Name: in vivo gene transfer to the rabbit common carotid artery endothelium
Uploaded: 2018-05-06T13:00:00.000+00:00
Duration: 10 min 18 s
Description: Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

我々 は AdVec、株式会社 HDAd 試薬、ジュリア Feyk 行政支援のため、手術のアドバイスを比較医学部獣医サービスを使用する許可のために感謝し、サポートします。この作品は、HL114541、ジョン l. ロック ジュニア公益信託によって支えられました。

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手術手技の特定の側面のメリットは特に注意。完全な露出と遺伝子の転送と切開の修復を容易にするため細かくは経由で総頸動脈の動員。ただし、脳血管攣縮を防ぐために、解剖中、頚動脈の直接操作を最小限必要があります。さらに、ガーゼで光の圧力を適用することによって停止する必要があります任意の出血は、動脈に隣接して鬱血は食塩を使用して領域を洗浄によってすぐにクリー?...

この方法の目標は、ウサギ頸動脈の血管内皮細胞に、遺伝子を導入することです。遺伝子の導入により遺伝子製品の生物学的役割の検討、正常動脈とウサギ人間の動脈疾患のモデルの両方。疾患モデルにおける遺伝子の過剰発現は、遺伝子 (とその蛋白質の製品) が治療薬1,,,234として約束を示すかどうかを明らかにできます。遺伝子発現カセットのシスエレメント規制要素を含めることができます5,6生体内で動脈の内皮細胞でこれらの要素の活動の評価。アクティブな式カセットを設計し、7生体内で大きな動脈内皮細胞の遺伝子の規則のメカニズムを調査する、特定のシスエレメント規制要素の活動の知識を使用できます。
ウサギは、人間の血管の生理と疾患のさまざまな側面の貴重なモデルです。ウサギは、人間の血管の多くの機能を共有します。たとえば、血液の基準値、止血の調節、血管軸張力は、ウサギとヒトの8間似ています。血管疾患の家兎モデルを複製を含む多くの人間の病気の主要な機能: 動脈瘤 (似たような幾何学的および流動特性)9, 脳血管攣縮 (血管内治療に類似した応答)10,11, と動脈硬化 (線維性被膜の脂質、マクロファージ、平滑筋細胞における豊富なコアを含む類似の機能と内膜肥厚斑)12,13。したがって、ウサギ モデルは、血栓症、脳血管攣縮、脳動脈瘤、糖尿病、血管狭窄、動脈硬化症8,13,14,など多くの血管疾患のため開発されている15,16。
血管生理学および病気の動物モデルの中から選択する研究者ウサギいくつか利点があります。齧歯動物に比べると、ウサギの大きな船は簡単に手術操作、血管内デバイスの使用および定量的測定のための組織の量を大きくを許可します。ウサギ、齧歯動物17よりは霊長類に近縁なはるかに近いしザイモグラム ウサギの大きい遺伝的多様性はより良い人間の遺伝の可変性を近似する.遺伝的多様性は前臨床研究は、特に重要なある-遺伝的に多様な人口に適用できる治療法を開発する彼らの自然狙いで。多くの場合ない他のすべてのモデル種ウサギ遺伝子が簡単に複製や合成ため高い被覆率でずっとウサギ ゲノムが配列として (7.48 x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]。(犬、豚、羊など) その他の大型動物モデルと比較して、ウサギは比較的高価なを購入して家と彼らが繁殖し、処理を容易にします。各家兎における特定の血管疾患モデルこの原稿8,12,18の範囲を超えているひと疾患のモデルとして、独自の利点と欠点があります。調査官は、これらの利点と欠点について、ウサギが特定の実験の質問に答えるための最高のモデルであるかどうかを確認ください。
デオキシリボ核酸 (DNA) の規制シーケンスを生体内で血管内皮細胞の導入は、複雑な生理学的環境でこれらの一連の活動の調査を実行できます。Transfected 内皮細胞の in vitro研究は、規制的 dna; の初期評価に役立ちますただし、組織培養モデルの表現のレベルは時々 再現されません研究が繰り返される生体内で5,19,20。生体外でシステムも培養血管細胞間タンパク質シグナル伝達、内皮細胞生理学、コミュニケーションの基本的な経路を調べる場合に便利にすることができます。ただしより複雑な経路や隣接血管細胞や免疫システムの複雑な人口に影響される規制のネットワーク最高、体内システム6,20で研究されています。記載方法は、そのまま容器、疾患の有無のコンテキスト内で血管内皮細胞の遺伝子発現調節を探索するためのプラットフォームを提供します。生体内のシステムは、また遺伝子式6の規制する免疫システムの貢献の生理学的および病理組織学的細胞のクロストークの調査と特定を許可します。
