これらの研究活動は、化学、バイオ、環境、受賞番号CBET-0853780とCBET-1159344と国立衛生研究所、国立環境健康科学研究所（NIEHS）の下で輸送（CBET）システムの国立科学財団部門によってサポートされていました下の賞番号1R43ES022567-01、国立がん研究所、がんイメージングプログラム賞番号CA127745-01アンダー。本研究で使用IVISルミナ機器は米軍国防大学研究計測プログラムから得られた。

1。細胞調製<ol><li>液体窒素凍結ストックから生物発光HEK293細胞のバイアルを回収し、10％ウシ胎児血清を補充したダルベッコ変法イーグル培地（DMEM）、抗生物質、抗真菌1×0.01 mMの非必​​須アミノ酸、およびT、0.01 mMのピルビン酸ナトリウムでそれらを成長させる37℃、5％CO 2で75組織培養フラスコ。 注：メディアと補足コンポーネントが細胞株によって異なりますので、それに応じて選択する必要があります。 </li><li>リフレッシュ中に到達するまで2〜3日ごとに〜80％合流。 注：必要な細胞の量は、実験計画に基づいて行われます。必要に応じて、より多くの細胞を継代培養することにより得ることができる。 </li><li>テストのために細胞を回収。 <ol><li>培地を除去し、リン酸塩で細胞を洗浄するには、優しく、フラスコを渦巻くことで緩衝生理食塩水（PBS）、その後破棄はPBSを過ごした。 </li><li> 2分間37℃でトリプシンとインキュベートを追加し、あるいは細胞までフラスコから切り離されました。 </li><li> PBS中剥がれた細胞を収集し、クリーンな遠心管に移す。 注：非接着細胞株を使用する場合は、遠心分離により細胞を分離し、PBSで1回洗浄する。 </li></ol></li><li>血球計または他の細胞カウントシステムを使用して総細胞数を決定します。 </li><li> 5分間300 xgで細胞懸濁液を遠心。上清を捨て、新鮮な、温めておいた培地で細胞を再懸濁します。 </li><li>不透明なマルチウェルプレートの各ウェルへの細胞のシード同数。ブラック24ウェルプレートの各ウェルに、1mlの容積のこの例では、プレート5×10 5細胞。ネガティブコントロールとして機能する3つのウェル中の培地のプレートが等量。 注：各ウェルに播種細胞数が柔軟であり、個々の実験のために最適化することができる。各生物発光細胞株については、実験的に細胞数とbiolumiとの間の関係を決定するnescent出力だけでなく、最小限の検出可能な細胞は、前に、毒性試験に数える。標準化された撮像条件で人口サイズの広い範囲（例えば、1×10 3〜1×10 6細胞）を横切ってこの、メジャー生物発光をしています。 </li><li>以下のように化合物（複数）のテストで細胞を扱う。 </li></ol> 2。化学準備<ol><li>濃厚原液としてテストされている化学物質を準備します。この例では、100mgの/ブレオマイシンファミリーの抗生mlの原液を使用しています。 注：有機溶剤、化学添加するための車両として使用されている場合は特に、潜在的な車両ベース毒性影響を最小限にするため、それは株式の濃度は、少なくとも1,000倍希望ポストアプリケーション濃度にすることをお勧めします。 </li><li>細胞に直接製造化学株式を追加します。この例では、線量の最終濃度100、200、300、および400μgのトリ/ mlとその他の抗生物質井戸をひだのある。対照として未処理細胞の三井戸のままにしておきます。 注意：ターゲット化学物質の最終濃度は、特定の実験的な目標に基づいて選択されるべきである。広範囲の濃度は、通常、毒性作用の完全なスペクトルを得るためにテストされる。 </li></ol> 3。イメージングとデータ解析<ol><li>すぐに化学添加後、画像取得と生物発光測定のために適切な機器のイメージングチャンバー内にプレートを配置。 </li><li>生物発光、各読書のため10分の積分時間を使って24時間にわたり15分毎に測定します。 注：この例で使用される積分時間ごとのプレート単位であり、任意の他のルミノメーターを用いごとウェルに基づいて測定のために調整することができる。積分時間は、選択された生物細胞株に基づいて調整することができる。生物発光は、細胞破壊や任意の外部刺激せずに自律的に生産されているので、読んでイングスは、具体的な実験の目的を達成するために任意の所望の時点で採取することができる。 </li><li>互換性のあるソフトウェアを使用して関心領域を識別することにより各ウェルから生物発光強度を定量化する。全光束（光子/秒）または各サンプルの平均輝度（フォトン/センチメートル/秒2 / steridian）のいずれかとして光出力を表示します。 </li></ol>

