1。 MEAの準備<ol><li>以前は18またはそのようなマルチチャンネルシステムとして、MEAメーカーのベンダーから直接購入し説明されているようにエレクトロポレーション実験で使用するためのMEAは、標準のフォトリソグラフィ技術を用いて製造することができる<a href="http://www.multichannelsystems.com" target="_blank">（http:/www.multichannelsystems.com/）</a>とAlpha MED科学株式会社<a href="http://www.med64.com/" target="_blank">（ http://www.MED64.com） 。</a>実験に用いたMEAは固体エレクトロニクス研究所（CSSER）のためのセンターによって運営されてアリゾナ州立大学のクリーンルーム施設で社内で作製した。実験に用いたMEAはよく1センチメートル内径50〜200μmのガラスの中から電極径サイズが4の配列で8で32インジウムスズ酸化物電極を持っていた。ガラスはよくポリジメチルシロキサン（PDMS）を使用して、MEAに接着した。 </li><li>セルの前にオートクレーブでMEAを殺菌するeeding。彼らはMEAの完全性を損​​なわない限り、このような70％エタノールに浸漬するように殺菌するための他の方法、別の場所で説明されているようにUV処理及び熱水処理を使用することもできます。 </li><li>層流フードにオートクレーブしたMEAを転送し、標準ポリスチレン滅菌シャーレに入れてください。 MEAに細胞を播種する前に、20分間紫外線をオンにすることにより、層流フードを殺菌。場合MEAは流フードはUV照射下にある間、無菌アルミ箔を使用したMEAを含むペトリ皿をカバーするUV光に敏感です。 </li></ol> 2。 MEAの播種細胞<ol><li>トリプシン/ EDTAおよびメッキ用1mlの細胞培地中で100から200細胞/μlの濃度になるようにそれらを上げるを使用して付着した哺乳動物細胞の稼働細胞株から細胞を回収する。私たちの実験では、培養したNIH3T3線維芽細胞株HeLa細胞株及びMEA上の一次皮質と海馬ニューロンに成功しています。 </li><li&gt; MEAのセルとのメディアの20〜30μlのドロップを置きます。インキュベーターにMEAとペトリ皿を転送します。セルのドロップサイズがMEA上のすべての電極を覆うように十分な大きさであることを確認することが重要です。 </li><li>細胞が層流フードへのインキュベーターからMEAとペトリ皿を移し、優しくよくMEAにあらかじめ温めメディアのμlを400から500まで（37℃）を追加播種後2時間。細胞がまだ取り付けられており、均等にMEA表面に広がっていることを保証するために、顕微鏡下で確認してください。インキュベーターにMEAの背中を転送します。メディアの添加前に2時間の待機期間は、細胞がMEA表面に付着するのに十分な時間を確保します。早すぎると細胞を播種後、細胞培地の添加は、細胞を洗い流すことができます。細胞は、MEAによく接着しない場合は、その表面には、細胞接着性を改善するために細胞接着因子などaspolylysine、フィブロネクチン、およびラミニンで治療することができます。私たちのHeLa細胞実験では、一般的にfibronectiとMEAの表面を処理1〜2時間のためのn（10μg/ ml）を。 </li><li>細胞を播種した後、8月12日時間は、任意の死んだ細胞を除去し、PBSで1から2回の洗浄を行います。必要に応じて細胞培地ごとに24〜48時間を交換することができます。典型的な実験では、細胞は、通常、播種後24〜48時間トランスフェクションのための準備が整いました。エレクトロポレーション効率の一貫性を維持するためには、文化はエレクトロポレーション前に、少なくとも80％コンフルエントになるまで待つことをお勧めします。 </li></ol> 3。 siRNAの部位特異的なトランスフェクション<ol><li>核酸溶液を調製します。 siRNAトランスフェクションは、200から400μlの最終容量を達成するために氷冷エレクトロポレーション緩衝液で1〜2μMのsiRNAのエレクトロポレーション溶液を調製します。このビデオの記事では、siRNAのアレクサ488共役スクランブル配列でHeLa細胞のトランスフェクションを実証している。 </li><li>インキュベータから無菌層流フードへの細胞とMEAを転送します。 MEAから細胞培地を取り除き、PBSで洗浄を行います。その後、私の200から400μlを加えるウェルへのCE冷たい1μMのsiRNAのエレクトロポレーション·ソリューションを提供します。 </li><li>ターゲット電極（電気パルスの最適なパラメータを決定するための手順4を参照してください）​​に陽極平方電気パルスを印加して下さい。私たちの実験では、MEAのボンドパッドと接触するように電気パルスとデュアル·インライン·パッケージ（DIP）16ピンクリップを生成するために実用的な楽器2414A波形発生器を使用していました。波形発生器は、個々の電極の選択性を提供するために、デマルチプレクサ、プリント回路基板を介して、DIPクリップに接続することができます。参照電極、セルメディアに鋼陰極を配置します。メディアの鉄鋼参照電極を削減しながら、それはそれはMEA表面に接触しないことを保証するために非常に重要です。接触すると、それは、細胞及びMEAへの損傷を引き起こす可能性があります。それは細胞上記1ミリメートルの位置に基準電極を下げることをお勧めします。 1ミリメートルの高さのスペーサーは、refの配置を支援するために十分に配置することができますレンス電極。外部参照電極を使用する別の方法は、カソード·アノードのペアとして隣接する電極を使用することです。 </li><li>直ちに、MEAを対象電極に電気パルスを印加した後、インキュベーターにMEAの背中を転送します。 5分の潜伏期間の後、静かにあらかじめ温めておいた（37℃）細胞培地でエレクトロポレーション緩衝液の75％を交換してください。その後、蛍光イメージングは​​、細胞に蛍光標識されたsiRNAの配信を確認するために行うことができます。 </li></ol> 4。エレクトロポレーションパラメータの最適化<ol><li>パルス振幅、パルス持続時間、パルス数の値の範囲については、エレクトロポレーション、デザインのエレクトロポレーション実験のために最適な電気パルスパラメータを決定する。 HeLa細胞を持つ私たちの経験では、我々は観察さマイルで成功したエレクトロポレーションにつながっ100μmの電極サイズの単一電圧3 V〜9 Vから振幅のパルス、ミリ秒、1〜10ミリまでの期間nimal細胞死。これは、1つのパラメータを同時に変化させることなどが一定に保たれていることを私たちの推薦である。パルス·パラメータのそれぞれについて、エレクトロポレーション効率を定量化するために、我々はステップ4.2から4.5に記載されてヨウ化プロピジウム（PI）は、細胞透過性色素その汚れ核酸材料、およびライブアッセイベースの手法を使用しています。 </li><li>ステップ1とステップ2で概説指示に従って、MEAで細胞培養を確立します。 </li><li>事前のエレクトロポレーションは、氷冷エレクトロポレーション緩衝液中で30μg/ mlのPI溶液の300から500μlとcalcienがPBSで溶液AM4μMの400μlを調製した。 </li><li>インキュベーターから層流フードへのMEAを転送します。 MEAから細胞培地を取り除き、PBSで洗浄を行います。その後、PIの氷冷エレクトロポレーション緩衝液の300から500μlを添加する。 </li><li>標的電極（電気を生成して適用するための我々の実験で使用する機器に必要なパルス振幅と持続時間との電気パルスを印加MEAにTRICパルスは、ステップ3.3）で説明されています。参照電極のどちらかだけでなく、MEAにおけるエレクトロポレーションバッファーに鋼電極を低くしたり、参考資料として隣接する電極を使用しています。選択的に異なる電極で変化する振幅または持続時間のパルスを印加することにより、複数の実験は、同じデバイス上で実行することができます。たとえば、32極、MEAに、8実験は8基、4電極から成るそれぞれに分割することにより、電極32を実行することができます。 8基の各々は、コントロールとして使用することができます0.5 Vの刻みと1グループに3 V型6 Vの範囲で変化する振幅の持続時間5ミリ秒の単一矩形電圧パルスで刺激することができる。 </li><li>すぐに適用した後に電圧パルスを5分間インキュベーターにMEAを転送します。その後、静かによくMEAからエレクトロポレーション緩衝液の75％を除去し、温かい細胞培地に交換してください。インキュベーターにMEAの背中を転送します。 </li><li> 2〜4時間の潜伏期間の後カルセインPBS中のソリューション午前4μMの300から500μlでよくMEAに細胞培地を交換してください。さらに30分間インキュベーターにMEAの背中を転送します。 </li><li>カルセインAMでの細胞のインキュベーション後、PBSで2回洗浄した後、PBSでよくMEAにソリューションをカルセインAMを交換してください。とカルセインAM（励起/発光 - 490 nm/520 nm）の - PI（535 nm/617 nmの励起/発光）用フィルター付き光学顕微鏡で用いた細胞の蛍光画像を取得します。エレクトロポレーションのために、PIの取り込みは、細胞が赤色蛍光を発するとカルセインAMが緑色蛍光を発するために生きている細胞を引き起こす原因となる。観察することができる3つの可能なシナリオは、1）、エレクトロポレーション、生き細胞（赤と緑の両方を発するでしょう）、2）細胞が生きているが、エレクトロポレーション（ちょうど緑色蛍光を発する）と3ではありません）細胞は死んによるエレクトロポレーション（ちょうど赤い蛍光を発するでしょう。） 。細胞生存率は電極上に生きている細胞の総数のパーセント、エレクトロポレーションEFFのように計算したiciencyはPI / siRNAでロードされたセルの総数のパーセントとして計算した。 </li></ol> 5。代表的な結果 図1は、PIのHeLa細胞の部位特異的な荷重を示します。これは、電極上の細胞のみがPI（ 図1B）の取り込みを示すことを観察することができます。ライブアッセイを行っポストエレクトロポレーションは、細胞（ 図1A）の実行可能性を評価するために使用された。 図1に示すように、例えばエレクトロポレーション効率と細胞生存率は、それぞれ81.8パーセントと96.1パーセントです。高い細胞生存率とエレクトロポレーション効率はエレクトロポレーションパルス·パラメータを最適化することによって達成することができます。最適化されたエレクトロポレーションパルス（8 V、1ミリ秒）のための蛍光標識siRNAを用いてHeLa細胞の部位特異的なトランスフェクションは、 図2に示されている。 8 VでsiRNAのエレクトロポレーションの効率は、1ミリ秒は74.4であることが判明した±16.0％、細胞生存率は87.9±8.4％（すべてのデータは平均値±標準偏差、N = 5のように表されている）であることが判明した。 <img alt="図1" src="/files/ftp_upload/4415/4415fig1.jpg" /> 図 200μmの電極（6 V、5ミリ秒）でHeLa細胞におけるPIの1部位特異的送達。 A）はCalcienベースのライブアッセイは2時間後にエレクトロポレーションを行った。生きている細胞が緑色蛍光で示されます。 B）は赤の蛍光は、電極上の細胞によるPIの取り込みを示しています。 C）は、AとBの重畳画像の白い矢印は、エレクトロポレーションと生きている細胞を示す。黒い矢印は死んでいる細胞を示す。 A、BおよびCの白丸は電極の概略を示します。 <img alt="図2" src="/files/ftp_upload/4415/4415fig2.jpg" /> 図2蛍光標識scrambl HeLa細胞のトランスフェクションsiRNAのE配列。 A）が100μmの電極上のHeLa細胞のイメージは、Alexa 488タグ付けたsiRNA（8 V、1ミリ秒）でトランスフェクションした。 B）は、細胞核のイメージは、DAPIで染色した。白丸は電極のエッジをマークします。

