title: Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.
abstract: Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.
authors.affiliation: Cellular Neurology Unit, NINDS, National Institutes of Health, Bethesda, MD, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Bakowska
authors.forename: M V
authors.initials: MV
authors.lastname: Di Maria
authors.forename: S M
authors.initials: SM
authors.lastname: Camp
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: P D
authors.initials: PD
authors.lastname: Allen
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D000229:Dependovirus / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism*; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D018139:Simplexvirus / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods