RAD Experiments RSS feed

Labeling study (CBIL and Kaestner's lab)

University of Pennsylvania Elisabetta Manduchi — Wed, 21 Aug 2002 17:40:07 EDT

Study aimed at evaluating three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere).

Pancreatic developmental time series

University of Pennsylvania Klaus Kaestner — Wed, 28 Aug 2002 15:17:52 EDT

Study to evaluate the expression of pancreas-specific genes at different developmental time points.

KO vs WT replicate study (CBIL and Kaestner's lab)

University of Pennsylvania Gregory R. Grant — Tue, 03 Sep 2002 00:28:01 EDT

Study aimed at evaluating the number of replicates necessary to determine the differentially expressed genes between HNF gamma knock out and wild type CD1 mice.

Mouse PancChip design, preliminary study

University of Pennsylvania Klaus Kaestner — Mon, 14 Oct 2002 11:27:47 EDT

Four expression studies were performed with Incyte GEM arrays using RNA from newborn and adult mouse pancreas, newborn mouse liver, a mouse insulinoma cell line, and human islets. In each case, clones in the top 15% expression level, as measured by fluorescence intensity, were identified.

Amplified RNA Study

University of Pennsylvania Peter F. Davies — Wed, 02 Apr 2003 10:16:38 EDT

Fidelity of TNF-alpha induced differential gene expression in amplified aRNA samples relative to unamplified RNA samples (human aortic endothelial cells, HAEC).

Stem Cell Molecular Signature Study

Affymetrix Lemischka, Ihor R. — Wed, 02 Apr 2003 13:43:21 EDT

Study to determine global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy, and to identify the shared expressed genes among the hematopoietic cells and neural and embryonic stem cells in mouse.

Large-scale analysis of the human and mouse transcriptomes

Genomic Institute of the Novartis Research Foundation John B. Hogenesch — Fri, 18 Apr 2003 08:43:36 EDT

Gene expression profiles of 47 human tissue samples and cell lines and 45 mouse tissue samples derived from dissection to describe the normal mammalian transcriptome.

Neuron specific mRNA complexity responses to Trauma Induced Apoptosis

University of Pennsylvania Tracy K. McIntosh — Fri, 16 May 2003 17:25:49 EDT

Study aimed at examining differential expression between single apoptotic neurons from both the CA3 and dentate and those in an un-injured brain.

Mouse PancChip 4.0 Validation

University of Pennsylvania Klaus Kaestner — Wed, 28 May 2003 11:02:38 EDT

Set of two two-channel assays to test and validate the PancChip 4.0 microarray. One assay hybridized adult mouse total RNA from the pancreas vs. liver. The other total adult mouse pancreas vs. islet RNA.

CellCycle

Elizabeth A. Winzeler — Wed, 09 Jul 2003 13:10:13 EDT

The purpose of this study was to examine gene expression from nine different stages of Plasmodium falciparum 3D7. Gene expression was analyzed using a custom-made Affymetrix type array containing probes to both coding and non-coding regions of the Plasmodium falciparum genome. The oligos w were designed from sequence data released as of July 2001. Note - this study represents the analysis of only a portion of the oligos on the array.

Transcriptional profiling of pig aortic endothelium

University of Pennsylvania Peter F. Davies — Mon, 28 Jul 2003 14:01:50 EDT

This study investigates the role of endothelial cell (EC) gene expression in the focal origin of atherosclerosis, particularly in response to local hemodynamics. Differential gene expression was profiled in EC isolated from athero-susceptible and athero-protected regions of the normal pig aorta. Specifically, a region of disturbed flow (DF, the inner aortic arch) was compared to a region of undisturbed flow (UF, descending thoracic aorta). Linearly amplified RNA was used to screen nylon filter arrays of 13,824 human cDNAs.

