Abstract
We described a protocol for dissecting the function of an important serine/threonine protein kinase, mammalian target of rapamycin (mTOR), in regulating the long-term undifferentiated growth of human embryonic stem cells (hESCs). The function of mTOR in hESCs was inactivated with a highly specific chemical inhibitor, rapamycin, and gene-specific small-hairpin RNAs, and the effects were evaluated under self-renewal or early differentiation conditions. We found that inactivation of mTOR impairs proliferation and enhances mesoderm and endoderm activities of hESCs. This protocol described a general strategy for studying the function of key genes and signaling events during hESC long-term self-renewal and early lineage specifications with pharmacological and genetic approaches.
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Acknowledgments
The authors would like to thank the Wang lab for helpful discussion. The authors’ work is supported by grants from the National Institute of Health (GM083812 and GM083601), the Illinois Regenerative Medicine Institute (IDPH 2006-05516), NSF CAREER award (0953267), NSF-EBICS award, the Beckman award from the University of Illinois, and the National Natural Science Foundation of China (30728022).
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Zhou, J., Li, D., Wang, F. (2012). Assessing the Function of mTOR in Human Embryonic Stem Cells. In: Weichhart, T. (eds) mTOR. Methods in Molecular Biology, vol 821. Humana Press. https://doi.org/10.1007/978-1-61779-430-8_23
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DOI: https://doi.org/10.1007/978-1-61779-430-8_23
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Publisher Name: Humana Press
Print ISBN: 978-1-61779-429-2
Online ISBN: 978-1-61779-430-8
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