Name: visualizing rna localization in xenopus oocytes
Uploaded: 2010-01-14T17:00:00
Description: Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

我们对RNA的本地化工作是由美国国立卫生研究院（R01GM071049）的拨款，以荷航的支持。

第1部分：荧光标记基因的转录。 <ol><li>含有RNA的本地化元素或其他相关序列的质粒DNA线性化和重悬在1μg/μL的DEPC处理过的H 2 O DNA模板必须有T7，SP6，T3 RNA聚合酶转录上游启动网站。 </li><li>无菌1.5ml离心管加入下列试剂： <table><tbody><tr><td> A. 10X Tx缓冲区（见M＆M的） </td><td> 2μL </td></tr><tr><td> B. 20X帽/ NTP的组合（见M＆M的） </td><td> 1μL </td></tr><tr><td> C. 1毫米的Alexa Fluor 546 - 14 - UTP（Invitrogen公?...

<ol>
	<li>Cohen, S., Au, S., Pant, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> JoVE. 24, (2009).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis+(Daudin).+I.+Stages+of+oocyte+development+in+laboratory+maintained+animals.">Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals.</a> J. Morphol. 136, 153-179 (1972).</li><li>Gagnon, J. A., Mowry, 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=RNA+transport+in+ovo:+Simultaneous+visualization+of+two+RNAs.">RNA transport in ovo: Simultaneous visualization of two RNAs.</a> Mol Reprod Dev. 76, 1115-1115 (2009).</li><li>Gautreau, D., Cote, C. A., Mowry, 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=Two+copies+of+a+subelement+from+the+Vg1+RNA+localization+sequence+are+sufficient+to+direct+vegetal+localization+in+Xenopus+oocytes.">Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.</a> Development. 124, 5013-5020 (1997).</li><li>Lewis, R. A., Gagnon, J. A., Mowry, 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=PTB/hnRNP+I+is+required+for+RNP+remodeling+during+RNA+localization+in+Xenopus+oocytes.">PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.</a> Mol Cell Biol. 28, 678-6786 (2008).</li><li>Messitt, T. J., Gagnon, J. A., Kreiling, J. A., Pratt, C. A., Yoon, Y. J., Mowry, 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=Multiple+kinesin+motors+coordinate+cytoplasmic+RNA+transport+on+a+subpopulation+of+microtubules+in+Xenopus+oocytes.">Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.</a> Dev Cell. 15, 426-436 (2008).</li><li>Mowry, K. L., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vegetal+messenger+RNA+localization+directed+by+a+340-nt+RNA+sequence+element+in+Xenopus+oocytes.">Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes.</a> Science. 255, 991-994 (1992).</li><li>Yoon, Y. J., Mowry, 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=Xenopus+Staufen+is+a+component+of+a+ribonucleoprotein+complex+containing+Vg1+RNA+and+kinesin.">Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin.</a> Development. 131, 3035-3045 (2004).</li></ol>

在这里，我们提出了一个可视化mRNA 的爪蟾卵母细胞的本地化的协议。这种方法，使用荧光标记的RNA转录，具有较高的信噪比比以前取得与地高辛标记的成绩单和比在原位（Mowry和麦尔登，1992年，Gautreau等，1997）为基础的方法更简单，速度更快。使用这种方法，我们可以设计RNA序列突变，并迅速在体内的功能测试。此外，通过使用额外的Alexa的双绞线荧光团，多个RNA的物种可以被可视...

A correction to the step 2 of Part 1 for article Visualizing RNA Localization in Xenopus Oocytes.

