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Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro.
I Hamaguchi1, N B Woods, I Panagopoulos
1Molecular Medicine and Gene Therapy, Department of Medicine, Lund University Hospital, Lund, Sweden.
Journal of Virology
|October 24, 2000
Summary
Lentivirus vectors efficiently transduce mouse embryonic stem cells and maintain transgene expression during differentiation, unlike oncoretroviruses. This enables studies of gene function in stem cell development and hematopoietic cell generation.
Area of Science:
- Gene therapy
- Stem cell biology
- Virology
Background:
- Murine embryonal stem cell virus (MESV) long terminal repeat (LTR) promoter drives transgene expression in undifferentiated ES cells, but not upon differentiation.
- Efficient gene delivery and sustained expression in differentiating stem cells are crucial for various applications.
Purpose of the Study:
- To evaluate the efficiency of a lentivirus vector, pseudotyped with vesicular stomatitis virus G protein (VSV-G), in transducing mouse embryonic stem (ES) cells.
- To compare transgene expression from a lentivirus vector with an internal phosphoglycerate kinase (PGK) promoter against an oncoretrovirus vector with an MESV LTR during ES cell differentiation.
- To assess the potential of lentivirus vectors for gene function studies during ES cell development and hematopoietic differentiation.
Main Methods:
- Transduction of CCE ES cells using VSV-G pseudotyped lentivirus and oncoretrovirus vectors expressing green fluorescent protein (GFP).
- Analysis of transduction efficiency via fluorescence-activated cell sorting (FACS).
- Differentiation of transduced ES cells into embryoid bodies (EBs) and hematopoietic cells in vitro, followed by assessment of GFP expression.
Main Results:
- VSV-G pseudotyping enabled highly efficient transduction of ES cells by both lentivirus (99.8% GFP+) and oncoretrovirus (86.7% GFP+) vectors.
- Oncoretrovirus vector LTR-driven expression was significantly reduced or silenced upon differentiation into EBs and hematopoietic cells.
- Lentivirus vector-driven GFP expression persisted throughout in vitro differentiation to EBs and in derived hematopoietic cells.
Conclusions:
- VSV-G pseudotyping is effective for high-efficiency ES cell transduction.
- Lentivirus vectors with internal promoters like PGK offer sustained transgene expression during ES cell differentiation, unlike oncoretrovirus LTRs.
- This system provides a valuable tool for analyzing lentivirus vector design and for gain-of-function studies in stem cell development and hematopoietic differentiation.