RNAの局在に関する私たちの仕事は、KLMにNIH（R01GM071049）からの助成金によってサポートされています。

パート1：蛍光標識mRNAの転写。 <ol><li> RNAの局在化の要素や他の関連配列を含むプラスミドDNAを線形化し、DEPC処理H 2 Oでの1μg/μLで再懸濁します。 DNAテンプレートは、T7、SP6、またはT3 RNAポリメラーゼによる転写のための上流のプロモーター部位を持っている必要があります。 </li><li>滅菌1.5mlチューブに下記の試薬を追加します。 <table><tbody><tr><td> A. 10X送信バッファ（M＆Mを参照） </td><td> 2μlの</td></tr><tr><td> B. 20Xキャップ/ NTPミックス（M＆Mを参照） </td><td> 1μL </td></tr><tr><td> C. 1mMののAlexa Fluor ® 546から14 - UTP（Invitrogen社） </td><td> 1μL </td></tr><tr><td> D. UTP、[α- 32 P]（1μCiの/μL）（パーキンエルマー） </td><td> 1μL </td></tr><tr><td> E. DEPC - H 2 O </td><td> 11μlの</td></tr><tr><td> F. 0.2 M DTT </td><td> 1μL </td></tr><tr><td> G. RNasin（プロメガ社製） </td><td> 1μL </td></tr><tr><td> H.リニアテンプレートDNA </td><td> 1μL </td></tr><tr><td> I. RNAポリメラーゼ（プロメガ） </td><td> 1μL </td></tr></tbody></table></li><li>静かに試薬を混合し、（10秒。微量遠心機で）短時間遠心します。 </li><li>フルオロフォアの光退色を防ぐためにアルミホイルでチューブをカバーし、37℃で2〜4時間インキュベートする。 </li><li>鋳型DNAを分解するために1μlの1 mg / mLのRNaseフリーのDNase（プロメガ）を追加します。 </li><li> 37℃で15分間インキュベーション℃に</li><li>反応を停止するには79μlの20 mMのEDTA（pH 8.0）を追加します。 </li><li> &quot;入力&quot;して保存とラベルを別々のチューブに1μlを取り外します。 </li><li>全長のmRNAを排除しながら取り込まれなかったヌクレオチドを​​含んでなる1ミリリットルG50カラムを介してスピンの反応（材料と方法を参照）、。 </li><li>エタノール沈殿によりRNAを濃縮する。 <ol class="lalpha"><li> 250μlの100％エタノール、10μlの7M CH 3 COONH 4、1μlのキャリアRNA（酵母tRNA、10μg/μLの）または1μlのグリコーゲン（20 mg / ml）を追加。 </li><li>よく混ぜ、-80℃で&gt; 30分間または15分間ドライアイス上で固体になるまで凍結する。 </li><li> 15分、上清をデカント用遠心機で最高速度で回転する。 </li><li> 75％エタノール150μlで洗浄する。 </li><li>遠心、上清をデカントで最高速度で3分間スピン。 </li><li> 37℃で3分間のペレットを乾燥させ、すべてのエタノールが蒸発していることを保証する。 </li></ol></li><li> 11μlのDEPC - H 2 Oに再懸濁し、&quot;組み込まれた&quot;とラベルを別々のチューブに1μlを削除します。 </li><li>パーセントの混入を決定し、50 nMの（計算のための材料と方法を参照）にRNAを希釈する。 </li><li> RNAは凍結/融解を避けるために、単一の使用のために、5μlのアリコートで凍結する必要があり、-80℃で数週間保存することができます</li></ol>第2部：マイクロインジェクションのための準備。 <ol><li> RNAの調製： RNA（50 nm）のアリコートを解凍し、70℃RNAを変性° Cを3〜5分間。任意の粒子を除去し、氷の上に保つために最高速度で室温の遠心機で10分間スピン。 </li><li>卵母細胞の準備： <ol class="lalpha"><li>外科的にアフリカツメガエルの雌（ナスコ）から卵巣を摘出する。 </li><li>コラゲナーゼ溶液25 mlを入れた50 mlコニカルチューブにアフリカツメガエルの卵巣のトリム部分（材料と方法を参照）。 </li><li> 15分間18℃で穏やかに振盪、または卵母細胞まで目に見えて卵巣から分離されています。バッチの変動にバッチに起因する、コラゲナーゼ処理時間が異なる場合がありますとdefoliculationを確保するために目視検査によって慎重に監視する必要があります。 </li><li>卵母細胞が、管に定着液を除去し、MBSHバッファ（材料と方法を参照）で卵母細胞を洗浄することができます。 </li><li> 3回の洗浄の合計のためにさらに2回洗浄を繰り返します。この卵母細胞の単離プロトコルは、Cohenら、2009年にはその詳細と似ています。 </li><li>標準的な解剖顕微鏡下MBSHバッファに手動でソートステージIII / IVの卵母細胞（デュモン、1972）。 IV期の卵母細胞は、顔料とわずかに大きく、斑点のある間III期の卵母細胞は、完全に不透明ですが白です。わずかに透明な卵母細胞は、完全に色素沈着や展示偏光顔料の分布が古すぎることは若すぎると卵母細胞です。 </li></ol></li><li>針の準備： 〜0.05 mmの外径に針を引いて、ベベル。 （私たちは、ドラモンド科学3.5インチ毛細血管（オーダー＃3 - 000 - 203 - G / X）、サッターインストゥルメント（株）マイクロピペットプラーとWPI社bevellerを使用してください。） </li></ol>パート3：マイクロインジェクション<ol><li>注入あたり2 NLを提供するDEPC - H 2 Oで針をキャリブレーション。我々は、フロントガス駆動型マイクロインジェクターを（材料と方法を参照）を使用して、私たちの針をロードし、マイクロメータを使用してドロップサイズを校正する。 </li><li>針にRNAをロードする。 </li><li> MBSHバッファーを含有する注射剤の皿にソートされた卵母細胞を置きます。私たちは、黒い発泡ゴムの層で皿の上に顕微注入する。薄い卵母細胞は白backgrouでよく目立つND。 </li><li>慎重には50nMでのRNAの2 NLで各卵母細胞を注入する。 </li><li> 、RNA追放するDEPC - H 2 Oで針をすすぎ、および注入のために、次のRNAをロードする。 </li></ol>パート4：卵の文化<ol><li>滅菌24ウェルプレート（シグマアルドリッチ社）のウェルにおける場所の卵。ウェルあたり千卵母細胞への我々は、文化まで。 </li><li>バッファを削除し、400μlのO ocyte C ulture M ediumと交換してください（OCM、材料と方法を参照してください）ウェル当たり。培養中に湿潤環境を維持するためにぬれたペーパータオルで気密プラスチック容器内の培養プレートを配置。 </li><li> 18℃で8〜48時間までの間の時間ポイントのために卵母細胞をインキュベートする。 </li></ol>パート5：卵母細胞の固定、脱水とストレージ<ol><li>ガラスバイアルに卵母細胞の生存、デッド卵母細胞と場所を整理する。我々は日常的に&gt; 90％の生存率を観察。 </li><li> 20分間MEMFA固定剤（材料と方法を参照）と岩の場所は、卵母細胞。アルミホイルで覆うことにより、光から卵母細胞を保護する。 </li><li>固定液を除去し、MBSH等量のバッファーと置き換えることにより、卵母細胞を洗浄してください。 2回の洗浄の合計のためにもう一度この洗浄を繰り返します。 </li><li>無水メタノール中に卵母細胞を洗う。 <ol class="lalpha"><li>ボリュームの半分を削除し、メタノールで交換してください。 </li><li>二度&quot;&quot;手順を繰り返します。 </li><li>ソリューションのすべてを削除する、メタノールで交換してください。 </li><li>メタノール洗浄を繰り返します。 </li></ol></li><li>卵母細胞は、画像への準備ができるまで-20℃で保存することができます。 </li></ol>パート6：共焦点顕微鏡によるイメージングRNAの局在<ol><li>イメージングの前に、撮像面をカバーするガラス底FluoroDish（WPI社）にマレーのクリアミディアム（2:1安息香酸ベンジル、ベンジルアルコール）を追加します。 </li><li>慎重に転送されるメタノールの量を最小限に抑え、メタノールからFluoroDishに卵母細胞を移す。 </li><li>倒立型共焦点顕微鏡でのイメージ（我々が正常にツァイスLSM510とライカTCS SP2を使用している）。 </li></ol><img src="/files/ftp_upload/1704/1704fig1.jpg" alt="図1" /> 図1：蛍光標識転写産物のマイクロインジェクションによる細胞内RNAの局在の可視化A.以下のマイクロインジェクションは、のAlexa Fluor 546で標識されたRNAは、最初は核に限定されているB.は、培養8時間後、蛍光標識されたコントロールRNAが均一に見ることができます。卵母細胞の細胞質に分布する。C.は、しかし、輸送用機械を採用する配列を含む蛍光標識したRNAは卵母細胞の植物極（下部に向かって）への細胞内局在性の過程で見ることができます。スケールバー= 50μmの。

