我々は、博士に感謝する。寛大Hep293TT細胞を提供するための組換えAAV3とAAV - EGFPプラスミド、それぞれ、博士ゲイルトムリンソンの彼らの親切なギフトのためのR.ジュードSamulskiとシャオシャオ。この研究は国立衛生研究所からの公衆衛生サービス助成金助成金R01 HL - 076901、R01 HL - 097088、およびP01 DK - 058327（プロジェクト1）（ASの）によって部分的にサポートされていました。

1。組換えアデノ随伴ウイルス3型（rAAV3）ベクターのパッケージング<ol><li>ウェブサイトにアクセスしてください：&quot; <a href="http://www.geguchadze.com/calc">http://www.geguchadze.com/calc</a> &quot;とDNAの量を計算する。 </li><li> FBSと抗生物質を含まない培地中でプレミックスプラスミドpHelper（アデノウイルスヘルパー）、pACG2c3（AAVヘルパー）とpdsAAV - CB - EGFP（するrAAVベクトル）。総量は10 15センチメートル2プレート用8750μLにする必要があります。 </li><li>ステップ1.2のDNA溶液にPEIの1250μL（0.1％M / V、Polysciences、カタログ番号23966、pH4.5）を直接追加します。数秒間ボルテックスDNA - PEIのソリューション。 </li><li>インキュベートDNA - PEIは、溶液を室温で5分（RT）のための複合体を形成する。 </li><li> 15センチメートル2板あたりのDNA - PEI溶液1mLを追加。 </li><li>新鮮な25mLの完全培地+10％FBS（C - DMEM）に置き換えて、4時間後に培地を取り外します。 </li><li>七十二時間後にトランスフェクション、スクレープ細胞と250 mLのコニカル遠心チューブに分注する。 </li><li> 4℃、3000rpmでスピン、℃で10分間。上清を捨て。 </li><li> RB TMS緩衝液（50mMトリス- HCl、150mMのNaCl、pH8.0）を5mLに細胞ペレットを再懸濁する。新しい15 mLコニカルチューブに移す。 </li><li> 37℃で10分間解凍するためのドライアイス - エタノール浴で凍結℃で10分間。 3回繰り返します。 </li><li> 4.8 M MgCl 2とベンゾナーゼ2μL（25 U /μL、Novagen社、カタログ番号70664〜3）の1μLを追加。 37渦とインキュベート℃で40分間。 </li><li> 40分間4℃、4,000 rpmでCをスピンダウンして（ライセートを清澄化）上清を集める。 </li><li> 15％の2 mLの25％の2 mLの40％、1.5 mLおよび13 mLのクイックシールの遠心管（ベックマン、カタログ番号342413）、上から下への負荷イオジキサノール勾配（OptiPrep、カタログ番号1114542）で60％のイオジキサノール1.5mLの。イオジキサノール勾配の上清（ステップ1.12）ロードする。 RB TMS Bufferでチューブを埋める。 </li><li> ° Cベックマン90Tiローターを使用して、16で1時間75000 rpmで遠心する。 </li><li> 40〜60％の境界で注射器を挿入することにより40％イオジキサノール画分を集める。 </li><li> 1つの50 mLコニカルチューブに40％のイオジキサノール分画を転送し、バッファーを40 mLのために構成する（20mMトリス、15mMのNaClを、pHは8.5）。 </li><li>ハイトラップQ HPカラム（GE Healthcare社、カタログ番号17-1154-01）を使用してろ過装置を組み立てます。以下のバッファーでカラムを洗浄する：バッファーを25mL（20mMトリス、15mMのNaClを、pH 8.5）に、緩衝液B（10mMトリス、500mMのNaCl、pH 8.5）に25mLの、バッファー50 mLの。バッファ内のウイルスのサンプルを40 mLの、バッファー50 mLの（20mMトリス、15mMのNaClを、pH 8.5）に、緩衝液C（20mMトリス、175 mMのNaCl、pH 8.5）に20mLの。 </li><li> 20 mLのアポロコンセントレータ（オービタルサイエンス）のバッファCを収集する。 4℃、3,000 rpmで遠心する℃で10分間を。 </li><li>フロースルーを捨て、4℃、3000rpmで冷却したPBSおよび遠心の20 mLを加え℃で10分間を。 </li><li>フロースルーを捨て、冷却したPBS 500μLを加える。 -80小アリコートで凍結シリコン処理したエッペンドルフチュー​​ブとストアへの膜と転送℃からベクトルを洗い流す</li></ol> 2。 rAAV3ベクトルの力価の決定<ol><li>氷上でエッペンドルフチュー​​ブに精製ウイルスの10μLを解凍。のddH 2 Oの40μLを追加</li><li>ベンゾナーゼの0.2μL（25 U /μL、Novagen社、カタログ番号70664）を追加し、37℃で1時間。 </li><li>別のエッペンドルフチュー​​ブにプラスミド標準（0.2 ng /μLの）50μLを取る。 </li><li> 65の各チューブとインキュベート℃で30分間に100 mMのNaOHを50μLを追加。 </li><li>少なくとも5分間、直ちに氷上で冷やします。 </li><li>バイオドットろ紙（Bio - Rad社製、カタログ番号1620161）に応じて転写膜（ミリポア、カタログ番号INYC00010）をカット。 20分のための3つのフィルター紙で10 × SSCでメンブレンを洗浄します。 </li><li>スロットブロットSF装置（バイオドット、猫＃170〜6542）を設定します。真空フラスコに接続し、スロットブロット装置に10倍のSSCの100μLを加える。 </li><li>ステップ2.5のサンプルのそれぞれに20 × SSCの100μLおよび6xローディング色素の1μLを追加。 </li><li>スロットブロット装置の膜上に各サンプル100μLをロードします。 </li><li>サンプルの各々に10倍のSSCの別の100μLを加える。 </li><li>すべてのサンプルがメンブレンを通過するまで、ステップ2.9および2.10を繰り返します。 </li><li>スロットブロット装置を分解して膜を取り除く。インスペクタのリンカSL - 1000 UV架橋剤に入れて。 &quot;エネルギー&quot;、&quot;最適な架橋&quot;（ショー1200）の電源を入れ、&quot;開始&quot;を押す。 </li><li>サケの精子の沸騰を1 mL（SS）DNA（フィッシャー、カタログ番号NC9753983）5分間。少なくとも5分間氷上で冷やします。 </li><li>のddH 2 O、20 × SSC、50 ×デンハルト溶液、20％SDS、1.25 mLを、ポリA 0.25mLのとステップ2.13からSS DNAを1mLの5 mLの15 mLのの27.5 mLを混合してプレハイブリダイゼーション溶液を調製します。 </li><li> 65でキャップガラスシリンダーとインキュベートでプレハイブリダイゼーション溶液で一晩ハイブリダイゼーションチャンバーのCの25 mLの膜を一時停止する。 </li><li>のddH 2 O10μlに50 ngの鋳型DNAを希釈する。 100℃沸騰℃で少なくとも5分間、直ちに氷上で5分間と寒さのため。 </li><li> 1分間、室温で3,000 rpmで遠心する。 10mMの3のdNTP（dCTPを欠いている）、ヘキサヌクレオチドミックスの2μL（Roche社、カタログ番号11277081001）、クレの1μL（Roche社、カタログ番号11008404001）とα-32 P - dCTPの5μLの2μLを加える。 37℃で10分間インキュベートする。 </li><li> 2分間、3,000 rpmでG - 50カラムを（GE Healthcare社、カタログ番号27-5330-01）心する。カラムにステップ2.17にプローブをロードする。 2分間、3,000 rpmで遠心し、フロースルーを収集する。 </li><li>放射能をカウント。 ° C、直ちに氷上で5分間と寒さのために少なくとも5分間、100℃のプローブを沸騰。 </li><li>膜とのプレハイブリダイゼーション溶液にプローブ（6 × 10 5 cpm / mlの）を追加します。 </li><li>ハイブリダイゼーションチャンバーで65℃で一晩インキュベートする。 </li><li>ハイブリダイゼーション溶液を捨て、15分間室温で2 × SSC 0.1パーセントSDSで膜をすすいでください。 </li><li>液を捨て、℃で30分間65℃で0.1 × SSC 0.1パーセントSDSで膜をすすいでください。 </li><li>液を捨て、室温で短時間で0.1 × SSCで膜をすすいでください。 </li><li> 6時間-80℃でフィルムを公開し、フィルムを開発する。典型的な結果を図1に示されています。 </li></ol> 3。 rAAV3ベクターによる形質導入し、ヒト肝癌細胞における導入遺伝子発現<ol><li> 96ウェルプレートの各ウェル内のシード1 × 10 4細胞（したHuh7またはHep293TTのどちらか）。少なくとも18時間加湿CO 2インキュベーター内でインキュベートする。 </li><li> 96ウェルプレートから培地を取り除く。 </li><li> FBSと抗生物質を含まない培地50μLで細胞を2回洗浄し、削除する。 </li><li> 5000 VGS /細胞でFBSと抗生物質とするrAAVベクターを含まない培地50μLを加える。 </li><li> 4時間CO 2インキュベーターで培養する。 </li><li>培地を捨てる。完全培地50μLで細胞を2回洗浄し、削除します。 </li><li>各ウェルに、C - DMEMを150μLを追加し、72時間CO 2インキュベーター内でプレートをインキュベートし、蛍光顕微鏡（DMI 4000B、ライカマイクロシステムズ）を用いてEGFPの発現を可視化する。 </li><li>ウイルス感染細胞の3つのウエルからの画像は、ImageJの解析ソフト（NIH、ベセスダ、MD、米国）によって定量的に解析されています。 </li><li>導入遺伝子の発現は、視野あたりの緑色蛍光の総面積（画素2）として評価される。典型的な結果を図2に示されています。 </li><li>導入遺伝子の発現は、フローサイトメトリーによって決定することができる。 </li></ol> 4。代表的な結果： 上記のプロトコルに続いて、一つは効率的にAAVの血清型のベクトルを生成することができます。典型的な収量は、ベクトルの株式を精製〜10 11 VGS /μLを含む〜500μLです。ベクトルの株式の純度は、ベンゾナーゼの消化がある場合とない場合の、定量的なDNAのスロットブロットでハイブリダイゼーションシグナルを比較することによって決定されます。効率的に細胞株 - 肝芽腫（HB）と肝細胞癌（HCC） - rAAV3ベクトル形質導入ヒト肝癌を精製。 <img src="/files/ftp_upload/2538/2538fig1.jpg" alt="図1" /> 図1。rAAV3ベクトルの力価を決定するための定量的DNAスロット-ブロット分析。ベンゾナーゼ（一番上の行）で消化し ​​たウイルス株を、精製の​​2倍希釈を32 P -標識したEGFP特異的DNAプローブを用いて定量的なDNAのスロットブロットで分析した。 AAV3ベクトルの力価は、1 ngの（中段）または膜にロードされたAAV - EGFPプラスミド基準の10ngの（一番下の行）との比較により決定した。数字は、DNAのコピーに対応しています。 <img src="/files/ftp_upload/2538/2538fig2.jpg" alt="図2" /> 図2組換えAAVヒト肝癌細胞におけるベクターによる導入遺伝子の発現。 Hep293TT細胞、最近設立されたヒト肝芽細胞の4行目は 、どちらかのモック感染（左パネル）だった、または2時間、37℃でscAAV2 - EGFPまたはscAAV3 - EGFPベクターのどちらかの5000 VGS /セルで形質導入。導入遺伝子の発現は72時間後の形質導入は、蛍光顕微鏡を用いて可視化した。

