私たちは、優れた研究室のサポートやゼブラフィッシュのケアのためのフィオナ·フォーリーに感謝します。この作品は、GWにAHAの博士号を取得する前のフェローシップ（11PRE5730027）とHJY（R01HL66292）と防衛庁（R01HL095690）へNHLBIの助成金をサポートされていました。

ステージ固有のゼブラフィッシュ胚注射の概要標的mRNAに結合し、その転写物からのタンパク質発現を破壊アンチセンスモルフォリノオリゴヌクレオチド（MO）は、広く研究がゼブラフィッシュ13,14で（機能喪失型）遺伝子のノックダウンで使用されています。遺伝子ツールは、LLCは、蛍光顕微鏡を用いて注入胚でMOを検出するためにカルボキシフルオレセイン（緑色の蛍光を発する）またはリサミン（赤色蛍光を発する）のいずれかでタグ付けされたMOを提供しています。ゼブラフィッシュの開発のさまざまな段階で卵黄セルにMOを注入することによって、それは、胚の特定の区画（ 図1A-F）にMOを提供するのは可能です。 1から4細胞の段階（0-1 HPF）との間で卵黄に注入されたMOは、グローバルなノックダウンを容易にするために、32細胞期15日まで存続する卵黄との接続を介してすべての胚細胞（ 図1A、D、D &#39;）に入ります。 MOはmidblast中に卵黄に注入ULAステージは（2.5から3 HPF）他のほとんどの胚細胞系譜を入力せずに、DFC / KV細胞系統12に特異的に細胞質の橋8およびノックダウン遺伝子機能を通じてそうDFCの前駆細胞（ 図1B、E、E &#39;）を入力することができます。卵黄12,16でも遺伝子の機能がDFC / KVで必要かどうかをテストしたりするための重要な制御として、それは後にドーム30パーセント被包段（約4.5 HPF）の間に注入することによって、卵黄細胞にMOを制限することもできますすべての細胞質の橋が（ 図1C、F、F &#39;）を閉鎖した。これらの注射はDFC / KV細胞17から24における遺伝子機能を解析するために組み合わせて使用されている。 KV内の流体の流れを評価するために、蛍光マイクロビーズは6から10体節のステージの間にKVの内腔に注入され（12-14 HPF）、その後すぐにビデオ顕微鏡（ 図1G-J）を用いて画像化。ビーズは赤または緑色蛍光（あるいはその両方）を放出が利用可能であるので、それは別のを使用することが可能である画像蛍光マイクロビーズおよびMOへのチャネル。 1。モルフォのステージ固有のインジェクション（MO） ゼブラフィッシュ胚にMOの注入は、以前25,26実証されている。ここでは、簡単にステージ固有のMO注入を説明します。すべての注射後、胚をシャーレに移し、28.5℃でインキュベートした。 <ol><li>グローバルMO注入：直ちに受精後の胚を採取し、注入プレートにロードします。キャピラリー針に蛍光MOを読み込み、マイクロインジェクター（ 例えばハーバード装置PLI-90ピコインジェクタ）に針をマウントします。針の先端を破って25,26に記載されているように 1 nlの体積を有する射出ドロップを作成するために注入設定（圧力および/ ​​または時間）を調整します。解剖顕微鏡を使って、卵黄にMOの1 nlを注入する（ 図1A）実験あたり≥50胚の。 4セルクワガタで注射を完了するには、迅速に作業電子。 </li><li> DFC-標的のMO注入：28.5にて直ちに受精後の胚を採取し、インキュベート℃に胚は256細胞期（約2.5 HPF）に達したときに、迅速に注入プレートにそれらをロードします（ 図 256セル、1,000細胞ステージ間≥100胚の卵黄中に蛍光MOの1 nlを注入する1B）。 </li><li>卵黄ターゲティングMO注入：28.5で受精直後、インキュベートした後に胚を収集℃にドームステージ（〜4 HPF）で、注入プレートに胚をロードしてから、ドームと30％の被包段（ 図1C）の間≥100胚の卵黄に蛍光MOの1 NLを注入します。 </li></ol> 2。分析のために注入胚を選択する<ol><li> MOは胚（8 HPF）は75％で被包段階に達する注入すると、蛍光MOの配布のための蛍光解剖顕微鏡下で受精卵と死亡胚を削除してから、画面生きた胚。その後、アロwは28.5で開発するために胚を選択℃を<ol><li>グローバルMOの注射については、蛍光均等にすべての胚細胞（;図2A図1D）全体に分布している胚を選択します。 </li><li> DFC-標的ミズーリ注射用、蛍光MOは卵黄に拡散しており、背胞胚マージン（DFCが）（;図2B図1E）に集中して現れる胚を選択します。この段階では、それは、基本的な卵黄で明るい蛍光による蛍光DFCを可視化することが困難な場合があります。蛍光ミズーリにDFC以外の胚細胞に取り込まれたまま、または注射部位（ 図2D）で集計していた胚を除外するように注意してください。 </li><li>卵黄をターゲットとしたMOの注射については、ミズーリ蛍光が均一に卵黄全体に分布し、任意の胚細胞（;図2C図1F）で観察されていない胚を選択します。繰り返しになりますが、蛍光MOが残存する胚を取り除く注射部位に集約されます。 </li></ol></li><li> 2-4-体節ステージ（〜11 HPF）、画面の胚より高い倍率を用いた蛍光顕微鏡下で二回目の間に。次に、選択した胚は28.5で開発することができ℃、 <ol><li>グローバルMOの注射の場合は、選択した胚は、MO蛍光が均等にKVとすべての胚細胞（;図2E図1D &#39;）で配布があることを確認します。 </li><li> DFC-標的ミズーリ注射については、慎重にのみKV細胞と卵黄（ 図1E &#39;）でMOの蛍光を有する胚を選択します。そのようなTG（Dusp6：d2EGFP）などのKV細胞でGFPを発現するトランスジェニック胚27は 、ミズーリが正常KV細胞（ 図2F）に配信された胚の識別に役立ちます。 KVの細胞は他の胚細胞のMO蛍光を持つ胚を廃棄します。 </li><li> 図：卵黄をターゲットとした、MO注入については、卵黄（ 図1F &#39;で排他的にミズーリ蛍光を有する胚を選択する2G）。 </li></ol></li></ol> 3。 KV内の流体の流れを分析するために取り付けた胚<ol><li>グローバルMOのKVで非対称流体の流れを分析するには、MOまたは卵黄ターゲティングMO注入胚をDFCはターゲティング、慎重dechorionate〜鋭いピンセットで4から6体節段階（〜11月12日HPF）との間に20胚。 </li><li> 42℃で乾燥したお風呂で液体として維持するための5 mlチューブ℃に1％低融点アガロースとアリコート50mlを準備</li><li>ガラスうつ病スライド（米国科学用品株式会社）への転送1つの胚と多くの水の可能な限り除去する。ただ（あまりアガロースがその後の画像に干渉することができます）胚をカバーするのに十分な暖かい1％低融点アガロースを追加することで、胚を固定。アガロースが固まるように、そのようなそのKVは（ 図1H）（背側）を向いている胚を配置するためにピンセットを使用しています。スライドごとに1つの胚で10胚をマウントします。次のステップで、すべての注射はCOMPLであることを確認するために迅速に働く10体節期（14 HPF）によりeted。 </li></ol> 4。 KVに蛍光マイクロビーズの注射<ol><li> 1.5mlのエッペンドルフチュー​​ブに滅菌水で1:50にMultifuorescent 0.5ミクロン径の微小球を（ポリサイエンス社）Fluoresbrite希釈します。 </li><li>チューブをタップして、徹底的にマイクロビーズの1:50希釈液を混合し、キャピラリーニードルに3μlをロードします。ミズーリ注射（ 例えばハーバード装置PLI-90ピコインジェクタ）に使用されるのと同じマイクロインジェクターを使用して、ピンセットで針の先端を破って、可能な限り小さい（&lt;0.5 NL）注射降下を生成するために注射の設定を調整します。 </li><li>解剖顕微鏡下では、最初にマウント胚のKVのルーメンと針の位置を合わせます。内腔に針を挿入し、ビーズの少量（&lt;0.5 NL）（ 図1G）を注入。小さな針の先端と小さな注入量は、有害KVを避けるために重要です。 </li><li>マウントされているすべての胚を注入します。胚はinjecteされたらdは、滅菌水の滴が乾燥からそれを防ぐためにアガロースの上に追加することができます。注射の過程で、ビーズはしばしば針の開口部をふさがれます。これは、針の先端を再分割することと、マイクロインジェクション装​​置の調節圧力または時間設定によって注入量を調整する必要があります。それは注射中に重大な損傷を引き起こすのに十分な鈍になった場合に、針を交換してください。 </li><li>画面直立蛍光化合物顕微鏡（ 例えばツァイスAxioImager M1）の下にビーズの正常な配信のために注入された胚は、20X対物レンズを使用。まず、KVの構造が明視野照明を（ 図1J）を使用して、無傷であるかどうかを判断し、次にビーズを観察する蛍光を使用します。ビーズが存在し、KVのルーメン内を移動させた胚を選択します。 KVの損傷やKVでないフローティングビーズと共に胚を破棄します。それはKVを逃すと周辺組織または基礎卵黄にビーズを送達するために簡単です。失敗した場合、マウントあの熱10胚および注入手順を繰り返します。 </li></ol> 5。 KVの流体の流れの可視化と分析<ol><li> KVの内側にビーズと、選択した各胚については、アガロースをカバーし、その後63X水浸対物レンズ（ 図1I）を使用して、直立蛍光化合物の顕微鏡下でKVを観察するために滅菌水の滴を追加します。あるいは、カバースリップは非水浸漬の目的および/または倒立顕微鏡で使用するために適用することができます。 </li><li> 10秒の動画を撮影するために（ 例えばツァイスAxioCamHSm）顕微鏡に搭載されたハイスピードカメラを使用しています。明または微分干渉コントラスト（DIC）チャネルを用いた蛍光チャネル（約70フレーム/秒）とKVの内腔を用いてビーズの両方を記録します。 </li><li>時間をかけてすべてのビーズの動きを可視化するために、ImageJのソフトウェア（無償でダウンロード中に蛍光ビーズのムービーを読み込む<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> ）ndはムービー内のすべての蛍光シグナルの最大投影を作成します。 ImageJの中で最大の投影を実行するには、クリックして &quot;イメージ→スタック→Zプロジェクトを。&quot; &quot;Zプロジェクト&quot;ウィンドウで、 &quot;最大輝度&quot;の投影タイプを持つすべてのスライスを投影。この画像は、ビーズが撮像されたKV（ 図3A-B）のDIC像に重ね合わせることができます。 </li><li>個々のビーズの動きはImageJを使用して追跡することができます。プラグイン（ &quot;マニュアルトラッキング&quot;）でダウンロードすることができます<a href="http://rsbweb.nih.gov/ij/plugins/track/track.html" target="_blank">http://rsbweb.nih.gov/ij/plugins/track/track.html</a> 。 ImageJの中に蛍光ビーズムービーを開き、マニュアルトラッキング機能を実行します。マニュアルトラッキングのウィンドウで、 &quot;時間間隔&quot;と &quot;x / yのキャリブレーション&quot;の &quot;ショーパラメータ&quot;と入力パラメータを確認してください。手動≥50フレームの映画の焦点面に残っているビーズを選択してから、胚あたり≥5ビーズを追跡します。各ビーズのパスと速度は目によって生成されeソフトウェア（ 図3 CD）。 </li></ol>

<ol>
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Biol. 348 (1), 22-33 (2010).</li><li>Matsui, T., 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=Canopy1,+a+positive+feedback+regulator+of+FGF+signaling,+controls+progenitor+cell+clustering+during+Kupffer's+vesicle+organogenesis.">Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (24), 9881-9886 (2011).</li><li>Shu, X., 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=Na,K-ATPase+alpha2+and+Ncx4a+regulate+zebrafish+left-right+patterning.">Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning.</a> Development. 134 (10), 1921-1930 (2007).</li><li>Esguerra, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ttrap+is+an+essential+modulator+of+Smad3-dependent+Nodal+signaling+during+zebrafish+gastrulation+and+left-right+axis+determination.">Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination.</a> Development. 134 (24), 4381-4393 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <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+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Yuan, S., Sun, Z. <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+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Molina, G., 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=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat. Chem. Biol. 5 (9), 680-687 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1995).</li><li>Schneider, I., Houston, D. W., Rebagliati, M. R., Slusarski, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+fluxes+in+dorsal+forerunner+cells+antagonize+beta-catenin+and+alter+left-right+patterning.">Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning.</a> Development. 135 (1), 75-84 (2008).</li><li>Clement, A., Solnica-Krezel, L., Gould, 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=The+Cdc14B+phosphatase+contributes+to+ciliogenesis+in+zebrafish.">The Cdc14B phosphatase contributes to ciliogenesis in zebrafish.</a> Development. 138 (2), 291-302 (2011).</li><li>Matsui, T., Bessho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+in+zebrafish.">Left-right asymmetry in zebrafish.</a> Cell Mol. Life Sci. , (2012).</li></ol>

特別な利害関係は宣言されません。

DFC / KV細胞系譜にMOをターゲットにステージ固有の注射を使用すると、遺伝子機能の細胞自律性を検討し、全体的な遺伝子ノックダウンによって引き起こされる多面的な表現型を回避するための有用なアプローチである。しかし、これらの注射は技術的に挑戦することができます。 1）MOは卵黄全体2）MOの拡散、注射部位で集計ままおよびDFC / KVセルまたは3に入る）MO：256セル、1,000細胞ステー...

