Abstract
The distinctive properties of human embryonic stem cells (hESCs) enable them to provide unique models to study the network of signaling pathways that regulate organogenesis, generate disease models, produce cells and tissues for therapies, and identify new drugs for treatment. Genetic modification of hESCs is a powerful tool to assist the above studies. Generation of lineage-specific fluorescent protein reporter hESC lines will greatly benefit investigators to monitor specific cell lineages in a live, easy, and timely manner. This technique will facilitate high throughput screening to identify molecules important in regulating specific cell fate commitment. In addition, such reporter cell lines enable researchers to enrich certain cell populations by fluorescent activated cell sorting (FACS) for either downstream biological analysis or in vivo applications. We have shown that hESCs can be stably transfected with a plasmid in which expression of the green fluorescent protein (GFP) is under the control of the Oct-4 promoter using chemical transfection. The expression pattern of transgenic Oct-4-GFP reflects that of endogenous Oct-4.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., and Bongso, A. (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M. (1998) Embryonic stem cell lines derived from human blastocysts. Science. 282, 1145–1147.
Niwa, H. (2007) How is pluripotency determined and maintained? Development. 134, 635–646.
Walker, E., Ohishi, M., Davey, R. E., Zhang, W., Cassar, P. A., Tanaka, T. S., et al. (2007) Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment. Cell Stem Cell. 1, 71–86.
Giudice, A. and Trounson, A. (2008) Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell. 2, 422–433.
Tomishima, M. J., Hadjantonakis, A. K., Gong, S., and Studer, L. (2007) Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library. Stem Cells. 25, 39–45.
Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., and Donovan, P. J. (2008) Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells. Stem Cells. 26, 1436–1443.
Gerrard, L., Zhao, D., Clark, A. J., and Cui, W. (2005) Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells. 23, 124–133.
Gallo, P., Grimaldi, S., Latronico, M. V., Bonci, D., Pagliuca, A., Gallo, P., et al. (2008) A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells. Gene Ther. 15, 161–170.
Alami, R., Greally, J. M., Tanimoto, K., Hwang, S., Feng, Y. Q., Engel, J. D., et al. (2000) Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice. Hum. Mol. Genet. 9, 631–636.
Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., et al. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686.
Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., and Carpenter, M. K. (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Noisa, P., Urrutikoetxea-Uriguen, A., Cui, W. (2011). Generation of Human Embryonic Stem Cells Carrying Lineage Specific Reporters. In: Nieden, N. (eds) Embryonic Stem Cell Therapy for Osteo-Degenerative Diseases. Methods in Molecular Biology, vol 690. Humana Press. https://doi.org/10.1007/978-1-60761-962-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-60761-962-8_6
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-961-1
Online ISBN: 978-1-60761-962-8
eBook Packages: Springer Protocols