この作品は、スロベニアの研究機関（プロジェクトJ2 - 9770、インフラセンターIP - 0510とプログラムP2 - 0249）によってサポートされていました。このビデオでは、&quot;エレクトロポレーションベースの技術と治療&quot;リュブリャナ、スロベニアの大学で電気工学の学部が主催する科学的なワークショップや大学院のコースのための補助教材を表します。著者は、親切にプラスミドDNAを提供するためにもDušaホジッに感謝。

1。細胞培養、プラスミドおよび実験用バッファの準備<ol><li>この実験ではチャイニーズハムスター卵巣細胞（CHO - K1）を使用しています。細胞は栄養混合2mMのL -グルタミン、10％ウシ胎児血清、400μlの/ lのゲンタマイシン（Sigma - Aldrich社ビックケミー社、Deisenhofenからすべて、ドイツ）、及び1 / mlの補足HAM - F12（PAA）で栽培されていますリットルcrystacilin（Pliva、ザグレブ、クロアチア）。細胞を37 24のためのインキュベーターで、加湿5％CO 2雰囲気中で℃で保存されています。 </li><li>増幅プラスミドpEGFP - N1（クロンテックラボラトリーズ社、マウンテンビュー、CA、米国）大腸菌のDH5α株に緑色蛍光タンパク質（GFP）をコードし、ハイスピー​​ドプラスミドマキシキット（キアゲン社、ヒルデン、ドイツ）でそれを分離する。プラスミドDNAの濃度は、（TE緩衝液にプラスミドを溶解）分光測光法260 nmで測定し、ゲル電気泳動によって確認されるべきである。 </li><li> isoosmolarリン酸ナトリウム緩衝液（10 mMののNa 2 HPO 4、10mMののNaH 2 PO 4、1mMのMgCl 2、250mMスクロース、pH7.4）を準備します。 </li><li>実験日に0.25％トリプシン/ EDTA溶液（Sigma - Aldrich社ビックケミー社、Deisenhofen、ドイツ）でトリプシン処理により細胞懸濁液を準備する。 4℃で1000rpm（180 × g）で5分間の遠心細胞° C（Sigma社、ドイツ）と5の細胞密度× 10 6細胞/ mlにisoosmolarリン酸ナトリウム緩衝液に再懸細胞ペレット。 </li></ol> 2。ハードウェア機器<ol><li>細胞は、高電圧プロトタイプ発生器に接続された集積電極をピペットチップに電界（図1）にさらされている。先端と電極の形状は比較的均一な電界を印加することができ、発電機は、異なる方向に電気パルスの配信が可能になります。先端と発電機は、リュブリャナ大学1、電気工学部、バイオサイバネティクスの研究室で開発されました。 </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="図1"/> 図1（b）に統合された電極を有するピペットチップの垂直方向と水平方向（）断面と写真。断面のグレー色で電極用プラスチック製ハウジングと黒のために使用されます。統合された電極を有するピペットの先端には4つの円筒状のロッド電極から構成されています。電極はステンレス鋼から成っている、その直径は1.4 mmである、隣接する電極の間隔は1ミリメートルであり、そして反対側の電極が離れて2ミリメートルです。電極を並列にプラスチック製の先端に接着し、その該当する長さは30mm 2であるされています。 3。遺伝子electrotransferプロトコル<ol><li>濃度10μg/ mlの中の細胞懸濁液にプラスミドpEGFP - N1を追加。 </li><li>電気パルスを適用する前に、室温で2〜3分間混合物をインキュベートする。 </li><li>アスパイア統合された電極を有するピペットチップへの細胞懸濁液100μl。 </li><li>最高の遺伝子electrotransferの有効性を達成し、細胞の生存を維持するために、電気パルスの最適なパラメータを使用する必要があります。この実験では、8の矩形パルス（1ミリ秒、1Hzの繰り返し周波数での振幅225 Vの持続時間と各）の列車は、高電圧プロトタイプのジェネレータを使用して、各サンプルに適用されます。二つの異なる電界のプロトコルは、（図2）が使用されています：第2のプロトコルパルスの代わりに電界の方向と向きを変えることによって配信されるのに対し、最初のプロトコルで、すべてのパルスは、同じ方向に配信されます。二番目のプロトコルは、異なる方向に電気パルスのアプリケーションを可能にする適切なパルス発生器、を使用することができます。 </li><li>直ちにパルス印加後、6ウェルプレートにピペットの先端から細胞を転送し、ウシ胎児血清（FCS - Sigma、米国）（サンプルの体積の25％）を追加します。 </li><li> 37℃で5分間細胞をインキュベート° C細胞膜の再シール性を可能にする。 </li><li>よく6で各サンプルにHAM - F12の2 mlを加えインキュベーターで、加湿5％CO 2雰囲気で37℃24時間のための細胞℃でインキュベートする。 </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="図2"/> 図2電界プロトコル：（a）すべてのパルスが同じ方向に配信されるが、（b）のパルスが交互に電界の方向と向きを変えることによって配信されます。 4。画像収集と遺伝子electrotransferの有効性の判定<ol><li>遺伝子electrotransferの有効性は、パルス印加後にGFPの24を発現する細胞の割合として決定されます。 </li><li>細胞は、モノクロメーターシステム（ポリクロームIV、Visitron、ドイツ）と発光は507 nmで検出されると生成される488 nmで励起光を（私たちの場合はツァイス200、Axiovert、ZRドイツ）蛍光顕微鏡を用いて観察している。画像は、撮像システム（MetaMorphイメージングシステム、Visitrを使用して記録されます。で、ドイツ）が、他の同じような買収のソフトウェアを使用することもできます。 </li><li> 20倍対物レンズの倍率で少なくとも5枚の画像を（位相コントラストおよび緑色蛍光）を取得。 </li><li>位相コントラスト画像と緑色蛍光画像でGFPを発現している細胞では細胞を数える。それぞれ対応するイメージ内のすべてのセルの数（図3）でGFPを発現している細胞の数を割ることによって遺伝子のelectrotransferの有効性の割合を決定します。 </li></ol> 5。代表的な結果： <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="図3"/> 図3。すべてのパルスが同じ方向に配信されると、パルスが交互に電界方向を変えると方向が提示されることによって配信されたときにときにGFPを発現する細胞の割合。細胞は、振幅225 V、持続時間1ミリ秒と1 Hzの繰り返し周波数で8パルスのパルス列にさらされた。結果は、蛍光顕微鏡によって得られた。グラフ内の各値は、3つの独立した実験±標準偏差の平均を表しています。電界の方向と向きGFPの増加を発現する細胞の割合を変化させることにより。

