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Journal of Virology, August 2002, p. 7554-7559, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7554-7559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR

Daniela Hüser, Stefan Weger, and Regine Heilbronn*

Department of Virology, Institute of Infectious Diseases, Free University of Berlin, Berlin, Germany

Received 20 February 2002/ Accepted 30 April 2002

Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.

* Corresponding author. Mailing address: Institut für Infektionsmedizin, Abteilung Virologie, Universitätsklinik Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 27, 12203 Berlin, Germany. Phone: 4930 84453696. Fax: 4930 84454485. E-mail: regine.heilbronn{at}ukbf.fu-berlin.de.

Journal of Virology, August 2002, p. 7554-7559, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7554-7559.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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