1887

Abstract

Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5·5×10 vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.

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1999-09-01
2019-10-19
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References

  1. Clark, K. R., Voulgaropoulou, F. & Johnson, P. R. ( 1996; ). A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Therapy 3, 1124-1132.
    [Google Scholar]
  2. Fan, D. S., Ogawa, M., Fujimoto, K. I., Ikeguchi, K., Ogasawara, Y., Urabe, M., Nishizawa, M., Nakano, I., Yoshida, M., Nagatsu, I., Ichinose, H., Nagatsu, T., Kurtzman, G. J. & Ozawa, K. ( 1998; ). Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic l-amino acid decarboxylase genes using two separate adeno-associated virus vectors. Human Gene Therapy 9, 2527-2535.[CrossRef]
    [Google Scholar]
  3. Flotte, T. R., Afione, S. A., Conrad, C., McGrath, S. A., Solow, R., Oka, H., Zeitlin, P. L., Guggino, W. B. & Carter, B. J. ( 1993; ). Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proceedings of the National Academy of Sciences, USA 90, 10613-10617.[CrossRef]
    [Google Scholar]
  4. Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. ( 1977; ). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology 36, 59-72.[CrossRef]
    [Google Scholar]
  5. Herzog, R. W., Yang, E. Y., Couto, L. B., Hagstrom, J. N., Elwell, D., Fields, P. A., Burton, M., Bellinger, D. A., Read, M. S., Brinkhous, K. M., Podsakoff, G. M., Nichols, T. C., Kurtzman, G. J. & High, K. A. ( 1999; ). Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nature Medicine 5, 56-63.[CrossRef]
    [Google Scholar]
  6. Hölscher, C., Hörer, M., Kleinschmidt, J. A., Zentgraf, H., Bürkle, A. & Heilbronn, R. ( 1994; ). Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants. Journal of Virology 68, 7169-7177.
    [Google Scholar]
  7. Inoue, N. & Russell, D. W. ( 1998; ). Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors. Journal of Virology 72, 7024-7031.
    [Google Scholar]
  8. Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sakaki, T., Sugano, S. & Saito, I. ( 1995; ). Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Research 23, 3816-3821.[CrossRef]
    [Google Scholar]
  9. Kaplitt, M. G., Leone, P., Samulski, R. J., Xiao, X., Pfaff, D. W., O’Malley, K. L. & During, M. J. ( 1994; ). Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nature Genetics 8, 148-154.[CrossRef]
    [Google Scholar]
  10. Li, J., Samulski, R. J. & Xiao, X. ( 1997; ). Role for highly regulated rep gene expression in adeno-associated virus vector production. Journal of Virology 71, 5236-5243.
    [Google Scholar]
  11. McCarty, D. M., Christensen, M. & Muzyczka, N. ( 1991; ). Sequences required for coordinate induction of adeno-associated virus p19 and p40 promoters by Rep protein. Journal of Virology 65, 2936-2945.
    [Google Scholar]
  12. Maeda, Y., Ikeda, U., Ogasawara, Y., Urabe, M., Takizawa, T., Saito, T., Colosi, P., Kurtzman, G., Shimada, K. & Ozawa, K. ( 1997; ). Gene transfer into vascular cells using adeno-associated virus (AAV) vectors. Cardiovascular Research 35, 514-521.[CrossRef]
    [Google Scholar]
  13. Niwa, H., Yamamura, K. & Miyazaki, J. ( 1991; ). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-199.[CrossRef]
    [Google Scholar]
  14. Ogasawara, Y., Urabe, M. & Ozawa, K. ( 1998; ). The use of heterologous promoters for adeno-associated virus (AAV) protein expression in AAV vector production. Microbiology and Immunology 42, 177-185.[CrossRef]
    [Google Scholar]
  15. Ogasawara, Y., Urabe, M., Kogure, K., Kume, A., Colosi, P., Kurzman, G. & Ozawa, K. ( 1999; ). Efficient production of adeno-associated virus vectors using split-type helper plasmids. Japanese Journal of Cancer Research 90, 476-483.[CrossRef]
    [Google Scholar]
  16. Orban, P. C., Chui, D. & Marth, J. D. ( 1992; ). Tissue- and site-specific DNA recombination in transgenic mice. Proceedings of the National Academy of Sciences, USA 89, 6861-6865.[CrossRef]
    [Google Scholar]
  17. Pereira, D. J., McCarty, D. M. & Muzyczka, N. ( 1997; ). The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection. Journal of Virology 71, 1079-1088.
    [Google Scholar]
  18. Surosky, R. T., Urabe, M., Godwin, S. G., McQuiston, S. A., Kurtzman, G. J., Ozawa, K. & Natsoulis, G. ( 1997; ). Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. Journal of Virology 71, 7951-7959.
    [Google Scholar]
  19. Tamayose, K., Hirai, Y. & Shimada, T. ( 1996; ). A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. Human Gene Therapy 7, 507-513.[CrossRef]
    [Google Scholar]
  20. Urabe, M., Hasumi, Y., Ogasawara, Y., Matsushita, T., Kamoshita, N., Nomoto, A., Colosi, P., Kurtzman, G. J., Tobita, K. & Ozawa, K. ( 1997; ). A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome. Gene 200, 157-162.[CrossRef]
    [Google Scholar]
  21. Wigler, M., Pellicer, A., Silverstein, S. & Axel, R. ( 1978; ). Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14, 725-731.[CrossRef]
    [Google Scholar]
  22. Yang, Q., Chen, F. & Trempe, J. P. ( 1994; ). Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. Journal of Virology 68, 4847-4856.
    [Google Scholar]
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