Erratum: Visualizing RNA Localization in Xenopus Oocytes

10X Tx缓冲 <ul><li class="none"> 60毫米氯化镁2 </li><li class="none"> 400毫米的Tris - HCl（pH值7.5） </li><li class="none"> 20毫米的精胺 - 盐酸</li></ul> 20X帽/ NTP的混合 <ul><li class="none"> 10 mM的CTP版</li><li class="none"> 10毫米ATP </li><li class="none"> 9 mM的双绞线</li><li class="none"> 2 mM的GTP </li><li class="none">摹第20毫米G（PPP）模拟（New England Biolabs公司） </li></ul> G - 50柱 <ul><li class="none">水合物5克经Sephadex G - 50珠（Sigma公司Aldrich公司）在100毫升去离子H 2 O。用DEPC处理30分钟。高压灭菌器。在室温下保存不完整的股票。在使用之前，添加以下的无RNase的解决方案： </li><li class="none"> 0.5毫升0.2 M EDTA </li><li class="none"> 1毫升的1 M Tris pH值8.0 </li><li class="none"> 0.5毫升，20％SDS </li><li class="none">商店完整的G - 50的解决方案，在4 ° C </li><li class="none">取下并丢弃从3毫升注射器（BD公司）柱塞和注射筒放入15毫升锥形管（康宁）。插入注射器用少量玻璃棉（一分钱的一半左右大小的插头）。 </li><li class="none">旋流完整的G - 50解决方案，以重悬珠。 </li><li class="none">加入2毫升的G - 50空列的解决方案。 </li><li class="none"> 1,000 × g的台式离心机1分钟旋转。 </li><li class="none">每一列添加200μLDEPC处理的去离子H 2 O 。自旋。 </li><li class="none">共有3个洗，重复洗两倍以上。 </li><li class="none">取出针筒到一个新的15毫升锥形管。 </li></ul> 胶原酶溶液 <ul><li class="none"> 75毫克胶原酶梭菌histolyticum（Sigma公司Aldrich公司） </li><li class="none"> 25毫升0.1 M的KPO 3 +（pH值7.4） </li></ul> MBSH缓冲区 <ul><li class="none"> 88毫米氯化钠</li><li class="none"> 1 mM的氯化钾</li><li class="none"> 2.4毫米碳酸氢钠3 </li><li class="none"> 0.82 mM的硫酸镁4 × 7H 2 O </li><li class="none"> 0.33毫米的Ca（NO 3）2 × 4H 2 O </li><li class="none"> 0.41 mM的氯化钙2 × 6H 2 O </li><li class="none"> 10毫米HEPES（pH值7.6） </li></ul> 卵母细胞培养介质 <ul><li class="none"> 50％L15培养基</li><li class="none"> 15毫米HEPES（pH值7.6） </li><li class="none"> 1 mg / ml的胰岛素</li><li class="none"> 100毫克/毫升的庆大霉素</li><li class="none"> 50 U /毫升制霉菌素</li><li class="none"> 50 U / ml青霉素</li><li class="none"> 50毫克/毫升链霉素</li></ul> MEMFA解决方案 <ul><li class="none"> 0.1 M MOPS（pH值7.4） </li><li class="none"> 2 mM的EGTA </li><li class="none"> 1毫米硫酸镁4 </li><li class="none"> 3.7％的甲醛</li></ul> 计算RNA的产量 <ul><li class="none">在“输入”和“收编”的样品，使用标准的闪烁计数器中确定的CPM。 </li><li class="none">法团（“法团”）/（10 ×“输入”） </li><li class="none">典型的掺入值范围〜0.03和0.10之间。 </li><li class="none">不同的聚合酶的最高理论产量： T7，T3，SP6的 - 2.64微克</li><li class="none">反应收率微克=（使用聚合酶的最高产量）×（法团） </li><li class="none">稀释的RNA，以50海里=（微克核糖核酸）/ 320 /（RNA的长度在基地）/（5X10 -8） </li><li class="none">通常的反应产量〜50纳米的RNA 50-100μL。 </li></ul>

visualizing rna localization in xenopus oocytes

RNA本地化是一个保守的机制，建立细胞极性。 VG1基因定位于非洲爪蟾卵母细胞和行为空间限制VG1蛋白基因表达的植物极。 VG1分布严格控制，这种方式是需要适当的生殖发育中的胚胎层规范。 RNA序列的mRNA的3&#39;UTR区的元素，VG1本地化元素（VLE）是必要的和足够的直接运输。要研究的认可和VG1基因在体内的运输，我们已开发出一种成像技术，可以通过一个简单的视觉读数跨因素的传输机制进行了广泛分析。 为了形象化RNA的本地化，我们合成荧光标记的VLE的RNA和microinject到个人的卵母细胞成绩单。后卵母细胞培养，让注入的RNA运输，卵母细胞共聚焦显微镜成像前固定和脱水。 mRNA的本土化模式的可视化提供了一个监测完整的RNA运输途径，并确定独联体范围内行事的成绩单和反式作用因子结合VLE（刘易斯等人，2008年的元素，在指导RNA的运输角色的读数Messitt等，2008）。我们已经扩展这种技术通过共同本地化与其他RNA和蛋白质（加尼翁和Mowry，2009年，Messitt等，2008），并结合马达蛋白和细胞骨架的破坏等，2008）（Messitt探测mRNA的本地化的内在机制，。

Watch this Scientific Journal Video about Visualizing RNA Localization in Xenopus Oocytes at JoVE.com

Visualizing RNA Localization in Xenopus Oocytes

可视化<em&gt;在体内</em&gt; RNA的运输是通过微量注射荧光标记的RNA转录成<em&gt;爪蟾</em&gt;卵母细胞，共聚焦显微镜。

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 ...

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

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

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 ...

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 ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

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

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

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 ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...