<ol>
	<li>U.S. Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+really+FDA+approved?.">Is it really FDA approved?.</a> FDA Consumer Health Information. 2, (2009).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Olson, H., 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=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharmacol. 32, 56-67 (2000).</li><li>Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+cell+culture+technology.">Current developments in cell culture technology.</a> Adv. Exp. Med. Biol. 745, 1-13 (2012).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+human+ADME/Tox+drug+properties+in+drug+discovery.">Screening for human ADME/Tox drug properties in drug discovery.</a> Drug Discov. Today. 6, 357-366 (2001).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+evaluate+ADMET+drug+properties.">In vitro approaches to evaluate ADMET drug properties.</a> Curr. Top. Med. Chem. 4, 701-706 (2004).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. M., 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=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

DM閉じる、SA RIPP、およびGS Sayler 490バイオテクノロジー社の創設者と所有者である

この方法は、生きた細胞が継続的に生涯にわたって監視することができインビトロ細胞毒性スクリーニングアッセイとして自律的に生物発光哺乳類細胞の使用方法を示します。このプロトコルは柔軟性があり、必要に応じて具体的な実験条件に適応するように変更することができる。例えば、ここに示された実験は、急性毒性を追跡するのに適しているが、増加した時間間隔（ ...

彼らは食品医薬品局（FDA）によって、消費者の使用が承認される前に米国では、人間の消費のために意図された医薬品やその他の製品は、広範な評価を必要とする。このテストを実行するための負担は、実質的に新規化合物の開発コストを増加させ、したがって、消費者にとってコストの上昇につながり現像剤1上に配置されている。伝統的にずっとこのスクリーニングのが人間のホストのプロキシとして機能するように、動物の科目を利用しているが、これはADME / Toxの（吸着、分布、代謝、排泄、および毒性に毎年費やさ推定28億ドルで、大規模な財政負担であることが証明された）2のみをスクリーニングし、動物モデルは確実に人間の毒性応答3を予測できないことを示唆している証拠。したがって、in vitroでのヒト細胞培養ベースのテストでは 、その相対的に低いの過去20年間の人気を得ているコスト、高スループット、および人間の生物学的利用能及び毒物学4のより良い表現。現在、細胞培養系毒性スクリーニング法は、細胞生存5を評価するために、細胞膜の完全性をプロービング、内因的に利用可能な細胞質酵素の活性のスクリーニング、またはミトコンドリアの活性のレベルを追跡し、そのようなATPレベルを測定するなどの様々なエンドポイントを採用、6。測定は、従って、単​​一の時点でデータを生成し、取り出すことができる前に、しかし、関係なく選択されたエンドポイント、これらの方法は、すべてのサンプルの破壊を必要とする。結果として、多数の試料を調製し、基本的な毒物学的動態研究のため、並列に処理し、再度新たな化合物の開発に要するコストと労力を添加する必要がある。また、使用したアッセイは、そのようなガウルシフェラーゼ7、 ウミホタル属ルシフェラーゼ8、 型Metridiaルシフェラーゼ9としてルシフェラーゼ分泌細胞溶解の必要性を排除し、エンドポイント測定用媒体の一部を必要とするが開発されてきたが、これらは依然として所定の時点でのサンプリングに限定されるものではなく外因性の光活性化基質の添加を必要としている。 基材のコストを排除するだけでなく、必要なサンプル破壊の不利益を回避するために、ヒト細胞株は同様である生きた細胞の連続的な監視を可能にするために完全な細菌の生物発光（ ルクス ）遺伝子カセット（luxCDABEfrp）を発現する遺伝子操作されている蛍光染料ベースのライブセルイメージングに、追加光子活性化と微視的調査手続きなし。この細胞株は、このように試料の破壊を回避し、構成的に外部刺激を必要とせずに連続的な、直接検出するための光信号を生成することができる。機構的に、これらの細胞から発生する生物発光シグナルが発生S luxAB形成されたルシフェラーゼ酵素が減少リボフラビンリン酸塩の存在（FRP遺伝子によってFMNからリサイクルさFMNH 2、中長鎖脂肪酸アルデヒド（内在性基質を用いたluxCDE遺伝子産物によって合成され、再生）の酸化を触媒するとき製品）と酸素分子10。宿主細胞中での発現カセットルクスしたがって、細胞破壊又は外因性基質を添加せずに製造され、検出される光が可能となる。同様に、 ルクス遺伝子および内因的に利用できるFMNとの間の相互作用、およびO 2補基質、及びFMNH 2〜FMNの変換をサポートすることができる環境を維持するための要件は、得られた生物発光信号のみが生きているから検出できることを保証、代謝的に活性な細胞。 これらの要件は、以前にそのルクス -BASを実証するために利用されていますED生物発光出力は、携帯人口規模11とその毒性化合物の曝露用量反応ファッション12 autobioluminescent生産を損なうと強く相関している。ここでは、毒性試験のためのautobioluminescent哺乳類細胞のアプリケーションを検証する代表例として知られているDNA損傷活性ブレオマイシンファミリーの抗生物質の自動毒性スクリーニングを実証するために以前に特徴付けautobioluminescentヒト胎児腎臓（HEK293）細胞ライン11を使用しています。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

