この作品は、衛生研究所（NIH）の国立研究所によってサポートされていましたCA127231と（SM）のデイモンラニヨン財団臨床研究者賞（CI 1502）を付与します。我々は彼らの機関やプロトコルの標準化へのこの技術の移植性を議論に彼の有用な洞察のためパラツキー大学オロモウツ、チェコ共和国からの教授ズデニェクドヴォルザークに感謝したいと思います。

1。酵母ベクターにおけるPXRとSRC-1融合の構築<ol><li>ヒトPXR LBD（107から434アミノ酸）およびヒトSRC-1完全長（アミノ酸1から1401）のPCR増幅。 <ol><li> SRC-1テンプレートとしてをpCMX-SRC1プラスミドを使用し、PXR LBDテンプレートとしてのpSG5-hPXRプラスミド8を使用してください。 </li><li>雪解けのPCRのSuperMix、DNAテンプレートとプライマーは（材料を参照）、氷上で保管してください。 </li><li> 0.25μgの/μLDNAテンプレート1μL、10 mMのプライマー対2μLそれぞれ、PCR法のSuperMix 45μL追加し、50μlに全量を、H 2 Oを加える。 </li><li> 2分2分、30秒、94℃で20サイクル、30秒、55℃、72℃、94℃の1サイクルでのPCRを設定し、拡張のための10分間の72℃での1サイクル。 1％アガロースゲル上で実行して、PCR産物を確認する。 </li></ol></li><li> PCR産物は、Y2Hベクターにクローニングする。 <ol><li> 1％アガロースゲルからPCR産物を精製する。ダイジェストPXR、LBD、PCR、およびY2HベクトルpSH2-1とBamHIとSalIで、NotΙとSalΙとSRC-1 PCRおよびY2HベクトルpGADNOTを消化。これを行うには、30μlに全量を最終的に1μgのDNAを、3μLの10×反応緩衝液、1μlの制限酵素、及びH 2 Oを加える。 </li><li> 1時間、37℃の水浴中で消化サンプルを置く。 1％アガロースゲル上で実行して消化を確認する。 </li><li> 1％アガロースゲルから消化サンプルを精製、ライゲーションはPCR産物とY2Hベクターを消化し、DH5αコンピテント細胞に形質転換する。 </li><li>コロニーをピックアップし、プラスミドDNAを分離します。 </li></ol></li><li>のNotI / SalI消化によってpGADNOT-SRC-1プラスミドDNAを識別をBamHI / SalI消化によってpSH2-1-PXRプラスミドDNAを識別します。配列決定により、すべての正のプラスミドDNAを確認してください。 </li></ol> 2。酵母ツーハイブリッドアッセイ<ol><li> 5ミリリットルのYPADに酵母株を接種し、一定の攪拌（30℃、220回転）で一晩インキュベートする。注： サッカロマイセス·セレビシエ株はCTY10-5Dた（MAT ADE2のTRP 1から901 LEU2-3、112 HIS3-200 GA L4 - GAL80 - URA3 :: のlexA-LacZ）ののlexAオペレータ10と統合されたGAL1-lacZ遺伝子が含まれています。ケトコナゾールに耐性の生酵母細胞を得ることではなく、アゾール耐性18〜20の原因は、CTY10-5D酵母の新規株としてトランスポーターの変化を運ばなかったものが最初に削除ERG3（ERG3の Δ）によって得られ、その後、追加、削除を導入したERG11（ERG3Δ/ ERG11Δ）遺伝子の相同組換えによる。構造の詳細は、以前に公開された原稿6に記載されている。 </li><li> （午前中）、次の日は、5ミリリットルのYAPD酵母（1:20）に接種し、一定の攪拌（30℃、220回転）により、OD 600〜0.2を得るために、インキュベートする。 </li><li> 2分間の微量2,000 rpmで細胞をペレットと上清を捨てる。その後PELLEを洗うオートクレーブ水で一回、0.1M LiAc形質との二度トン。 </li><li>最後の洗浄後、上澄み液を廃棄し、細胞ペレットに以下の試薬 ​​を追加します。