<table border="1"><tbody><tr><td>試薬/材料の名称</td><td>会社</td><td>カタログ番号</td></tr><tr><td>標準コントロールオリゴリサミンタグ付け</td><td>遺伝子ツール、LLC </td><td></td></tr><tr><td>カスタムRock2bモルホリノオリゴ</td><td>遺伝子ツール、LLC </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent 0.5ミクロンのマイクロスフェア</td><td>ポリサイエンス社</td><td> 24054 </td></tr></tbody></table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

例えば、心臓、脳、腸などの内臓が正常な機能にとって重要である1左右（LR）非対称性を開発しています。運動性繊毛はLRの確立に関与しているマウス、カエル、ゼブラフィッシュ2月6日を含む、脊椎動物の胚の非対称性。これらの&#39;のLR繊毛は&#39;器官7を開発するためのLRパターニング情報を提供すると考えられている左側側板中胚葉、内カスケードのシグナル伝達に保存された非対称節点（TGF-βスーパーファミリー）をトリガすることが必要である非対称の流体の流れを生成します。このように、LRのパターニングのメカニズムを理解するためには、それはLR繊毛細胞の組織、LRの繊毛の運動性と長さと堅牢な非対称フローを生成する能力を制御する遺伝子を同定することが不可欠である。 ゼブラフィッシュ胚に、LR繊毛でクッパー胞（KV）2,4,5に配置されています。 KVはmonociliated上皮細胞の単層で構成されていますそれは液体で満たされた腔を囲みます。運命のマッピングはKVが被包ステージ8,9の間に背の胚盤葉縁に移行背先駆細胞（DFCが）として知られている〜20から30細胞群に由来することが示されている。初期の体節の段階では、DFCは、クラスタと繊毛上皮細胞への分化は、胚10,11の尾芽にKVを形成する。ゼブラフィッシュの光学的透明性と迅速な開発とDFCはインの組み合わせを特定して追跡する能力ゼブラKV LR繊毛細胞を研究するための優れたモデル系胚作る。 興味深いことに、DFC / KV細胞系列の前駆細胞は卵黄細胞と2 HPF 8後の近い他の胚細胞の間に細胞質の橋のに対し、最大4時間、受精後（HPF）に卵黄細胞の間に細胞質の橋を保持します。これらの細胞質の橋を生かし、我々は、モルホリノoligonucleを提供するためにステージ固有の注射戦略を策定DFCは、ノックダウン、これらのセル12における標的遺伝子の機能にもっぱらotides（MO）。この手法は、遺伝子の機能が野生型胚の文脈で発展DFC / KV系統でノックダウンさせたキメラ胚を作成します。 KVにおける不斉の流体の流れを分析するために、我々は、KVルーメンとビデオ顕微鏡2を使用してレコードビーズ運動に蛍光マイクロビーズを注入します。流体の流れを簡単に可視化され、時間をかけてビーズの変位を追跡することにより定​​量することができる。 ここでは、ステージ固有のDFC-標的遺伝子ノックダウン技術と流れを可視化するためのKVへの蛍光ビーズの注入を使用して、我々は、KVの発達と機能の間の特定の遺伝子の役割を特徴付けるための効果的なアプローチを提供するプロトコルを提案する。

ステージ固有のMO注入胚の特定区画における遺伝子機能を解析する上で有効なアプローチを提供します。 図1は、DFC / KV細胞における遺伝子機能をテストするために、流体の流れを可視化する蛍光ビーズを導入する方法使用済みの注射戦略のフローチャートを提示KVインチ成功した段階特異注入胚の蛍光MOの分布は、 図1D-Fに、 図2でライブ胚における模式?...

Watch this Scientific Journal Video about Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle at JoVE.com

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

クッパー胞の繊毛で生成された流体の流れ（KV）はゼブラフィッシュ胚の左右パターン形成を制御します。ここでは、KVの細胞に特異的に遺伝子の機能を調節するための手法について説明します。また、流体の流れを可視化するためにKVに蛍光ビーズを提供する方法を示しています。

クッパー胞における遺伝子機能と繊毛発生する流体の流れの可視化の分析

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

analysis-gene-function-visualization-cilia-generated-fluid-flow

Research

JoVE Journal

Biology

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

14.8K ビュー.  Upstate Medical University. クッパー胞の繊毛で生成された流体の流れ（KV）はゼブラフィッシュ胚の左右パターン形成を制御します。ここでは、KVの細胞に特異的に遺伝子の機能を調節するための手法について説明します。また、流体の流れを可視化するためにKVに蛍光ビーズを提供する方法を示しています。

ビデオ: Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

遺伝子機能を解析するためにゼブラフィッシュ胚のマイクロインジェクション

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

シチュヌチュ・ニュードゥス・コロミック流体のハイブリダイゼーションで

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

涙腺幹細胞のための3次元、無血清培養システム

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

ナノフローサイトメトリーによる単一細胞外小胞膜貫通タンパク質の特性評価

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...