Microarray Analysis of MIN6 cells treated with Exendin-4 over time
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ABSTRACT: The aim of the study was to investigate the effect of Exendin-4 on MIN6 cells allowing for the elucidation of the various transcriptional programs initiated by this multifunctional peptide hormone. MIN6 cells were treated with Exendin-4 for 24, 48 and 72 hours. After these time points RNA was isolated and used for hybridization (on Cy3) against matched control MIN6 cells (on Cy5) using the Mouse PancChip 5.0 . Three assays were carried out for each time point.
Project description:MafA and MafB transcription factors have been shown to be key regulators of insulin and glucagon transcription. MafB is essential for alpha and beta cell differentiation, as MafB deficient mice produced fewer insulin+ and glucagon+ cells during development, with MafA expressed in remaining insulin+ cells. In contrast, beta cell development was reported to be normal in a total MafA knock out, although the animals developed beta cell dysfunction and diabetes as adults. However, we have found that MafB expression is elevated during development and retained in adult insulin+ cells after conditional removal of MafA in the pancreas. These studies will evaluate the broader significance of these insulin and glucagon regulators in alpha and beta cell development and function. Our efforts will focus on determining if the concerted actions of MafA and MafB factors are significant to beta cell formation, and we specifically plan to: Determine how alpha and beta cell differentiation is affected in MafA/MafB compound mutant mice during pancreas development. cDNA microarray studies (pancchip 6.0) with wild type, MafAKO, MafB-/-, and MafAKOMafB-/- mutant E18.5 pancreata will be performed to comprehensively identify genes controlled by MafA and MafB in developing alpha and beta cells.
Project description:Cytokines IL-1 and IFN-gamma have been shown to be the primary effectors of beta-cell destruction during the immune response. The aim of this study was to identify the gene expression changes that are associated with resistance of beta cells to cytokines. INS-1 rat insulinoma cells were grown in increasing doses of cytokines until a resistance phenotype had developed. RNA was prepared from these cytokine resistant sublines as well as the original, cytokine-sensitive parental INS-1 cells in Dr. Chris Newgard's lab, quantified and sent frozen in water. 3-7 biological replicates per condition were sent for the following three conditions: (i) 834/40, Cytokine-sensitive (CS), (ii) 833/15 and 833/117, Cytokine resistant grown in the absence of cytokines (CRu), (iii) 833/15C and 833/117C: Cytokine Resistant Grown in the Presence of Cytokines (CRt)
Project description:Diabetes mellitus results from an inadequately functioning beta-cell mass. In the adult pancreas, beta-cell mass is dynamic, increasing to meet metabolic demands and decreasing with metabolic or injury insults. Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor agonist that augments beta-cell mass by increasing beta-cell neogenesis and proliferation and by reducing apoptosis. We utilized a cDNA microarray approach to identify genes that are differentially regulated during islet growth after Ex-4 treatment or a partial pancreatectomy (Ppx). Mice underwent 50% Ppx or sham operation and received Ex-4 or vehicle every 24 hours. cDNA prepared from total pancreatic RNA isolated at 12, 24 and 48 hrs after surgery was hybridized to the PancChip 4.0 microarray.
Project description:The purpose of this study was to identify direct targets of the glucocorticoid receptor (GR) using an orthogonal analysis. An expression study of mouse livers in the presence or absence of exogenous glucocorticoid complemented a genome-wide location analysis on chromatin from the same livers. These were hybridized to the BCBC Mouse PancChip 5.0 and the Mouse PromoterChip BCBC-3.0 respectively.
Project description:The aim of this study was to test, in vivo, if Foxa2 inhibits HNF6-mediated transcription in the liver. We utilized hepatocyte-specific gene ablation of Foxa2 and the Mouse PromoterChip BCBC 3.0 and Mouse PancChip 5.0 cDNA microarrays to investigate HNF6 binding to its target promoters in vivo in the presence or absence of Foxa2. For the mouse promoter microarray analysis, chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2loxP/loxP Alfp.Cre and control mouse livers. Along with sheared genomic DNA (common reference), the immunoprecipitated DNA was amplified, labeled and hybridized to the Mouse PromoterChip BCBC 3.0. For microarray analysis of gene expression, liver RNAs were isolated from three Foxa2loxP/loxP Alfp.Cre and three control mice. RNAs were reverse transcribed, labeled, and hybridized to the Mouse PancChip 5.0. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2 and that the expression levels of HNF6 targets are not influenced by Foxa2.
Project description:Comparison of two microarray platforms: the Mouse PancChip 5.0 and the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The aim of this experiment was to determine the ability to identify differentially expressed genes in islet and pancreas RNA, and the sensitivity of the two platforms, using the same source material in a carefully controlled manner. RNA was extracted from adult mouse pancreas (n=5) and highly purified islet samples (n = 5). All samples were amplified once. 5 biological replicates (islets vs. pancreas) in a dye swap experimental design were hybridized to the PancChip. 3 biological replicates, using the same amplified RNA hybridized to the PancChip, of both the pancreas and islet samples were also hybridized to the GeneChip. All data were normalized using appropriate methods and differential expression between islet and pancreas was determined using PaGE with a 10% FDR. The study revealed that the PancChip is a highly cost effective alternative to the Affymetrix 430-2, that the PancChip is up to 60% more sensitive than the Affymetrix 430-2 GeneChip and that 80% (7,000) of the probe sets on the PancChip show expression in either Islets or Pancreas, while only 25% (6,800) show expression on the Affymetrix GeneChip.
Project description:The BCBC Promoter Chip 5B was used to identify genomic targets of Pdx-1 binding. Genomic DNA from mouse NIT-1 insulinoma cells was immunoprecipitated with a Pdx-1 Antibody, PCR amplified, and labeled to the chip. All hybridizations were versus a common reference sample which was a labeled IgG IP. Additionally a Pdx1 SACO library for NIT-1 cells was generated.
Project description:In order to identify the subset of genes directly regulated by Foxa2 in the liver, we performed genome-wide location analysis. Chromatin immunoprecipitation (ChIP) samples from livers of wild type and Foxa2 liver-conditional null mice (Foxa2loxP/loxPAlfp.Cre) were hybridized to a mouse enhancer/promoter microarray with more than 36,000 elements (BCBCPromChip 5). This analysis identified 574 enhancer and promoter regions, corresponding to 484 unique genes, as occupied by Foxa2 in the adult liver.
Project description:The goal of this experiment was to analyze expression changes in the pancreas at embryonic days 12.5 and 13.5 between wild type and Nkx2.2 null mice. We know that Nkx2.2 is essential for pancreatic endocrine differentiation and development. At these early time points which are critical for endocrine cell specification, we would like to identify a transcriptional program that Nkx2.2 regulates. We would also like to identify direct and functional transcriptional targets of Nkx2.2.
Project description:The aim of this experiment was to use microarray analysis to compare the response of wild type (WT) and C/EBPbeta deficient (KO) mice during a partial hepatectomy time course in hopes of identifying transcriptional targets of C/EBPbeta. In addition, the WT time course alone was examined to analyze mammalian cellular proliferation in vivo. In the partial hepatectomy model, quiescent hepatocytes reenter the cell cycle and progress in a synchronous fashion. This allows for the elucidation of regulatory networks operative in mammalian cell cycle. (Identification of transcriptional networks during liver regeneration (2004) Journal of Biological Chemistry)