240μLのPEG 3500 50％W / V、36μlの1 M LiAc形質、50μL2 mg / mlの沸騰したサケ精子のssDNA、H 2 Oを34μL。 、ミックス1μgのPSH-PXRおよび1μgのpGADNOT-SRC-1のDNAを加え、混合する渦。 </li><li> 30分間（30℃で220 rpm）を、丸底チューブをポリスチレン転換を攪拌、混合するミックスを転送します。 42℃の水浴にチューブを移動し、さらに30分間インキュベートする。 </li><li> 2分間2,000 rpmでチューブを遠心分離し、上澄み液を除去します。オートクレーブ水で一回ペレットを洗浄。 </li><li>チューブにピペット100μlの滅菌水、タッピング、ピペッティング優しい指で細胞を再懸濁し、-His/-Leuドロップアウトミディアム1リ​​ットル-His/-Leuドロップアウト中につき（製剤のプレート上にプレート：20グラムのブドウ糖、 1.7グラムのYNB、0.67グラムCSM-His/-Leu、5gの硫酸アンモニウム、1.5％寒天）及びセシ形質転換体を単離するための3〜4日間、30℃でubate。 </li></ol> 3。のX-galフィルターアッセイ<ol><li>ペトリ皿の部分のサイズにニトロセルロース膜をカット。向きをマークするためにニトロセルロース膜にラベルを付けます。 </li><li>酵母コロニーにpremarkedニトロセルロース膜を配置し、膜が平板培地の表面に接触していることを確認してください。 </li><li>膜を取り外し、3mmの濾紙上にコロニー側を上に置き、15分間-80℃で膜、ろ紙を置き。 </li><li> X-gal溶液を作る。追加50μL（1Lバッファが16.1グラムHPO 4 7H 2 O、5.5グラムののNaH 2 PO 4、H 2 O、0.75グラム2のNaが含まれているZバッファの5ミリリットルに20 mg / mlのX-galおよび15μlのβ-メルカプトエタノール塩化カリウム、硫酸マグネシウム4•7H 2 O 0.25グラム）。 Zバッファを4℃に維持した場合、これらの試薬を添加する前に室温にそれをもたらす。 </li><li> 3mmの濾紙の二つの円を配置シャーレ内と5ミリリットルのX-gal溶液でそれらを浸す。ペトリ皿から過剰の緩衝液を排出し、コロニー側を上にしてニトロセルロース膜を配置します。 </li><li>膜は、ろ紙との完全な接触をしているし、それが完全に濡れていることを確認して蓋を閉じます。ホイルにペトリ皿をラップし、一晩に30分間37℃でインキュベートする。 </li></ol> 4。液体のβ-galアッセイ<ol><li> OD 600 0.7に-His/-Leuドロップ液体培地中の酵母を育てる。 </li><li> 2分間3,000 rpmで1.5 mlのマイクロ遠心チューブと遠心分離機に1ミリリットル媒体搬送。上清を捨てる。 </li><li> 、ペレットを再懸濁させ、Zバッファの1ミリリットルを追加します。2分間、再び3000 rpmで遠心分離します。上清を捨てる。 </li><li>その後、50μlのクロロホルムと20μlの0.1％SDSを加え、混合し、細胞ペレットを再懸濁する2MEとZバッファの150を添加する。精力的に15秒間のサンプルをボルテックスする。 </li><li> ONPGソリューション（Z bufに1 mg / mlの700μlのを追加します。FER）。タイマーを設定し、黄色が全反応時間を開発し、注意することができるように十分に長い、30℃でインキュベートする。 </li><li>反応を停止し、420 nmの吸光度を測定するために、1MのNa 2 CO 3の500を添加する。ブランクは700μlのONPG水溶液を加えた500μlの1 MのNa 2 CO 3である。 </li></ol>ミラーユニット=（*千A420）/（A600 *分*のml）に次のようにミラーユニットを計算する A420：420 nmにおける吸光度の単位; A600：600 nmにおける吸光度の単位、 分 ：分単位での反応時間; ミリリットル ：中の反応容積

