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Journal of Virology, October 2007, p. 11290-11303, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00963-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

DNA Palindromes with a Modest Arm Length of {gtrsim}20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice{triangledown}

Katsuya Inagaki,1 Susanna M. Lewis,2 Xiaolin Wu,3 Congrong Ma,1 David J. Munroe,3 Sally Fuess,4 Theresa A. Storm,4 Mark A. Kay,4 and Hiroyuki Nakai1*

Department of Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,1 Program in Genetics and Genome Biology, Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1L8, Canada,2 Laboratory of Molecular Technology, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702,3 Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California 943054

Received 4 May 2007/ Accepted 30 July 2007

Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of {gtrsim}20 bp (total length, {gtrsim}40 bp) are a significant target for rAAV integration. Up to ~30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of {gtrsim}20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

* Corresponding author. Mailing address: Department of Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, W1244 BSTWR, 200 Lothrop St., Pittsburgh, PA 15261. Phone: (412) 648-8958. Fax: (412) 624-1401. E-mail: nakaih{at}pitt.edu

{triangledown} Published ahead of print on 8 August 2007.

Journal of Virology, October 2007, p. 11290-11303, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00963-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Inagaki, K., Piao, C., Kotchey, N. M., Wu, X., Nakai, H. (2008). Frequency and Spectrum of Genomic Integration of Recombinant Adeno-Associated Virus Serotype 8 Vector in Neonatal Mouse Liver. J. Virol. 82: 9513-9524 [Abstract] [Full Text]  
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  • Fattah, F. J., Lichter, N. F., Fattah, K. R., Oh, S., Hendrickson, E. A. (2008). Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells. Proc. Natl. Acad. Sci. USA 105: 8703-8708 [Abstract] [Full Text]  
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