We thank Yumeng (Helen) Xia (Rutgers University) for manuscript editing. The work on BASL polarity formation is supported by grants from the U.S. National Institute of General Medical Sciences to J.D. (R01GM109080) and Rutgers University.

1.プラスミド構築<ol><li>以前に1,6に記載されているような技術をクローニングすることによって構築物を生成します。 <ol><li>最初BASLのコード配列を増幅するために適切なプライマーを用いた高忠実度DNAポリメラーゼを使用し、YDAとMPK6とエントリーベクターにそれらをサブクローン。 7での詳細なプロトコルを検索します。 </li><li> MPK6とYDAのドミナントネガティブバージョン（DNmpk6 8とDNyda 4、それぞれ）を生成するには、テンプレートとしてMPK6とYDAのエントリープラスミドを使用して、部位特異的突然変異誘発法9により、点変異を導入します。 </li></ol></li><li>タバコ一過性アッセイのための構築物を構築するには、pH35CG 10にBASLを統合するために、標準的なLR（attL部位のx attRの組換え）の反応（7における詳細な手順を）行い、pXNGWとpXCGWにDNydaとDNmpk6はそれぞれ、11ベクトル 。 <ol><li>酵素消化と配列決定によって得られたプラスミドを確認してください。一度です3日- uccessful、GV3101 12株と2 28℃で選択プレート上で生育するアグロバクテリウム・ツメファシエンスにバイナリー構築物を形質転換します。 </li></ol></li></ol> アグロバクテリウム浸潤液の調製<ol><li>構築pH35CG、pXNGWとpXCGW用を100μg/ mlのルリア-ベルターニ（LB）適切な抗生物質を含む培地10mlに、いくつかの新たに形質転換されたアグロバクテリウムのコロニーを接種する（ゲンタマイシンを25μg/ mlで、リファマイシンを25μg/ mlで、スペクチノマイシン、同じ濃度P19のためのカナマイシンを50μg/ mlのゲンタマイシン及びリファマイシン（トランスジェニックサイレンシングに対してタンパク質を発現する）13の。 </li><li>約16時間、200回転の速度で28℃のシェーカーでアグロバクテリウム培養液を成長した後、OD 600（1.5達する見込み- 1.8）を測定する（ 図2A-B）を 。 10分間、2500×gでの文化をスピンダウン。再懸濁し、WAMgCl 2 10mMの（浸透液）で細胞ペレットをshに。 </li><li>最終OD 600 = 0.5（ 図 2C-D）に到達するために、再度の文化をスピンダウンし、浸透液の適切な量の再懸濁ペレット。 3時間 - 植物を浸潤する前に、1、室温での細胞の混合物を残します。この工程は、細胞の恒常性を維持するのに役立ちます。 </li></ol> 3.タバコ工場、リーフ・インジェクション<ol><li>葉注射のために（16時間光/ 8時間暗のサイクルで23°C）標準植物成長チャンバー内で5週齢の14から4成長タバコ植物（ ベンサミアナタバコ）を 、使用してください。 注：この段階では、タバコ植物は、典型的には6葉（2大、2培地、および2小）を有します。 2中型の葉は葉の注入（二つの小さなものは、多くの場合、あまりにも若いうち二つの大きなものは、あまりにも古いです）のために最も最適です。 </li><li>浸潤の日の前に、水tobac共同植物は土壌を飽和させます。 </li><li> 3時間 - 浸潤の日に、1のために暗闇の中で植物をプレインキュベートします。このステップでは、葉の表皮細胞内に浸透するとき大幅にアグロバクテリウムを容易にする、表皮内の気孔が広く開くことができます。 </li><li>日付で標識された色のテープを使用して、潜入する葉にフラグを設定。マーク黒の太いシャーピーで浸潤する分野（オプション;浸透した領域が感染された後に表示されています。）。 </li><li> アグロバクテリウムの再懸濁液の等しい体積を取り、穏やかに旋回（ 図2E）によって100×15ミリメートルペトリ皿にそれらを混ぜます。注：私たちのケースでは、我々はDNmpk-cYFPのCFP-BASLの1ミリリットル、DNyda-nYFPの1ミリリットルと1ミリリットルでのp19の1ミリリットルを混合しました。 </li><li>浸潤の過程では、浸潤混合物で3ミリリットルの無針注射器を埋める（1〜2ミリリットル）およびタバコの葉の下側（背軸）（ 図2F）に押し込みます。浸潤が、それは便利ですプッシュする一方の手を使用すると、他の手は優しく（押圧力に抗して、向軸）上側をサポートします。 注：浸潤混合物が正常に注入されると、感染した領域は表皮に容易に認識になります。しかし、浸潤が原因で、過酷な注入（組織損傷）又は不十分なプッシュ（無液体の浸透）に失敗することがあります。各試験のために、我々は、（異なる植物からの）は、2つの独立した葉の中の少なくとも2回の注射を示唆しています。 </li><li>バック成長チャンバ内に感染した植物を置きます。