(マウス) を中心に生殖系列の遺伝子導入は、内皮細胞に遺伝子発現を導くために代わるアプローチです。このアプローチは、内皮特異的プロモーターまたは規制地域21,22を介したターゲットを生涯発現を提供できます。しかし、トランスジェニック マウスの生成は時間がかかり、高価なトランスジェニック数行をしばしばテスト目的のセルのタイプおよび達成十分な導入遺伝子発現レベルで実験的に遺伝子のターゲットを確認する必要があります。マウス システムの結果は、ひずみに依存をすることができます。内皮細胞をターゲットとした遺伝子と遺伝子導入マウスモデルは多くの利点を持っている: 遺伝子導入を実現するために、すべての実験動物の手術を実行する必要はありません、実験マウスでの多数の他の利用可能なトランスジェニック マウス飼育することができます遺伝的・形態の相互作用をテストするため、様々 な表現型の特性を促進するマウスの蛋白質と反応する抗体があります。しかし、血管系、22遺伝子プロダクトを演技するサイトを決定する困難を通して遺伝子発現の結果通常生殖線を介して血管内皮細胞に遺伝子のターゲットします。これは遺伝子プロダクトが分泌されているときに特にを通して血管内皮細胞から分泌される遺伝子プロダクトが動物内のサイトの任意の数で生物学的活性を持つことが。本稿で説明した方法には、技術的な専門知識、専門設備が必要ですより時間がかかると内皮特異トランスジェニック マウス ラインの開発よりも安価にことです。それは大きな動脈セグメントの内皮細胞に選択的にタンパク質の機能の評価を可能にし (実験によって異なることが全身的要因を排除対コントロールとして対側頸動脈の使用が可能動物-たとえば、血圧やコレステロール値-自由な変数として)。
遺伝子療法は血管疾患の治療のための有望なアプローチ、特に慢性疾患治療遺伝子23の持続的または多分生涯式、単一アプリケーションできますので。遺伝子療法の治療の約束は、しばしばターゲットに比較的簡単なターゲットは、血液媒介ウイルスのベクトルの多くは肝肝24,25に、体細胞遺伝子の動物モデルで検討されています。ただし、血管疾患の影響を受けやすい肝臓を対象とした遺伝子治療は全身蛋白質過剰発現を達成しなければなりません。これは通常、有害または致命的26をすることができますベクトルの大量投与を必要です。また、タンパク質昇給も無名の実験結果の解釈を複雑にあるターゲットを副作用のリスクの全身のレベルを増加しました。本稿で説明したように血管内皮細胞をターゲット ローカル遺伝子治療は注入のベクトルが導入された動脈セグメントを超えて広く配布されていないとなし、ローカル血管効果を達成することができますので全身的副作用を避けることができます。全身血漿蛋白質の変化。27また、ベクトルのはるかに低い量は堅牢な肝伝達を達成するために必要であるより動脈セグメントを変換するが必要です。肝臓から遺伝子発現は、おそらくのために細胞のターン オーバー、高度な遺伝子発現が維持される場合、繰り返し投与を必要とする、時間の経過とともに減少する報告されています。28対照的に、血管内皮細胞の低離職率は、チョウ飼育家兎では少なくとも 48 週間と 24 週間、少なくともウサギのコレステロールの動脈硬化病変における安定した表現を提供します。1,27
ウサギ共通頸動脈血管内皮細胞への遺伝子導入のこの方法が適切なかどうかを決定するには、利点と欠点 (表 1) が具体的な研究目標の文脈で考慮必要があります。このメソッドの利点があります: ザイモグラムのウサギは人間の遺伝的多様性代表より近交系マウス (前臨床の仕事のために重要);ウサギは簡単に操作できる大型船舶と分析のためのより多くの組織を提供します。メソッドは、生殖細胞内皮トランスジェニック マウスのターゲットよりもはるかに多くすばやく内皮細胞をターゲットとした遺伝子発現を実現できます。ベクトル用量を遺伝子発現の; のモデル変数のレベルに簡単に調整できます。大きな動脈の内皮細胞に固有のプロセスを調べることができます。ローカル血管遺伝子により制御されていない変数として全身的要因を排除するコントロールとして使用する同じ動物で、反対側の頸動脈。デメリット: 特別な設備と専門知識が必要です。実験するより少ない遺伝子組み換え背景がネズミよりもウサギで利用できます。より少なく豊富なマウス蛋白質 (バイオアセッテイ遺伝子蛋白質の実験結果の解釈に重要なことができる他の抗原) の対のウサギに抗体があります。