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+PDH+deficiency+using+AAV8-siRNA+vector-mediated+knockdown+of+pyruvate+dehydrogenase+E1&#945;.">An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1&#945;.</a> Mol Genet &#38; Metabol. 101, 183-191 (2010).</li><li>Petrs-Silva, H., Dinculescu, A., Li, Q., Deng, W. T., Pang, J. J., Min, S. H., Chiodo, V., Neeley, A. W., Govindasamy, L., Bennett, A., Agbandje-McKenna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+properties+of+tyrosine-mutant+AAV2+vectors+in+the+mouse+retina.">Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina.</a> Mol Ther. , (2011).</li></ol>

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非病原性ヒトパルボウイルス、アデノ随伴ウイルス（AAV）に基づいて組換えベクターを開発し、遺伝子治療臨床試験5の数で現在使用されていますされています。以前は、10の最も一般的に使用されるAAV血清型の、一般的にAAV3ベクトルの導入効率は、in vitroおよび in vivo 1 の両方、特に低いことが報告されている。しかし、我々の最近非常によくAAV3ベクトル形質導入ヒト肝癌細胞株は、のようなフローサイトメトリー2で定量的に決定した観察、とAAV3はこれらの結合とエントリの細胞共受容体としてのヒト肝細胞増殖因子受容体（hHGFR）を利用しているセル3は 、強く、特に一般的にヒト肝細胞、およびヒト肝癌細胞に対してAAV3の選択的な組織親和性を示唆している。しかし、AAV3ベクターはまた、効率的に2正常ヒト肝細胞を形質導入するので、in vivoでのがんの遺伝子治療のアプリケーションでそれらの潜在的な利用に悪影響を及ぼす可能になります。この潜在的な問題を回避する一つの可能​​な戦略は選択的に腫瘍ではなく、正常な肝細胞によって産生される遺伝子産物を識別することによって癌細胞の転写標的を伴います。例えば、以前の研究は、正常な肝細胞は一般に非常に少量のを生成するのでα-フェトプロテイン（AFP）の血清レベルは、特定の存在を追跡するためのマーカー、進行、および/または肝臓癌の特定の種類の再発として使用されていることが示されているこのタンパク質。実際、我々は肝癌細胞ではなく、正常な肝細胞2、および研究において導入遺伝子の発現を標的とするAFPプロモーターを含むAAV3ベクトルを使用している現在、肝癌のマウス異種移植モデルでは、このアプローチの有効性をテストするために進行中です。私たちのさらなる研究では、我々は様々なAAVの血清型のベクターの導入効率が大幅に6月14日ウイルスキャプシドの表面に露出したチロシン残基の部位特異的突然変異誘発により増強することができることを観察した。 6 7の表面に露出したチロシンもAAV3に保存されているので、我々はこれらの残基の部位特異的突然変異誘発を実施し、チロシンに変異がAAV3ベクトルの導入効率が大幅にヒト肝癌細胞（未発表データ）に増強されることを観察した。研究はまた、ヒト肝と肝細胞癌のためのマウス異種移植モデルにおけるチロシン変異AAV3ベクトルの安全性と有効性を評価するために現在進行中であり、そして成功した場合、最適なチロシン変異AAV3の血清型のベクターは、ヒトの肝臓を標的とするために有用であることが証明されるかもしれない潜在的な遺伝子治療のためのがん。

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high-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors

非病原性ヒトパルボウイルスに基づいて組換えベクターは、アデノ随伴ウイルス2（AAV2）を開発し、遺伝子治療臨床試験の数で現在使用されていますされています。さらに最近では、追加のAAV血清型の数も様々な大小の動物モデル1で組織親和性選択を示すことが示されている、単離されている。 10の最も一般的に使用されるAAV血清型のうち、AAV3は群を抜いて試験管内でだけでなく、in vivoでの細胞や組織を形質導入における最も効率的です。 AAV3がバインディングの共受容体の細胞としてのヒト肝細胞増殖因子受容体を使用しているため非常に効率的に細胞株 - 肝芽腫（HB）と肝細胞癌（HCC） - しかし、私たちの最近発表された研究で、我々はAAV3ベクターはヒト肝癌を伝達することが文書化されているこれらの細胞2,3のとエントリー。 この記事では、レポーター遺伝子を有する組換えAAV3ベクターによるヒト肝癌細胞の高効率伝達を達成するために必要な手順を説明します。治療用遺伝子を有する組換えAAV3ベクターの使用は、最終的にヒトの肝癌の潜在的な遺伝子治療につながる可能性があります。

Watch this Scientific Journal Video about High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors at JoVE.com

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

この記事では、我々はヒト肝癌細胞を標的とするための最も効率的なベクターとしてアデノ随伴ウイルス3型（AAV3）の識別について説明します。

組換えアデノ随伴ウイルスの血清型3つのベクトルによる肝癌細胞の高効率形質導入

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

high-efficiency-transduction-liver-cancer-cells-recombinant-adeno

Research

JoVE Journal

Medicine

13.1K ビュー.  University of Florida. この記事では、我々はヒト肝癌細胞を標的とするための最も効率的なベクターとしてアデノ随伴ウイルス3型（AAV3）の識別について説明します。

ビデオ: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

医学

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

培養細胞における癌関連シグナル伝達を問い合わせるための多重ルシフェラーゼベースのスクリーニング·プラットフォーム

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

組換えアデノ随伴ウイルスベクターの定位注射を介して、パーキンソン病のα-シヌクレインベースのラットモデルの開発

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

中リコンビナーゼ媒介カセット交換による組換えヒト多能性幹細胞の迅速かつ効率的な生成<em&gt; AAVS1</em&gt;軌跡

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

心臓の遺伝子がマウスでの編集の CRISPR アデノ随伴ウイルスを介した配信

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

小動物モデルにおけるアデノ随伴ウイルスベクターの結膜下投与

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

ダブルヨウ素酸濃度グラジエント遠心分離によるAAV単離

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

著者スポットライト:前臨床応用のための効率的なアデノ随伴ウイルス単離(英語)

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.