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Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

Hamaguchi, I LU ; Woods, N B LU ; Panagopoulos, I LU ; Andersson, E ; Mikkola, H ; Fahlman, C ; Zufferey, R ; Carlsson, L ; Trono, D and Karlsson, S LU (2000) In Journal of Virology 74(22). p.84-10778
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... (More)

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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author
; ; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Animals, Cell Differentiation, Gene Expression, Gene Expression Regulation, Viral, Gene Transfer Techniques, Genetic Vectors, Green Fluorescent Proteins, HIV-1/genetics, Hematopoietic Stem Cells/physiology, Humans, Luminescent Proteins/genetics, Mice/embryology, Retroviridae/genetics, Transcription, Genetic, Transduction, Genetic, Transgenes, Vesicular stomatitis Indiana virus/genetics, Virus Integration
in
Journal of Virology
volume
74
issue
22
pages
84 - 10778
publisher
American Society for Microbiology
external identifiers
  • pmid:11044122
ISSN
0022-538X
DOI
10.1128/jvi.74.22.10778-10784.2000
language
English
LU publication?
yes
id
50866922-8e82-41cb-bf0a-ca87e02c252d
date added to LUP
2019-11-25 13:40:11
date last changed
2020-08-25 04:02:53
@article{50866922-8e82-41cb-bf0a-ca87e02c252d,
  abstract     = {<p>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.</p>},
  author       = {Hamaguchi, I and Woods, N B and Panagopoulos, I and Andersson, E and Mikkola, H and Fahlman, C and Zufferey, R and Carlsson, L and Trono, D and Karlsson, S},
  issn         = {0022-538X},
  language     = {eng},
  number       = {22},
  pages        = {84--10778},
  publisher    = {American Society for Microbiology},
  series       = {Journal of Virology},
  title        = {Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro},
  url          = {http://dx.doi.org/10.1128/jvi.74.22.10778-10784.2000},
  doi          = {10.1128/jvi.74.22.10778-10784.2000},
  volume       = {74},
  year         = {2000},
}