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
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...

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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?...

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。

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</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?...

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 ...

Research

JoVE Journal

Biology

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Microinjection of Xenopus Laevis Oocytes

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 ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Visualizing RNA Localization in Xenopus Oocytes

Visualizing RNA Localization in Xenopus Oocytes

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

Fertilization of Xenopus oocytes using 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

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

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 ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...