Abstract
The major limitations in gene transduction to embryonic stem (ES) cells are (1) low efficiency of gene delivery and (2) suppression of gene expression after integration into the host genome. A human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector has been demonstrated to be an excellent tool for stable and efficient gene expression in ES cells. Here, we introduce a protocol for lentiviral vector-mediated transgene expression in murine ES cells. Using lentiviral vectors expressing LacZ, green fluorescent protein, and Cre recombinase, we demonstrate the efficiency and utility of the vectors in ES cell study.
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References
Gassmann M., Donoho G., and Berg B. (1995) Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 92, 1292–1296.
Camenisch, Gruber M., Donoho G., Van Sloun P., Wenger R. H., and Gassmann M. (1996) A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells. Nucleic Acids Res. 24, 3707–3713.
Niwa H., Burdon T., Chambers I., and Smith A. (1998) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–C2060.
Hamaguchi I., Woods N. B., Panagopoulos I., et al. (2000) Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro. J. Virol. 74, 10,778–C10,784.
Pfeifer A., Ikawa M., Dayn Y., and Verma I. M. (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA 99, 2140–C2145.
Lois C., Hong E. J., Pease S., Brown E. J., and Baltimore D. (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–C872.
Iwakuma T., Cui Y., and Chang L. J. (1999) Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261, 120–132.
Zennou V., Petit C., Guetard D., Nerhbass U., Montagnier L., and Charneau P. (2000) HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101, 173–185.
Sirven A., Pflumio F., Zennou V., et al. (2000) The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 96, 4103–4110.
Srinivas S., Watanabe T., Lin C. S., et al. (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4.
Zaiss A. K., Son S., and Chang L. J. (2002) RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy. J. Virol. 76, 7209–7219.
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© 2006 Humana Press Inc.
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Oka, M., Chang, LJ., Costantini, F., Terada, N. (2006). Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 329. Humana Press. https://doi.org/10.1385/1-59745-037-5:273
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DOI: https://doi.org/10.1385/1-59745-037-5:273
Publisher Name: Humana Press
Print ISBN: 978-1-58829-498-2
Online ISBN: 978-1-59745-037-9
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