Beta cell specific ablation of Foxa2 (HNF-3b) in mice

University of Pennsylvania Klaus Kaestner — Mon, 29 Sep 2003 15:37:44 EDT

Study to further characterize the genetic and functional consequence of the beta cell-specific ablation of Foxa2 (hepatocyte nuclear factor 3 beta, HNF-3b) in mice to better define the role of this gene in glucose homeostasis. The study involved 8 two-channel assays on PancChip 4.0, each consisting of a competitive mutant vs control hybridization. 4 control and 4 Foxa2loxP/loxP;Ins.CRE RNA samples were hybridized to the array using a dye-swap design. (The results from one of these assays had to be discarded from the analyses, so 7 assays were used.)

GeneNote

Department of Molecular Genetics Orit Shmueli — Tue, 07 Oct 2003 10:37:48 EDT

The motivation for the present study was to generate a novel data set, GeneNote (Gene Normal Tissue Expression), in order to portray the complete picture of gene expression profiles in healthy human tissues. We used the complete Affymetrix GeneChip HG-U95 set, which includes 62,839 probe-sets, representing a nearly complete set of human genes. The intensity results of two replicates were processed and analyzed to yield the complete transcriptome for the following human tissues: brain, spinal cord, heart, skeletal muscle, bone marrow, thymus, lung, liver, spleen, pancreas, prostate and kidney.

Suspension Differentiation of fibroblast-like pancreatic progenitors

Stem Cell Institute Lucas Chase — Wed, 12 Nov 2003 12:52:19 EDT

The purpose of this microarray study was to analyze the initial changes in gene expression that occurred during the first 48 hours of a pancreatic progenitor differentiation scheme. The experiment compared both progenitors cultured normally in 10% SCM with nicotinamide as well as progenitors cultured in suspension for 48 hours in 10% SCM with nicotinamide. For the described conditions, 4 sample pairs were analyzed by microarray, for a total of 8 hybridizations. Each biological set was comprised of one normal and one suspension culture sample. Each set was hybridized only once.

Panc-1 Cell Differentiation

NIDDK Intramural Marvin Gershengorn — Tue, 02 Dec 2003 13:18:51 EDT

This study (Experiment) consists of 16 arrays.The purpose of our microarray experiment was to assess changes in gene expression during this differentiation program. The basic experiment was to compare gene expression in control cells cultured in SCM and in cells that had been cultured in SFM for 24 hours. Three biological replicates were prepared for each condition and one of the biological replicates was analyzed by microarray in triplicate, for a total of 5 array hybridizations for each time point. In the specific case of Affymetrix arrays, duplicate biological samples were also prepared from cells that had been exposed to SFM for 8 hours, 3 days, and 8 days.

Human pancreatic cultures

Harvard University Susan Bonner-Weir — Mon, 15 Dec 2003 17:54:44 EDT

Human pancreatic cultures: 3 time points for 4 pancreases, and 2 of subpopulations for each of two other pancreases.

Identification of Tissue Restricted and Interferon alpha Regulated Transcripts in Human Islet Tissue

Columbia Unversity Paul E. Harris — Thu, 18 Dec 2003 16:46:13 EDT

The purpose of this study was to identify mRNA transcripts encoding tissue restricted cell surface proteins on human islet tissue that, in turn, might serve as markers for determination of healthy, transplanted and/or diseased islet cell mass. Using oligonucleotide microarrays and clinical grade human islets, we obtained the gene expression profiles of cultured normal islet tissue and compared them to profiles of islets treated with interferon alpha 2 beta, islet-depleted exocrine pancreas, pooled whole pancreas, pooled liver and pooled kidney tissue. Data set filtering and comparisons of gene expression patterns revealed a small set of genes corresponding to transmembrane or membrane associated proteins with limited tissue distributions (present in Islets of Langerhans and, often, central or peripheral nervous system tissues, but absent from exocrine pancreas, liver, kidney and other tissues) with possible utility as novel islet markers. Under the influence of IFN alpha, some of these transcripts show differential expression as confirmed by real time PCR. In addition, we found significant differential expression of two sets of transcripts expressed in islets but with broad tissue distributions, non classical MHC class I b (HLA-G and F) and MHC Class II locus (e.g. HLA-DR alpha and HLA-DQ alpha and beta).

