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Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP switching system

Division of Genetic Therapeutics, Jichi Medical School¹ and CREST, Japan Science and Technology Corporation (JST)², 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan
Avigen Inc., 1201 Harbor Bay Parkway, #1000, Alameda, CA 94502, USA³
Laboratory of Molecular Genetics, Institute for Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan⁴

Author for correspondence: Keiya Ozawa (at Jichi Medical School). Fax +81 285 44 8675. e-mail kozawa{at}ms.jichi.ac.jp

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 (5·5x10⁸ 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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