This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

全ての実験手順は、バージニア州制度動物実験委員会の大学によって承認されました。 注： 図1は、実験手順の概略を示しています。 卵母細胞の調製<ol><li>事前プライムX.卵母細胞の単離の前に妊馬血清性性腺刺激ホルモン（PMSG）の150 Uと女性約1週間をアフリカツメガエル 。 29 G針と1ccの無菌注射器と背側リンパ嚢に1 mlの150 U / mLのPMSGを注入。 </li><li>卵母細胞注入と卵母細胞アニマルキャップアッセイのためのソリューションを準備します。 <ol><li>準備のCa ++ / Mg ++を含まないOR 2（OR2-）：82のNaCl、2.5mMのKClを、1.5mMのの Na 2 HPO 4、50mMのHEPES pHは7.2。 </li><li> OR2-プラス1 mMのCaCl 2および1mM MgCl 2：OR 2 を準備します。 </li><li> 60％リーボビッツL15、0.4 mg / mlのBSA、100μg/ mlのゲンタマイシン、pHが7.8：OCMを準備します。 </li><li>前1X MBS削減する：88のNaCl、1 mMの塩化カリウムを、0.7 mMのCaCl 2を、1mMのMgSO 4を、5 mMのHEPES（pHは7.8）、2.5 mMの炭酸水素ナトリウム3。 </li><li> 1X MBSで3％フィコールを準備します。 </li><li> 110のNaCl、2 mMの塩化カリウム、1mMのカルシウム（NO 3）2、1mMのMgSO 4を、0.1mMのEDTA、1 mMの炭酸水素ナトリウム、2 mMののNa 3 PO 4、pHが7.4：1×NAMを準備します。 </li></ol></li><li>用溶液中カエルを配置することによって、0.1×MBSにおけるエチル3-アミノベンゾエートメタンスルホン酸塩（MS222）（第1 Lの0.1×MBSで希釈し、次いで、10 mlの95％エタノール中に0.3グラムのMS222を溶解）の0.03％溶液中で雌を麻酔10から15分、または応答しなくなるまで。 <ol><li>その麻酔を確認することは、（不完全麻酔下のカエルは、手足を動かしたり身体を裏返し）応答しない保証するために、その背面に麻酔をかけたカエルを回して完了です。 </li></ol></li><li>外科鉗子で卵巣組織を単離し、メスの刃で肌と体壁を通って腹部切開を行うことによって、卵巣断片を単離はさみ。 OR2-に卵巣の断片を置きます。 24ミリメートル湾曲した針に3-0絹縫合糸で体​​壁を閉じて、同じ縫合で別々に皮膚を閉じます。水に1グラム/ Lの水族館塩中の女性の回復を許可します。 </li><li> （ - 20卵母細胞10）個、新鮮なOR2-への転送細かい鉗子を使用して、小さなに卵巣組織を引き裂きます。 </li><li> 、1時間シェーカー上で穏やかに攪拌新鮮なコラゲナーゼに転送し、さらに1時間攪拌することによりOR2-で2.0 mg / mlのコラゲナーゼAのDefolliculate卵巣断片。 </li><li>ウォッシュは、より小さな卵母細胞を捨てるOR2での[Ca ++ / Mg ++をを含む ] 10回、卵母細胞。 </li><li> OCMで卵母細胞を2回洗浄し、新鮮なOCMへの転送。目視検査時に大きさによって第6期の卵母細胞を分離し、未成熟な（小さい）卵母細胞を捨てる：第6期の卵母細胞は、動物半球であっても色素沈着と未成熟卵母細胞よりも大きく、直径が約1.2〜1.4ミリメートルです。 </li><li> 20℃のI - 18に卵母細胞を維持nは注射前にOCM。注：アガロースでコーティングされたペトリ皿を皿に卵母細胞の付着を最小限にするために使用されてもよいです。 </li></ol>図書館転写物の2注入<ol><li> （例えば、神経板ステージ14 10）開発の適切な段階で抽出したRNAを用いた指向性cDNAライブラリー15を準備します。 cDNAライブラリーを購入するか、以下の4つのステップで概要につき市販のキットを使用して1つを構築します。 <ol><li>ポリ（A）+ RNA（材料の表を参照）を濃縮するために製品を使用し、製造業者の指示に従って、約5μgのmRNAを分離してください。注：cDNAライブラリーを作製するために使用される転写物が（所望の組織の顕微解剖以下のような神経板など）の誘導組織に限定することができる、または特定の段階で胚全体からのものであってもよいです。 </li><li>製造業者の指示に従って、市販のキットを用いてcDNAを生成します。 </li><li> vectoに約20 ngのcDNAをを連結rは、市販のキットまたは他の適当なベクターに含まれています。適当なプラスミドベクターは、5 &#39;βグロビンおよび3のポリ（A）配列（PCS2 +、pTnT、pCS105）などのmRNA安定性のための配列を含みます。 </li><li>熱ショック形質転換のために、供給業者の推奨するプロトコルを使用して有能な細菌細胞へのライゲーションを変換します。 注：また、オープンリーディングフレーム（ORFeome）クローンの回収は、商業的に入手可能であり、注射のための転写物を生成するために使用することができます。 </li></ol></li><li> 10 4複雑（4月10日から5月10日までの総複雑さ）に10 3のプラスミドの10のプールを準備します。万コロニー各 - これは、千と10のプレートを表しています。 <ol><li> 37℃で18時間、および7ミリリットルLBのガラススプレッダーと穏やかな圧力によってそれぞれのコロニーを収集 - プレートライブラリ文化10 15 cmのLB-アンピシリンプレート上に、12を成長させます</li><li> 0.5ミリリットルからグリセロールストックを準備します（-20＆で0.2 mlの滅菌グリセロールおよびストアに追加＃730; C）、製造者の指示に従って、標準的な市販のDNAミニプレップキットを用いてDNAを調製するために、残りの6.5ミリリットルを使用しています。 </li></ol></li><li> 1.5時間-酵素1 37℃で16を消化し ​​、適切な制限付き- （2.0μgの1.0）プールされたプラスミドDNAを線形化。使用されるRNAポリメラーゼキットの仕様に水にエタノール沈殿および再懸濁が続くフェノール/クロロホルム抽出と直線化DNAを単離します。製造業者の指示以下の市販のRNAポリメラーゼキットとセンスRNAを合成します。 </li><li>約20μmの直径の（ガラスキャピラリーチューブと針プラーを使用して）マイクロインジェクション用の針を準備します。較正済み接眼マイクロメータとの複合顕微鏡の針の先端を測定します。注：ニードルプラーの設定が細かい鉗子で壊れたときに所望のチップサイズを生成するように細かいチップで針を製造するために経験的に決定されなければなりません。 </li><li>準備（に粘土を押してくださいマイクロインジェクションの間に所定の位置に卵母細胞を保持するための平行溝と皿の底に均一な層）粘土で内張り35×10ミリメートルペトリ皿。炎で先端に融合したショッピングモールプローブまたはパスツールピペットで溝を作ります。粘土の代替として、エラストマー金型17とのくぼみを作 ​​るアガロース料理を準備します。粘土が並ぶ料理の行で1X MBSで3％フィコール（約2ミリリットル）に広口径ピペットで転送卵母細胞。 </li><li>マイクロインジェクターを用いて、1 ngの/ NL RNAと針を記入し、（卵母細胞の細胞質のアップ描画防ぐために）わずかな正の圧力を生成するためにバランスを調整します。 </li><li>赤道域で約20 NL RNAを卵母細胞に注入します。その後、フィコール-MBSに残る1X MBSに優しく転送するために注入した卵母細胞のための1時間を許可します。アニマルキャップアッセイの前に20℃で24時間 - 8インキュベートします。 </li></ol> 3.アニマルキャップアッセイ<ol><li> 3/4倍NAMを準備し、細かい鉗子、ヘアループ、湾曲したカバースリップフラグメント、粘土ライニングDISを得ます卵母細胞を保持するカップ状のくぼみとHES。 （ - 2ミリメートル×2 - 約1〜4 mm）の小さな断片にカバーガラスを離れて壊すことにより、湾曲したカバースリップフラグメントを作成し、エッジポリッシュまで炎を通過すると垂れ、湾曲した部分を生成します。 </li><li> Xを肥やしますアフリカツメガエルの卵のin vitroまたは自然交配やステージ原腸する培養による18 -前段階でソートするに0.1X MBS中（10室温で11時間後に受精を）。 10の胚-ミッド原腸胚（11.5ステージ11）を収集。 </li><li> NAM 3/4倍で、ハーフ、フル約ペトリ皿に胚を移します。細かい鉗子の二組を使用して、原腸胚から受精（卵黄）膜を除去します。 </li><li>粘土が並ぶ皿でNAM（約2ミリリットル）3/4倍に転送する卵母細胞との溝は上述したように、個々の胚に対応するために、感想を生産、粘土の個々の印象に固定化します。 </li><li>移植胚粘土が並ぶお料理にとgオフアニマルキャップをカット微細鉗子の二組を使用してastrulae。だけではなく、赤道組織18アニマルキャップ外胚葉を分離するように注意してください。 </li><li>卵母細胞を接触させることをアニマルキャップの内面と各卵母細胞の動物半球上のアニマルキャップを置きます。 8時間以上 - 6のための卵母細胞の表面に露出した内部層に開いたままにすることができます（カバースリップの連絡先などの動物のキャップを粘土を外胚葉を平坦化、湾曲したガラスカバースリップフラグメントを塗布し、ガラスに下向きの圧力を加えることによって組換え体を一緒にホールド）。 注意：別の方法として、その内面を上に向けて、粘土のインデントにアニマルキャップを配置し、キャップに卵母細胞を置きます。粘土の小さな拡張子の組換え体を固定します。 </li><li> 20℃での培養制御胚がアッセイに所望の段階に達するまで。 </li><li>カバーガラス/粘土を除去し、鉗子とヘアループと外胚葉を単離することにより、組換えセパレート。 </li><li> MEMFAで1時間（3.8％のため、外胚葉断片を修正MEM中のホルムアルデヒド[0.1 M MOPS pHは7.4、2 mMのEGTA、および1mMのMgSO 4]）。 </li><li> -20℃でエタノールとストアにMEMFAからの断片（およびコントロール胚）を転送します。 </li></ol> in situハイブリダイゼーション （ISH） ではにより外胚葉における応答の解析4。 <ol><li> ISHのためのソリューションを準備します。 <ol><li>準備し、1×PBS：0.01Mのリン酸緩衝生理食塩水、塩化ナトリウム0.138 M、pH7.4の</li><li>準備をPBS-Tweenで（PTW）：1×PBS、0.1％のTween-20 </li><li> 2％BSA、2％のポリビニルピロリドン、2％フィコール：100×デンハルト溶液を調製します</li><li>ハイブリダイゼーションバッファーを準備します：50％ホルムアミド、5×SSC、1 mg / mlのトルラ酵母RNA、1 / mlのヘパリン、1×デンハルト液、0.1％Tween-20を、0.1％CHAPS、10mMのEDTA、DEPC-H 2 Oを</li><li> 100 mMのマレイン酸、150mMのNaCl、pHが7.5：マレイン酸バッファー（MAB）を準備</li><li>準備MAB +ブロック：MAB、2％ブロッキング試薬（溶解する60℃の熱） </li><li>準備アルカリホスファターゼ（AP）緩衝液：100mMトリスpHは9.5、50のMgCl 2、100mMのNaCl、0.1％トゥイーン、のdH 2 O </li></ol></li><li> RNAプローブを準備します。 <ol><li> 1.5mlチューブ（50μlの反応）に追加、商業RNAポリメラーゼキットを使用し、ミックスのNTP掘る：25.5μlのDEPC-H 2 O、10μlの5X転写バッファー、2.5μlの10倍DIG-NTPミックス、5μlの100 mMのDTT、2μLのRNAsin、2μlの直線化DNAテンプレート（〜を1μg/μl）を、3μlのRNAポリメラーゼと90分間37℃でインキュベートします。 </li><li> 2μlのRNAポリメラーゼを加え、60分間37℃でインキュベートします。 </li><li> 1％アガロースゲル上での反応の2μLを確認してください。 </li><li> 1μlのRQ1 RNaseフリーのDNaseを追加し、20分間37℃でインキュベートします。 </li><li> 50μlのDEPC-H 2 O、25μlの10 M酢酸アンモニウムおよび313μlのエタノールを添加することによって、プローブを沈殿させます。 -20℃で一晩（O / N）で保存し、その後20分間13,800×gで遠心分離することによりRNAを回収します。簡単にスピンし、500μlの75％エタノールで洗浄し、削除しますエタノール、空気乾燥さペレットを許します。 50μlのDEPC-H 2 Oを追加</li><li> 〜0.5μgの/μlの最終濃度になるようにハイブリダイゼーションバッファーを追加します。 </li></ol></li><li>ハイブリダイゼーションのために組織を準備します。