Gene expression in normal mouse tissues

University of Colorado Health Sciences Center John Hutton — Mon, 12 Jan 2004 12:41:03 EDT

Gene expression was studied using PancChip5.0 in 15 tissues. Most tissues were from the adult mouse, except for fetal pancreas (e18.5) and placenta. Adult tissues studied were: adrenal gland, pancreas, brain, heart, kidney, liver, lung, ovary, parotid gland, pituitary gland, small intestine, stomach and testis. All were studied in duplicate except for the pituitary, the liver, and the stomach. All samples were pooled samples. Tissues were taken from 3 males and 3 females, with the exception of the gonadal tissues, and the fetal tissues were sex was not determined.

LCM human islet beta cells

Harvard University Gordon Weir — Wed, 14 Jan 2004 19:07:42 EDT

Fresh frozen sections of islets obtained by surgery were laser capture microdissected using autofluorescence to guide selection of beta cell areas of the islet. RNA was extracted and amplified with 2 rounds of T7 linear amplification. Two technical replicates were hybridized to Affymetrix U95Av2 arrays.

Nestin positive cells vs human islets

Massachusetts General Hospital/HHMI Joel F. Habener — Wed, 21 Jan 2004 10:38:03 EDT

A monolayer of human nestin positive cells grown in culture using bFGF and EGF was compared to hand picked isolated islets.

GNF large-scale survey of human and mouse tissues

Genomic Institute of the Novartis Research Foundation John B. Hogenesch — Mon, 26 Jan 2004 12:15:40 EDT

Gene expression profiles of 79 human normal and diseased tissues using Affymetrix U133A and custom Affymetrix GNF1B arrays as well as 53 normal mouse tissues using custom Affymetrix GNF1M chips.

Microarray analysis of in vitro differentiation of adult human pancreatic progenitor cells

Massachusetts General Hospital/HHMI Joel F. Habener — Fri, 30 Jan 2004 16:28:40 EDT

We have developed an in vitro culture system that allows us to expand progenitor cells from human islet preparations and differentiate them into insulin-producing cells. We noticed however, that cultures from individual islet preparations had very heterogeneous outcomes, from good differentiation to almost none. We therefore speculated that our progenitor cell cultures contained different kinds of cells and that the true endocrine progenitor cells are present in the successful cultures, but not in the unsuccessful ones. To address this issue and to begin to identify markers for the true endocrine progenitor cells we compared global gene expression between a very successful culture (final insulin-expression 10% of islets) and an unsuccessful one (final insulin-expression 0%). We also included RNA from freshly isolated islets for control purposes. The cultures from donor A yielded substantial differentiation, while the cultures from donor B showed no successful differentiation.

Cytotoxicity Study of Rat INS1 cell lines

Duke University Christopher B. Newgard — Wed, 04 Feb 2004 11:22:05 EDT

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)

GSIS Study of Rat INS1 cell lines

University of Pennsylvania Klaus Kaestner — Fri, 06 Feb 2004 13:23:04 EDT

The aim of this study was to identify the gene expression changes associated with glucose responsiveness in Rat INS-1 derived cell lines. RNA was prepared from cell lines which were shown to be highly glucose responsive and also lines which were shown to be poorly glucose responsive. Dr. Chris Newgard's lab quantified the RNA and sent it frozen in water. 3 biological replicates per condition were sent for the following conditions: (i) 832/13 and 833/15, robust glucose responsiveness. (ii)832/1 and 832/2 showed poor glucose responsiveness.

Lipotoxicity Study of Rat INS1 cell lines

University of Pennsylvania Klaus Kaestner — Wed, 18 Feb 2004 11:34:03 EDT

This experiment was designed to study the effects of lipid culture on INS-1 derived glucose-responsive 832/13 cells. After 48-hr culture with lipids, a dramatic decrease in glucose-responsiveness is observed. In order to detect some earlier changes, RNA was also collected at earlier time points (12hr and 24hr). To compensate for any changes associated with cell density, control cells (1%BSA culture) were included for each time point. RNA was prepared from INS-1 cells in Dr. Chris Newgard's lab, quantified and sent frozen in water. Six biological replicates were sent for the following conditions: (i) 1%BSA (12, 24, and 48 Hours) (ii) 1mM-Oleate/Palmitate (12, 24, and 48 Hours)

