サポート情報

技術情報

記事ID : 9183
研究用

rAVE™ 参考文献/発現ベクター詳細情報


参考文献

  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.rAVEpdf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.rAVEpdfファイル(150kb)

rAVE™ 発現ベクター詳細情報

  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
products_GDC_20030512_ravehippocampus.gif
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rAVETE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。

products_GDC_20030512_ravecassette.gif

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。

products_GDC_20030512_ravepurity.gif

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。

作業内容 実稼働日数
1.pAAVベクターへの目的遺伝子のクローニング 6日間
2.DNA増幅 (construct and helpers) 2日間
3.AAVパッケージング 7日間
4.精製と透析 3日間
5.力価測定と品質検査 2日間
6.HAタグの付加 10日間
7.お客様の遺伝子のPCRクローニング 10日間

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!!

  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
  4. Janson CG, During MJ. Viral vectors as part of an integrated functional genomics program. Genomics. 2001 Nov;78(1-2):3-6.rAVE
  5. Tenenbaum L, Hamdane M, Pouzet M, Avalosse B, Stathopoulos A, Jurysta F, Rosenbaum C, Hanemann CO, Levivier M, Velu T. Cellular contaminants of adeno-associated virus vector stocks can enhance transduction. Gene Ther. 1999 6(6):1045-53.
  6. Lai YK, Rolling F, Baker E, Rakoczy PE. Kinetics of efficient recombinant adeno-associated virus transduction in retinal pigment epithelial cells. Exp Cell Res. 2001 267(2):184-92.
  7. Owen R IV, Lewin AP, Peel A, Wang J, Guy J, Hauswirth WW, Stacpoole PW, Flotte TR. Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism. Hum Gene Ther. 2000 11(15):2067-78.
  8. Seo BB, Wang J, Flotte TR, Yagi T, Matsuno-Yagi A. Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells. J Biol Chem. 2000 275(48):37774-8.
  9. Paul D, Qazilbash MH, Song K, Xu H, Sinha BK, Liu J, Cowan KH. Construction of a recombinant adeno-associated virus (rAAV) vector expressing murine interleukin-12 (IL-12). Cancer Gene Ther. 2000 7(2):308-15.
  10. Fu H, Samulski RJ, McCown TJ, Picornell YJ, Fletcher D, Muenzer J. Neurological correction of lysosomal storage in a mucopolysaccharidosis IIIB mouse model by adeno-associated virus-mediated gene delivery. Mol Ther. 2002 5(1):42-9.
  11. Yamano S, Scott DE, Huang LY, Mikolajczyk M, Pillemer SR, Chiorini JA, Golding B, Baum BJ. Protection from experimental endotoxemia by a recombinant adeno-associated virus encoding interleukin 10. J Gene Med. 2001 3(5):450-7.
  12. Ponnazhagan S, Mukherjee P, Wang XS, Qing K, Kube DM, Mah C, Kurpad C, Yoder MC, Srour EF, Srivastava A. Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation. J Virol. 1997 71(11):8262-7.
  13. Nathwani AC, Hanawa H, Vandergriff J, Kelly P, Vanin EF, Nienhuis AW. Efficient gene transfer into human cord blood CD34+ cells and the CD34+CD38- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV. Gene Ther. 2000 7(3):183-95.
  14. Handa A, Muramatsu S, Qiu J, Mizukami H, Brown KE. Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors. J Gen Virol. 2000 81 Pt 8:2077-84.
  15. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol. 1996 70(11):8098-108.
  16. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci U S A. 1996 93(24):14082-7.
  17. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad Sci U S A. 2000 97(25):13714-9.
  18. Byun J, Heard JM, Huh JE, Park SJ, Jung EA, Jeong JO, Gwon HC, Kim DK. Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol. 2001 33(2):295-305.
  19. Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, Samulski RJ. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol. 2002 76(2):791-801.
  20. Larson PJ, High KA. Gene therapy for hemophilia B: AAV-mediated transfer of the gene for coagulation factor IX to human muscle. Adv Exp Med Biol. 2001;489:45-57.
  21. Maeda Y, Ikeda U, Shimpo M, Ueno S, Ogasawara Y, Urabe M, Kume A, Takizawa T, Saito T, Colosi P, Kurtzman G, Shimada K, Ozawa K. Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors. J Mol Cell Cardiol. 1998 30(7):1341-8.
  22. Su H, Lu R, Kan YW. Adeno-associated viral vector-mediated vascular endothelial growth factor gene transfer induces neovascular formation in ischemic heart. Proc Natl Acad Sci U S A. 2000 97(25):13801-6.