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Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer.</a> Langmuir. 21, 8360-8367 (2005).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsystem+for+transfection+of+exogenous+molecules+with+spatio-temporal+control+into+adherent+cells.">Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells.</a> Biosensors and Bioelectronics. 22, 863-870 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-chip+for+spatially+controlled+transfection+of+nucleic+acid+payloads+into+cells+in+a+culture.">Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture.</a> Lab on a Chip. 7, 1004-1011 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+array+(MEA)+platform+for+targeted+neuronal+transfection+and+recording.">Microelectrode array (MEA) platform for targeted neuronal transfection and recording.</a> Biomedical Engineering, IEEE Transactions on. 55, 827-832 (2008).</li><li>Jain, T., McBride, R., Head, S., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+introduction+of+nucleic+acids+into+mammalian+cells+grown+in+microwell+arrays.">Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays.</a> Lab on a Chip. 9, 3557-3566 (2009).</li><li>Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+high-throughput+electroporation+microchip+applicable+for+siRNA+delivery.">An efficient and high-throughput electroporation microchip applicable for siRNA delivery.</a> Lab Chip. 11, 163-172 (2010).</li><li>Li, Z., Wei, Z., Li, X., Du, Q., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+high-throughput+electroporation+chips.">Efficient and high-throughput electroporation chips.</a> , (2011).</li><li>Jain, T., Papas, A., Jadhav, A., McBride, R., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+surface-bound+siRNAs+in+microwell+arrays.">In situ electroporation of surface-bound siRNAs in microwell arrays.</a> Lab Chip. 12, 939-947 (2012).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporationfor+Gene+Transfer+In+Vivo.">Single-Cell Electroporationfor Gene Transfer In Vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Akaneya, Y., Jiang, B., Tsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+gene+silencing+by+local+electroporation+in+targeting+brain+region.">RNAi-induced gene silencing by local electroporation in targeting brain region.</a> J. Neurophysiol. 93, 594 (2005).</li><li>Lee, W. G., Demirci, U., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+electroporation:+challenges+and+perspectives+for+clinical+applications.">Microscale electroporation: challenges and perspectives for clinical applications.</a> Integr. Biol. 1, 242-251 (2009).</li><li>Boukany, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nature Nanotechnology. 6, 747-754 (2011).</li></ol>

特別な利害関係は宣言されません。

このビデオの記事では、siRNAのスクランブル配列とHeLa細胞の部位特異的なトランスフェクションのためのMEAの使用例を示します。この技術の利点の一つは、主要な細胞株を含む種々の細胞株への適用である。私たちの研究室では以前にE18の日齢のラットおよびスクランブルsiRNA配列とGFPプラスミド18,19とNIH-3T3細胞から主海馬ニューロン培養の部位特異的なトランスフェクションのため...

<table border="1"><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>細胞培地： アドバンストMEM L-グルタミン、200mMの ペニシリン/ストレプトマイシン ウシ胎仔血清</td><td> GIBCO /インビトロジェン Himedia研究所/ VWR ロンザグループ株式会社 GIBCO /インビトロジェン</td><td> 12492-013 95057-448 09-757F 16000-044 </td><td>細胞培地組成： アドバンスMEM中で2％のFBS、2％L-グルタミンおよび2％Pennstrep </td></tr><tr><td>トリプシンEDTA </td><td>メディアテック株式会社</td><td> 25から053-CL </td><td></td></tr><tr><td> PBS </td><td>メディアテック株式会社</td><td> 21から040-CV </td><td></td></tr><tr><td> sequをスクランブルタグ付けアレクサ488とローダミンリファレンスのsiRNA </td><td>キアゲン社</td><td> 1027292 </td><td></td></tr><tr><td>エレクトロポレーション緩衝液</td><td>バイオラッドラボラトリーズ</td><td> 165-2677 </td><td></td></tr><tr><td>波形発生器</td><td>実利的な</td><td> 2414A </td><td>希望するパルスを供給することができます任意波形/パルス·ジェネレータを使用することができます。 </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

