Abstract
RNA interference methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering small interfering RNA (siRNA) into mesenchymal stem cells and neural stem cells. In this study, we used three transfection reagents (Lipofectamine RNAiMAX, Oligofectamine and Lipofectamine 2000) to deliver siRNA into human embryonic stem (hES) cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and Oct4, a key regulator of pluripotency. As a result, siRNA can be delivered into hES cells more efficiently by Lipofectamine RNAiMAX compared with the other two transfection reagents and high efficient knockdown of target genes was obtained by Lipofectamine RNAiMAX even at a low concentration of siRNA. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.
Elbashir, S. M., Harborth, J., Lendeckel, W., et al. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498.
McManus, M. T., & Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nature Reviews. Genetics, 3, 737–747.
Liu, Y. P., Dambaeva, S. V., Dovzhenko, O. V., et al. (2005). Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells. Stem Cells and Development, 14, 487–492.
Chen, S., Choo, A., Wang, N. D., et al. (2007). Establishing efficient siRNA knockdown in mouse embryonic stem cells. Biotechnology Letters, 29, 261–265.
Zaehres, H., Lensch, M. W., Daheron, L., et al. (2005). High-efficiency RNA interference in human embryonic stem cells. Stem Cells, 23, 299–305.
Ren, C. P., Zhao, M., Shan, W. J., et al. (2005). Establishment of human embryonic stem cell line stably expressing Epstein-Barr virus-encoded nuclear antigen 1. Acta Biochimica et Biophysica Sinica (Shanghai), 37, 68–73.
Ren, C. P., Zhao, M., Yang, X. Y., et al. (2006). Establishment and applications of epstein-barr virus-based episomal vectors in human embryonic stem cells. Stem Cells, 24, 1338–1347.
Zhao, M., Ren, C. P., Yang, H., et al. (2007). Transcriptional profiling of human embryonic stem cells and embryoid bodies identifies HESRG, a novel stem cell gene. Biochemical and Biophysical Research Communications, 362, 916–922.
Zaehres, H., & Daley, G. Q. (2006). Transgene expression and RNA interference in embryonic stem cells. Methods in Enzymology, 420, 49–64.
Nichols, J., Zevnik, B., Anastassiadis, K., et al. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95, 379–391.
Hay, D. C., Sutherland, L., Clark, J., et al. (2004). Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells, 22, 225–235.
Masui, S., Nakatake, Y., Toyooka, Y., et al. (2007). Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nature Cell Biology, 9, 625–635.
Boyer, L. A., Lee, T. I., Cole, M. F., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 122, 947–956.
Lim, L. S., Loh, Y. H., Zhang, W., et al. (2007). Zic3 is required for maintenance of pluripotency in embryonic stem cells. Molecular Biology of the Cell, 18, 1348–1358.
Wang, Z. X., The, C. H., Kueh, J. L., et al. (2007). Oct4 and Sox2 directly regulate expression of another pluripotency transcription factor, Zfp206, in embryonic stem cells. Journal of Biological Chemistry, 282, 12822–12830.
Matin, M. M., Walsh, J. R., Gokhale, P. J., et al. (2004). Specific knockdown of Oct4 and beta2-microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells, 22, 659–668.
Hyslop, L., Stojkovic, M., Armstrong, L., et al. (2005). Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells, 23, 1035–1043.
Babaie, Y., Herwig, R., Greber, B., et al. (2007). Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells, 25, 500–510.
Niwa, H., Miyazaki, J., & Smith, A. G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet, 24, 372–376.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 30200140) and the Natural Science Foundation of Hunan Province, China (Grant No. 07JJ3038). We gratefully acknowledge Dr. J. A. Thomson and WiCell Research Institute for providing us hES cells.
Author information
Authors and Affiliations
Corresponding author
Additional information
Ming Zhao and Hong Yang contributed equally to this work.
Rights and permissions
About this article
Cite this article
Zhao, M., Yang, H., Jiang, X. et al. Lipofectamine RNAiMAX: An Efficient siRNA Transfection Reagent in Human Embryonic Stem Cells. Mol Biotechnol 40, 19–26 (2008). https://doi.org/10.1007/s12033-008-9043-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-008-9043-x