Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase

BMC Biotechnol. 2008 Aug 9;8:60. doi: 10.1186/1472-6750-8-60.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Nucleotidyltransferases / metabolism*
  • DNA, Circular / genetics*
  • Fungal Proteins / metabolism
  • Genetic Vectors / genetics*
  • Genome, Viral / genetics*
  • Lentivirus / genetics*
  • Mutagenesis, Insertional / methods*
  • Transfection / methods*

Substances

  • DNA, Circular
  • Fungal Proteins
  • DNA Nucleotidyltransferases
  • FLP recombinase