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).

動物実験は、ベルン州Kantonstierarztの地方委員会、KantonalerVeterinärdienstベルン（BE85 / 15）によって承認されています。 アフリカツメガエル卵母細胞の調製<ol><li>厳密に20°Cに維持され、水に12時間/ 12時間の明/暗サイクルでカエル（ アフリカツメガエル）を維持します 。 </li><li>女性のカエル9から卵巣のローブを削除します。 注：ローブの除去は、再生のための刺激である、と多くの場合、卵母細胞の品質は手術で改善されます。卵母細胞は、卵黄膜、毛包細胞の層、および血管21を含む結合組織で覆われています。全体の構造は、「毛包」と呼ばれます。 </li><li>ペニシリンおよびストレプトマイシンを補充した密滅菌バース生理食塩水7（MBS）に卵母細胞を含む卵胞が充てんされてい卵巣のローブを置きます（88のNaCl、1 mMの塩化カリウム、2.4 mMのNaHCO 3、0.82ミリモルのMgSO 4、0.41ミリモルのCaCl 2、0.33ミリモルのCa（NO 3）2、10mMの4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸; HEPES（pHは7.5、NaOHで）、100μg/ mLのペニシリン、および100μg/ mLのストレプトマイシン）。 </li><li>シングルアウトステージV-VI 21包。ピンセットで卵巣葉を持ちます。卵胞の上に白金耳（ 図1）を下げ、静かに卵巣組織から卵胞を含む結合組織を破壊するためにループを撤回。 注：（ -直径1.2ミリメートル1.0）と暗い色素性動物半球と黄色がかった植物半球間の良好なコントラストによりステージV-VI 21包は、その大きさによって特徴付けられます。 </li><li>プラスチック製パスツールピペットを用いて直径35mmのペトリ皿（1.5ミリメートルの開口径に背をカット）に（ すなわち、完全な球形と無傷の色素沈着を持つ）のみ健康そうな卵胞を転送します。 </li></ol>注：ここで説明するマイクロインジェクションシステムはKressmannとBirnstiel 22によって報告されたことに由来します。 <ol><li> in vitro転写による受容体、ポリ（A +）尾部、およびRNA定量の添加は、ゲル電気泳動23によってα4β2δGABAのサブユニットをコードするそれぞれのcDNAからcRNAをを準備します。 </li><li>マイクロピペットプラーを使用してホウケイ酸ガラスキャピラリー（1.0ミリメートル、外径（OD）、0.58ミリメートル、内径（ID）、長さ100mm）からマイクロインジェクションピペットを準備します。ベベルチップで15ミクロン - 12の先端径を作成するために、マイクロマニピュレーターとミクロフィラメントを使用して、顕微鏡下でガラスキャピラリーの先端を折り。 </li><li> 10 mLシリンジを使用して、パラフィンオイルとマイクロインジェクションピペットを埋め戻し、その後、手製のMICR上にマイクロインジェクションピペットをマウントoinjection装置（ 図2）。 注：手製油油圧式射出装置は、フットペダルスイッチによって制御グリルモーターで構成されています。追加のスイッチは、モータの回転モードを変更します。このモーターは、10μLのガラスシリンジのプランジャーを前進または後退させるマイクロメータねじ（0.5ミリメートル/ターン）を駆動します。注射は約3回転の速度であり、0.5ターンは卵胞に50 nLの注入に対応しています。注射器の先端は、それ自体がタイゴンチューブの非常に短い一片によって注射針に接続されている厚肉ポリテトラフルオロエチレンチューブに接続されています。注射キャピラリーをマイクロマニピュレーターによって制御されるドリルチャックによって保持されています。システム全体は、パラフィン油で満たされています。それらは、注入システムが損なわれるように、気泡は、避けるべきです。セットアップは、冷光源で点灯されます。実体顕微鏡は、光制御のために必要とされます。卵胞はstereomicrで冷光下で可視化されていますOSCOPE（40X倍率）。注射セットアップのパフォーマンスは、 アフリカツメガエル卵母細胞への放射性トレーサーの注入によってテストされています。 45の容積 - 55 nLのは、1回の注射送達されるべきです。 </li><li>滅菌プラスチックチップでピペットを用いて、耐湿性の熱可塑性（2×2センチ）のクリーン内側に滅菌水の液滴を配置することによって、セットアップをテストします。