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。

著者が明らかに何もありません。

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 border="0" cellpadding="0" cellspacing="0" style="width:888px;" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Disposables</td>
			<td style="width:181px;"></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">3mL syringe with 24G needle</td>
			<td>Becton Dickinson</td>
			<td>309571</td>
			<td style="width:199px;">2x for gene transfer surgery; 3x for harvest surgery</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">1mL syringe with 27G needle</td>
			<td>Becton Dickinson</td>
			<td>309623</td>
			<td style="width:199px;">6x for gene transfer surgery; 1x for harvest surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">20mL syringe, luer lock</td>
			<td>Nipro Medical Corp</td>
			<td>JD+20L</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheters, 24G x 3/4&#34;</td>
			<td>Terumo Medical Products</td>
			<td>SROX2419V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">19G needle</td>
			<td>Becton Dickinson</td>
			<td>305187</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">21G needle</td>
			<td>Becton Dickinson</td>
			<td>305165</td>
			<td style="width:199px;">For 20 mL syringe of saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Gauze 4&#34; x 4&#34;</td>
			<td>Dynarex</td>
			<td style="width:149px;">3242</td>
			<td style="width:199px;">~10-15 per surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-244</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 silk suture</td>
			<td>Covidien Ltd.</td>
			<td>S-182</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">7-0 polypropylene suture</td>
			<td>CP Medical</td>
			<td>8648P</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">5-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">3-0 polyglycolic acid suture</td>
			<td>CP Medical</td>
			<td>398A</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alcohol swabs</td>
			<td>Covidien Ltd.</td>
			<td>6818</td>
			<td style="width:199px;">For placement of I.V. line</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Catheter plug</td>
			<td>Vetoquinol</td>
			<td>411498</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Ketamine HCl, 100 mg/mL</td>
			<td>Vedco Inc.</td>
			<td>05098916106</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Xylazine, 100 mg/mL</td>
			<td>Akorn Inc.</td>
			<td>4821</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Lidocaine HCl, 2%</td>
			<td>Pfizer</td>
			<td>00409427702</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Bupivacaine HCl, 0.5%</td>
			<td>Pfizer</td>
			<td>00409161050</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Beuthanasia D-Special</td>
			<td>Intervet Inc.</td>
			<td>NDC 00061047305</td>
			<td style="width:199px;">Harvest surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Buprenorphine HCl, 0.3 mg/mL&#160;</td>
			<td>Patterson Veterinary</td>
			<td>12496075705</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Saline IV bag, 0.9% sodium chloride</td>
			<td>Baxter</td>
			<td>2B1309</td>
			<td style="width:199px;">2x for gene transfer surgery; can use vial of sterile saline in place of one</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Heparin&#160; (5000 U/mL)</td>
			<td>APP Pharmaceuticals</td>
			<td>NDC 63323-047-10</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Fentanyl patch, 25 mcg/hr&#160;</td>
			<td>Apotex Corp.</td>
			<td>NDC 60505-7006-2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:359px;">Gene transfer vector</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;">Dilute 350 &#181;L per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Instruments</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Metzenbaum needle holder 7&#34; straight</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7900</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Operating scissors 6.5&#34; straight blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6828</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Needle holder /w suture scissors</td>
			<td>Miltex</td>
			<td style="width:149px;">8-14-IMC</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo scissors</td>
			<td>Roboz</td>
			<td>RS-5658</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Castroviejo needle holder, 5.75&#34; straight with lock</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6412</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Stevens scissors 4.25&#34; curved blunt/blunt</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5943</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Alm retractor 4&#34; 4X4 5mm blunt prongs</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6514</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Backhaus towel clamp 3.5&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">4x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Micro clip setting forceps 4.75&#34;</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-6496</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Micro vascular clips, 11 mm</td>
			<td>Roboz</td>
			<td style="width:149px;"></td>
			<td style="width:199px;">2x for gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surg-I-Loop</td>
			<td>Scanlan International</td>
			<td style="width:149px;">1001-81M</td>
			<td style="width:199px;">5 cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Bonaccolto forceps, 4&#8221; (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5210</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Dumont #3 forceps Inox tip size .17 X .10mm</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5042</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Graefe forceps, 4&#8221; (10 cm) long serrated straight, 0.8mm tip</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-5280</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; straight, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7110</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Halstead mosquito forceps,&#160; 5&#34; curved, 1.3mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7111</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Jacobson mosquito forceps 5&#34; curved extra delicate, 0.9 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7117</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Kantrowitz forceps, 7.25&#34; 90 degree delicate, 1.7 mm tips</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-7305</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Tissue forceps 5&#34;, 1X2 teeth, 2 mm tip width</td>
			<td>Roboz</td>
			<td style="width:149px;">RS-8162</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11092-12</td>
			<td style="width:199px;">2x</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Adson forceps, 12 cm, serrated, straight</td>
			<td>Fine Science Tools</td>
			<td style="width:149px;">11006-12</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery handpiece and electrode</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">HPAC-1; R-F11</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical Suite Equipment</td>
			<td></td>
			<td style="width:149px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Circulating warm water blanket and pump</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Forced air warming unit</td>
			<td>3M</td>
			<td style="width:149px;">Bair Hugger Model 505</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">IV infusion pump</td>
			<td>Heska</td>
			<td style="width:149px;">Vet IV 2.2</td>
			<td style="width:199px;">Gene transfer surgery only</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Isoflurane vaporizer and scavenger</td>
			<td>Multiple vendors</td>
			<td style="width:149px;">Catalog number not available</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary multi-parameter monitor</td>
			<td>Surgivet</td>
			<td style="width:149px;">Surgivet Advisor</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Veterinary electrosurgery unit</td>
			<td>MACAN Manufacturing</td>
			<td style="width:149px;">MV-9</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Surgical microscope</td>
			<td style="width:181px;">D.F. Vasconcellos</td>
			<td style="width:149px;">M900</td>
			<td style="width:199px;">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, ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

ラットの総頚動脈で外頚静脈グラフトの介在：洗練された顕微鏡下手術の訓練

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

局所脳虚血への早期虚血耐性を誘導するために適切な前処理刺激として二国間の総頚動脈閉塞

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

再狭窄の研究のための頸動脈内ステント留置のマウスモデル

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成体マウス静脈性高血圧モデル：外頸静脈吻合部の総頸動脈。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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 ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

ラット頸動脈における血管バルーン傷害と管腔内投与

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

持続的な障害と皮質虚血を研究するために高齢ラットにおける一般的な頸動脈閉塞と永久遠位中大脳の実行

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...