foxA1 and beta cell function

University of Pennsylvania Klaus Kaestner — Wed, 03 Mar 2004 10:41:22 EDT

The aim of this experiment was to use microarray analysis to examine the phenotype of the foxA1 (HNF3alpha) null mouse. foxA1 has a central role in the regulatory control of islet genes essential for glucose homeostasis in vivo. Previous studies have shown that the foxA1 null mouse demonstrates severe postnatal growth retardation followed by death between P2 and P12. These mutant mice are hypoglycemic despite unchanged expression of foxA1 target genes involved in hepatic gluconeogenesis. foxA1 is known to bind to and transactivate the proglucagon gene promoter and mice null for this gene have a 70% reduction in pancreatic proglucagon gene expression and plasma glucagon levels are reduced markedly. Marco Vatamaniuk from Klaus Kaestners were provided for both the WT and Null.

HNF4alpha in beta cell function

University of Pennsylvania Klaus Kaestner — Fri, 05 Mar 2004 13:47:33 EDT

The aim of this experiment was to use microarray analysis to examine the phenotype of dis-regulated insulin secretion and abnormal beta cell growth in HNF4 alpha null mice. These mice show impaired glucose tolerance and elevated fasting and fed plasma insulin levels. Rana Gupta from Klaus Kaestnerstudy.

Atherogenic risk factors and regional endothelial cell gene expression

University of Pennsylvania Peter F. Davies — Mon, 29 Mar 2004 17:49:05 EDT

Atherosclerosis preferentially develops in susceptible regions of the arterial system that are defined by the regional vascular hemodynamics. Previous work in adult castrate male pigs revealed the coexistence of pro- and anti-atherosclerotic profiles in endothelial cell gene expression in disturbed flow regions of arteries in the absence of risk factors for atherogenesis. This study investigates the impact of gender, high fat/high cholesterol diet and regional hemodynamics on arterial endothelial cell gene expression profiles in sexually mature intact pigs.

Global expression analysis of gene regulatory pathways during endocrine pancreatic development

Guoqiang Gu — Fri, 09 Apr 2004 17:01:07 EDT

To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.

Expression profiles of novel pancreatic cell lines.

Vanderbilt University Mark Magnuson — Wed, 28 Apr 2004 18:11:09 EDT

We studied the expression profiles of two different cell lines that were generated in our laboratory from tumors found in mice expressing the SV40 T antigen under the control of the upstream glucokinase promoter. The first cell line, called GKP2 cells, was derived from an insulinoma and expresses insulin and pdx1 along with other markers of beta cells. The second cell line, named GKP4 cells, was derived from periductal tumors that did not express insulin or pdx1 but expressed genes of the endocrine lineage, such as Isl1 and Nkx2.2. Interstingly, a small fraction of the GKP4 cells can spontaneously turn on the expression of insulin and pdx1, indicating that they could be islet cell progenitors. The aim of our project was to identify the genes expressed specifically in GKP4 cells. To do so, we compared the gene expression profiles of GKP4 cells with GKP2 cells using DNA microarray.

Microarray Analysis of MIN6 cells treated with Exendin-4 over time.

Massachusetts General Hospital/HHMI Anna L. Nolan — Fri, 30 Apr 2004 09:07:12 EDT

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.

Side-specific valvular endothelial gene expression

University of Toronto Craig Simmons — Tue, 15 Jun 2004 11:33:44 EDT

Differential transcriptional profiles from endothelial cells isolated from opposite sides of aortic valves from healthy adult male pigs (N=8, from slaughterhouse). Samples were hybridized to a human array and paired by animal.