<table><tbody><tr><td>&lt;strong&gt;Disposables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3mL syringe with 24G needle</td><td>Becton Dickinson</td><td>309571</td><td>2x for gene transfer surgery; 3x for harvest surgery</td></tr><tr><td>1mL syringe with 27G needle</td><td>Becton Dickinson</td><td>309623</td><td>6x for gene transfer surgery; 1x for harvest surgery</td></tr><tr><td>20mL syringe, luer lock</td><td>Nipro Medical Corp</td><td>JD+20L</td><td></td></tr><tr><td>Catheters, 24G x 3/4&quot;</td><td>Terumo Medical Products</td><td>SROX2419V</td><td></td></tr><tr><td>19G needle</td><td>Becton Dickinson</td><td>305187</td><td>Gene transfer surgery only</td></tr><tr><td>21G needle</td><td>Becton Dickinson</td><td>305165</td><td>For 20 mL syringe of saline</td></tr><tr><td>Gauze 4&quot; x 4&quot;</td><td>Dynarex</td><td>3242</td><td>~10-15 per surgery</td></tr><tr><td>3-0 silk suture</td><td>Covidien Ltd.</td><td>S-244</td><td></td></tr><tr><td>5-0 silk suture</td><td>Covidien Ltd.</td><td>S-182</td><td>Gene transfer surgery only</td></tr><tr><td>7-0 polypropylene suture</td><td>CP Medical</td><td>8648P</td><td>Gene transfer surgery only</td></tr><tr><td>5-0 polyglycolic acid suture</td><td>CP Medical</td><td>421A</td><td>Gene transfer surgery only</td></tr><tr><td>3-0 polyglycolic acid suture</td><td>CP Medical</td><td>398A</td><td>Gene transfer surgery only</td></tr><tr><td>Alcohol swabs</td><td>Covidien Ltd.</td><td>6818</td><td>For placement of I.V. line</td></tr><tr><td>Catheter plug</td><td>Vetoquinol</td><td>411498</td><td>Gene transfer surgery only</td></tr><tr><td>Ketamine HCl, 100 mg/mL</td><td>Vedco Inc.</td><td>05098916106</td><td></td></tr><tr><td>Xylazine, 100 mg/mL</td><td>Akorn Inc.</td><td>4821</td><td></td></tr><tr><td>Lidocaine HCl, 2%</td><td>Pfizer</td><td>00409427702</td><td></td></tr><tr><td>Bupivacaine HCl, 0.5%</td><td>Pfizer</td><td>00409161050</td><td></td></tr><tr><td>Beuthanasia D-Special</td><td>Intervet Inc.</td><td>NDC 00061047305</td><td>Harvest surgery only</td></tr><tr><td>Buprenorphine HCl, 0.3 mg/mL&amp;nbsp;</td><td>Patterson Veterinary</td><td>12496075705</td><td>Gene transfer surgery only</td></tr><tr><td>Saline IV bag, 0.9% sodium chloride</td><td>Baxter</td><td>2B1309</td><td>2x for gene transfer surgery; can use vial of sterile saline in place of one</td></tr><tr><td>Heparin&amp;nbsp; (5000 U/mL)</td><td>APP Pharmaceuticals</td><td>NDC 63323-047-10</td><td>Gene transfer surgery only</td></tr><tr><td>Fentanyl patch, 25 mcg/hr&amp;nbsp;</td><td>Apotex Corp.</td><td>NDC 60505-7006-2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Gene transfer vector</td><td></td><td></td><td>Dilute 350 &amp;micro;L per artery; 2 x 10&lt;sup&gt;11&lt;/sup&gt; vp/mL for adenovirus; gene transfer surgery only</td></tr><tr><td>&lt;strong&gt;Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metzenbaum needle holder 7&quot; straight</td><td>Roboz</td><td>RS-7900</td><td>Gene transfer surgery only</td></tr><tr><td>Operating scissors 6.5&quot; straight blunt/blunt</td><td>Roboz</td><td>RS-6828</td><td></td></tr><tr><td>Needle