1. ¹Cellular Neurology Unit, NINDS, National Institutes of Health, Bethesda, MD, USA
2. ²Albany Medical School, Albany, NY, USA
3. ³Division of Pulmonary and Critical Care, Asthma and Allergy Center, Johns Hopkins School of Medicine, Baltimore, MD, USA
4. ⁴Department of Anesthesia, Brigham and Women's Hospital, Boston, MA, USA
5. ⁵Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Charlestown, MA, USA
6. ⁶Neuroscience Program, Harvard Medical School, Boston, MA, USA

Correspondence: Dr XO Breakefield, Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, CNY 6205, Building 149, 13th Street, Charlestown, MA 02129, USA

Received 5 December 2002; Accepted 27 February 2003.

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.