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

Genomic Insertion of Lentiviral DNA Circles Directed by the Yeast Flp Recombinase

Free PMC article

Genomic Insertion of Lentiviral DNA Circles Directed by the Yeast Flp Recombinase

Brian Moldt et al. BMC Biotechnol. .
Free PMC article


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.


Figure 1
Figure 1
Strategy for Flp-directed integration of lentiviral DNA circles. (A) Schematic representation of vectors utilized for lentiviral transduction and generation of Sleeping Beauty vector-tagged cell lines. The lentiviral vector, pLV/FRT-hygro, is a third generation SIN vector containing an ATG-deficient FRT-hygro fusion gene located between the packaging signal (ψ) and the central polypurine tract (cPPT, not shown) flanked downstream by a selectable marker expression cassette. In the lentiviral SIN vector pLV/PGK-puro the PGK-driven Flpx9 expression cassette is located downstream from ψ and cPPT sequence (the latter not shown). The SB transposon docking vector, pSBT/RSV-FGIP, contains the left and inverted repeats of SB (LIR and RIR, represented by white arrows) and an expression cassette containing the RSV promoter driving a fusion gene consisting of an ATG-FRT-tagged eGFP gene, an IRES element, the puromycin-resistance gene, and a polyadenylation signal. (B) Schematic representation of site-directed integration of LV DNA circles. After reverse transcription of the viral RNA, circular forms of the viral genomic DNA are generated by either non-homologous end joining (2-LTR) or homologous recombination (1-LTR). These circles are normally considered dead-end products of reverse transcription but the FRT site will enable DNA circles to become substrates for Flp recombination. In cells harboring an engineered FRT site flanked by a promoter and a start codon, Flp-mediated insertion of the virus-derived (i) 1-LTR circles and (ii) 2-LTR circles will generate a functional hygromycin B resistance expression cassette. To block the normal viral integration machinery, integration-defective lentiviral vectors (IDLVs), carrying an inactive viral integrase protein (harboring the D64A mutation), were utilized as carriers of viral RNA. Puro, puromycin resistance gene; LTR, long terminal repeat; FRT, Flp recombination target site.
Figure 2
Figure 2
Blockage of the normal viral integration machinery increases the formation of LV DNA circles in transduced cells. HEK-SB/FGIP cells were infected with integration-proficient or integration-deficient lentiviral vectors. Hirt DNA was harvested 24 hours post-transduction and subsequently used for Q-PCR analysis to determine the amount of linear and circular lentiviral DNA. Transductions were performed in triplicates and presented as mean value + SD.
Figure 3
Figure 3
Episomal lentiviral DNA can act both as a substrate for Flp recombination and as a source of Flp recombinase for site-directed genomic integration. (A) Flp-mediated integration of plasmids into FRT-tagged HEK-derived cell line. The HEK/FGIP1 cell line was transfected with pLV/FRT-hygro and pCMV-Flp or a negative control and selected for hygromycin B resistance. (B) Flp-mediated integration of lentiviral DNA circles. HEK/FGIP1 cells were transfected with pCMV-Flp or a negative control one day prior to transduction with the IDLV/FRT-hygro vector. (C) Transient expression of Flp recombinase from integration-defective LV vectors is sufficient for site-directed insertion of donor plasmid. HEK/FGIP1 cells were transduced with IDLV/PGK-Flp or a negative control and on the following day transfected with pLV/FRT-hygro. (D-E) IDLV-encoded Flp catalyzes site-directed genomic integration of lentiviral DNA circles. HEK/FGIP1-6 cells were co-transduced with IDLV/FRT-hygro and IDLV/PGK-Flp or a negative control. Transfections/transductions were performed in triplicates (except for in panel E in which n = 1) and presented as mean value + SD.
Figure 4
Figure 4
Southern blot and PCR analysis confirm precise insertion of lentiviral derived DNA circles (A) Southern blot analysis of representative hygromycin B-resistant clones containing inserted lentiviral circles. Genomic DNA was digested with XbaI, which cuts within the duplicated FRT sites, generating a ~5400/5630-bp fragment (1-LTR and 2-LTR insertions, respectively) recognized by the indicated hygro probe. Length differences between 1- and 2-LTR insertions could be resolved on a shorter exposure of the membrane (not shown). (B) PCR-based characterization of vector insertions in transduced cells. Genomic DNA from ten (taken from (3B)) and twelve (taken from (3D)) hygromycin B-resistant clones was used for PCR analysis to confirm site-specific insertion into an engineered FRT site (panel I–II and V–VI), integration of circular DNA substrates containing one or two LTRs (panel III and VII), and finally to verify that unspecific insertions of IDLV/FRT-hygro or IDLV/PGK-Flp had not occurred (panel IV and VIII–IX). N and P indicate negative (HEK/FGIP1) and positive (pLV/FRT-hygro or pLV/PGK-Flp) control clones, respectively.

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