和其功能特性的表达被这里描述，包括卵母细胞，用的cRNA，去除滤泡细胞层，并在电生理实验快溶液变更显微注射的隔离。的过程被在这个实验室5,6-优化，从常规使用7-9的那些偏离。传统上，裸卵准备用在RT延长胶原酶处理卵巢裂片的，并且这些裸露的卵母细胞显微注射用的mRNA。采用优化方法，多元膜蛋白已表达并研究了该系统中，如重组GABA A受体10-12，人重组氯通道13，锥虫钾通道14，和一个肌醇转运15,16。 这里详细描述的方法可以应用于在爪蟾卵母选择的任何蛋白质的表达，并迅速解变化可以用来研究其它配体门控离子通道。

爪蟾卵母细胞进行机械挑出使用铂环（ 图1）。的卵母细胞显微注射用的mRNA编码GABA A受体亚单位α4，β2，δ，0.5:0.5:2.5飞摩尔/卵母细胞（ 图2）。后4天，滤泡细胞层除去（ 图3）。卵母细胞电压钳制在-80毫伏和暴露于1μM的3α，21-二羟基5α孕-20-酮（THDOC），一种有效的正变构调节剂的存在增加γ氨基丁酸（GABA）的浓度GABA A受体。 图4A示出记录在这样一个实验原始电流痕迹。 图4B示出了根据不同的GABA的浓度引起电流幅度。这允许这个亚基朝向GABA组合的灵敏度的测定。的单个曲线分别安装和标准化I MAX，随后取平均值。使用的方程为一 （c）= I 最大 /（1+（EC 50 / C）n），其中c是GABA，EC 50的GABA的浓度（以1μMTHDOC的存在下）的引发一半的浓度-maximal电流幅度， 我 max是最大电流幅值，I是电流振幅，n为希尔系数。在αβ4 2δGABA的EC 50 A受体总额为0.41±0.12μM，和n为0.76±0.04。 <img alt="图1" src="/files/ftp_upload/55034/55034fig1.jpg" /> 图1:铂环。铂丝圈在协议步1.4提及。铂是一种可以不产生碎片被弯曲的金属，它不沾生物材料。 <a href="http://ecsource.jove.com/files/ftp_upload/55034/55034fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55034/55034fig2.jpg" /> 图2:自制显微注射设备。该装置中的协议步骤2. 一个 ，烤架电动机被描述; B，螺旋测微计; C，注射器和活塞之间的弹簧; D，10μL玻璃注射器; 即 ，厚壁聚四氟乙烯管; 男 ，手钻夹头; 克 ，注射针; H，体视显微镜;而我 ，电机控制。 <a href="http://ecsource.jove.com/files/ftp_upload/55034/55034fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.计划显示卵母细胞的Defolliculation。卵母细胞先在水浴胶原酶溶液在36℃孵育20分钟。的卵母细胞，然后在改性Barth的培养基洗涤。最终培养步骤是在在RT高渗EGTA溶液4分钟。其结果是，结缔组织和毛囊细胞被除去，得到的方式向裸露的卵母细胞。 <a href="http://ecsource.jove.com/files/ftp_upload/55034/55034fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55034/55034fig4.jpg" /> 从两电极电压钳实验图4.当前的痕迹。 一个， 从PRES一个GABA浓度响应曲线的电流迹线从非洲爪蟾卵母细胞表达αβ4 2δGABA 受体获得1μMTHDOC的ENCE。的条表示的GABA /1μM的THDOC灌注的时间段。的GABA的浓度增加被施加到的卵母细胞，并测定相应的电流振幅。 GABA的浓度以上的棒表示。 B， 在αβ4 2δGABA 受体的平均浓度响应曲线。各个曲线是第一归拟合最大电流幅值和随后平均。数据显示为平均值±SD，N = 3。 <a href="http://ecsource.jove.com/files/ftp_upload/55034/55034fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

xenopus-oocytes-optimized-methods-for-microinjection-removal

Nanyang Technological University

Research

JoVE Journal

Biology

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K 次观看.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

视频: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

生物化学

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 ...

生物学

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 ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

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 ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and &#947;-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

The protocol demonstrates that by performing microtransplantation of synaptic membranes into Xenopus laevis oocytes, it is possible to record consistent and reliable responses of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and &#947;-aminobutyric acid receptors.

突触膜的微移植以重新激活人突触受体以进行功能研究

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal ...

神经科学

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

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