このようなsiRNAおよびshRNAなどの真核生物とRNAi剤のその後の開発におけるRNAi経路の発見は、機能ゲノミクスおよび治療 ​​のための特定の遺伝子の1-8を黙らせる強力な方法を実現しました。 RNAiに基づく研究に関与する主な課題は、標的細胞へのRNAiの薬剤の送達である。このようなバルクエレクトロポレーションおよび化学的トランスフェクション法のような従来の非ウイルス送達技術は、多くの場合、配達上必要な空間的制御を欠いており、9月12日貧弱トランスフェクション効率を与える。このようなカチオン性脂質、カチオン性ポリマーとナノ粒子のような化学的トランスフェクション法の最近の進歩は非常に強化されたトランスフェクション効率が13をもたらしました。しかしながら、これらの技術は、まだ非常に小型化されたハイスループット技術、単一細胞研究と細胞間相互作用の調査を受けることができます配信を正確に空間的制御を提供するために失敗する。 ntは&quot;遺伝子送達で&gt;最近の技術の進歩は。接着細胞のハイスループットトランスフェクションの14から23、マイクロエレクトロポレーションを使用しているかの過半数を有効にしたマイクロスケールエレクトロポレーション（最大単セルまで）配達を正確に時空間制御を提供しており、示されている24から26までの高効率19を達成する。さらに、エレクトロポレーションベースのアプローチは、化学ベースのトランスフェクションの方法で必要に応じて、siRNAやDNA複合体とのインキュベーションの長時間（通常4時間）を必要とし、裸の​​siRNAやDNAの直接エントリにつながらない細胞質への分子。結果遺伝子の発現としては、トランスフェクション27後6時間、早ければ達成することができます。私たちのラボは、以前に付着哺乳動物細胞培養17-19にサイト固有のトランスフェクションのための微小電極アレイの使用（MEA）を実証してきました。 MEAベースのアプローチでは、遺伝的ペイロードの配信は、ローカライズされたマイクロスケールelectroporatiを介して実現されるセルの上に。選択された電極への電気パルスの印加は、刺激電極の領域に存在する細胞のエレクトロポレーションにつながる局所的な電界を発生させる。微小電極を独立して制御するには、トランスフェクションオーバー空間的、時間的な制御を提供し、また、同一の培養実験のスループットを向上させ、文化·ツー·文化の変動を減少させる上で実行される複数のトランスフェクションベースの実験を可能にします。 ここでは、実験装置やエレクトロポレーションを用いて蛍光標識スクランブル配列のsiRNAを用いて接着したHeLa細胞の標的とトランスフェクションのためのプロトコルを記述します。同じプロトコルはまた、プラスミドベクターのトランスフェクションのために使用することができます。また、ここで説明されたプロトコルは、簡単にマイナーな修正と哺乳動物細胞株の多様性に拡張することができます。事前定義されたおよびカスタムの両方の電極パターンとMEAの商業的利用は、この手法アクセシブルトンを作る基本的な細胞培養用器材のoほとんどの研究室。

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

記事の詳細は微小電極アレイ（MEA）を使用して付着した哺乳動物細胞培養におけるsiRNAのスクランブル配列の部位特異的なトランスフェクションするためのプロトコル。

微小電極アレイを用いたsiRNAの付着細胞への高効率、サイト固有のトランス体（MEA）

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

13.4K ビュー.  Arizona State University. 記事の詳細は微小電極アレイ（MEA）を使用して付着した哺乳動物細胞培養におけるsiRNAのスクランブル配列の部位特異的なトランスフェクションするためのプロトコル。

ビデオ: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

上皮成長因子受容体をターゲットとしたゼラチンベースのエンジニアNanovectorsを使用してヒト膵臓癌細胞における遺伝子デリバリーとトランスフェクション

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

生物工学

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

サイト固有の細菌染色体工学：ΦC31インテグラーゼを介したカセット取引所（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

単一の合成Curliオペロンを作成して付着細菌を工学

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

シングルセルおよび3D閉じ込めにおける単一胚を注文：ハイコンテントスクリーニングのための新しいデバイスを

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

siRNAの治療を送達するための多孔質シリコンマイクロ粒子

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

カチオン性およびアニオン性リポソームを用いてCNSへの治療用のsiRNAの送達

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 つの補完的な手法を使用して胎盤をターゲットとした薬剤投与の安全性の総合評価

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

神経細胞のコミュニケーションと軸索の信号伝搬特性を研究するためマイクロ電極アレイとのインタ フェース

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

インビボニューラル記録のためのTi3C2 MXene微小電極アレイの作製

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

ヒトiPSC由来心筋細胞、心臓線維芽細胞、内皮細胞を用いた3次元心臓ミクロ組織アレイの作製

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...