holder /w suture scissors</td><td>Miltex</td><td>8-14-IMC</td><td>Gene transfer surgery only</td></tr><tr><td>Castroviejo scissors</td><td>Roboz</td><td>RS-5658</td><td></td></tr><tr><td>Castroviejo needle holder, 5.75&quot; straight with lock</td><td>Roboz</td><td>RS-6412</td><td>Gene transfer surgery only</td></tr><tr><td>Stevens scissors 4.25&quot; curved blunt/blunt</td><td>Roboz</td><td>RS-5943</td><td></td></tr><tr><td>Alm retractor 4&quot; 4X4 5mm blunt prongs</td><td>Roboz</td><td>RS-6514</td><td>2x</td></tr><tr><td>Backhaus towel clamp 3.5&quot;</td><td>Roboz</td><td></td><td>4x</td></tr><tr><td>Micro clip setting forceps 4.75&quot;</td><td>Roboz</td><td>RS-6496</td><td>Gene transfer surgery only</td></tr><tr><td>Micro vascular clips, 11 mm</td><td>Roboz</td><td></td><td>2x for gene transfer surgery only</td></tr><tr><td>Surg-I-Loop</td><td>Scanlan International</td><td>1001-81M</td><td>5 cm length</td></tr><tr><td>Bonaccolto forceps, 4&amp;rdquo; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td><td>Roboz</td><td>RS-5210</td><td></td></tr><tr><td>Dumont #3 forceps Inox tip size .17 X .10mm</td><td>Roboz</td><td>RS-5042</td><td></td></tr><tr><td>Graefe forceps, 4&amp;rdquo; (10 cm) long serrated straight, 0.8mm tip</td><td>Roboz</td><td>RS-5280</td><td></td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; straight, 1.3mm tips</td><td>Roboz</td><td>RS-7110</td><td>2x</td></tr><tr><td>Halstead mosquito forceps,&amp;nbsp; 5&quot; curved, 1.3mm tips</td><td>Roboz</td><td>RS-7111</td><td></td></tr><tr><td>Jacobson mosquito forceps 5&quot; curved extra delicate, 0.9 mm tips</td><td>Roboz</td><td>RS-7117</td><td></td></tr><tr><td>Kantrowitz forceps, 7.25&quot; 90 degree delicate, 1.7 mm tips</td><td>Roboz</td><td>RS-7305</td><td></td></tr><tr><td>Tissue forceps 5&quot;, 1X2 teeth, 2 mm tip width</td><td>Roboz</td><td>RS-8162</td><td></td></tr><tr><td>Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td><td>Fine Science Tools</td><td>11092-12</td><td>2x</td></tr><tr><td>Adson forceps, 12 cm, serrated, straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Veterinary electrosurgery handpiece and electrode</td><td>MACAN Manufacturing</td><td>HPAC-1; R-F11</td><td></td></tr><tr><td>&lt;strong&gt;Surgical Suite Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Circulating warm water blanket and pump</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Forced air warming unit</td><td>3M</td><td>Bair Hugger Model 505</td><td>Gene transfer surgery only</td></tr><tr><td>IV infusion pump</td><td>Heska</td><td>Vet IV 2.2</td><td>Gene transfer surgery only</td></tr><tr><td>Isoflurane vaporizer and scavenger</td><td>Multiple vendors</td><td>Catalog number not available</td><td></td></tr><tr><td>Veterinary multi-parameter monitor</td><td>Surgivet</td><td>Surgivet Advisor</td><td></td></tr><tr><td>Veterinary electrosurgery unit</td><td>MACAN Manufacturing</td><td>MV-9</td><td></td></tr><tr><td>Surgical microscope</td><td>D.F. Vasconcellos</td><td>M900</td><td>Needs ~16x magnification</td></tr></tbody></table>

