title: Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells.
abstract: Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors.
authors.affiliation: Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama, 244-0813, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsunoda
authors.forename: T
authors.initials: T
authors.lastname: Hayakawa
authors.forename: N
authors.initials: N
authors.lastname: Sakuragawa
authors.forename: H
authors.initials: H
authors.lastname: Koyama
mesh_terms: D002411:Cation Exchange Resins; D004279:DNA, Viral / Q000378:metabolism; D000229:Dependovirus / Q000187:drug effects / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells / Q000821:virology*; D006801:Humans; D008055:Lipids; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*; D015678:Viral Structural Proteins / Q000235:genetics; D016662:Virus Integration / Q000235:genetics*
chemical_list: D002411:Cation Exchange Resins; D004279:DNA, Viral; D008055:Lipids; C086724:Lipofectamine; D015678:Viral Structural Proteins