Recombination Occurs in Lentiviral Vectors Designed To Express Both EGFP and EYFP in Human Hematopoietic Cells.

Recombinant lentiviruses are widely used vectors for in vitro and in vivo long-term gene transfer. Many of the initial gene transfer studies were conducted using single reporter genes. However, for many gene delivery applications, expression and detection of two genes are useful. The goal of this study was to explore the feasibility of directing the independent expression of two marker genes from a single HIV-1 derived lentiviral vector. We used the enhanced green fluorescent protein (EGFP) and the enhanced yellow fluorescent protein (EYFP) as markers. Because of spectral overlap of these fluorescent proteins, we modified the optical bench of a FACScalibur flow cytometer (BDIS), with insertion of Omega Optical filters, in order to detect the two emitted fluorescences. To independently co-express the marker genes, we tested two different promoter associations in the same lentiviral backbone. In the first case, the EF1α promoter drove EGFP transcription, and EYFP was translated from spliced RNA transcribed from the LTR promoter. In the second case, two internal promoters : EF1α and CMV were inserted in a SIN (Self Inactivating) vector. Different vectors were generated from the pTRIP deltaU3 EF1α EGFP and pTRIP EF1α EGFP lentiviral vectors (gifts from P. Charneau), with transcriptional units and viral elements in variable positions. Following transduction of hematopoietic and non-hematopoietic cell lines, FACS and PCR analyses allowed to quantify transduction and integration efficiencies, respectively. Addition of EYFP in the original single gene lentiviral vector resulted in a decrease in the overall percentage of fluorescent cells. Moreover, vectors using splice mechanism produced weak co-expression of both genes (less than 4% of double positive cells). However, one of the SIN vector that contains two internal promoters transduced 32 to 100% of Nalm6 human pre-B cells, depending on the MOI; of these fluorescent cells, 12 to 78% were double positive. The size of the provirus was assessed in transduced cells, using southern blotting. Cells transduced with vectors coding for the two marker genes contained DNA sequences with sizes below the expected values; these observations suggest that recombination occurred within the vector sequence during or after transduction. This was further confirmed by southern blotting of sorted single and double positive cells; again, bands with lower than expected sizes were detected. We conclude that two highly homologous sequences can lead to recombination when inserted in close positions into a lentiviral backbone. This conclusion was reinforced by the observation that substitution of the DsRed2 coding sequence to EYFP in the same lentiviral vector that also encodes EGFP produced 100% double positive cells following transduction.