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利害の対立が宣言されていません。

私たちの修正Y2Hアッセイでは、PXR 6上のケトコナゾールとの相互作用に重要な残基を同定した。 SRC-1はコアクチベーターである（及びpGADNotベクターにクローニングした）ので、我々はまた、SRC-1は、これが活性化プロフィールを変更および/または漏出性に影響を与えるかどうかをPSHベクター系にクローン化されたときにla​​cZ発現を活性化し得るかどうかを試験した酵母ツーハイブ?...

酵母ツーハイブリッド（Y2H）アッセイが広くタンパク質-タンパク質相互作用を発見するために使用され、より最近では、タンパク質-タンパク質相互作用複合体7、図8、図9、図10、図11を破壊する新規な小分子の発見のために。しかし、このアッセイの従来のアプローチは、創薬または「ヒット」のために使用、改変された時にはまだ相互作用し、改変された残基11の尋問を可能にすること、タンパク質-タンパク質表面内の化学薬品化合物のアロステリック相互作用残基の検出を可能にしない。実際に、このような方法（単数または複数）は、開発することが可能であれば、タンパク質 - タンパク質相互作用の破壊のための重要なアロステリック相互作用残基のハイスループット評価に扱いやすい酵母系を可能にする。薬物発見の文脈において、タンパク質と化合物の相互作用を確立するための最も直接的な方法は、構造決定（タンパク質-阻害剤複合体、例えば crystalization）を含むであろう。これらのメソッドは中出しですbersome、精巧なリソースを使用し、それはすべてのタンパク質上の構造研究を行うことは技術的に実現不可能である。 扱いやすい遺伝的薬剤スクリーニング系は、細菌、1,2および哺乳動物ツーハイブリッドなどの他のモデル系において確立されている。しかしながら、これらのシステムは、Y2Hのような最適化お​​よび代替システムがまだほとんどの薬物発見で試験する必要がある。乏しい感度および特異法13を用いて相互作用の信頼性を含む限界があるが、単一のY2Hアッセイは、相互作用残基に関する特定の質問に答えるように改変することができる。核内受容体研究の分野において、Y2Hは、相互作用タンパク質14を定義するために使用されてきたが、これらのタンパク質相互作用はほとんど/アンタゴニストは、核受容体-タンパク質複合体と相互作用するリガンドた性質を定義するために使用されていない。したがって、我々の研究室では、特にありません受容体タンパク質のために、メソッドを定義することに力を注い逆Y2Hベースの検出プラットフォームを使用して残留物を相互作用新規リガンド/アンタゴニストを発掘だろ基づく方法を、プロテオミクスに容易に適用できる。 ケトコナゾールはPXRとその活性化因子SRC-1を破壊という我々の以前の知見に基づいて、我々は小説はPXR 6にケトコナゾール作用する残基を定義して、質問するために私達を可能にするY2H系を逆に開発しました。我々の方法は、DNA結合および転写活性化に関与する分離可能なドメインからなる酵母GAL4タンパク質の特性に基づく。タンパク質はGAL4活性化ドメインとの融合体として発現される完全長クチベーターSRC-1（ステロイド受容体コアクチベーター1）しながら、PXR LBDタンパク質はAD（LexAのDNA結合ドメイン（DNA-BD）との融合体として発現される）。 PXR及びSRC-1融合タンパク質間の相互作用は、酵母genomに統合されたレポーター遺伝子β-LacリZを含有するGAL4結合部位の転写活性化をもたらすE。ケトコナゾール、PXRアンタゴニストは、PXR及びSRC-1の相互作用15、16、17を破壊し、我々はX-gal活性のためのフィルター上のコロニーを染色した後、ケトコナゾールの存在下又は非存在下でPXR及びSRC-1との相互作用を検出することができる。 Y2Hの原理は、図1に示されており、実験手順を図2にまとめる。

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	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast Strain CTY10-5d erg3&#916;/erg11&#916;</td>
			<td>Our lab</td>
			<td></td>
			<td>CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3&#916;/erg11&#916;) genes6 .</td>
		</tr>
		<tr>
			<td>YPD Growth Medium</td>
			<td>BD Biosciences</td>
			<td>630409</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-His/-Leu Complete Supplement Mixture</td>
			<td>MP Biomedicals</td>
			<td>4250-412</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG (o-Nitrophenyl &#914;-D- Galactopyranoside).</td>
			<td>Sigma-Aldrich</td>
			<td>N1127</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Fisher</td>
			<td>BP-1615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicated Salmon Sperm DNA boiled (10 mg/ml)</td>
			<td>Life Technology</td>
			<td>156-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Acros Organics</td>
			<td>61177</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoconazole</td>
			<td>Sigma-Aldrich</td>
			<td>K1003</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>Acros Organics</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td></td>
		</tr>
		<tr>
			<td>50% PEG-3350 solution, filter-sterilized</td>
			<td>Sigma-Aldrich</td>
			<td>P-3640</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Whatman</td>
			<td>10402091</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Fisher</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

<table border="1">
	<tbody>
		<tr>
			<td>5&#39;-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD forward primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-AGAGTCGACTCAGCTA CCTGTGATGCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD reverse primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 forward primer for pGADNOT</td>
		</tr>
		<tr>
			<td>5&#39;-GCGGTCGACTTATTCAGTCA GTAGCTG -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 reverse primer for pGADNOT</td>
		</tr>
		<tr>
			<td>Platinum PCR Supermix</td>
			<td>Invitrogen</td>
			<td>11306-016</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>our lab</td>
			<td>R0136</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>our lab</td>
			<td>R0138</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>our lab</td>
			<td>R0189</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists

薬物代謝および炎症の重要な調節因子として、プレグナンX受容体（PXR）は、代謝、炎症（ 例えば脂肪肝） の1,2を結ぶ疾患の病態生理に重要な役割を果たしている。 PXRのアゴニストリガンドの同定に多くの進歩があったが、薬物様拮抗薬およびPXR 3,4,5上のそれらの結合部位の限られた記述があります。重大な障壁を効率的PXRは1998年にクローニングされ、特徴付けられたという事実にもかかわらず、拮抗薬との構造の研究のために、完全長のタンパク質を精製することができないことであった。当研究室では、PXR 6に結合残基、アンタゴニスト、ケトコナゾールのを定義するのに基づく新規のハイスループット酵母ツーハイブリッドアッセイを開発しました。我々の方法は、ケトコナゾールと対話することが期待PXRのAF-2表面上の単一変異の効果を救うだろ突然変異のライブラリを作成する必要があります。救助または「機能獲得型」セコndは突然変異はPXRにケトコナゾールの遺伝的相互作用および表面残基（複数可）に関する結論が実現可能であるようにすることができる。従って、我々はそのコアクチベーター、SRC-1との相互作用PXR変異体の高スループットツーハイブリッド酵母画面を開発しました。酵母は抗真菌薬、ケトコナゾールの研究に対応するように変更されたこのアプローチを、使用して、我々は、ケトコナゾールに結合することができないのクローンが濃縮されたPXRに特定の変異を実証できた。逆論理によって、我々は、元の残基はケトコナゾールとの直接的な相互作用残基であると結論付けている。このアッセイは、核内受容体表面上の結合部位アンタゴニストをスクリーニングするための新規な、扱いやすい遺伝的アッセイを表す。このアッセイは、酵母、ならびに標準的な構造生物又はプロテオミクスベースの方法を使用して研究することができない細胞タンパク質（単数または複数）に関係なく、その細胞傷害能の任意の薬物に適用することができる。潜在的な落とし穴は、データの解釈（相補メソッド便利）、信頼を含めるシングルY2H法、酵母を取り扱うまたは酵母ツーハイブリッドアッセイを実施する専門知識、およびアッセイの最適化にある低インピーダンス。

我々は、PXRとステロイド受容体コアクチベーター1（SRC-1）の関連性の比色読み取りを検出できるかどうかを確認するためにアッセイを行った。酵母は有意なステロール生を有するので、予め酵母におけるlacZ発現は、追加の外因性リガンドを必要とせずに誘導することができることが示されている。私たちは、lacZ発現（青色コロニー）もPXRとSRC-1で形質転換された酵母株が誘導されることがわ...

Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

ケトコナゾールに結合し、プレグナンX受容体（PXR）の活性化に拮抗する。 PXR変異体の酵母ハイスループットスクリーニングは、ケトコナゾールが結合するための独自の領域を規定する。この酵母ベースの遺伝的方法は、表面結合部位と会合したリガンドを有する新規な核内受容体相互作用を検出します。

相互作用するリガンドおよび/またはアンタゴニストと、哺乳動物核内受容体残基を同定するために逆酵母ツーハイブリッドシステム

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

reverse-yeast-two-hybrid-system-to-identify-mammalian-nuclear

Research

JoVE Journal

Biology

12.7K ビュー.  Albert Einstein College of Medicine. ケトコナゾールに結合し、プレグナンX受容体（PXR）の活性化に拮抗する。 PXR変異体の酵母ハイスループットスクリーニングは、ケトコナゾールが結合するための独自の領域を規定する。この酵母ベースの遺伝的方法は、表面結合部位と会合したリガンドを有する新規な核内受容体相互作用を検出します。 

ビデオ: Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

P2X2リガンド依存性陽イオンチャネルと相互作用するタンパク質を特定するためのプロテオミクス

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

生物学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

マイクロインジェクションによる哺乳動物細胞におけるmRNA核外輸送の速度論の解析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

学部の教育研究所の酵母エストロゲンの画面に最適化されたを使用して個人的な心配プロダクトのエストロゲンのリガンドを検出

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

蛍光標識した CXC ケモカイン Ligand 12 CXC ケモカイン受容体 4 を混乱させる化合物を識別するためにフロー フローサイトメトリーを用いた測定

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

エピジェネティック修飾と生体力学的手がかりを組み合わせて哺乳類多能性細胞を生成する2段階戦略

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...