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公司） </td><td> 1μL </td></tr><tr><td> D.双绞线，[α- 32 P]（1μCi/μL）（Perkin Elmer公司） </td><td> 1μL </td></tr><tr><td> E. DEPC - H 2 O </td><td> 11μL </td></tr><tr><td> F. 0.2中号数码地面电视</td><td> 1μL </td></tr><tr><td> G. RNasin（Promega公司） </td><td> 1μL </td></tr><tr><td> H.线性DNA模板</td><td> 1μL </td></tr><tr><td>一RNA聚合酶（Promega公司） </td><td> 1μL </td></tr></tbody></table></li><li>轻轻混合试剂，并短暂离心​​（10秒。微量）。 </li><li>在37 ° C孵育2-4小时，覆盖铝箔，以防止荧光团的漂白管。 </li><li>添加1μL1 mg / ml的无RNase的DNA酶（Promega公司），以降低模板DNA。 </li><li>孵育15分钟，在37 ° C。 </li><li>加入79μL20 mM的EDTA（pH 8.0）中终止反应。 </li><li>标示为“输入”并保存到一个单独的管，取出1μL。 </li><li>自旋通过1毫升G50列的反应（见材料与方法），其中将包括非法人核苷酸，而不含全长基因。 </li><li>集中乙醇沉淀的RNA： <ol class="lalpha"><li>加入250μL100％的乙醇，10μL7M CH 3 COONH 4，1μL载体RNA（酵母tRNA，10μg/μL的）1μL糖原（20毫克/毫升）。 </li><li>拌匀，和冻结，直到固体在-80 ° C下30分钟或15分钟干冰。 </li><li>以最大速度离心15分钟，倒出上清液旋转。 </li><li>洗干净的75％乙醇150μL。 </li><li> 3分钟以最大的速度旋转的离心，倒出上清液。 </li><li>沉淀在37 ° C干燥3分钟，确保所有的乙醇蒸发。 </li></ol></li><li>重悬在11μLDEPC - H 2 O，并取出1μL到一个单独的管，标示为“收编” 。 </li><li>确定％掺入稀释到50纳米的RNA（见材料及计算方法）。 </li><li>应冻结在5μL等份的RNA，为一次性使用，避免冻结/解冻周期，几个星期可以储存在-80 ° C </li></ol>第2部分：显微注射准备。 <ol><li> RNA制备： 解冻RNA（50海里）的分装，变性的RNA在70 ° 3-5分钟彗星。在房间温度最高速度离心旋转，以消除任何微粒，并保持在冰上10分钟。 </li><li>卵母细胞的制备： <ol class="lalpha"><li>手术去除卵巢爪蟾女性（NASCO）。 </li><li>修剪成50毫升含有胶原酶溶液25毫升的锥形管件（见材料与方法） 的爪蟾卵巢。 </li><li>在18 ° C，15分钟后，轻轻摇动或直到明显从卵巢中分离的卵母细胞。由于一批一批变异，胶原酶治疗时间可能会有所不同，应仔细监测通过目视检查确保defoliculation。 </li><li>允许卵母细胞在管解决，消除解决方案，并用MBSH缓冲区的卵母细胞（见材料与方法）。 </li><li>共有3个洗，重复洗两倍以上。这种卵母细胞隔离协议，是相似的，在科恩等人，2009年的详细。 </li><li>手动排序阶段III / IV期的卵母细胞在MBSH缓冲区（杜蒙，1972年），一个标准的解剖显微镜下。第三阶段卵母细胞是完全不透明的，但白色的，而Ⅳ期的卵母细胞稍大，色素斑点。略带透明的卵母细胞，都太年轻，卵母细胞，充分色素或展示极化色素分布过​​于陈旧。 </li></ol></li><li>针的制备： 拉和斜面针〜0.05毫米外径。 （我们使用3.5英寸的毛细血管德拉蒙德科学（订单＃3 - 000 - 203 - G / X），一个苏特仪器有限公司微量车夫和一个WPI公司beveller。） </li></ol>第3部分：显微注射<ol><li>校准用DEPC - H 2 O针，每次注射2 NL提供。我们前面加载我们的针使用天然气驱动的微量（见材料与方法），并使用千分尺校准墨滴大小。 </li><li>装入针的RNA。 </li><li>将整理成卵母细胞注射含有MBSH缓冲盘。我们microinject碟子上了一层黑色泡沫橡胶。苍白的卵母细胞的立场以及在一个白色backgrouND。 </li><li>小心2 NL的RNA注入每个卵母细胞，在50纳米。 </li><li>驱逐的RNA，用DEPC - H 2 O冲洗针，并加载注射下一RNA。 </li></ol>第4部分：卵母细胞培养<ol><li>在一个无菌24孔板（西格玛爱秩序）以及卵母细胞。我们文化的每口井千卵母细胞。 </li><li>删除的缓冲区，并更换与400μLØ ocyte彗星ulture中号edium器（OCM，材料与方法）每口井。培养板内放置一个不透气的塑料容器用湿纸巾保持在潮湿的环境文化。 </li><li>在18 ° C为8和48小时不等的时间点培养的卵母细胞。 </li></ol>第5部分：固定，脱水和存储的卵母细胞<ol><li>理清任何死卵母细胞，并将其放置在玻璃小瓶中幸存的卵母细胞。我们经常观察&gt; 90％的生存。 </li><li> MEMFA固定液（见材料与方法）和20分钟的岩石卵母细胞。用铝箔纸覆盖，防止光线照射的卵母细胞。 </li><li>洗卵母细胞，通过去除固定液和更换同等体积MBSH缓冲区。再次重复此一共有两个洗的洗。 </li><li>洗净放入无水甲醇卵母细胞： <ol class="lalpha"><li>取出量的一半，用甲醇代替。 </li><li>重复的步骤“的”两次。 </li><li>删除所有的解决方案，用甲醇代替。 </li><li>重复甲醇洗。 </li></ol></li><li>卵母细胞可以储存在-20 ° C，直到准备好形象。 </li></ol>第6部分：共聚焦显微镜成像RNA的本地化<ol><li>在成像之前，加美利小号结算中（2:1苯甲酸苄酯苄醇）玻璃底部FluoroDish（WPI的公司），覆盖成像表面。 </li><li>仔细的卵母细胞转移甲醇FluoroDish，减少体积的甲醇转移。 </li><li>一个倒置的共聚焦显微镜图像（我们已经成功地使用了蔡司LSM510的Leica TCS SP2）。 </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="图1" /> 图1：最初是可视化的亚细胞RNA的本地化注射荧光标记的成绩单A.经过显微注射，RNA的Alexa Fluor 546标记的限制，以细胞核 B.经过8小时的文化，一个荧光标记的对照RNA可以看到均匀分布在卵母细胞的细胞质中。然而，荧光标记的RNA序列招聘运输机械，可以在亚细胞定位的卵母细胞（向底部）的植物极的过程中看到。比例尺= 50微米。

<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>

使用实验动物的实验布朗大学规定的准则和法规的规定执行。

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 ...

visualizing-rna-localization-in-xenopus-oocytes

Research

JoVE Journal

Biology

Visualizing RNA Localization in Xenopus Oocytes

11.5K 次观看.  Brown University. 可视化在体内 RNA的运输是通过微量注射荧光标记的RNA转录成爪蟾卵母细胞，共聚焦显微镜。

视频: Visualizing RNA Localization in Xenopus Oocytes

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

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 ...

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 ...

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

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

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

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

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

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

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

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

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. ...

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 ...

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

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 (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

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 ...