title: Positive and negative effects of adeno-associated virus Rep on AAVS1-targeted integration.
abstract: Adeno-associated virus type 2 integrates preferentially into the AAVS1 locus on chromosome 19 of the human genome. It was reported previously that transfection with two plasmids, one for Rep and the other carrying a transgene flanked by inverted terminal repeats (ITRs), enables preferential integration of the latter into AAVS1. Aiming at increasing the frequency of AAVS1-specific integration, the Rep- to transgene-plasmid ratio necessary to achieve a higher frequency of site-specific integration was examined. 293 cells were co-transfected with the Rep78 plasmid and an ITR-flanked Neo gene at different ratios. G418-resistant clones were selected randomly. Extensive Southern blot analysis showed an optimum range of Rep78 expression. In that range, approximately 20 % of clones harboured the Neo gene at AAVS1. Excess Rep expression, however, resulted in 'abortive' integration of the Neo gene, a rearrangement of AAVS1 without transgene integration. Rep78 appeared to cause abortive integration more extensively than Rep68. Deleterious effects of the Rep protein on the AAVS1 locus should be considered to develop an improved AAVS1-targeted system.
authors.affiliation: Department of Virology, Jichi Medical School, Tochigi 329-0498, Japan.|CREST, Japan Science and Technology Corporation (JST), Tochigi 329-0498, Japan.|Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi, Tochigi 329-0498, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Urabe
authors.affiliation: CREST, Japan Science and Technology Corporation (JST), Tochigi 329-0498, Japan.|Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi, Tochigi 329-0498, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Kogure
authors.affiliation: CREST, Japan Science and Technology Corporation (JST), Tochigi 329-0498, Japan.|Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi, Tochigi 329-0498, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kume
authors.affiliation: Department of Intractable Diseases, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Department of Virology, Jichi Medical School, Tochigi 329-0498, Japan.
authors.forename: Kiyotake
authors.initials: K
authors.lastname: Tobita
authors.affiliation: CREST, Japan Science and Technology Corporation (JST), Tochigi 329-0498, Japan.|Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-Kawachi, Tochigi 329-0498, Japan.
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D002460:Cell Line; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D006801:Humans; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2