  23. Jung SC, Han IP, Limaye A, Xu R, Gelderman MP, Zerfas P, Tirumalai K, Murray GJ, During MJ, Brady RO, Qasba P. Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice. Proc Natl Acad Sci U S A. 2001 98(5):2676-81. rAVE
  24. During MJ, Kaplitt MG, Stern MB, Eidelberg D. Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation. Hum Gene Ther. 2001 12(12):1589-91. rAVE
  25. Leone P, Janson CG, Bilaniuk L, Wang Z, Sorgi F, Huang L, Matalon R, Kaul R, Zeng Z, Freese A, McPhee SW, Mee E, During MJ, Bilianuk L. Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann Neurol. 2000 48(1):27-38. rAVE
  26. Mastakov MY, Baer K, Xu R, Fitzsimons H, During MJ. Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol Ther. 2001 3(2):225-32.rAVE
  27. Davidson BL, Stein CS, Heth JA, Martins I, Kotin RM, Derksen TA, Zabner J, Ghodsi A, Chiorini JA. Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc Natl Acad Sci U S A. 2000 97(7):3428-32.
  28. Halbert CL, Rutledge EA, Allen JM, Russell DW, Miller AD. Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J Virol. 2000 74(3):1524-32.
  29. Zabner J, Seiler M, Walters R, Kotin RM, Fulgeras W, Davidson BL, Chiorini JA. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol. 2000 74(8):3852-8.
  30. Lipshutz GS, Gruber CA, Cao Y, Hardy J, Contag CH, Gaensler KM. In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression. Mol Ther. 2001 3(3):284-92.
  31. Flotte TR, Laube BL. Gene therapy in cystic fibrosis. Chest. 2001 120(3 Suppl):124S-131S.
  32. Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR, McNamara SC, Gibson RL, Ramsey BW, Carter BJ, Reynolds TC. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Hum Gene Ther. 2001 12(15):1907-16.
  33. Sarra GM, Stephens C, de Alwis M, Bainbridge JW, Smith AJ, Thrasher AJ, Ali RR. Gene replacement therapy in the retinal degeneration slow (rds) mouse: the effect on retinal degeneration following partial transduction of the retina. Hum Mol Genet. 2001 10(21):2353-61.
  34. McGee Sanftner LH, Rendahl KG, Quiroz D, Coyne M, Ladner M, Manning WC, Flannery JG. Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina. Mol Ther. 2001 3(5 Pt 1):688-96.
  35. Jomary C, Grist J, Milbrandt J, Neal MJ, Jones SE. Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells. Mol Vis. 2001 7:36-41.
  36. During MJ, Xu R, Young D, Kaplitt MG, Sherwin RS, Leone P. Peroral gene therapy of lactose intolerance using an adeno-associated virus vector. Nat Med. 1998 4(10):1131-5. rAVE
  37. During MJ, Symes CW, Lawlor PA, Lin J, Dunning J, Fitzsimons HL, Poulsen D, Leone P, Xu R, Dicker BL, Lipski J, Young D. An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy. Science. 2000 287(5457):1453-60.rAVE
  38. Schwaab R, Oldenburg J. Gene therapy of hemophilia. Semin Thromb Hemost. 2001 27(4):417-24.
  39. Duan D, Yan Z, Yue Y, Ding W, Engelhardt JF. Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlates with myoblast differentiation. J Virol. 2001 75(16):7662-71.
  40. Sanchez-Pernaute R, Harvey-White J, Cunningham J, Bankiewicz KS. Functional effect of adeno-associated virus mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum of 6-OHDA-lesioned rats. Mol Ther. 2001 4(4):324-30.
  41. Kirik D, Rosenblad C, Bjorklund A, Mandel RJ. Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system. J Neurosci. 2000 20(12):4686-700.
  42. Leone P, Janson CG, McPhee SJ, During MJ. Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease. Curr Opin Mol Ther. 1999 1(4):487-92. rAVE
  43. Cottard V, Mulleman D, Bouille P, Mezzina M, Boissier MC, Bessis N. Adeno-associated virus-mediated delivery of IL-4 prevents collagen-induced arthritis. Gene Ther. 2000 7(22):1930-9.
  44. Akagi K, Sandig V, Vooijs M, Van der Valk M, Giovannini M, Strauss M, Berns A. Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 1997 25(9):1766-73.
  45. Meuwissen R, Linn SC, van der Valk M, Mooi WJ, Berns A. Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene. Oncogene. 2001 20(45):6551-8.
