Moldt, B., Staunstrup, N. H., Jakobsen, M., Yáñez-Muñoz, R. J. and Mikkelsen, J. G. (2008) Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase. BMC Biotechnology, 8
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BACKGROUND: Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription. RESULTS: We demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette. CONCLUSION: Our data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile.
This is a Submitted version This version's date is: 2008 This item is not peer reviewed
https://repository.royalholloway.ac.uk/items/c45abbd7-e6a7-807b-7552-ce6a39a65bd5/3/
Deposited by Research Information System (atira) on 25-Jul-2012 in Royal Holloway Research Online.Last modified on 25-Jul-2012
Moldt, Brian Staunstrup, Nicklas H Jakobsen, Maria Yáñez-Muñoz, Rafael J Mikkelsen, Jacob G Research Support, Non-U.S. Gov't England BMC biotechnology BMC Biotechnol. 2008 Aug 9;8:60.