Expression and recombination of the EGFP and EYFP genes in lentiviral vectors carrying two heterologous promoters

Abstract

Expressing two genes in the progeny of stem and progenitor cells that are transduced with a unique viral vector is desirable in certain situations. We tested the ability of two lentiviral vectors to transduce human cells of hematopoietic origin and concomitantly express two reporter genes, either EGFP (enhanced green fluorescent protein) and DsRed2, or EGFP and EYFP (enhanced yellow fluorescent protein), from two internal promoters. The vectors were generated from the pTRIP deltaU3 EF1alpha EGFP lentiviral vector. Following transduction of hematopoietic and non-hematopoietic cell lines, we performed FACS, PCR and Southern blot analyzes to quantify transduction, integration efficiencies and size of integrated lentiviral vectors, respectively. The detection of DsRed2 fluorescence appeared unexpectedly low in human cells of hematopoietic origin. Alternatively, a modification in the flow cytometry assay allowed us to distinguish between the two overlapping fluorescence signals emitted by EGFP and EYFP, when transduced cells were excited with a 488-nm laser beam. However, the low frequency of double-positive EGFP+ EYFP+ cells, and the existence of single-positive, mostly EGFP- EYFP+, cells, prompted us to search for recombinations in the vector sequence. Southern blotting of DNA obtained from transduced cells indeed demonstrated that recombination had occurred between the two closely related EGFP and EYFP sequences. These observations suggest that recombination occurred within the EGFP and EYFP genes, which differ by only four amino acids. We conclude that the insertion of two highly homologous sequences into a lentiviral backbone can favor recombination.