Abstract
Embryonic stem (ES) cells are an important source of stem cells in tissue engineering and regenerative medicine because of their high self-renewal capacities and differentiation potentials. However, the detailed molecular mechanisms controlling the differentiation and renewal programs in ES cells remained unclear. One of the difficulties in understanding these mechanisms substantially results from the low efficacies of gene manipulation by delivering exogenous gene expression or knockdown of endogenous gene expression with small interfering RNA (siRNA) in ES cells. Here we describe an optimized protocol for efficiently transfecting mouse ES cells by Effectene, a liposome-based method. The high transfection efficiency in mouse ES cells is demonstrated in this chapter by (1) achieving a percentage of enhanced green fluorescence protein (EGFP) expression in >98% embryoid bodies after introducing plasmids encoding the protein and (2) decreased SOX-2 and Oct-3/4 expression and subsequent morphological evidence of cell differentiation after introducing siRNA expression for suppressing SOX-2 and Oct-3/4, which are known to be essential for maintenance of stem cell properties in mouse ES cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Evans, M. J., Kaufman, M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.
Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634–7638.
Bugeon, L., Syed, N., Dallman, M. J. (2000) A fast and efficient method for transiently transfecting ES cells: Application to the development of system for conditional gene expression. Transgenic Res 9, 229–232.
Ward, C. M., Stern, P. L. (2002) The human cytomegalovirus immediate early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells. Stem Cells 20, 472–475.
Lakshmipathy, U., Pelacho, B., Sido, K., Linehan, J. L., Coucouvanis, E., Kaufman, D. S. et al. (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22, 531–543.
Schaniel, C., Li, F., Schafer, X., Moore, T., Lemischka, I.R., Paddison, P. J. (2006) Delivery of short hairpin RNAs-triggers of gene silencing-into mouse embryonic stem cells. Nat Methods 3, 397–400.
Hough, S. R., Clements, I., Welch, P. J., Wiederholt, K. A. (2006) Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. Stem Cells 24, 1467–1475.
Chen, S., Choo, A., Wang, N. D., Too, H. P., Oh, S. K. (2007) Establishing efficient siRNA knockdown in mouse embryonic stem cells. Biotechnol Lett 29, 261–265.
Ko, B.S., Chang, T.C., Shyue, S. K., Chen, Y.C., Liou, J.Y. (2009) An efficient transfection method for mouse embryonic stem cells. Gene Therapy 16, 154–158.
Acknowledgments
This work was supported by grants from Taiwan, National Health Research Institutes (NHRI-97A1-CVPP03-014) and National Science Council (NSC97-3111-B-400-004).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Liou, JY., Ko, BS., Chang, TC. (2010). An Efficient Transfection Method for Mouse Embryonic Stem Cells. In: Zhang, B., Stellwag, E. (eds) RNAi and microRNA-Mediated Gene Regulation in Stem Cells. Methods in Molecular Biology, vol 650. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-769-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-60761-769-3_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-768-6
Online ISBN: 978-1-60761-769-3
eBook Packages: Springer Protocols