特に断りのない限り、説明した各溶液交換（約4ml）でトップにバイアルを埋めます。 <ol><li>水平ロッカー上、10分ごとに75％エタノール/ PTW、その後、50％エタノール/ PTWにバイアルおよび移植胚からエタノールを除去します。 </li><li>ロッカーに各5分間PTWで3回洗浄します。 </li><li> 10μgのPTWで/μlのプロテ​​イナーゼK処理に移します。垂直ロックチューブ15分。 </li><li>垂直ロックチューブ - 0.1 MトリエタノールアミンpHは7.8で10分間2回ずつ洗浄します。 </li><li>チューブに12.5μLに無水酢酸を加え、垂直方向に5分を揺すります。 5分間の追加の12.5μlの無水酢酸で繰り返します。 </li><li>ロッカーに縦PTW 5分で洗浄します。 </li><li>ロッカーに4％パラホルムアルデヒド20分でREFIX。ヒートハイブリダイゼーションバッファー60℃に。 </li><li>ロッカーに各5分間PTWで3回洗浄します。 </li><li>各チューブからPTWのすべてが、〜1ミリリットルを削除し、250μlのHybをバッファに追加します。穏やかに混合する管を渦巻きます。垂直ロックチューブ5分。 </li><li> 60℃Hybをバッファー（0.5ml）で交換し、60℃10分で静かに攪拌します。新鮮なHybを緩衝液で交換して、60℃2〜4時間で攪拌します。 </li><li> 60℃（3分）にヒートプローブ（Hybをバッファー中の0.5μg/μlので1ミリリットル）。 Hybを緩衝液を除去し、チューブにプローブを追加します。 O / Nは、60℃で穏やかに撹拌します。 </li></ol></li><li>抗体のための組織を準備します。 <ol><li>暖かいHybをバッファと、2×SSC + 0.1％Tween-20を60℃にソリューションを提供しています。 </li><li> Hybをバッファ（ - 3X再利用2、-20℃のプローブを保存）とプローブ溶液を交換してください。 10分間60℃で洗浄します。 </li><li> 2X SSC-Tween中60℃（20分攪拌しながら、それぞれ）で3回洗浄します。 </li><li> 0.2X SSC-Tween中60℃（20分攪拌しながら、それぞれ）で3回洗浄します。 </li><li>ロッカーにそれぞれ水平に15分間（RT）MABで2回洗浄します。 </li><li> 1ミリリットルのMAB +ブロックを追加します。ロッカーの上に縦に2時間を洗ってください。 </li><li> 1ミリリットルのMAB + 1 / 2,000の抗ジゴキシゲニンAPを含むブロックに転送します。 4 CO / Nで垂直方向にロック。 </li></ol></li><li>色試薬のために組織を準備します。 <ol><li> MABと抗体溶液を交換してください。ロッカーにそれぞれ水平にMAB 5分間で3回洗浄します。 </li><li>ロッカーにそれぞれ水平にMAB 1時間で3回洗浄します。 </li><li>ロッカー上の各水平APバッファ10分間で2回洗浄します。 </li><li>複数のウェルプラスチックトレーに組織を転送し、APのバッファを削除し、BMパープル試薬（〜1ミリリットル）を追加します。反応は、O / Nに暗い5分で進行させます。 </li><li>適切な染色が達成されると、PTWのバイアルに組織を移します。ロッカー上PTWで2回各々10分間洗浄します。 </li><li>ロッカーの4 CO / Nでブアン液中で固定してください。 </li><li> RTで3 70％エタノール/ 30％PTW洗浄でブアンを洗浄します。トンを続行Oイメージ落射顕微鏡に取り付けられたカメラを使用してキャプチャ、漂白に必要な18の場合。 </li></ol></li></ol> 5.兄妹の選択およびクローニング<ol><li>最も高い活性（外胚葉組織の最大の応答）とプールを選択します。 </li><li> （上記のプロトコルにセクション2を使用して）前のステップからのコロニーを、最も高い活性を有するプールのグリセロールストックを（適切な密度にLBで希釈）と、10分の1程度で新たに10のプレートをプレートアウト滴定します。 </li><li>活動が単一コロニーにトレースされるまで、プールのサイズを減らします。 </li><li>ベクター標準プライマーを用いてDNA配列を得ます。 </li></ol>