Mouse pancreas development (Hutton/Juhl)

University of Colorado Health Sciences Center John Hutton — Tue, 06 Jul 2004 11:16:33 EDT

Pancreas study with 5 sub-studies: (i) 14 assays (7 done on Affymetrix MGU74Av2 and 7 on MOE430 2.0) looking at 7 different time points in pancreas development, (ii) 2 assays (done on Affymetrix MGU74Av2) looking at tumorgenic cell lines alphaTC and betaTC, (iii) 8 assays (6 done on Affymetrix MGU74Av2 and 2 done on MOE430 2.0) looking at Ngn3 mutant and wildtype pancreas at 3 different embryonic time points in pancreas development, (iv) 3 assays (done on Affymetrix MGU74Av2) looking at embryonic e12.5, newborn pancreas and adult islets, (v) 3 assays (done on Affymetrix MGU74Av2) looking at e11.5 separated pancreatic epithelium and mesenchyme or the intact e11.5 pancreas.

Affymetrix MOE430v2 vs Mouse PancChip 5.0 comparison

University of Pennsylvania Klaus Kaestner — Thu, 07 Oct 2004 14:46:53 EDT

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.

C/EBPbeta Hepatectomy Time Course

University of Pennsylvania Linda Greenbaum — Mon, 25 Oct 2004 14:13:49 EDT

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)

Overexpression of Neurogenin3 in Mpac mouse ductal cells

Caroline Mrejen — Wed, 03 Nov 2004 14:04:59 EDT

We have developed an in vitro model of Ngn3-dependent differentiation by infecting pancreatic duct cell lines with an Ngn3-expressing adenovirus. We used glass microarrays containing 18,000 mouse cDNAs and compared gene expression patterns in mPAC L20 cells infected with either AdCMV-gal (control) or AdCMV-ngn3. All comparisons were performed 48 h after viral infection, meaning that genes differentially expressed in Ngn3-expressing mPAC cells could include genes whose expression was directly or indirectly controlled by Ngn3.

Pancreatic Growth after Partial Pancreatectomy and Exendin-4 Treatment

University of Pennsylvania Doris A. Stoffers — Wed, 13 Jul 2005 15:16:49 EDT

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.

Cyclophosphamide-induced beta Cell Destruction in NOD Mice

Joslin Diabetes Center and Harvard Medical School Christophe Benoist — Wed, 19 Oct 2005 14:27:44 EDT

Type 1 diabetes appears to progress in a highly regulated manner and insulitis can persist for long periods of time before the terminal destruction of beta cells. To study the final stage of diabetogenesis, BDC2.5/NOD mice were treated with cyclophosphamide to induce type 1 diabetes. Pancreatic islets were analyzed using the Affymetrix MU74v2A microarray platform before treatment (Eight Samples at Day 0) and as treatment progressed (Four Samples at Day 1, Three Samples at Day 2, and Three Samples at Day 3).

Global profiling of double stranded RNA- and IFN-gamma-induced genes in rat pancreatic beta cells.

Universite Libre de Bruxelles Decio L. Eizirik — Wed, 19 Oct 2005 14:37:36 EDT

Viral infections and local production of IFN-gamma might contribute to beta-cell dysfunction/death in Type 1 Diabetes. Double stranded RNA accumulates in the cytosol of viral-infected cells, and exposure of purified rat beta cells to dsRNA (polyinosinic-polycytidylic acid, PIC) in combination with IFN-gamma results in beta-cell dysfunction and apoptosis. To elucidate the molecular mechanisms involved in PIC + IFN-gamma-effects, we determined the global profile of genes modified by these agents in primary rat beta cells. FACS-purified rat beta cells were cultured for 6 or 24 h in control condition or with IFN-gamma, PIC or a combination of both agents. The gene expression profile was analyzed in duplicate with the Affymetrix RG U34A microarray.

Cytokine induced dysfunction and apoptosis in insulin producing INS1 Cells

Universite Libre de Bruxelles Decio L. Eizirik — Wed, 19 Oct 2005 14:48:52 EDT

Locally released cytokines contribute to beta cell dysfunction and apoptosis in Type 1 diabetes. In vitro exposure of insulin producing INS 1E cells to the cytokines interleukin (IL) 1beta + interferon (IFN) gamma leads to a significant increase in apoptosis. To characterize the genetic networks implicated in beta cell dysfunction and apoptosis and its dependence on nitric oxide (NO) production, we performed a time course analysis using the Affymetrix RG U34A microarry. INS 1E cells were exposed in duplicate to IL 1beta + IFN gamma for six different time points (1, 2, 4, 8, 12, and 24 h) with or without the inducible NO synthase blocker.

