rAVE™　参考文献／発現ベクター詳細情報

• アデノ随伴ウイルスベクター 遺伝子デリバリー試薬 rAVE gene delivery reagents
• rAVE™ 発現ベクター カスタムサービス
• rAVE™ レポーター遺伝子ベクター

1. Monahan PE, Samulski RJ. Adeno-associated virus vectors for gene therapy: more pros than cons? Mol Med Today. 2000 6(11):433-40.
2. Monahan PE, Samulski RJ. AAV vectors: is clinical success on the horizon? Gene Ther. 2000 7(1):24-30.
3. Janson CG, McPhee SW, Leone P, Freese A, During MJ. Viral-based gene transfer to the mammalian CNS for functional genomic studies. Trends Neurosci. 2001 Dec;24(12):706-12.rAVE→pdfファイル(113kb)
4. Janson CG, During MJ. Viral vectors as part of an integrated functional genomics program. Genomics. 2001 Nov;78(1-2):3-6.rAVE→pdfファイル(150kb)

1. rAAVの使用例
2. rAVE™ の作製
3. rAVE™発現カセットについて
4. rAVE™ ベクターの純度
5. 各作業における納期の目安
6. どのように供給されるか？
7. 参考文献

1. rAAVの使用例：

rAAVベクターは、遺伝子デリバリーツールとして非常に有用です。10年以上にもわたって研究されているrAAVのバイオロジーはベクターと併せて良く理解され特徴づけられており、様々な組織や細胞に使用されています。導入に適したセルライン・組織を下表の通りまとめました。また、様々な適用に関する情報も下記よりお選びいただけます。

セルライン	組織	in vivo アプリケーション
HEK293 5,6,7,8,18	Muscle 15,16,17,18,19,20,38	Targeted gene therapy of disease
HeLa 5,6,18	Heart 21,22,29,50	cystic fibrosis 31,32
RPE 5	Brain 24,25,26,27,30,41,49,52,54	hemophilia 20,38
Rho O 7	Liver 11,23,30	Duchenne's Muscular Disease 19,39
143B (human) 8	Lung 28,29,30,31,32	Parkinson's Disease 24,40,41
Fibroblasts 9,10	Retina 19, 33, 34, 35	Canavan Disease 25,42
COS 11	Alimentary tract 36, 37	ischemia 18,21
Primary cell cultures 10, 55	Spleen 11	rheumatoid arthritis 43
Hematopoietic cells 12,13,14		hypertension 50,51
rAVE-GFPベクター注入4週間後 脳(海馬)におけるGFP発現(緑)		Knock-outs
		cre-lox 44,45,46,47,48
		antisense AAV 49,50,51
		Generation of disease models 45, 52
		Functional genomics 3,4,53
		Regulatable expression systems 33,53,54
		Genetic vaccines 36,55,56,57
		Anterograde tracing58

2. rAVE™ の作製：

GeneDetect社は、何年もrAAV開発に携わってきた経験をいかして高レベルに導入遺伝子を発現するように最適化したrAAVストックを提供しています。このrAVE™ベクターを使用して、3つのプラスミドをHEK293 cellsのトリプル形質導入にも成功しました。シス活性AAVプラスミドは、目的の遺伝子の両端にAAV ITR(inverted terminalrepeats) を含むrAVE™発現カセットを含んでいます。それに対し、他の2つのプラスミドはウイルスパッケージングに必要な他のアデノウイルスヘルパー機能と構造的なAAVカプシドタンパク質をコードする遺伝子を含んでいます。アフィニティーカラム精製改良法^59,60により、高精製度のrAVE^TEベクターストックを作製しています。

3. rAVE™発現カセットについて：

高レベルに遺伝子を発現させるための秘訣は、最適化された遺伝子発現カセットにあります。

rAVEカセットは、AAV ITRに隣接しています。導入遺伝子は、CAGプロモーターによって発現されます。調節エレメントであるSARとWPREは導入遺伝子の発現を促進します。HAタグが必要であれば、遺伝子の5'末端あるいは3'末端に付けることが別途可能です。各エレメントの大きさは、bpで示し、AAVには4.7kbのパッケージング制限があります。このため導入遺伝子の大きさは1600bpより小さい必要があります。

Q.構築された発現ベクターによる遺伝子発現と内因性の遺伝子発現とをどのようにして区別することができるでしょう？
A.目的の遺伝子を簡単に検出する為に、ご要望に応じて5'もしくは3'末端にHAタグ配列を付加する事が可能です。

4. rAVE™ ベクターの純度：

すべての rAVE™ ベクターは、革新的なAAV技術を用いて最高のスタンダードに精製されています。AAV2では、平衡化ヘパラン硫酸プロテオグリカンを用いてウイルス粒子のアフィニティー精製を行います。

3種類の異なるrAAV精製グレードのSDS-PAGE分析
a=クルード
b=塩化セシウム精製
c=アフィニティー精製

GeneDetect社で最適化/開発した精製プロトコールに従って、一貫して95%の精製レベルにrAVE™ ベクターを作ることが可能です。その結果、より効率の高いベクターを作ることが可能になりました。クルードなAAV調整液でないかぎり、アフィニティー精製したAAV2は、パッケージング中に生じた導入遺伝子のコンタミは起こりません。ベクターストック内のこのような混入物は、多くの場合、偽形質導入の問題を引き起こします⁷⁰。rAVE™ ベクター産物を使用することで、これらの問題を排除することができます。

全ての rAVE™ ベクター をシッピングする前に、純度テストが行われ、定量PCRによってサンプル(＞1x10¹⁰ genomic particles/ml)の確認をします。

5. 各作業における納期の目安：

下記の 目安は、実稼働日数(working days)を示しています。通常、お客様のDNAを受け取ってから5-6週間以内でお届けできます。ただし、追加サービスによって異なります。

6. どのように供給されるか？

rAVEベクターは、1mM MgSO₄含有ＰＢＳの0.3mL中に供給されます。

7. 参考文献：

文献リスト中にrAVE印がついている文献には、rAVE遺伝子デリバリー試薬が使用されています！！

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