Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression Profiling Identifies Novel Gene Targets and Functions for Pdx1 in the Duodenum of Mature Mice

ABSTRACT: Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, less is known about the function of Pdx1 in the small intestine. We aim to investigate the role of Pdx1 in mature proximal small intestine by profiling the expression of genes differentially regulated in response to Pdx1 inactivation restricted to the intestinal epithelium in mice. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1flox/flox;VilCre mice, by crossing mutant mice homozygous for loxP site-flanked Pdx1 alleles with transgenic mice expressing Cre recombinase under the control of the mouse villin 1 gene promoter. Total RNA was isolated from the first five centimeters of the small intestine from adult Pdx1flox/flox;VilCre and littermate control mice. Microarray analysis was performed to investigate genome-wide transcriptional profiles in the proximal small intestine.

ORGANISM(S): Mus musculus

SUBMITTER: Chin Chen

PROVIDER: E-GEOD-29048 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Expression profiling identifies novel gene targets and functions for Pdx1 in the duodenum of mature mice.

Chen Chin C Sibley Eric E

American journal of physiology. Gastrointestinal and liver physiology 20111201 4

Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, the functions of Pdx1 in mature small intestine are less known. We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated in response to inactivation of Pdx1 specifically in the intestinal epithelium. Pdx1 was conditionally inactivated in the intestinal epithelium of ...[more]

PMID: 22135308

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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

Expression Profiling Identifies Novel Gene Targets and Functions for Pdx1 in the Duodenum of Mature Mice

Publications

Expression profiling identifies novel gene targets and functions for Pdx1 in the duodenum of mature mice.

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