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CEA_SGF:E00004#TetOFF_Stat3F

ABSTRACT: The goal of this study was to identify the target genes of Stat3 involved in murine ES cell self-renewal. To achieve this goal, we used the Gs2 ES cell line expressing an inducible dominant negative mutant of Stat3 (Stat3F) in wich Y705 is mutated to phenilalanine. The Gs2 ES cells are routinely maintained in the presence of LIF 1000 U/ml and tetracycline 1µg/ml (LIF/ON). In this condition, Stat3F is not expressed and the cells are maintained in an undifferentiated phenotype. Upon tetracycline removal but inthe presence of LIF (Tet OFF condition) Stat3F is expressed and the cells differentiate despite the presence of LIF. We thus performed microarray analysis of the transcriptome of ES cells after 16h, 24h and 48h after Tetracycline removal (OFF16; OFF24 and OFF48) respectively)and compared them to Gs2 ES cells maintained in the continuous presence of LIF and tetracyclin (LIF/ON). Keywords: time-course

ORGANISM(S): Mus musculus

PROVIDER: GSE1151 | GEO | 2004/07/01

SECONDARY ACCESSION(S): PRJNA87279

REPOSITORIES: GEO

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Project description:This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org).The experiment was conducted at the Unyversity of Bath, Bath, UK. Aim is to identify genes regulated by PI3K-dependent signaling in undifferentiated ES cells. Materials and methods: E14tg2a cell line was used. Cells were cultured in Knock-out DMEM supplemented with 15% (v/v) Knock-out serum replacement, 1000U/ml LIF, 2mM glutamine, 50 ?M 2-mercaptothanol and 1x NEAA (as described in Handbook of work methods, 4.3.1.1). Cells were plated at 5x105 per 10cm gelatin-coated TC dish for 24h. ES cells were then starved for 24h in media lacking LIF and pre-incubated for 30 minutes with 5 <B5>M LY294002 (the concentration of this PI3K inhibitor that we have shown to perturb self-renewal in the presence of LIF and which knocks down PI3K signals) or DMSO alone. Cells were then stimulated with 103U/ml mLIF (Chemicon) for 2h (which gave maximal activation of STAT3), 24h, 48h or 72h.</p>\n<h3>Relationships between samples</h3>\n <p ALIGN="JUSTIFY">Samples were either maintained in LIF or LIF plus LY294002 (PI3K inhibitor) for the required treatment times.</p>\n<h3>Treatments</h3>\n <p ALIGN="JUSTIFY">Treatment times with PI3K inhibitor LY294002 at 5 <B5>M <96> 2h (n=5), 24h (n=3), 48h (n=3), 72h (n=3). DMSO was used as vehicle alone treatment control.</p>\n<h3>Culture conditions</h3>\n <p ALIGN="JUSTIFY">Gelatin-coated TC dishes (Nunc)</p>\n<h3>RNA isolation method</h3>\n <p ALIGN="JUSTIFY">RNA Easy with on-column DNase treatment</p>\n<h3>Experimental description</h3>\n <p ALIGN="JUSTIFY">This information is included above. Briefly, E14tg2a ES cells were either maintained in LIF or LIF plus LY294002 (5 <B5>M) for 2, 24, 48 or 72 hours, after which RNA was extracted from each sample and sent for expression profiling. During this time-course, only 72h time points plus LY294002 showed phenotypic evidence of differentiation. </p>\n

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CEA_SGF:E00004#TetOFF_Stat3F

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