in vivo gene transfer to the rabbit common carotid artery endothelium

このメソッドの目的は、分離セグメント両方ウサギ総頚動脈の内皮に transgene を導入することです。方式により、内皮発現遺伝子の生物学的役割を決定して大きな動脈の DNA 配列の生体内での転写活性を定量化する捜査焦点の内皮細胞選択的遺伝子導入血管内皮細胞。メソッドは、血管内腔に遺伝子発現ウイルスのベクトルを提供するウサギ頸動脈と、切開の手術の分離を使用します。ルーメン内容の後続の吸引と、ルーメンのベクトルの短い潜伏期間は効率的かつ耐久性のある検出可能な伝達や表現の外の血管内皮細胞での遺伝子発現を達成するために十分な分離動脈セグメント。メソッドによりマウスレニンの生物活性の評価正常動脈とひと血管疾患のモデルの両方可能性がありますいずれかのターゲット (e.g他のサイトへの遺伝子導入による全身効果を回避しながら。肝臓)、生殖系列の遺伝子導入による血管内皮細胞に遺伝的構造を提供する方法。法の適用は、熟練した外科医と麻酔、設備の整った手術室の購入および住宅のウサギ、費用の必要性と遺伝子導入ベクターの構築とその利用に関する専門知識の必要性によって制限されます。この方法で得られた結果を含める: 動脈構造、セル、細胞外のマトリックス、または血管運動機能の変化を遺伝子関連増加または減少動脈の炎症。血管内皮細胞のアポトーシス; の変化進行、遅滞、または内膜肥厚や動脈硬化などの病気の回帰。メソッドことができますを含む検索結果を提供する内皮細胞の遺伝子発現を変更するネイティブおよび合成 DNA 規制シーケンスの能力の測定: レベル導入遺伝子の mRNA、遺伝子蛋白質のレベル、遺伝子のレベル酵素活性。

このメソッドを実装すると、自信を持って、予備実験がオペレーターが内腔内皮細胞に主に発現との効率的かつ再現可能な遺伝子導入を実現できることを確立する必要があります。我々 の経験でこれは最も簡単に β-ガラクトシダーゼを表すベクトルを使用して評価されます。定量的逆転写ポリメラーゼの鎖と β-ガラクトシダーゼ mRNA (メッセンジャー RNA) の測定と同...

