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

Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells , it is difficult to obtain stable cell lines that express them constitutiv ely. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determi ne the function of the induced Rep proteins, the packaging capacity was exa mined after supplementation with a plasmid expressing small Rep and Cap pro teins. A significant amount of recombinant AAV (5.5 x 10(8) vector particle s per 10 cm dish) was produced by transfection with a vector plasmid and in fection with Cre-expressing recombinant adenovirus, indicating that the lar ge Rep proteins retained the function required for packaging. These finding s indicate that large Rep protein expression can be strictly regulated by t he Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.