<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>

実験動物を用いた実験は、ブラウン大学が定めるガイドラインおよび規則に従って行った。

ここでは、 アフリカツメガエルの卵母細胞におけるmRNAの局在を可視化するためのプロトコルを提示している。蛍光標識RNA転写物を使用して、このメソッドは、以前にジゴキシゲニン標識転写産物を得て、より簡単かつ迅速にその場ベースのアプローチよりもよりも、対ノイズ比の高い信号（モウリーとメルトン、1992、ガウトリューら、1997）があります。この方法を使用して、我々は...

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バッファ <ul><li class="none"> 60mMのMgCl 2の </li><li class="none"> 400mMのトリス- HCl（pH7.5）で</li><li class="none"> 20 mMのスペルミジン塩酸</li></ul> 20倍キャップ/ NTPミックス <ul><li class="none"> 10mMのCTP </li><li class="none"> 10 mMのATP </li><li class="none"> 9mmのUTP </li><li class="none"> 2mMのGTP </li><li class="none"> 20 mMのG（PPP）Gキャップアナログ（ニューイングランドバイオラボ） </li></ul> G - 50カラム <ul><li class="none"> 100ミリリットル脱イオンH 2 Oの水和物5グラムセファデックスG - 50ビーズ（シグマアルドリッチ） 30分間DEPC -扱います。とオートクレーブ。室温で不完全な在庫を保管してください。ご使用の前に、以下のRNaseフリーのソリューションを追加します。 </li><li class="none"> 0.5ミリリットルの0.2 M EDTA </li><li class="none"> 1mlのMトリスpH8.0 </li><li class="none"> 0.5ミリリットル20％SDS </li><li class="none"> 4℃で完全なG - 50ソリューションを保存する</li><li class="none">取り外して廃棄プランジャーを3 mlのシリンジ（BD Biosciences社）から、15 mlのコニカルチューブ（コーニング）にシリンジの注射筒を配置。グラスウール（ペニーの半分のサイズ約プラグ）の少量の注射器を差し込みます。 </li><li class="none">渦巻き完全なG - 50ビーズを再懸濁させるソリューション。 </li><li class="none">空の列に2ミリリットルG - 50溶液を加える。 </li><li class="none">卓上遠心機で1,000 × gで1分間スピン。 </li><li class="none">各カラムに200μlのDEPC処理脱イオンH 2 Oを追加。スピン。 </li><li class="none"> 3回の洗浄の合計のためにさらに2回洗浄を繰り返します。 </li><li class="none">新鮮な15 mlコニカルチューブにシリンジバレルを取り外します。 </li></ul> コラゲナーゼ溶液 <ul><li class="none"> クロストリジウムヒストリ （シグマアルドリッチ）から75mgのコラゲナーゼ</li><li class="none"> 25ミリリットルの0.1 M KPO 3 +液（pH7.4） </li></ul> MBSHバッファ <ul><li class="none"> 88 mM NaClを</li><li class="none"> 1 mMの塩化カリウム</li><li class="none"> 2.4mmのNaHCO 3を </li><li class="none"> 0.82 mMのMgSO 4を X 7H 2 O </li><li class="none"> 0.33 mMのカルシウム（NO 3）2 X 4H 2 O </li><li class="none"> 0.41 mMのCaCl 2を X · 6H 2 O </li><li class="none"> 10mMのHEPES（pH7.6）で</li></ul> 卵母細胞の培養液 <ul><li class="none"> 50パーセントL15培地</li><li class="none"> 15mMのHEPES（pH7.6）で</li><li class="none"> 1 mg / mLのインスリン</li><li class="none"> 100 mg / mlのゲンタマイシン</li><li class="none"> 50 U / mlのナイスタチン</li><li class="none"> 50 U / mlペニシリン</li><li class="none"> 50 mg / mlストレプトマイシン</li></ul> MEMFAソリューション <ul><li class="none"> 0.1 MのMOPS（pH7.4）に</li><li class="none"> 2mMのEGTA </li><li class="none"> 1 mMのMgSO 4を </li><li class="none"> 3.7％ホルムアルデヒド</li></ul> コンピューティングRNAの収量 <ul><li class="none"> &quot;入力&quot;と標準シンチレーションカウンターを使用して、&quot;法人&quot;のサンプルでCPMを決定します。 </li><li class="none">設立=（&quot;法人&quot;）/（10 ×&quot;入力&quot;） </li><li class="none">典型的な取り込み値は〜0.03と0.10の範囲です。 </li><li class="none">別のポリメラーゼの最大理論収量： T7、T3、SP6 - 2.64μgの</li><li class="none"> μg単位反応収率=（使用されるポリメラーゼの最大収率）X（取り込み） </li><li class="none"> 50nMの=（μgのRNA）/ 320 /（拠点におけるRNAの長さ）にRNAを希釈する/（5 × -8） </li><li class="none">反応は、通常利回り〜50 nmでRNAの50〜100μL。 </li></ul>