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RNA acts early in Xenopus. embryos to promote formation of a vegetal dorsalizing center.</a> Cell. 67, 753-765 (1991).</li><li>Smith, W., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+noggin.,+a+new+dorsalizing+factor+localized+in+the+Spemann+organizer+in+Xenopus+embryos.">Expression cloning of noggin., a new dorsalizing factor localized in the Spemann organizer in Xenopus embryos.</a> Cell. 70, 829-840 (1992).</li><li>Plautz, C., Zirkle, B., Deshote, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+induction+of+anterior+head+ectodermal+properties+in+Xenopus.+embryos+are+mediated+by+transcriptional+cofactor+ldb1.">Early stages of induction of anterior head ectodermal properties in Xenopus. embryos are mediated by transcriptional cofactor ldb1.</a> Developmental Dynamics. 243 (12), 1606-1618 (2014).</li><li>Servetnick, M., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+neural+and+lens+competence+in+Xenopus.+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus. ectoderm: evidence for an autonomous developmental timer.</a> Development. 112, 177-188 (1991).</li><li>Nieuwkoop, P., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li><li>Lemaire, P., Garrett, N., Gurdon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+Siamois.,+a+Xenopus.+homeobox+gene+expressed+in+dorsal-vegetal+cells+of+blastulae+and+able+to+induce+a+complete+secondary+axis.">Expression cloning of Siamois., a Xenopus. homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.</a> Cell. 81, 85-94 (1995).</li><li>Lustig, K., Kroll, K., Sun, E., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+cloning+of+a+Xenopus+T.-related+gene+(Xombi.)+involved+in+mesodermal+patterning+and+blastopore+lip+formation.">Expression cloning of a Xenopus T.-related gene (Xombi.) involved in mesodermal patterning and blastopore lip formation.</a> Development. 122, 4001-4012 (1996).</li><li>Hsu, D., Economides, A., Wang, X., Eimon, P., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus.+dorsalizing+factor+Gremlin+identifies+a+novel+family+of+secreted+proteins+that+antagonize+BMP+activities.">The Xenopus. dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.</a> Molecular Cell. 1 (5), 673-683 (1998).</li><li>Lustig, K., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+oocyte+expression+assay+to+reconstitute+inductive+signaling.">Use of an oocyte expression assay to reconstitute inductive signaling.</a> Proc. Natl. Acad. Sci. USA. 92, 6234-6238 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning.">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE Science Education Database. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests.">Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> JoVE Science Education Database. , (2015).</li><li>Viczian, A., Zuber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Determination+Using+the+Animal+Cap+Transplant+(ACT)+Assay+in+Xenopus+laevis.">Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis.</a> J. Vis. Exp. (39), e1932 (2010).</li><li>Sive, H., Grainger, R., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of.Xenopus laevis.: A laboratory manual. , (2000).</li><li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+activation+during+early+stages+of+lens+induction+in+Xenopus.">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li><li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxe+view+of+lens+development+and+disease.">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li><li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism.">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li><li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project.">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li><li>Karpinka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenbase,+the+Xenopus.+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li><li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis.">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469 (2013).</li></ol>

