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- Hamaguchi, I, et al.
(författare)
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Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro
- 2000
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 74:22, s. 84-10778
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Tidskriftsartikel (refereegranskat)abstract
- The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.
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| 3. |
- Mikkola, H, et al.
(författare)
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Lentivirus gene transfer in murine hematopoietic progenitor cells is compromised by a delay in proviral integration and results in transduction mosaicism and heterogeneous gene expression in progeny cells
- 2000
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 74:24, s. 8-11911
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Tidskriftsartikel (refereegranskat)abstract
- Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were used to transduce murine Lin(-) c-kit(+) Sca1(+) primitive hematopoietic progenitor cells. Following transduction, the cells were plated into hematopoietic progenitor cell assays in methylcellulose and the colonies were scored for GFP positivity. After incubation for 20 h, lentivirus vectors transduced 27.3% +/- 6.7% of the colonies derived from unstimulated target cells, but transduction was more efficient when the cells were supported with stem cell factor (SCF) alone (42. 0% +/- 5.5%) or SCF, interleukin-3 (IL-3), and IL-6 (53.3 +/- 1.8%) during transduction. The, vesicular stomatitis virus glycoprotein-pseudotyped MGIN oncoretrovirus control vector required IL-3, IL-6, and SCF for significant transduction (39.3 +/- 9.4%). Interestingly, only a portion of the progeny cells within the lentivirus-transduced methylcellulose colonies expressed GFP, in contrast to the homogeneous expression in oncoretrovirus-transduced colonies. Secondary plating of the primary GFP(+) lentivirus vector-transduced colonies revealed vector PCR(+) GFP(+) (42%), vector PCR(-) GFP(-) (46%), and vector PCR(+) GFP(-) (13%) secondary colonies, indicating true genetic mosaicism with respect to the viral genome in the progeny cells. The degree of vector mosaicism in individual colonies could be reduced by extending the culture time after transduction and before plating into the clonal progenitor cell assay, indicating a delay in the lentiviral integration process. Furthermore, supplementation with exogenous deoxynucleoside triphosphates during transduction decreased mosaicism within the colonies. Although cytokine stimulation during transduction correlates with higher transduction efficiency, rapid cell division after transduction may result in loss of the viral genome in the progeny cells. Therefore, optimal transduction may require activation without promoting intense cell proliferation prior to vector integration.
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- Woods, N B, et al.
(författare)
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Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells
- 2000
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Ingår i: Blood. - 0006-4971. ; 96:12, s. 33-3725
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Tidskriftsartikel (refereegranskat)abstract
- The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)
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