UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

Abstract

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, 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 UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

Using a lentiviral Tet-regulated miR-E shRNA dual color vector to evaluate gene function in human leukemic stem cells in vivo

“…UMG LV6 MLL-AF9 mBlueberry2 was generated by swapping the AgeI-BsrGI mBlueberry2 fragment from the pLKO.1 mBlueberry2 vector into the UMG LV6 MLL-AF9 GFP vector (18), thereby replacing GFP with mBlueberry2. Generation of lentiviral viruses and transductions were performed as described previously (19).…”

Ferritin Heavy Subunit Silencing Blocks the Erythroid Commitment of K562 Cells via miR-150 up-Regulation and GATA-1 Repression

“…Stable transduction was achieved, as previously reported [ 19 , 23 ]. Transient transfections were performed using the Amaxa Nucleofactor kit (Lonza, Basel, Switzerland) [ 37 ]. FHC stable knock-down was verified by Western Blot analysis and RT-qPCR while FHC transient knockdown was checked at 24, 48 and 72 h by RT-qPCR analysis.…”

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

“…2 × 10 6 K562 cells were incubated in 5 ml of RPMI 10% FBS containing 100 nM TAT-GFP or TAT-BMI-1 for 20 mins at 37°C. Cytoplasmic and nuclear extracts were prepared as described by Chiarella et al [ 67 ] using hypotonic lysis (containing 10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, protease inhibitors (P8849 Sigma) for cytoplasmic proteins, and high-salt buffer (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, protease inhibitors) to recover nuclear proteins. The enrichment of the cytosolic proteins was confirmed by Western blotting with mouse anti-actin (A4700 Sigma)1:10000; that of nuclear proteins by rabbit anti-HDAC1 1:10000 (H3284 Sigma).…”