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Homologous recombination is required for AAV-mediated gene targeting by A. Vasileva is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) __Nature Genet__., **18**, 325–330; D. W. Russell and R. K. Hirata (2000) __J. Virol__., 74, 4612–4620; R. Hirata __et al__. (2002) __Nat. Biotechnol__., **20**, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency

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Author

A. Vasileva

Publisher

Oxford University Press

Published

2006

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)