UMG-lenti: novel dual-promoter lentiviral vectors for efficient transgene- and reporter protein expression in primitive haematopoietic progenitors.

Lentiviral vectors are widely used to investigate the biological effects of regulatory proteins and candidate oncogenes by stably enforcing their expression in diverse target cells. In these studies it is essential to be able to monitor and/or isolate the cell subpopulation(s) infected by the virus, and hence expressing the relevant transgene. This can be achieved through the insertion in the vectors of sequences encoding selectable markers or reporter proteins whose expression can be revealed by flow cytometry. To ensure simultaneous expression of transgene and selectable/ reporter gene, intra-ribosomal entry site (IRES) sequences are often inserted between the two cDNAs to generate bi-cistronic mRNAs whose transcription is directed by a single promoter. However, depending on the different transgenes and on the target cell types, the expression of the cDNA located downstream of the IRES sequences (typically encoding the reporter protein) is frequently inconsistent and this makes the recognition of the infected, transgene-expressing cells, problematic. To circumvent this problem, we have designed novel lentiviral vectors (named UMG-LV5 and -LV6) where the expression of the transgene is driven by the UbiC promoter and that of the reporter protein, EGFP, by the 170-bp-long minimal promoter of the WASP gene. These vectors, harbouring two distinct transgenes (ZNF521 or MSI2), were tested in a variety of human haematopoietic cell lines as well as in primary human CFD34+ cells in comparison with the FUIGW vector that contains the expression cassette UbiC-transgene-IRES-EGFP. In all cells studied, infection with UMG-LV5 and -LV6 resulted in a transgene expression slightly lower than that of FUIGW, but a considerably stronger expression of EGFP, that allowed to easily distinguish the transduced cells from the non-transduced counterpart. These vectors are therefore valuable new tools for gene transfer-based studies in haematopoietic stem and progenitor cells. Supported by: AIRC, PhD Programme in Molecular Oncology and POR Calabria FSE 2007/2013 (HEMMAS project).