哺乳動物細胞ベースのインビトロアッセイは、広く毒性試験のための動物実験の代替として用いられているが、原因ホタルルシフェラーゼに基づくスクリーニング法の破壊的な性質のために必要とされる並列の試料調製の高い金銭的および時間コストに限定されている。このビデオは、目的の化合物の細胞毒性効果を監視するための安価で容易な方法としては、ルシフェリン基質の破壊的な添加を必要としない自律的に生物発光哺乳動物細胞の利用が記載されている。安定的に完全な細菌の生物発光（luxCDABEfrp）遺伝子カセットを発現する哺乳動物細胞は、自律的に高価であり、おそらくは干渉ルシフェリン基板と、外部エネルギー源による励起、又は伝統的である試料の破壊を添加せずにピークが490nmでその光信号を生成する光学イメージング手順の間に行われるS。外部刺激からのこの独立性は結果の信号のみをアクティブ代謝中に検出されていることを意味し、もっぱら細胞上の生物発光反応を維持するための負担を配置します。生物生産の変化は細胞の成長と代謝に悪影響を示すものであるので、この特徴は、 ルクス発現細胞株細胞毒性に対するbiosentinelとして使用するための優れた候補になります。同様に、必要なサンプル破壊の自律的な性質や不足が毒物曝露の期間を通じて、リアルタイムで同じサンプルを繰り返しイメージングを可能にし、自動化された方法で、既存の画像処理装置を使用して複数のサンプル間で実行することができます。

本研究では、autobioluminescent HEK293細胞の動態を、抗生物質露光（ 図1）に応答して24時間かけて連続的にモニターした。に結合し、DNA 13を切断することにより、生きた細胞を殺すことが知られているブレオマイシンファミリーのメンバーであり、この抗生物質の毒性効果は、擬似カラー画像から直接可視化することができる未処理の細胞と比較して生物生産の減少を介して?...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

細菌性生物発光遺伝子カセットを発現する哺乳動物細胞（<em&gt;ルクス</em&gt;）自律的に光を生成する。化学露光により得られた生物力学を容易に高スループット自動化に適合させることができる安価な、連続的なリアルタイム毒性スクリーニングツールこれらの細胞を作り、細胞の成長および代謝に対する治療効果を反映することが実証されている。

細胞毒性の継続とリアルタイム監視のための自律的に生物発光哺乳類細胞

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K ビュー.  Oak Ridge National Laboratory. 細菌性生物発光遺伝子カセットを発現する哺乳動物細胞（ルクス）自律的に光を生成する。化学露光により得られた生物力学を容易に高スループット自動化に適合させることができる安価な、連続的なリアルタイム毒性スクリーニングツールこれらの細胞を作り、細胞の成長および代謝に対する治療効果を反映することが実証されている。

ビデオ: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

哺乳動物細胞をトリプシン処理し、継代培養

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

定量リアルタイムPCR（定量PCR）アレイを用いたプレマイクロRNAとマイクロRNAのプロファイリング

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

蛍光共鳴エネルギー移動によるリガンド - 受容体相互作用のリアルタイム監視

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

単分子バイオセンサーを用いた生細胞中のシグナル伝達事象をリアルタイムで監視するための顕微鏡のFRET

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

蛍光顕微鏡法による、複数世代にわたってライブ哺乳類細胞におけるプラスミドの複製を監視

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

プローブベースのリアルタイムPCRは、マイクロRNAの定量的測定のためのアプローチ

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

細菌、酵母、ヒト細胞における一過性形成分子シャペロンアセンブリのモニタリング

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...