一般的に、タンパク質発現は、浸潤、次の48時間後に検出可能です。 </li></ol> 4.共焦点イメージング<ol><li> （ - 4ディスク/領域を製造することができる通常3）（ 図2G）48時間後の浸潤では、注入された領域からリーフディスクを切除するために穴パンチャーを使用します。静かにスライド（上向きの背軸側）にリーフディスクを転送し、水滴でそれらをマウントするためにピンセットを使用してください。あまりにもHAR押す避けてくださいカバースリップ上のdが絞ら/損傷を受けた細胞は、共焦点顕微鏡（ 図2H）の下でうまく表示されませんので。 </li><li>共焦点イメージングの前に、発現レベルを確認するために、エピ蛍光化合物顕微鏡に搭載されたサンプルをスキャンします。注意：私たちの経験に基づいて、中間の発現レベルを示す細胞が最高の共焦点顕微鏡下で表現されます。 </li><li>共焦点顕微鏡上の40X（NA 1.3）レンズを開始します。蛍光タンパク質の培地レベルを発現する細胞が中心に残るように任意の位置にスライドを調整します。それぞれ540 nmの、 - 500 nmおよび514 nmの/ 520 - CFPとYFPの励起/発光スペクトルは、458nmで/ 480です。 </li><li>レーザー強度と最もバランスのとれた蛍光強度/ノイズ比のためのスマートゲインを調整後、画像をプレビューする「ライブ」ボタンを有効にします。結像品質を改善するために、走査画素および/またはフレーム/ライン平均値を増加させます。最後に、REAに焦点調節ノブを調整chの中央値は焦点面とは、画像を撮影するために、「取得」をクリックします。 注：CFPとYFPの画像は、それぞれ、「順次」走査モードのオプションをオンまたはオフチェックすることにより、連続的にまたは同時に捕捉することができます。 </li><li> Z投影が「XYZ」の撮影モードを選択し、セルの詳細なビューのために所望される場合、走査範囲の深さを定義する（10 - は15μm）にし、標的細胞の上から下へ画像を収集。 .TIFFファイルとして選択し、「名前の変更」に画像ファイルを右クリックして、ライカのソフトウェアで得られた画像をコンパイルし、輸出画像。 </li><li>画像30 - 定量分析のための40の細胞。 </li></ol> 5.画像処理および定量分析<ol><li>画像を処理し、定量を行うためにフィジーソフトウェア（http://fiji.sc/Fiji）を使用します。 <ol><li>蛍光強度のグラフをプロットします。 <ol><li>より良いCFP / YFPの発現があるかどうかを視覚化するために、均一に分布し、細胞周囲に沿ってCFPとYFPの信号強度を示すためにフィジーを使用しています。これを達成するために、最初のイメージを起動するには「ファイル」と「開く」を選択し、フィジーを起動します。ツールメニューの「行」をクリックし、右クリックして「セグメント化された行」を選択します。関心領域を決定し、CFPまたはYFP信号（ 図3A-C）をトレースする細胞周辺に沿って線を引きます。 注：フィジーは、セグメント化された線に沿って、絶対蛍光強度値を測定します。 </li><li>セグメント化された線に沿ってCFP / YFPのレベルを表す位置と強度値でグラフを生成するには、ドロップダウンメニュー「分析」と「プロット・プロファイル」を選択します。必要であれば、同等の強度のグラフを生成するために、Excelや他のグラフィックソフトに（メニュー「コピー」を選択することで）強度値を複製します。 </li></ol></li><li>極性の程度を定量化。 <ol><li>約20 represenを収集3回の反復実験からtative細胞はタバコ細胞でのCFPとYFPの極性度を評価しました。 （Lとして定義される）低い蛍光強度を超える（Hとして定義される）高い蛍光強度の比に基づいて、極性度を計算します。注：定量方法は、図3で説明されています。 <ol><li> HとLの値を測定するには、「測定」をドロップダウンメニューから「分析」して、[選択]をクリックし、（上記の）細胞周辺での関心領域に沿ってセグメント化された線を引きます。 「結果」ウィンドウがポップアップ表示された場合、値が定量化を行うために「平均」を使用します。 </li><li> CFPとYFPの比較的均一な発現を示す細胞から、 例えば 、CFP-BASL（ 図3A）またはYFPがDNyda-nYFPとDNmpk6-cYFP（ 図3B）の共発現から補完、測定することにより、HとLの値を取得2はランダムに同じ長さで、周辺のセグメントを選択しました。 </li> &lt;李&gt;は、この場合には、蛍光タンパク質の明白な極性蓄積を示す細胞から、CFP-BASL、DNyda-nYFPとDNmpk6-cYFP（ 図3C）、の共発現は、濃縮された蛍光信号と周辺領域からH値を収集しますおよび低/無蛍光の蓄積と地域からLsを。 </li><li> 20細胞を一緒正規分布のための試験からのH / Lの結果の比率をプール。 （正規分布の場合） スチューデントのt検定によりp値または（非正規分布の場合）コルモゴロフ-スミルノフ（KS）テストを計算します。 </li></ol></li></ol></li></ol></li></ol>