Cytokine-induced and nuclear factor-kappa B-dependent genes in primary rat beta-cells

Universite Libre de Bruxelles Decio L. Eizirik — Wed, 19 Oct 2005 16:22:05 EDT

Type 1 diabetes mellitus results from an autoimmune destruction of pancreatic beta-cells. Based on findings suggesting NF-kappa B plays a role in beta cell apoptosis, we blocked NF-kappa B activation in cytokine-exposed FACS sorted beta cells by a recombinant adenovirus (AdI kappa B((SA)2)) containing an inhibitor of NF kappa B alpha (I kappa Bac) super-repressor (S32A/S36A). The expression profile was then analyzed with the Affymetrix RG U34a microarray.

Identification of novel cytokine induced genes in rat pancreatic beta-cells

Universite Libre de Bruxelles Decio L. Eizirik — Wed, 19 Oct 2005 16:29:32 EDT

Type 1 diabetes is an autoimmune disease resulting from the selective destruction of insulin-producing beta-cells. Beta cell culture for 6-9 days in the presence of IL-1beta and interferon (INF)-gamma leads to apoptosis. Primary rat beta-cells were FACS purified and exposed for 6 or 24 h to control condition, IL-1beta + INF-gamma, or IL-1beta alone (24 h only). The gene expression profile was analyzed by hybridization in duplicate to the Affymetrix RG U34A microarray.

Transcriptional Networks in Liver: HNF6 function is largely independent of Foxa2

University of Pennsylvania Klaus Kaestner — Tue, 29 Nov 2005 19:00:59 EDT

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.

Human Tissue Survey From Massively Parallel Signature Sequencing (MPSS)

Ludwig Institute for Cancer Research C. Victor Jongeneel — Tue, 20 Dec 2005 10:24:17 EDT

Massively parallel signature sequencing (MPSS) was used to sample the transcriptomes of 32 normal human tissues resulting in the documentation of gene expression patterns of almost 20,000 genes. Differentiation is driven largely by changes in the transcriptional program of cells, through regulatory and epigenetic events. This study provides a comprehensive snapshot of cell populations from fully differentiated tissues resulting in valuable information about genes which are specialized (tissue specific) or required for all cells. MPSS samples the transcripts which are present in an mRNA population in essentially an unbiased fashion.

Human pancreatic islets from normal and Type 2 diabetic subjects

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 09 Jan 2006 13:41:36 EDT

Human pancreatic islets isolated from 7 people with normal glucose tolerance, and 5 people with type 2 diabetes. All 12 people were organ donors after either cerebrovascular accident or intracerebral haemorrhage. Normals were required to maintain glucose at least 6.1mM in intensive care. Diabetic subjects were all at least 10 years from diagnosis and not insulin-requiring. For every subject, RNA was isolated, cRNA was made and hybridized to the U133A and U133B Affymetrix arrays (total of 24 arrays). No samples were pooled.

Gene Expression Survey of Transcription Factors using RT-PCR

University of Texas Southwestern Medical Center Galvin H. Swift — Thu, 12 Jan 2006 15:02:08 EDT

A global survey of RNA from 14 fetal and 12 adult human organs by RT-PCR determined the expression patterns of 790 genes encoding DNA-binding transcription factors. The data can be sorted to identify sets of transcription factors with expression relatively restricted to a given organ or to particular organ groups. These data are a resource to help define the spectrum of transcription factor control, contribute to the elucidation of transcription factor cascades responsible for the development and maintenance of each organ, and provide a baseline to study the effects of disease or developmental defects.

