Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinaseReport as inadecuate




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BMC Biotechnology

, 8:60

First Online: 09 August 2008Received: 11 April 2008Accepted: 09 August 2008

Abstract

BackgroundCircular 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.

ResultsWe 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.

ConclusionOur 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-8-60 contains supplementary material, which is available to authorized users.

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Author: Brian Moldt - Nicklas H Staunstrup - Maria Jakobsen - Rafael J Yáñez-Muñoz - Jacob G Mikkelsen

Source: https://link.springer.com/article/10.1186/1472-6750-8-60







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