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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-LVs efficiency in non hematopoietic cells.(A) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (B) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.
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pone-0114795-g008: UMG-LVs efficiency in non hematopoietic cells.(A) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (B) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.

Mentions: The fragment of the WASP regulatory region used in the construction of the UMG-LV5 and UMG-LV6 vectors has been shown to direct the expression of the reporter gene in a tightly hematopoietic-specific manner [33] and therefore it would be expected to be functionally silent in other cell types. However, when used to infect human and murine non-hematopoietic cell lines derived from various tissues (human embryonyc kidney cells HEK293T, mouse mesenchymal stromal cells MS-5, mouse embryonic fibroblasts NIH3T3, and human medulloblastoma cells DAOY) UMG-LV6 promoted in all cases strong expression of both EGFP (Fig. 8A, 8B) and of transgene (Fig. 8B), comparable to those induced by UMG-LV11, suggesting that the functional interaction with the adjacent UBC promoter may overcome the tissue-specificity of the WASP regulatory element.


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-LVs efficiency in non hematopoietic cells.(A) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (B) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114795-g008: UMG-LVs efficiency in non hematopoietic cells.(A) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (B) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.
Mentions: The fragment of the WASP regulatory region used in the construction of the UMG-LV5 and UMG-LV6 vectors has been shown to direct the expression of the reporter gene in a tightly hematopoietic-specific manner [33] and therefore it would be expected to be functionally silent in other cell types. However, when used to infect human and murine non-hematopoietic cell lines derived from various tissues (human embryonyc kidney cells HEK293T, mouse mesenchymal stromal cells MS-5, mouse embryonic fibroblasts NIH3T3, and human medulloblastoma cells DAOY) UMG-LV6 promoted in all cases strong expression of both EGFP (Fig. 8A, 8B) and of transgene (Fig. 8B), comparable to those induced by UMG-LV11, suggesting that the functional interaction with the adjacent UBC promoter may overcome the tissue-specificity of the WASP regulatory element.

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