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UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Chiarella E, Carrà G, Scicchitano S, Codispoti B, Mega T, Lupia M, Pelaggi D, Marafioti MG, Aloisio A, Giordano M, Nappo G, Spoleti CB, Grillone T, Giovannone ED, Spina R, Bernaudo F, Moore MA, Bond HM, Mesuraca M, Morrone G - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.

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.

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UMG-LV11 promotes efficient transgene- and reporter gene expression in human hematopoietic cell lines.The cell lines indicated were infected as detailed in materials and methods with FUIGW, UMG-LV5 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521. As a control, void FUIGW vector was used. (A) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (B) Nuclear and cytosolic extracts were analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.
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pone-0114795-g004: UMG-LV11 promotes efficient transgene- and reporter gene expression in human hematopoietic cell lines.The cell lines indicated were infected as detailed in materials and methods with FUIGW, UMG-LV5 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521. As a control, void FUIGW vector was used. (A) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (B) Nuclear and cytosolic extracts were analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.

Mentions: In bicistronic vectors, the translational efficiency is known to be variable in a manner that depends on the cell type and on the nature of the genes flanking the IRES element. In particular, it has been reported that while the cap-dependent translation of the upstream cDNA is relatively consistent, the IRES-dependent translation of the downstream gene is significantly influenced by the gene located upstream of the IRES [32]. We therefore asked whether inverting the positions of the cDNAs for the reporter protein and for the protein of interest, relative to the IRES sequence, may result in a more robust expression of both proteins. To this end, we constructed a new vector - named UMG-LV11 – where the EGFP cDNA was inserted upstream of the IRES, whereas the multiple cloning site for insertion of the transgene was downstream. The cDNA for ZNF521 was subcloned in UMG-LV11, and this vector was assayed on three haematopoietic cell lines in comparison with UMG-LV5-ZNF521. As shown in Fig. 4A, UMG-LV11-ZNF521 induced a strong expression of EGFP in all cell lines tested, fully comparable to that of UMG-LV5-ZNF521, although it displayed a slightly lower transduction efficiency.


UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Chiarella E, Carrà G, Scicchitano S, Codispoti B, Mega T, Lupia M, Pelaggi D, Marafioti MG, Aloisio A, Giordano M, Nappo G, Spoleti CB, Grillone T, Giovannone ED, Spina R, Bernaudo F, Moore MA, Bond HM, Mesuraca M, Morrone G - PLoS ONE (2014)

UMG-LV11 promotes efficient transgene- and reporter gene expression in human hematopoietic cell lines.The cell lines indicated were infected as detailed in materials and methods with FUIGW, UMG-LV5 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521. As a control, void FUIGW vector was used. (A) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (B) Nuclear and cytosolic extracts were analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264771&req=5

pone-0114795-g004: UMG-LV11 promotes efficient transgene- and reporter gene expression in human hematopoietic cell lines.The cell lines indicated were infected as detailed in materials and methods with FUIGW, UMG-LV5 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521. As a control, void FUIGW vector was used. (A) Flow-cytometric analysis of EGFP expression in cells exposed to the relevant vectors. The percentages of EGFP-positive cells are indicated. (B) Nuclear and cytosolic extracts were analyzed by Western blotting for FLAG-ZNF521 and EGFP expression respectively. HDAC1 was used as a control for the amounts of extract loaded.
Mentions: In bicistronic vectors, the translational efficiency is known to be variable in a manner that depends on the cell type and on the nature of the genes flanking the IRES element. In particular, it has been reported that while the cap-dependent translation of the upstream cDNA is relatively consistent, the IRES-dependent translation of the downstream gene is significantly influenced by the gene located upstream of the IRES [32]. We therefore asked whether inverting the positions of the cDNAs for the reporter protein and for the protein of interest, relative to the IRES sequence, may result in a more robust expression of both proteins. To this end, we constructed a new vector - named UMG-LV11 – where the EGFP cDNA was inserted upstream of the IRES, whereas the multiple cloning site for insertion of the transgene was downstream. The cDNA for ZNF521 was subcloned in UMG-LV11, and this vector was assayed on three haematopoietic cell lines in comparison with UMG-LV5-ZNF521. As shown in Fig. 4A, UMG-LV11-ZNF521 induced a strong expression of EGFP in all cell lines tested, fully comparable to that of UMG-LV5-ZNF521, although it displayed a slightly lower transduction efficiency.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Haematopoiesis and Stem Cell Biology, Dept. of Experimental and Clinical Medicine, University of Catanzaro Magna Græcia, 88100, Catanzaro, Italy.

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.

Show MeSH
Related in: MedlinePlus