The authors declare that they have no competing financial interests.

コンピテント外胚葉における応答を誘導することができる遺伝子の機能的クローニングのためにここに記載された方法は、遺伝子産物の広い範囲を同定することができます。この方法は、発現クローニング技術と組織誘導のアッセイを組み合わせることにより、過去の作品を発展させ。我々は、RNA注入後、直接的または間接的に、誘導因子の産生の供給源としてアフリカツメガエル卵?...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table><tbody><tr><td>12/101 Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>12/101</td><td>Monoclonal antibody for detection of muscle tissue</td></tr><tr><td>20x SSC Buffer</td><td>Sigma</td><td>S6639</td><td>for ISH</td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td>for ISH</td></tr><tr><td>Anti-Dig-AP</td><td>Roche</td><td>11093274910</td><td>for ISH</td></tr><tr><td>Aurum Plasmid Mini Kit</td><td>Bio-Rad</td><td>732-6400</td><td>Plasmid DNA purification</td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td>for ISH</td></tr><tr><td>BM Purple</td><td>Roche</td><td>11442074001</td><td>for ISH</td></tr><tr><td>Boekel Hybridization Oven</td><td>Fisher Scientific</td><td>13-245-121</td><td>for ISH</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma</td><td>HT10132</td><td>for ISH</td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td>for OCM</td></tr><tr><td>CHAPS</td><td>Sigma</td><td>C3023</td><td>for ISH</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>Defolliculation of oocytes</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C121800</td><td>Dejelly embryos</td></tr><tr><td>DEPC-H2O</td><td>Fisher Scientific</td><td>BP5611</td><td>for ISH</td></tr><tr><td>Dig-RNA Labeling Mix</td><td>Roche</td><td>11277073910</td><td>for ISH probes</td></tr><tr><td>Dumont #5 forceps</td><td>World Precision Instruments</td><td>500233</td><td>for Vitelline envelope removal</td></tr><tr><td>Ethyl 3-aminobenzoate</td><td>Sigma</td><td>A5040</td><td>MS222 anesthetic</td></tr><tr><td>Ficoll PM 400</td><td>Sigma</td><td>F4375</td><td>for Injection media</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td>for ISH</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma</td><td>G1914</td><td>for OCM</td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>4878</td><td>3.5&quot; long, I,D, 0.530 mm</td></tr><tr><td>Glass sample vials</td><td>Fisher Scientific</td><td>06-408B</td><td>for ISH</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Hair affixed in pasteur pipette for tissue manipulation</td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td>for ISH</td></tr><tr><td>Injector Nanoliter 2010</td><td>World Precision Instruments</td><td>Nanoliter 2010</td><td>Microprocessor-controlled microinjector</td></tr><tr><td>Instant Ocean</td><td>Carolina</td><td>972433</td><td>Aquarium Salt for frog recovery</td></tr><tr><td>IRBG XGC Xenopus verified full-length cam cDNA</td><td>Source Bioscience</td><td>989_IRBG</td><td>cDNA library&amp;nbsp;</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/ml Ampicillin</td><td>Teknova</td><td>L5004</td><td>150 mm pre-poured LB-Amp plates for sib selection</td></tr><tr><td>LB Luria Broth</td><td>Teknova</td><td>L8650</td><td>LB for collecting colonies in sib selection from plates and dilution of cultures</td></tr><tr><td>Magnetic mRNA Isolation Kit</td><td>New England BioLabs</td><td>S1550S</td><td>for isolation of poly(A)-enriched RNA</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375</td><td>for ISH</td></tr><tr><td>Manual Microfil Micromanipulator</td><td>World Precision Instruments</td><td>M3310R</td><td>Manual micromanipulator</td></tr><tr><td>Nutating Mixer</td><td>Fisher Scientific</td><td>22-363-152</td><td>Rocker for ISH</td></tr><tr><td>Permoplast</td><td>Nasco</td><td>SB33495M</td><td>Clay for injection and dissection dishes</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>P5368</td><td>for ISH</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877</td><td>to stimulate oocyte development</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>PVP40</td><td>for ISH</td></tr><tr><td>Programmable Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Micropipette needle puller</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td>for ISH</td></tr><tr><td>pTnT Vector</td><td>Promega</td><td>L5610</td><td>cDNA library construction</td></tr><tr><td>Riboprobe Combination System</td><td>Promega</td><td>P1450</td><td>in vitro transcription</td></tr><tr><td>Superscript Full Length cDNA Library Construction Kit</td><td>Life Technologies</td><td>18248013</td><td>kit for cDNA library construction</td></tr><tr><td>Sutures, 3-0 silk</td><td>Fisher Scientific</td><td>19-037-516</td><td>Suture thread and needle for post-oocyte removal</td></tr><tr><td>Torula RNA</td><td>Sigma</td><td>R3629</td><td>for ISH</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>T1502</td><td>for ISH</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td>for ISH</td></tr><tr><td>Universal RiboClone cDNA Synthesis System</td><td>Promega</td><td>C4360</td><td>alternative kit for cDNA library construction</td></tr><tr><td>Xenopus Full ORF Entry Clones - ORFeome Collaboration</td><td>Source Bioscience</td><td>5055_XenORFeome</td><td>ORFeome Clones</td></tr><tr><td>XL2-Blue Ultracompetent Cells</td><td>Agilent Technologies</td><td>200150</td><td>cells for transformation of cDNA library</td></tr></tbody></table>

functional cloning using a xenopus oocyte expression system

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

mRNAの発現に応答して、卵母細胞に注入し、応答動物キャップ組織は、in situハイブリダイゼーション （ 図2および表1）によってOTX2の発現についてアッセイした。OTX2、レンズプラコードを介して神経管閉鎖の推定レンズ外胚葉（PLE）で表され肥厚19。 OTX2も PLE外側前方神経外胚葉ならびに非神経外胚葉ヘッドで表現され?...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

用いた機能クローニング<em&gt;アフリカツメガエル</em&gt;卵母細胞発現システム

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

functional-cloning-using-a-xenopus-oocyte-expression-system

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K ビュー.  Shepherd University. We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

ビデオ: Functional Cloning Using a Xenopus Oocyte Expression System

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 ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

遺伝学

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

幹細胞様<em&gt;アフリカツメガエル</em&gt;胚外植片を早期神経発達の特徴を研究するために<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

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

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

キセノプス・レービス胚における眼芽へのDNAのマイクロインジェクションとGFPのイメージングは、無傷で光学軸軸樹園を発現し、生きているキセノプス・タマジャクシ

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

トランスジェニックの生産アフリカツメガエル制限酵素を介した統合と核移植によって

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

マウスパルテノジェネティックハプロイド胚性幹細胞の精子代替としての応用

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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