この液滴への注入ピペットの先端を浸し、その後、プランジャー（注入用ピペット内の負圧）を後退させるモーターを回します。 注：水はピペットを入力して、油で目に見えるインタフェースを形成すべきです。 </li><li>液滴からのピペットの先端を撤回し、注入ピペットの内側に正の圧力を加えます。水滴は、注入用ピペットの先端に形成すべきです。 </li><li>植物極はナイロンメッシュ（G：0.8ミリメートル）の隙間に上向きでそれらを並べることにより、卵胞の注入の準備をPeとの底に接着MBSで覆われたトライディッシュ（60ミリメートル）。 </li><li>熱可塑性のクリーン内側に滅菌プラスチックチップでピペットを用いて、mRNAの2μLを分注します。注入ピペットの内部に負圧を印加することによって、注入ピペットへのmRNAを取ります。 </li><li>マイクロマニピュレーターを用いて、個々の卵胞の上に注入ピペットを置きます。 </li><li>植物極の中心に注射針を挿入し、ピペットの内側に正の圧力を加えることによって0.6μL/分の流速でのmRNAの50 nLのを注入します。 mRNAのエスケープを避けるために、卵胞から注入ピペットチップを取り外す前に、10秒 - 5待ちます。 注：mRNAは動物半球に位置する核への注入を避けるために、植物（黄色っぽい）極に注入する必要があります。 </li><li> MBSの2 mLを満たした新しいペトリ皿（35ミリメートル）に注入された卵胞を転送します。 18℃に設定したワインクーラーに皿を置きます。 1のために注入された卵胞を培養します -7日記録する前に、新たに発現したタンパク質の同一性に応じて。 </li></ol> 3.卵胞のストリップ（図3） 注：上記のように、血管24を含んでいる卵黄層、卵胞細胞、および結合組織で覆わ卵母細胞として知られている「毛包」。細胞表面へのソリューションのアクセスを妨げることなく、機械的安定性を提供卵黄層を除く全ての層は、電気生理学的実験の前に除去しなければなりません。周囲の細胞層なしの卵胞は、以前「剥皮」卵母細胞25と呼ばれています。この工程は、通常の電気生理学的実験と同じ日に行われます。 <ol><li>トランスファー10は、MBS 0.5mLの、1mg / mlのコラゲナーゼ、および0.1mg / mLのトリプシンインヒビターを含むホウケイ酸ガラス管に卵胞を注入しました。 36℃に維持した水浴中でチューブを浸します。インキュベート20分間の卵胞と時折チューブを振ります。 </li><li> RTでMBSの1 mLを含む第二の管にそれらを転送することにより卵胞をすすぎます。約10秒間チューブでそれらを残します。 </li><li> 4mMのエチレングリコールビス（2-アミノエチルエーテル）を含む二重濃縮MBS 0.5 mLを含む第3チューブに卵胞を転送- Nは、N、N &#39;、N&#39;-四酢酸（EGTA）、及び4分のためにそれらをインキュベートRT。時折チューブを振ります。 注：高張液（二重濃縮MBS）は、それにより卵母細胞の卵胞細胞層を剥離、卵胞細胞層内で卵母細胞の収縮を誘導するために使用されます。 EGTAは、コラゲナーゼ活性を阻害するために添加されます。 </li><li> RTでMBSの1 mLを含む第二の管にそれらを転送することにより卵胞をすすぎます。約10秒間チューブでそれらを残します。 </li><li> MBSの2 mLを含むペトリ皿（直径35mm）に卵母細胞を転送します。実体顕微鏡下で、によって卵胞から外封筒を分離単純に白金耳を用いて、離れて裸の卵母細胞をプッシュ。 注：卵母細胞の外側エンベロープは、培養皿にしっかりと付着し、容易に卵母細胞から分離することができます。いくつかの卵母細胞は自発的にそれらの外側エンベロープを失い、裸化卵母細胞として回収されます。この手順では、卵母細胞の球状の外観を保持し、比較的安定した準備になります。 </li></ol>卵母細胞の周り4.高速解法の変更<ol><li> 9,26を説明したように、電圧クランプ実験を行います。 </li><li>実験で必要とされる灌流スイッチを回して重力供給灌流システムのソリューションを変更します。 <ol><li> 6 mLの1.35ミリメートルの内径を有するガラスキャピラリーを通して/分、卵母細胞の周りのアゴニスト濃度の急速な変化を可能にするために、卵母細胞の表面から約0.4mmに配置された河口に灌流液を適用します。 注：変化率は以前に推定されています0.5未満の27で70％D。 </li></ol></li></ol>

