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Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks

Porteus, Matthew H. and Cathomen, Toni and Weitzman, Matthew D. and Baltimore, David (2003) Efficient Gene Targeting Mediated by Adeno-Associated Virus and DNA Double-Strand Breaks. Molecular and Cellular Biology, 23 (10). pp. 3558-3565. ISSN 0270-7306. http://resolver.caltech.edu/CaltechAUTHORS:PORmcb03

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Abstract

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.


Item Type:Article
Additional Information:Copyright © 2003, American Society for Microbiology. Received 18 November 2002/ Returned for modification 6 January 2003/ Accepted 3 March 2003 We thank Elissa Denney for excellent technical support over the course of this work. We thank Dan Miller and David Russell for sharing unpublished results. This work was supported by the Howard Hughes Medical Institute (Physician Postdoctoral Fellow Award), Burroughs-Wellcome Fund (Career Development Award), and NIH grant KO8 HL70268-01 to M.H.P.
Record Number:CaltechAUTHORS:PORmcb03
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:PORmcb03
Alternative URL:http://dx.doi.org/10.1128/MCB.23.10.3558-3565.2003
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2137
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:10 Mar 2006
Last Modified:26 Dec 2012 08:47

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