<ol>
	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.</a> Biophys J. 63, 1026-1031 (1992).</li><li>Rols, M., Delteil, C., Serin, G., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+electrotransfection+of+mammalian+cells.">Temperature effects on electrotransfection of mammalian cells.</a> Nucleic Acids Res. 22, 540-540 (1994).</li><li>Golzio, M., Teissie, J., Rols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization+effect+on+mammalian+cell+permeabilization+and+gene+delivery+by+electric+field.">Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.</a> Biochim Biophys Acta. 1563, 23-28 (2002).</li><li>Haberl, S., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mg+ions+on+efficiency+of+gene+electrotransfer+and+on+cell+electropermeabilization.">Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization.</a> Bioelectrochemistry. 79, 265-271 (2010).</li><li>Rols, M., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+mammalian+cells+to+macromolecules:+control+by+pulse+duration.">Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.</a> Biophys J. 75, 1415-1423 (1998).</li><li>Kanduser, M., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+gene+electrotransfer+using+high-and+low-voltage+pulses-An+in+vitro+study.">Mechanisms involved in gene electrotransfer using high-and low-voltage pulses-An in vitro study.</a> Bioelectrochemistry. 74, 265-271 (2009).</li><li>Pavlin, M., Flisar, K., Kanduser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrophoresis+in+gene+electrotransfer.">The role of electrophoresis in gene electrotransfer.</a> J Membr Biol. 236, 75-79 (2010).</li><li>Sersa, G., Cemazar, M., Semrov, D., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+electrode+orientation+improves+the+efficacy+of+electrochemotherapy+of+solid+tumors+in+mice.">Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice.</a> Bioelectrochem Bioenerg. 39, 61-66 (1996).</li><li>Faurie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-mediated+gene+transfer+and+expression+are+controlled+by+the+life-time+of+DNA/membrane+complex+formation.">Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation.</a> J Gene Med. 12, 117-125 (2010).</li></ol>

