UMG-Lenti: novel dual-promoter lentiviral vectors that ensure efficient transgene- and reporter protein expression in human hematopoietic stem and progenitor cells

Lentiviral vectors are widely used to investigate the biological effects of regulatory proteins and/or of candidate, leukaemia-associated oncogenes by stably enforcing their expression in haematopoietic stem and progenitor cells. For these studies it is critical to be able to monitor and/or sort the infected cells, that is usually accomplished using fluorescent proteins, such as eGFP, encoded by the modified viral genome. Currently the most widely-used strategy to ensure co-expression of transgene and selectable/ reporter gene is to insert between the two cDNAs an IRES sequence, thus generating bi-cistronic mRNAs whose transcription is directed by a single promoter. However, in many instances, only the gene upstream of the IRES is expressed strongly whereas the expression of the cDNA located downstream of the IRES sequences (typically encoding the reporter protein) is often inconsistent; this makes the detection of the transgene-expressing cells problematic. To overcome these limitations, we developed novel lentiviral vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UbiC promoter and that of the reporter protein, eGFP, by the minimal regulatory element (-170/+33) of the Wiskott-Aldrich syndrome protein (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 CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UbiC transgene IRESE eGFP. Both UMG-LV5 and UMG–LV6, yielded slightly lower transgene expression than FUIGW, but a considerably stronger expression of eGFP, that allowed to easily distingush between transduced and non-transduced cells. These vectors can therefore be considered new powerful tool for gene transfer based studies in haematopoietic stem and progenitor cells.