Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of pancreatic buds from wild type and Ipf1/Pdx1 knock-out mouse embryos

ABSTRACT: Identification of genes regulated by IPF1/PDX1 in early pancreatic progenitor cells, analysis of Ipf1/Pdx1 global null mutants e10.5 embryonic pancreatic buds compared to wt

INSTRUMENT(S): G2565BA DNA microarray scanner [Agilent]

ORGANISM(S): Mus musculus

SUBMITTER: Cecilia Williams

PROVIDER: E-MEXP-1047 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene array identification of Ipf1/Pdx1-/- regulated genes in pancreatic progenitor cells.

Svensson Per P Williams Cecilia C Lundeberg Joakim J Rydén Patrik P Bergqvist Ingela I Edlund Helena H

BMC developmental biology 20071123

<h4>Background</h4>The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in beta-cells leads to impaired beta-cell function and diabetes. Although several putative target genes have been linked to the beta-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells.<h4>Results</h4>Microarra ...[more]

PMID: 18036209

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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

Transcription profiling of pancreatic buds from wild type and Ipf1/Pdx1 knock-out mouse embryos

Publications

Gene array identification of Ipf1/Pdx1-/- regulated genes in pancreatic progenitor cells.

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