Landscape of Mouse Gene Expression

University of Toronto Timothy Hughes — Wed, 01 Feb 2006 11:05:48 EDT

Custom-built DNA oligonucleotide microarrays were used to generate an expression data set for nearly 40,000 known and predicted mouse mRNAs across 55 diverse tissues. Hybridizations were performed in duplicate with fluor reversal: that is, each mRNA sample was examined in duplicate, once in the Cy3 channel and once in the Cy5 channel, on separate arrays. Each array was hybridized with two samples simultaneously, each from an individual tissue. 3 Arrays were poor quality in both channels and were excluded, 19 arrays were poor quality in one channel. The Raw data for those which failed in one channel is included in this study but these are not used in the analysis (processed data).

PANDER Induced Cell-Death Networks in Pancreatic Islets

Childrens Hospital of Philadelphia Bryan A. Wolf — Mon, 06 Feb 2006 15:07:12 EDT

Expression profiling using the mouse PancChip version 5.0 was used to elucidate the genetic mechanisms of PANDER-induced cell death in Pancreatic Islets. Murine islets were treated with PANDER for 48 or 72 h (n=4 and n=3 respectively). Following linear amplification, the RNA was matched for purity using Quantitative PCR.

Brown preadipocyte IRS knockout profiling 1

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 20 Feb 2006 14:08:29 EDT

Brown preadipocytes were grown to confluence and synchronized by overnight serum starvation. Four independent RNA samples were analyzed from each IRS KO cell line and three independent clones of WT cells were separately analyzed as controls. Splitting of the samples resulted in a total 28 microarrays. 15 mg of adjusted cRNA were hybridized to Affymetrix U74A-v2 arrays.

Fat and Normal adipocytes from insulin receptor knockout mice sorted into small and large cells

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 20 Feb 2006 14:08:29 EDT

Mice with fat-specific disruption of the insulin receptor gene (FIRKO mice) have low fat mass, and are protected against obesity and obesity-related glucose intolerance. FIRKO mice also exhibit polarization of adipocytes into populations of large and small cells. Other PubMed identifiers for this study: 15131120,12110165,15131119

Low and High Fat Diet on Mice of Two Genetic Backgrounds (B6 vs. 129) - Liver

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 20 Feb 2006 14:08:29 EDT

Both environmental and genetic factors play important roles in the development of the metabolic syndrome. To elucidate how these factors interact under normal conditions, C57Bl/6 (B6) and 129S6/SvEvTac (129) mice were placed on a low-fat or high-fat diet. Liver samples were extracted and hybridized to Affymetrix Genome U74 (version 2) arrays.

Mouse skeletal muscle - controls, streptozotocin diabetes and insulin treated

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 20 Feb 2006 14:08:29 EDT

Metabolic abnormalities underlying diabetes are primarily the result of the lack of adequate insulin action and the associated changes in protein phosphorylation and gene expression. Affymetrix oligonucleotide microarrays were used to study the changes in the transcriptional program of mouse skeletal muscle in insulin-deficient diabetes. Mice which were made diabetic by streptozotocin treatment were compared to controls. Also, the reversibility of these changes was ascertained by treating a subset of the diabetic mice with insulin.

Skeletal Muscle Insulin Receptor Knockout - Control, Streptozotocin Diabetic and Insulin Treated

Joslin Diabetes Center and Harvard Medical School Ronald C Kahn — Mon, 20 Feb 2006 14:08:29 EDT

The targeted muscle insulin receptor knockout (MIRKO) model was used, in which there is a complete absence of the insulin-receptor signaling in skeletal muscle but normal insulin and glucose levels. By comparing skeletal muscle gene-expression profiles from MIRKO mice and their controls (lox/lox) under three different metabolic conditions (namely, in the basal state, after streptozotocin (STZ)-induced diabetes, and after STZ-induced diabetes rendered euglycemic with insulin treatment), we can address the following three important questions. (i) What is the direct effect of the loss of insulin signaling on gene expression in skeletal muscle? (ii) What is the contribution of the metabolic and other changes that accompany diabetes to induce indirect changes in gene expression? (iii) How are these pathways regulated and implicated in the pathophysiology of diabetes?

Liver - ob/ob mice