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利害の衝突が宣言されていません。

一般的な使用およびシグナル伝達のためのタバコ一過性発現系の可能な修正：蛍光タンパク質の過剰発現（FP）が正常に植物細胞内でタンパク質の細胞内局在の高速検査に使用されてきたタバコの表皮中のタンパク質をタグ付き。しかし、 植物体中のタンパク質複合体の動的シグナリング分布を研究することは、複雑な細胞内のコンテキスト、一過性のタンパク質-タンパ...

タンパク質は、予測不可能な細胞環境で発生するほぼすべての生物学的プロセスにおいて重要な役割を果たして複雑な階層型ネットワークとフォーム複合体内で相互作用します。しかし、成長の応答に対する植物の開発から、だけですぐにも効率的に細胞内レベルでこれらのダイナミックシグナル伝達事象を認識し、モニターすることができない便利なツールの欠如がありました。 タバコ葉表皮における一過性タンパク質発現システムは、生細胞内で蛍光タンパク質を可視化するための利点を訴えています。このシステムは、翻訳後タンパク質修飾とタンパク質の局在の迅速な検査を可能にする半in vivoでの条件を提供します。補完YFP信号を検査することによって、二分子蛍光相補性（BIFC）アッセイは、植物細胞内でのタンパク質 - タンパク質相互作用の可能性を通知します。他の方法に比べて、 例えば、酵母2ハイブリッド（Y2H）と共同-immunoprecipitation（共同IP）、BIFCは、タンパク質 - タンパク質相互作用は、細胞内レベルで発生する可能性があるコンパートメントを可視化する強力な手段を提供します。 非対称細胞分裂（ACD）は、組織/器官の形成のための新たな細胞型の生成中に細胞集団を幹維持するので、真核多細胞を促進するために不可欠な機構である。 シロイヌナズナ気孔発生は、植物にACDを研究するためのモデルシステムとして用いられてきました。前駆細胞、メリステモイド母細胞は、二つの異なる娘細胞を生成するために非対称的に分割し、メリステモイドは（ガード細胞のペアに終了する前に、細胞様の分裂幹受ける）と気孔系統の地上セル（SLGC）は（分裂と分化することができます舗装細胞）、それぞれ（ 図1）。気孔ACDでは、気孔系統における非対称の新規タンパク質のブレイキング（BASL）は分割非対称性を駆動するためにpremitotically偏光されて、どの株式会社リュド物理的な非対称性および細胞運命非対称性1。 MAPKKK YODAとのMAPK、MPK3と6から成るMAPKカスケードは、気孔分割パターニングと運命の採用2,3、4,5のための中心です。 最近、Zhang ら 。 ACD 6気孔シロイヌナズナのYDA-MAPKシグナル伝達経路への極性タンパク質BASLをリンクされています。標準的なYODA-MAPK経路は、MPK3 / 6を通じて、BASLをリン酸化し、その偏光を活性化させます。足場としてリン酸化BASL機能やタンパク質複合体を形成し、細胞表層6でシグナリングを集中するYODA（YDA）とMPK3 / 6を募集しています。 MAPKコンポーネントとBASLとYDA-MAPK経路の間に正のフィードバックループの偏光は、植物細胞中のタンパク質の偏光のための新規メカニズムを表します。局所的に濃縮されたMAPKシグナル伝達は密接に気孔ACD 6（ 図1）における細胞運命の分化にリンクされると仮定されています。 kの一つこのモデルをサポートEY実験データがBASL 6の発現により誘導されたMAPKの空間的な再分配を実証したタバコアッセイから来ました。 MAPK分子は、しばしば、細胞の内部にどこでも発見されたため、MAPKシグナル伝達が起こる場合、一般的には、監視することは容易ではありません。本研究では、シグナリングリレーが発生する場所を示唆するために、上流キナーゼおよび下流のものとの間の相互作用を可視化するために、分割YFPシステムを利用しました。我々はさらに、どのように補完YFPが空間的に共同によって変調することができるかどうかを可視化する（タンパク質 - タンパク質相互作用を示唆する）、分割YFPペアと第3のタンパク質を（CFPタグ付き）を共発現することによりBIFCシステムの使用を拡張しましたCFPタンパク質を発現しました。そうすることによって、我々はタバコ表皮の細胞表層で偏パターニングに均一な分布からYDAとMPK6、間の相互作用の空間的な再編成を誘導されたCFP-BASLの共発現を示しましたら細胞。このシステムは、したがって、細胞は、内部または外部刺激（ 例えば、他のタンパク質の同時発現、化学的応用、病原体攻撃または環境の変化などによりチャレンジされたときの条件の下で、植物細胞中で動的シグナル伝達事象を監視するために開発される可能性があります。 ）。

<table>
	<tbody>
		<tr>
			<td>Phusion DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D/TOPO</td>
			<td>Life Technologies</td>
			<td>K2400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickChange II XL Site-directed Mutagenesis Kit</td>
			<td>Agilent Technology</td>
			<td>200521</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>AMRESCO</td>
			<td>J106-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>AMRESCO</td>
			<td>J673-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G3632-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>R3501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>K4000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S4014-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>25x75mm Slide</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
		</tr>
		<tr>
			<td>24x50 mm Cover glass</td>
			<td>VWR</td>
			<td>48393-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica&#160;</td>
			<td>TCS SP5 II</td>
			<td>&#160;LAS AF Lite software</td>
		</tr>
		<tr>
			<td>40 x objective lens</td>
			<td>Leica</td>
			<td>HCX&#160;Plan APO&#160;</td>
			<td>HCX&#160;Plan APO&#160;, NA 1.30</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>SORVALL 6+</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 5 Forceps</td>
			<td>Canemco &#38; Marivac</td>
			<td>205EQA-Spec</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging spatial reorganization of a mapk signaling pathway using the tobacco transient expression system

生きた細胞内での動的なシグナル伝達イベントの可視化が課題でした。我々は、植物細胞内でのシグナル伝達の空間的な分布を監視するタンパク質 - タンパク質相互作用を試験から、タバコ表皮細胞における生体分子蛍光相補性（BIFC）アッセイを確立した一過性発現系を拡大しました。このプロトコルでは、我々は相互作用し、 シロイヌナズナ MAPKKK YODAとMAPK6との間のシグナリングは、原形質膜で起こることを示すためにBIFCアッセイを使用しました。足場タンパク質のBASLを共発現させた場合、YODA-MAPKの相互作用は、再分配と空間的にCFP-BASLとの共偏します。この修​​正されたタバコの発現システムは、ローカリゼーションおよび動的な変更（4日未満）と蛍光タンパク質の色（少なくとも3）の倍数に対応することができるシグナル伝達の迅速な検査を可能にします。我々はまた、タンパク質の分布（非対称の空間的局在性、または「分極」を定量化する詳細な方法を提示し;）タバコ細胞インチこの高度なタバコ発現系は、生植物細胞の動的シグナリング事象の迅速な試験のために広く使用される可能性を有しています。

過活動MAPKシグナル伝達によって誘導される細胞死を防止するために、YDA（DNyda）及びMPK6（DNmpk6）のキナーゼ不活性バージョンは、タバコ細胞における共発現アッセイに使用しました。 BIFCによって明らかになったDNydaとDNmpk6の間の相互作用も、CFP-BASL自体はいずれも、不均一な分布パターン（ 図3A-B）を生成しました。 CFP-BASLがDNyda-DNmpk6の相互作用ペアに?...

Watch this Scientific Journal Video about Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System at JoVE.com

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

タバコ一過性発現系を使用したMAPKシグナル伝達経路の空間再編のイメージング

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

The overall goal of this procedure is to visualize dynamic spatial distribution of protein

imaging-spatial-reorganization-mapk-signaling-pathway-using-tobacco

Research

JoVE Journal

Biology

9.7K ビュー.  Rutgers, The State University of New Jersey. The overall goal of this procedure is to visualize dynamic spatial distribution of protein

ビデオ: Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

の可視化線虫（Caenorhabditis elegans）</em親油性生体染色色素、DIIを使用する>キューティクルの構造

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

発現クローニングによりGrb2のシグナル伝達複合体を研究するために高コンテンツイメージングワークフロー

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

シングル酵母細胞中のMAPK誘導発現の時間的な定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

マウスにおける流体力学的遺伝子送達によるタンパク質の一過性発現

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.