Watch this Scientific Journal Video about In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium at JoVE.com

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

このメソッドは、ウサギの頚動脈の血管内皮細胞に、遺伝子を導入することです。遺伝子させず正常動脈または疾患モデルでの生物学的役割の検討ができます。メソッド、また規制的 dna の活性の測定に役立ちます。

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 ...

in-vivo-gene-transfer-to-the-rabbit-common-carotid-artery-endothelium

Research

JoVE Journal

Medicine

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

9.0K ビュー.  University of Washington. このメソッドは、ウサギの頚動脈の血管内皮細胞に、遺伝子を導入することです。遺伝子させず正常動脈または疾患モデルでの生物学的役割の検討ができます。メソッド、また規制的 dna の活性の測定に役立ちます。

ビデオ: In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

部分的ライゲーションによるマウス頸動脈における乱流誘発アテローム性動脈硬化症と頸動脈内皮からのRNA単離の方法における簡便な方法のモデル

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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 ...

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 ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

加速動脈硬化家兎モデル: 内腸骨動脈バルーン傷害の方法論的視点

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

耐久性のある遺伝子発現により一般的な頚動脈移植に頸静脈の家兎モデル

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病性ウサギにおける後肢虚血の前臨床モデル

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

静脈移植を用いた血管形成術を模したウサギ静脈間位置モデルによる動脈血圧下の内膜過形成の評価

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

カフ法 による マウス頸動脈への組織改変血管移植片の移植

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

生物工学

Carotid Artery and Jugular Vein Interposition: A Technique to Induce Venous Intimal Hyperplasia in Rabbit Model

Source: Nishio, H., et al. <a href="https://www.jove.com/v/60931">A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure</a> J. Vis. Exp. (2020).
This video demonstrates the interposition of the rabbit carotid artery with the jugular vein to induce venous intimal hyperplasia by subjecting the veins to arterial pressure. This model will help investigate molecular and genetic interventions to attenuate the progression of intimal hyperplasia after revascularization surgeries.

頸動脈と頸静脈の介在:ウサギモデルにおける静脈内膜過形成を誘発する技術

Source: Nishio, H., et al. A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under ...

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

要約

さらに動画を探す

この動画の章

部分的ライゲーションによるマウス頸動脈における乱流誘発アテローム性動脈硬化症と頸動脈内皮からのRNA単離の方法における簡便な方法のモデル

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

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

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

加速動脈硬化家兎モデル: 内腸骨動脈バルーン傷害の方法論的視点

耐久性のある遺伝子発現により一般的な頚動脈移植に頸静脈の家兎モデル

糖尿病性ウサギにおける後肢虚血の前臨床モデル

静脈移植を用いた血管形成術を模したウサギ静脈間位置モデルによる動脈血圧下の内膜過形成の評価

カフ法によるマウス頸動脈への組織改変血管移植片の移植

頸動脈と頸静脈の介在:ウサギモデルにおける静脈内膜過形成を誘発する技術

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

要約

さらに動画を探す

この動画の章

部分的ライゲーションによるマウス頸動脈における乱流誘発アテローム性動脈硬化症と頸動脈内皮からのRNA単離の方法における簡便な方法のモデル

そのような自閉症や統合失調症のような一般的な疾患でのde novo変異を同定するための戦略

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

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

加速動脈硬化家兎モデル: 内腸骨動脈バルーン傷害の方法論的視点

耐久性のある遺伝子発現により一般的な頚動脈移植に頸静脈の家兎モデル

糖尿病性ウサギにおける後肢虚血の前臨床モデル

静脈移植を用いた血管形成術を模したウサギ静脈間位置モデルによる動脈血圧下の内膜過形成の評価

カフ法 による マウス頸動脈への組織改変血管移植片の移植

頸動脈と頸静脈の介在:ウサギモデルにおける静脈内膜過形成を誘発する技術

カフ法によるマウス頸動脈への組織改変血管移植片の移植