visualizing rna localization in xenopus oocytes

RNAの局在は、細胞極性の確立に保存されたメカニズムです。 VG1 mRNAはアフリカツメガエルの卵母細胞と空間的にVG1蛋白質の遺伝子発現を制限する行為の植物極に局在化する。この方法でVG1分布の厳密な制御は、発生中の胚の適切な胚層の仕様が必要です。 mRNAの3&#39;UTRのRNA配列の要素は、VG1ローカライズ要素（VLE）は必須と直接輸送するのに十分です。 in vivoで VG1 mRNAの認識と輸送を調べるため、我々は、単純な視覚的な読み出しを経由してトランスファクター向けトランスポートメカニズムの広範な分析を可能にするイメージング技術を開発した。 RNAの局在を可視化するために、我々は、蛍光標識VLEのRNAを合成し、個々の卵母細胞にその写しを顕微注入する。注入されたRNAの輸送を可能にするために卵母細胞の培養後、卵母細胞は、前の共焦点顕微鏡によるイメージングに固定し、脱水です。 mRNAの局在パターンの可視化は、RNAのトランスポートの完全な経路を監視し、シス作用転写内の要素とVLEに結合するトランス作用因子のためにRNAの輸送を指示するの役割を識別するための読み出しを提供する（ルイスら、2008、 Messittら、2008）。我々は、追加のRNAやタンパク質（ギャニオンとモウリー、2009年、Messittら、2008）との共局在を介し​​てこの手法を拡張しており、モーター蛋白質の崩壊と細胞骨格との組み合わせで（Messittら、2008）プローブにmRNAの局在のメカニズム。

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

Visualizing RNA Localization in Xenopus Oocytes

の可視化<em&gt; in vivoで</em&gt; RNAのトランスポートは、蛍光標識RNA転写物のマイクロインジェクションによってに行われます<em&gt;アフリカツメガエル</em&gt;卵母細胞、共焦点顕微鏡に続く。

可視化RNAの局在にアフリカツメガエル卵母細胞

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 ビュー.  Brown University. の可視化 in vivoで RNAのトランスポートは、蛍光標識RNA転写物のマイクロインジェクションによってに行われますアフリカツメガエル卵母細胞、共焦点顕微鏡に続く。

ビデオ: 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.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

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.

アフリカツメガエル卵母細胞のマイクロインジェクション

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.

の受精アフリカツメガエル卵母細胞ホストの転送方式を使用して

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.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

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.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

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精製によってクロマチンの単離（チャープ）

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.

のNa、KとH、K-ATPaseのインによって陽イオン輸送を測定する<em&gt;アフリカツメガエル</em原子吸光法による&gt;卵母：アイソトープアッセイの代替

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.

顔面移植<em&gt;アフリカツメガエル</em&gt;胚

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.

ザ·<em&gt;アフリカツメガエル</em蛍光測定で&gt;卵母細胞のカットオープンワセリンギャップ電圧クランプ法

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.

アフリカツメガエル卵母細胞：マイクロインジェクションのために最適化された方法、濾胞細胞層の除去、および電気生理学的実験における高速解法の変更

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 ...

可視化RNAの局在にアフリカツメガエル卵母細胞

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