title: 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.
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Chiarella
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Carrà
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Scicchitano
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Bruna
authors.initials: B
authors.lastname: Codispoti
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Mega
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Lupia
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pelaggi
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Marafioti
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Aloisio
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Giordano
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Nappo
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Cristina B
authors.initials: CB
authors.lastname: Spoleti
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Grillone
authors.affiliation: Laboratory of Molecular Oncology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Emilia D
authors.initials: ED
authors.lastname: Giovannone
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Spina
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bernaudo
authors.affiliation: Dept. of Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, 10065, United States of America.
authors.forename: Malcolm A S
authors.initials: MA
authors.lastname: Moore
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Heather M
authors.initials: HM
authors.lastname: Bond
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Mesuraca
authors.affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Morrone
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins