Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptional profiling of mouse pancreatic beta-cells, embryonic stem cells and 10 other normal adult tissues.

ABSTRACT: To gain insights into how pancreatic beta-cells are programmed in vivo, we profiled key histone methylations (H3K4/K27me3) in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 other adult tissues (all under normal, untreated conditions). For these cells we also purified RNA to analyze tissue specfic genome wide transcription levels in relation to histone modifications.

ORGANISM(S): Mus musculus

SUBMITTER: joris van arensbergen

PROVIDER: E-TABM-877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program.

van Arensbergen Joris J García-Hurtado Javier J Moran Ignasi I Maestro Miguel Angel MA Xu Xiaobo X Van de Casteele Mark M Skoudy Anouchka L AL Palassini Matteo M Heimberg Harry H Ferrer Jorge J

Genome research 20100415 6

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ec ...[more]

PMID: 20395405

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Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.

Dataset Information

Transcriptional profiling of mouse pancreatic beta-cells, embryonic stem cells and 10 other normal adult tissues.

Publications

Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program.

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