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The authors have nothing to disclose.

この資料に記載された方法は、伝統的に7-9使用されるものから外れます。 1〜2時間コラゲナーゼ処理8に卵巣のローブを露出するための規格です。損傷を受けていない、裸に卵母細胞を分離します。およびmRNAは、市販の注入装置を使用してそれらを注入します。卵母細胞は、コラゲナーゼの高濃度に長時間暴露によって損傷される可能性が高い1）：これは古典的な手順は以下...

アフリカツメガエル卵母細胞は、広く（ - 3、およびその中の参考文献参考文献2）発現系として使用されます。彼らは、適切に組み立て、それらの形質膜に機能的に活性な多サブユニットタンパク質を組み込むことができます。このシステムを使用して、機能的に変異した、キメラ、または連結されたタンパク質の特性を研究するため、および潜在的な薬物をスクリーニングするために、単独で、または他のタンパク質と組み合わせて膜タンパク質を調査することが可能です。 他の異種発現システムより卵母細胞を使用する利点は、巨細胞の簡単な取り扱い、外来遺伝情報を発現する細胞の高い割合、浴灌流によって、卵母細胞の環境の簡単な制御、および膜電位の制御を含みます。 この発現系の欠点は、多くの研究室17〜20において観察された季節変動です。この変化の理由明確にはほど遠いです。さらに、卵母細胞の質は、多くの場合、強く変化することが観察されます。伝統的な方法7-9は卵巣ローブの分離、いくつかの時間のためのコラゲナーゼに卵巣ローブの露出、裸に卵母細胞の選択、および卵母細胞のマイクロインジェクションが含まれています。ここで、代替、高速手順の数は、私たちは、卵母細胞の品質の無い季節変動や変化の少ない、30年以上にわたってこの発現システムで動作することが許可されていることが報告されています。 改変及び改良された卵母細胞の単離のためにここに記載された方法、cRNAをマイクロインジェクションし、及び濾胞細胞層の除去は、 アフリカツメガエル卵母細胞における選択の任意のタンパク質の発現のために使用することができます。卵母細胞の周囲の培地の高速解の変化のための非常に簡単な方法は、任意のリガンド依存性イオンチャネルのキャリアの研究に適用することができます。

<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

タンパク質の異種発現系としてアフリカツメガエル卵母細胞は、第一のガードンらによって記載されました。 1、広くその発見以来使用されている（文献2から3、およびその中の参考文献）。外国のチャンネル発現のための卵母細胞は、魅力的な特徴は、内因性イオンチャネル4の貧しい豊富です。この発現系は、それらのリガンド依存性イオンチャネルの中で、多くのタンパク質の特徴付けのために有用であることが証明されました。 アフリカツメガエル卵母細胞におけるGABA A受容体の発現およびそれらの機能的特徴付けは、卵母細胞、cRNAを、濾胞細胞層を除去しながら、微量注入、および電気生理学的実験で高速なソリューションの変化の分離を含め、ここに記載されています。手順は、この研究室5,6で最適化し、日常的に7-9を使用したものから逸脱しました。伝統的に、露出した卵母細胞を調製しますRTで卵巣ローブの長期のコラゲナーゼ処理、およびこれらの裸化卵母細胞にmRNAをマイクロインジェクションしています。最適化された方法を使用して、多様な膜タンパク質が発現されており、そのような組換えGABA A受容体10-12、ヒト組換えクロライドチャネル13、トリパノソーマカリウムチャネル14、及びミオの -イノシトールトランス15、16のように、このシステムを研究しました。 ここに詳述した方法は、 アフリカツメガエル卵母細胞における選択の任意のタンパク質の発現に適用することができ、迅速な溶液交換は、他のリガンド依存性イオンチャネルを研究するために使用することができます。

アフリカツメガエル卵母細胞は、機械的に白金耳（ 図1）を使用して選び出しました。 0.5：2.5のfmol /卵母細胞（ 図2）卵母細胞は、mRNAは、受容体サブユニットは4、β2、δ、0.5をαGABAをコードをマイクロインジェクションしました。 4日後、卵胞細胞層（ 図3）を除去しました。電圧は、卵母細胞を-80mVでク?...

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.

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 ...

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

Research

JoVE Journal

Biology

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

20.1K ビュー.  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. 

ビデオ: 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.

操作と<em&gt;インビトロ</emの&gt;成熟<em&gt;アフリカツメガエル</em細胞質内精子注入し&gt;卵母細胞、胚発生を研究するために

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

発生生物学

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.

の共焦点分析を用いました<em&gt;アフリカツメガエル</em&gt; WntおよびShhのモルフォゲン勾配の調節を調査するために、

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.

電圧クランプ<em&gt;アフリカツメガエル</em&gt;蛍光性の非天然アミノ酸を用いた卵母細胞

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

生化学

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.

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 ...

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 ...

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 ...

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.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

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.

機能研究のためのヒトシナプス受容体を再活性化するためのシナプス膜の微小移植

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

神経科学

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.

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

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

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

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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

可視化RNAの局在にアフリカツメガエル卵母細胞

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

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

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

機能研究のためのヒトシナプス受容体を再活性化するためのシナプス膜の微小移植

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、アフリカツメガエル

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

要約

さらに動画を探す

この動画の章

操作と

の共焦点分析を用いました

電圧クランプ

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

可視化RNAの局在にアフリカツメガエル卵母細胞

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

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

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

機能研究のためのヒトシナプス受容体を再活性化するためのシナプス膜の微小移植

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、アフリカツメガエル