  46. South S, Bogulavsky J, Franklin S, Martin D, Vissel B, Sailer A, Malkmus S, Masuyama T, Horner P, Kaspar B, Gage F, Tonegawa S, Yaksh T, Heinemann S, Inturissi CE. A conditional knockout (KO) of the NMDAR1 subunit in spinal cord dorsal horn attentuates hyperalgesia. Soc. Neurosci Abstr. 2001, 53.4
  47. Bengoechea TG, Lin W, Kaspar B, Harada N, Domingez B, Gage FH, Lee KL. A conditional mouse mutation system for spatiotemporal removal of the neurotrophin receptor P75 to study its role in the adult basal forebrain. Soc. Neurosci. Abstr. 2001, 28.3.
  48. Greene RW, Lee JS, Gray J, Beaudet M, Estabrooke IV, Thompson MA, Saper CB, Scammell TE. Anatomically specific deletion of the adenosine A1 receptor using an adeno-associated viral vector. Soc. Neurosci. Abstr. 2001, 523.5.
  49. Klein RL, McNamara RK, King MA, Lenox RH, Muzyczka N, Meyer EM. Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer. Brain Res. 1999 832(1-2):136-44.
  50. Kimura B, Mohuczy D, Tang X, Phillips MI. Attenuation of hypertension and heart hypertrophy by adeno-associated virus delivering angiotensinogen antisense. Hypertension. 2001 37(2 Part 2):376-80.
  51. Phillips MI. Gene therapy for hypertension: sense and antisense strategies. Expert Opin Biol Ther. 2001 1(4):655-62.
  52. Senut MC, Suhr ST, Kaspar B, Gage FH. Intraneuronal aggregate formation and cell death after viral expression of expanded polyglutamine tracts in the adult rat brain. J Neurosci. 2000 20(1):219-29.
  53. Rossi FM, Guicherit OM, Spicher A, Kringstein AM, Fatyol K, Blakely BT, Blau HM. Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat Genet. 1998 20(4):389-93.
  54. Haberman RP, McCown TJ, Samulski RJ. Inducible long-term gene expression in brain with adeno-associated virus gene transfer.Gene Ther. 1998 5(12):1604-11.
  55. Manning WC, Paliard X, Zhou S, Pat Bland M, Lee AY, Hong K, Walker CM, Escobedo JA, Dwarki V. Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D. J Virol. 1997 71(10):7960-2.
  56. Brockstedt DG, Podsakoff GM, Fong L, Kurtzman G, Mueller-Ruchholtz W, Engleman EG. Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration. Clin Immunol. 1999 92(1):67-75.
  57. Xin KQ, Urabe M, Yang J, Nomiyama K, Mizukami H, Hamajima K, Nomiyama H, Saito T, Imai M, Monahan J, Okuda K, Ozawa K, Okuda K. A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus. Hum Gene Ther. 2001 12(9):1047-61.
  58. Chamberlin NL, Du B, de Lacalle S, Saper CB. Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS. Brain Res. 1998 793(1-2):169-75.
  59. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999 6(6):973-85.
  60. Clark KR, Liu X, McGrath JP, Johnson PR. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther. 1999 10(6):1031-9.
  61. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol. 1998 72(2):1438-45.
  62. Qing K, Mah C, Hansen J, Zhou S, Dwarki V, Srivastava A.Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med. 1999 5(1):71-7.
  63. Rutledge EA, Halbert CL, Russell DW. Infectious clones and vectors derived from adeno associated virus (AAV) serotypes other than AAV type 2. J Virol. 1998 72(1):309-19.
  64. Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, Samulski RJ. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol. 2002 Jan;76(2):791-801.
  65. Xu L, Daly T, Gao C, Flotte TR, Song S, Byrne BJ, Sands MS, Parker Ponder K. CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum Gene Ther. 2001 12(5):563-73.
  66. Donello JE, Loeb JE, Hope TJ. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. J Virol. 1998 72(6):5085-92.
  67. Paterna JC, Moccetti T, Mura A, Feldon J, Bueler H. Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain. Gene Ther. 2000 7(15):1304-11.
  68. Auten J, Agarwal M, Chen J, Sutton R, Plavec I. Effect of scaffold attachment region on transgene expression in retrovirus vector-transduced primary T cells and macrophages. Hum Gene Ther. 1999 10(8):1389-99.
  69. Agarwal M, Austin TW, Morel F, Chen J, Bohnlein E, Plavec I.Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells. J Virol. 1998 72(5):3720-8.
  70. Alexander IE, Russell DW, Miller AD. Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results. Hum Gene Ther. 1997 8(16):1911-20.
  • カタログ請求
  • お問い合わせ

商品は「研究用試薬」です。人や動物の医療用・臨床診断用・食品用としては
使用しないように、十分ご注意ください。