利害の衝突は宣言されません。

遺伝子electrotransferは短く、高電圧電気パルス3を適用することにより、細胞へのDNAの転送を可能にし、その安全性、有効性およびアプリケーションの容易さのためのウイルスベクターの安全な代替を表す汎用性の高いバイオ技術です。今日遺伝子electrotransferが広く4報告されているこの方法を用いて細胞と第一段階のすべてのタイプI臨床試験をトランスフェクトするために使用...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> HAM - F12 </td><td> PAA </td><td> E15 - 016 </td><td>培地</td></tr><tr><td> L -グルタミン</td><td>シグマアルドリッチ</td><td> G7513 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td> PAA </td><td> A15 - 151 </td><td></td></tr><tr><td>ゲンタマイシン</td><td>シグマアルドリッチ</td><td> G1397 </td><td>抗生物質</td></tr><tr><td> crystacilin </td><td> Pliva </td><td> 625110 </td><td>抗生物質</td></tr><tr><td> pEGFPを- N1 </td><td>クロンテックラボラトリーズ</td><td> 6085から1 </td><td>プラスミドDNA </td></tr><tr><td>ハイスピー​​ドプラスミドマキシキット</td><td>キアゲン</td><td> 12662 </td><td></td></tr><tr><td>のNa 2 HPO 4 </td><td>メルク</td><td> F640786 933 </td><td></td></tr><tr><td>のNaH 2 PO 4 </td><td> TKI Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl 2の </td><td>シグマアルドリッチ</td><td> M - 8266 </td><td></td></tr><tr><td>スクロース</td><td>シグマアルドリッチ</td><td> 16104 </td><td></td></tr><tr><td>トリプシン/ EDTA溶液</td><td>シグマアルドリッチ</td><td> T4174 </td><td></td></tr><tr><td>ピペットチップ</td><td>カスタムは行わ</td><td></td><td></td></tr><tr><td>電気パルス発生器</td><td>カスタムは行わ</td><td></td><td></td></tr><tr><td> 6ウェルプレート</td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> 15 ml遠心チューブ</td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75センチメートル2培養フラスコ</td><td> TPP </td><td> 90076 </td><td></td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

遺伝子electrotransferは小分子には、透過性化、細胞膜の不安定化を引き起こす短いし、強烈な電気パルスのアプリケーションによって細胞に遺伝子を提供するために使用される物理的な方法であり、DNAのような大きな分子の転送が可能になります。それは、その安全性、有効性と適用の容易さに起因するウイルスベクターに代わるものを、表します。遺伝子electrotransfer異なる電気パルスプロトコルは最大の遺伝子のトランスフェクションを達成するために使用されているため、そのうちの一つは、パルスの配信中に電界の方向と向きを変えている。電界の方向と向きを変更すると、細胞へのDNAのエントリのための有能な膜面積を増加させる。このビデオでは、我々はすべてのパルスが同じ方向に配信されると、パルスが交互に電界の方向と向きを変えることによって配信される際に遺伝子のelectrotransferの効果の違いを示しています。統合された電極と電気パルス印加時に別の方向に電界が変化することができる高電圧プロトタイプジェネレータ、とこの目​​的のためのヒントについては、使用されていました。遺伝子electrotransferの有効性は、すべてのセルの数で割った値、緑色蛍光タンパク質を発現する細胞の数などのパルス印加後24時間に決定されます。結果は、電気パルスの配信時の電界の向きが変更されたときに遺伝子導入が増加することを示している。

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

細胞の生存率には影響されていない間の電界の向きは、パルス印加中に変更されたときにエレクトロポレーションによる遺伝子導入は、約2倍に向上しています。遺伝子導入の増加は、細胞へのDNAのエントリのための有能な作られている膜面積の増加によって引き起こされる。

電気パルスのアプリケーションの間に電界の方向と向きを変更すると、プラスミド遺伝子導入を向上 in vitroで</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

changing-direction-orientation-electric-field-during-electric-pulses

Research

JoVE Journal

Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

10.4K ビュー.  University of Ljubljana. 細胞の生存率には影響されていない間の電界の向きは、パルス印加中に変更されたときにエレクトロポレーションによる遺伝子導入は、約2倍に向上しています。遺伝子導入の増加は、細胞へのDNAのエントリのための有能な作られている膜面積の増加によって引き起こされる。

ビデオ: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

免疫不全マウスにおけるヒト網膜芽細胞腫の腫瘍の確立と伝播

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

医学

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

前臨床モデルにおける虚血性心不全のための遺伝子導入

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

マウスの永久中大脳動脈結紮の応用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

がんの生産と活性酸素種の検出（ROS）

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSDの、ニューラル血液学的および行動的相関関係を明らかにするための動物モデルにおけるバイオマーカー

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

加水分解性架橋剤を介してアデノウイルスベクターテザーを使用した金属ステント表面からの血管の遺伝子導入

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

小説<em&gt; in vivoで</em&gt;遺伝子導入技術と<em&gt; in vitroで</em筋骨格障害における骨損失の検討のための&gt;細胞ベースのアッセイ

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

顕微鏡法によるIn Vitro創傷が治癒過程における上皮移行の評価

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

生体内でウサギの一般的な頚動脈内皮細胞への遺伝子導入

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...