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Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP
Yoji Ogasawara1, Hiroaki Mizukami1, Masashi Urabe1
1Division of Genetic Therapeutics, Jichi Medical School1 and CREST, Japan Science and Technology Corporation (JST)2, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan.
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 x 10(8) 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.