title: Assessing the function of mTOR in human embryonic stem cells.
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.
authors: Department of Cell and Developmental Biology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. jiaxi@life.uiuc.edu
authors: Jiaxi
authors: J
authors: Zhou
authors: Dong
authors: D
authors: Li
authors: Fei
authors: F
authors: Wang
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000201:enzymology*; D004707:Endoderm / Q000166:cytology / Q000187:drug effects; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects; D051379:Mice; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus