title: Differential gene expression patterns of the developing and adult mouse cornea compared to the lens and tendon

status: Public on Jul 23 2008

city: Baltimore

first: Shukti

last: Chakravarti

address: 720 Rutland Ave

institute: Johns Hopkins University School of Medicine

summary: The cornea continues to mature after birth to develop a fully functional, refractive and protective barrier tissue. Here we investigated the complex biological events underlying this process by profiling global genome-wide gene expression patterns of the immature postnatal day 10 and seven week-old adult mouse cornea. The lens and tendon were included in the study to increase the specificity of genes identified as up regulated in the corneal samples. Notable similarities in gene expression between the cornea and the tendon were in the mesenchymal extracellular matrix collagen (types I, III, V, VI) and proteoglycan (lumican, decorin and biglycan) genes. Expression similarities in the cornea and lens were limited to certain epithelial genes and the crystallins. Approximately 76 genes were over expressed in the cornea samples that showed basal expression levels in the lens and tendon. Thirty-two of these were novel with no known functions in the cornea. These include genes with a potential role in protection against oxidative stress (Dhcr24, Cdo1, Akr1b7, Prdx6), inflammation (Ltb4dh, Wdr1), ion-transport (Pdzk1ip1, Slc12a2, Slc25a17) and transcription (Zfp36l3, Pdzk1ip1). Direct comparison of the cornea of two ages showed selective up regulation of 50 and 12 genes in the P10 and adult cornea, respectively. Of the up regulated P10 genes several encode extracellular matrix collagens and proteoglycans that are stable components of the adult cornea and their high transcriptional activity at P10 indicate a period of active corneal growth and matrix deposition in the young cornea. Much less is known about the genes selectively over expressed in the adult cornea; some relate to immune response and innervations (Npy), and possibly to electron transport (Cyp24a1, Cyp2f2) and others of yet unknown functions in the cornea (Rgs10, Psmb8, Xlr4)). This study detected expression of genes with known functions in the cornea, providing additional validation of the microarray experiments. Importantly, it identified several novel genes whose functions have not been investigated in the cornea.

overall_design:
Briefly, the corneas were dissected free of the limbus from postnatal day 10 (P10) and seven-week old (adult) mice. To enrich for stromal contribution, the epithelium was scraped off and the cornea placed in TRIzol (Invitrogen Life Technologies, Carlsbad, CA). The scraping off of the epithelium reduced, but did not abrogate epithelial contribution, as judged by the expression of known epithelial markers. The lens and the flexor digitorum longus (FDL) tendon were removed from seven-week old adult mice for isolation of total RNA. For the adult cornea three independent preparations of total RNA was generated. For all the other tissues two independent preparations of total RNA were used. The adult and the P10 microarrays were used as baselines in our previous study of corneal cells in culture to identify genes differentially expressed by cultured cells of the stroma (Chakravarti et al. 2004).
The output fluorescence of the scanned images was analyzed first using the Affymetrix Microarray Suite 5.0 software to compute absolute expression, background calculation and detection call (present, absent or marginal). Background signal was between 42 to 55 arbitrary units, while the noise to signal ratio was between 1.42 to 1.78 arbitrary units. The expression data from all nine arrays were further analyzed as follows. The model-based DNA-Chip Analyzer (dChip) software (http://www.dchip.org/) was used to normalize the data from the image files for array-to-array comparison (Li and Wong 2001). This software uses an invariant-set algorithm as a basis for normalization and a model-based expression index to calculate the expression value for each transcript. To identify gene expression patterns that were statistically significantly different between any two given tissue types, we used the following criteria: (1) fold change ≥ 2; (2) difference of mean expression index ≥ 100 arbitrary units; (3) t-statistic, p < 0.05.

first: Feng

last: Wu


title: The Cancer Genome Atlas (TCGA) Consortium Integrated DNA Methylation Analysis

status: Public on Jan 01 2009

city: Ghent

first: Leander

last: Van Neste

address: Coupure Links 653

department: Molecular Biotechnology

country: Belgium

institute: Ghent University

summary:
Aberrant hypermethylation of CpG dinucleotides located in CpG islands within the promoters of key cancer genes is an epigenetic abnormality associated with heritable transcriptional gene silencing and inactivation in cancer. The genes involved include important tumor suppressors affecting key pathways for tumor initiation and progression. These methylated sequences can serve as potentially valuable markers for cancer risk assessment, diagnosis, prognosis, and prediction of therapeutic responses. In addition, many key cancer genes may be targeted by both epigenetic and genetic alterations and, thus epigenetic analysis can help focus the search for mutations, and vice versa. Studies of major cancer types suggest that any individual patient’s tumor may harbor at least 300 or more DNA hypermethylated genes.  In TCGA, a pilot project is underway to begin defining these genes for GBM via genomic approaches.

The approach in the epigenetic pilot is a two-tiered one which, first, involves pharmacological treatment of both well established human GBM cell lines, and a cell line grown as a neurosphere to enrich for tumor propagating cells, with a DNA methylation inhibitor (5-aza-2’-deoxycytidine, DAC) or a histone deacetylation inhibitor (trichostatin A) followed by an expression transcriptome analysis as previously described (Schuebel et. al.). This has resulted in identification of more than 3,700 total candidate genes. In the second tier, the top candidates are then analyzed on a custom Illumina GoldenGate array with the capacity to monitor methylation at a single CpG dinucleotide in the CpG islands of 1,498 gene promoters for the high throughput analysis of TCGA GBM samples.
Keywords: Microarray, Hypermethylome, DNA-hypermethylation, DAC, TSA, Epigenetic, TCGA, The Cancer Genome Atlas, GBM, Glioblastoma, Glioblastoma multiforme, Brain

overall_design:
Cell culture and treatment. For drug treatments, log phase U87MG, T98G, D54MG and the human glioblastoma derived neurosphere cell line HSR-GBM1 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with 5M 5aza-deoxycytidine (DAC) (Sigma; stock solution: 1mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300nM Trichostatin A (Sigma; stock solution: 1.5mM dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs.
Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen kit according to the manufacturers instructions, including a DNAase step. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturers instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies.
Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform and packages from Bioconductor bioinformatics software project. The log ratio of red signal to green signal was calculated and Loess normalization as implemented in the limma package from Bioconductor.

first: Joo-Mi

last: Yi

first: Kornel

middle: E

last: Schuebel

first: Daniel

middle: J

last: Weisenberger

first: Leslie

last: Cope

first: Fei

last: Pan

first: James

middle: G

last: Herman

first: Peter

middle: W

last: Laird

first: Stephen

middle: B

last: Baylin


title: Transcription elongation factor ELL2 influences splicing versus poly(A) site choice in the Ig heavy chain gene

status: Public on Jun 01 2008

city: Pittsburgh

first: kathleen

last: martincic

state: PA

address: 200 Lothrop Street

department: Immunology

country: USA

institute: University of Pittsburgh

summary: Processing of Immunoglobulin heavy chain (IgH) mRNA is a paradigm for competition between splicing and polyadenylation.  In plasma cells pre-mRNA is polyadenylated mainly at the promoter-proximal secretory site while B-cells utilize a cryptic 5’ splice site in the last secretory-specific exon; these are mutually exclusive events for all IgH pre-mRNAs.  Transcription elongation factor ELL2, more abundant in plasma cells relative to B-cells, was down-modulated by overexpression of heterogenous ribonucleoprotein F, a condition which reduced production of secretory IgH mRNA.  Transfection of B-cells with ELL2 and the IgH reporter showed an accelerated use of the secretory poly(A) site, positioned in competition with the splice to M1; a small interfering RNA to ELL2 reduced expression of IgH secretory mRNA.  Co-transcription factors ELL1 and PC4 were ineffective at driving secretory-poly(A) site use.  ELL2 had little effect on poly(A) site choice with reporters containing tandem-linked poly(A) sites.  Shorter forms of ELL2 protein result from both internal initiation at M186 and protein processing.  An alternative splicing reporter driven by IgH or non-Ig promoters revealed that ELL2 and its M186 initiated form were able to accelerate exon skipping.  Therefore, ELL2 influences IgH pre-mRNA processing through facilitating skipping of the alternative splice to the membrane form.

overall_design: AxJ plasma cells were stably transfected to overexpress hnRNP-F, -H or empty vector. Clones showing high overexpression levels of F or H by western blot were selected. The IgH sec to mb ratios of these clones were determined. A global gene expression analysis was performed on mRNA from two clones from hnRNP-F, which demonstrated a lower sec:mb ratio, and one from each of the controls: overexpression of hnRNP-H, or transfection with empty vector, or A20 B-cells, using Affymetrix gene micro array technology.

first: Kathleen

last: Martincic

first: Serkan

middle: A

last: Alkan

first: Alys

last: Cheatle

first: Christine

last: Milcarek


title: Gene expression analysis in 13 patients with mitochondrial ATP synthase deficiency (Agilent)

status: Public on Aug 01 2008

city: Prague

first: Viktor

last: Stranecky

address: Ke Karlovu 2

department: 1st Faculty of Medicine

country: Czech Republic

institute: Charles University

summary:
Defects of mitochondrial functions lead in humans to vast array of usually multisystemic pathologies and several hundreds of diseases resulting from various defects of mitochondria biogenesis and maintenance, defects of respiratory chain complexes (OXPHOS) or defects of individual mitochondrial proteins are known. We used Agilent Whole Human Genome Microarray for gene expression profiling of  genetically heterogeneous group of 13 patients with biochemically proven ATP synthase deficiency. Gene expression data analysis allowed classification of patients into several distinct groups, provided information on subgroup and patient specific gene expression profiles, defined candidate disease causing genes and gave basic information on pathogenic mechanisms associated with ATP synthase deficiency.
Keywords: ATP synthase, mitochondrial biogenesis, ROS, gene expression, microarray, human

overall_design: Two-condition experiment, patients vs. controls cells. Biological replicates: 9 control, 13 patients, independently grown and harvested.

first: Alena

last: Čížková

last: Stránecký

first: Stanislav

last: Kmoch


title: Analysis of HeLa cells after transfection with miR-1, miR-124 and miR-181.

status: Public on Jul 31 2008

city: Cambridge

first: Whitehead

last: Institute

state: MA

address: 9 Cambridge Center

department: Genome Technology Core

institute: Whitehead Institute

summary: Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells.  Results were compared to protein down-regulation at 48 hours measured by SILAC-MS.

first: Daehyun

last: Baek

first: Judit

last: Villén

first: Chanseok

last: Shin

first: Fernando

last: Camargo

first: Steven

last: Gygi

first: David

last: Bartel


title: Myeloma IRF4 target genes - Agilent

status: Public on Jul 07 2008

city: Bethesda

first: Louis

middle: M.

last: Staudt

state: MD

address: 9000 Rockville Pike, Bldg 10, Rm 4N114

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

summary:
The goal of this gene expression study was to identify genes whose expression depends on the transription factor IRF4 by knocking down it's expression using shRNA in two IRF4+ myeloma cell lines.
Keywords: time series design

overall_design: Two myeloma lines were analyzed over a time course of IRF4-targeted shRNA induction. There are 3 time courses: two using KMS12 (biological replicates) and one with SKMM1. Within each time course there are technical replicates.


title: 22RV1 prostate cancer cell line transfected with SPINK1 siRNA vs control siRNA

status: Public on Jun 10 2008

city: Ann Arbor

first: Scott

last: Tomlins

state: MI

address: 1400 E. Medical Center Dr., 5410 CCGC

department: Pathology

institute: University of Michigan

summary:
ETS gene fusions have been characterized in a majority of prostate cancers, however key molecular alterations in ETS negative cancers are unclear.  Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier-expression exclusively in a subset of ETS rearrangement negative cancers (~10% of total cases).  We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier-expression can be detected non-invasively in urine and observed that SPINK1 outlier-expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier-expression model, and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement negative prostate cancers.
Keywords: Genetic Modification

overall_design: 22RV1 cells were infected with non-targeting siRNA or siRNA against SPINK1. For reported hybridizations, the reference channel is 22RV1 cells infected with non-targeting siRNA. Duplicate hybridizations were performed with duplicate dye flips, for a total of four arrays. Over and under-expressed signatures were generated by filtering to include only features with significant differential expression (PValueLogRatio < 0.01) in all hybridizations and Cy5/Cy3 ratios > or < 1 in all hybridizations.

first: S

first: A

middle: M

last: Chinnaiyan


title: mouse lung resistance or sensitivity to cigarette smoke

status: Public on Jul 10 2008

city: Genoa

first: Paolo

last: Fardin

address: Largo G. Gaslini 5

department: Molecular Biology

country: Italy

institute: Istiuto Giannina Gaslini

summary: We have investigated the effects of cigarette smoke exposure in three different strains of mice. DBA/2 and C57Bl/6J are susceptible to smoke and develop different lung changes in response to chronic exposure, while ICR mice are resistant to smoke and do not develop emphysema. The present study was carried out to determine early changes in the gene expression profile of mice exposed to cigarette smoke with either a susceptible or resistant phenotype.

overall_design: Three strains of mice were exposed to smoke from three cigarette/day, 5d/wk for 4 weeks. Microarray analysis was carried out on total RNA extracted from the lung utilizing the Affymetrix platform.

first: Eleonora

last: Cavarra

first: Annamaria

last: Ricciardi

first: Silvia

last: Fineschi

first: Giovanna

last: De Cunto

first: Fabio

last: Sallustio

first: Michele

last: Zorzetto

first: Maurizio

last: Luisetti

first: Ulrich

last: Pfeffer

first: Giuseppe

last: Lungarella

first: Luigi

last: Varesio


title: Gene expression following acute methamphetamine administration in selectively bred mice

status: Public on Aug 09 2008

city: Chicago

last: Rosenberg

state: IL

department: Committee on Neurobiology

institute: University of Chicago

summary:
This study examined NAc gene expression in a pair short-term selected lines bred for high or low response to methamphetamine (MA).  We sought to identify candidate genes preferentially expressed among individuals showing either large or small acute responses to MA.  Additionally, we sought to identify genes and networks differentially expressed by MA exposure within these divergent lines.

Keywords: Short-term selected lines (STSLs), saline (SAL), methamphetamine (MA), nucleus acumbens (NAc)

overall_design: STSLs generated from C57BL/6J x DBA/2J stock were bred for high (HMACT) or low (LMACT) acute MA response.  Mice were given a challenge injection of MA or SAL, and decapitated 1 hr later.  NAc gene expression was assessed.

first: Abraham

last: Palmer


title: IFN-g counteracts YopH mediated immune evasion in Yersinia enterocolitica infection in mice

status: Public on Apr 17 2008

city: Tuebingen

first: Erwin

last: Bohn

address: Elfriede-Aulhorn-Strasse 6

department: Institutefor Medical Microbiology and Hygiene

country: Germany

institute: University of Tuebingen

summary:
Background:
Yersinia outer protein (Yop) H is a secreted virulence factor of Yersinia enterocolitica which inhibits phagocytosis of Y. enterocolitica and promotes virulence of Y. enterocolitica (Ye) in mice. The aim of this study was to address whether and how YopH affects the innate immune response against Ye in mice.

Results:
For this purpose mice were infected with wild type Ye (pYV+) or a YopH-deficient Ye mutant strain (DyopH). CD11b+ cells were isolated from infected spleen and subjected to gene expression analysis using microarrays. Despite attenuation of DyopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and DyopH infections at either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes including IFN-g and IL-6 are high after pYV+ infection but low after sublethal DyopH infection. In line with these findings,  infection of IFN-gR-/- and IL-6-/- mice with pYV+ or DyopH revealed that these cytokines are dispensable for control of DyopH, but not pYV+ infection. Consistently, in bacteria killing assays with BMM in vitro, stimulation of BMM with IFN-g is required for killing of pYV+ but not DyopH.

Conclusion:
In conclusion, this data suggest that IFN-g counteracts YopH-mediated virulence mechanisms of Ye which in Ye wild type infection contribute to evasion of the innate immune response including killing by macrophages.
Keywords: Comparison of gene expression due to bacterial virulence factors

overall_design: In this study microarray analyses were performed to define differences in gene expression of cells associated with innate immune response (CD11b+ cells) after infection of mice with a sublethal and lethal infection with wildtype Yersinia enterocolitica compared to uninfected mice. In addition, we wanted to investigate whether differences in gene expression can be defined which are due to the virulence factor YopH.  Moroeover, we were interested whether gene expression pattern of sublethal and lethal infected mice are different. Allover all five samples were compared. Number of replicates 1.

first: Gianluca

last: Matteoli

first: Edda

last: Fahl

first: Philipp

last: Warnke

first: Steffen

last: Müller

first: Michael

last: Bonin

first: Ingo

last: Autenrieth


title: Genes regulated by miR-34b/c and DAC

status: Public on Apr 30 2008

city: Sapporo

first: Hiromu

last: Suzuki

state: Hokkaido

address: S1, W17, Chuo-ku

department: Department of Molecular Biology

country: Japan

institute: Sapporo Medical University

summary:
We found frequent epigenetic silencing of microRNA-34b/c in human colorectal cancer. Introduction of miR-34b/c into a colorectal cancer cell line induced significant changes in gene expression profile. We also found overlap between the genes downregulated by miR-34b/c and those downregulated by DAC.
Keywords: dose response

overall_design: A colorecal cancer cell line HCT116 was transfected with miR-34b or -c precursor or negative control. Also, HCT116 was treated with 5-aza-2'-deoxycytidine (DAC) or mock. Genes up- or downregulated by miR-34b/c and those by DAC was compared.

first: Minoru

last: Toyota

first: Takashi

last: Tokino


title: Autoregulation of Th1-mediated inflammation by twist1 2nd part

status: Public on May 24 2008

city: Berlin

first: Pawel

last: Durek

address: Charitéplatz 1

institute: Deutsches Rheuma-Forschungszentrum

summary:
Gene expression profiling of repeatedly activated compared to recently activated Th1 cells to identify genes that play a role in chronic inflammatory disorders and may qualify as diagnostic or therapeutic targets;

Upon activation under appropriate costimulatory conditions, naive T helper (Th) cells differentiate into Th2 or Th17 cells, each characterized by the expression of specific effector cytokines. In response to a repeated stimulation with antigen, Th cells develop a stable memory for the expression of those cytokines as well as for other secreted or membrane-associated factors. The stable memory for the expression of proinflammatory effector functions may explain the resistance of Th effector cells to conventional immunosuppressive therapy, and the inability of immunosuppression to cure chronic inflammation. The imprinting of the functional memory is based on epigenetic modifications and expression of distinct transcription factors. In this project, we compare the transcriptomes of once and repeatedly activated murine Th1 cells, to identify genes that induce and maintain the functional memory and control the persistence of pathogenic memory Th1 cells. This in turn might help to discriminate pathogenic versus protective cells in immunopathology and present novel targets for the diagnosis and therapy of chronic inflammatory disease.

overall_design:
Genes differentially expressed in once versus four times stimulated Th1 cells. In vitro polarization of murine naïve DO11.10 T cells towards Th1 direction (5 ng/ml recombinant murine IL-12, 5 μg/ml anti-IL-4 antibody) with antigenic stimulation (ova323-339 and irradiated splenic APCs). The transcriptional profiles of resting one week old Th1 (Th1 1w) cells and resting 4 week old Th1 (Th1 4w) cells were compared using Affymetrix Murine Genome (MG) U74V2A GeneChip arrays.
10 µg of total RNA from each cell sample was reverse transcribed using T7-(d)T24 primer and SuperScript II reverse transcriptase
cDNA extraction with a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate
Biotinylated cRNA was transcribed with the MEGAscript high yield transcription kit (Ambion), fragmented, and the hybridization cocktail was prepared (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control)
probes were subsequently hybridised with the GeneChip U74Av2 for 16 hrs at 45 oC
after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody
TH1_1w_C1;  TH1_1w_C2;  TH1_1w_C3TH1_4w_C1;  TH1_4w_C2;  TH1_4w_C3
1w: 1 week in culture; 4w: 4 weeks in culture; C1-3: Culture or Experiment No.

first: Uwe

last: Niesner

first: Inka

last: Albrecht

first: Marko

last: Janke

first: Cornelia

last: Doebis

first: Christoph

last: Loddenkemper

first: Maria

middle: H

last: Lexberg

first: Katharina

last: Eulenburg

first: Stephan

last: Kreher

first: Juliana

last: Koeck

first: Ria

last: Baumgrass

first: Kerstin

last: Bonhagen

first: Thomas

last: Kamradt

last: Enghard

first: Jens

middle: Y

last: Humrich

first: Sascha

last: Rutz

first: Ulf

last: Schulze-Topphoff

first: Orhan

last: Aktas

first: Sina

last: Bartfeld

first: Helena

last: Radbruch

first: Ahmed

middle: N

last: Hegazy

first: Max

last: Loehning

first: Daniel

middle: C

last: Baumgart

first: Rainer

last: Duchmann

first: Martin

last: Rudwaleit

last: Haeupl

first: Inna

last: Gitelman

first: Veit

last: Krenn

first: Joachim

middle: R

last: Gruen

first: Jochen

last: Sieper

last: Zeitz

first: Bertram

last: Wiedenmann

first: Frauke

last: Zipp

first: Alf

last: Hamann

first: Michal

last: Janitz

first: Alexander

last: Scheffold

first: Gerd

last: Burmester

first: Hyun

middle: D

last: Chang

first: Andreas


title: Effect of loss of CREB and CREM on kainate-induced gene expression in the hippocampus (MG-U74A).

status: Public on Apr 03 2008

city: Heidelberg

first: Jan

last: Rodriguez Parkitna

address: Im Neuenheimer Feld 581

institute: German Cancer Research Center

summary:
Ablation of the Creb1 gene in forebrain neurons was performed using the Cre/loxP system, with the recombinase expressed from the Camk2alfa promoter. Mice were crossed into the Crem KO background to prevent compensation of CREB loss by CREM overexpression.
Our goal was to analyze how loss of CREB will affect acitivity-regulated transcription induced by strong stimulation, i.e. kainate.
Keywords: Treatment x Genotype

overall_design: Animals were injected i.p. with either 20 mg/kg kainate or saline and sacrificed after one hour by cervical dislocation. Expression profiling was performed using total RNA isolated from the hippocampus.

first: Thomas

last: Lemberger


title: Genes up-regulated by VE-cadherin expression and clustering at junctions

status: Public on Jul 06 2008

city: Milan

first: Elisabetta

last: Dejana

address: Via Adamello, 16

institute: IFOM-IEO-CAMPUS

summary: In order to identify genes regulated by VE-cadherin expression, we compared a mouse VE-cadherin null cell line (VEC null) with the same line reconstituted with VE-cadherin wild type cDNA (VEC positive). The morphological and functional properties of these cell lines were described previously [Lampugnani,M.G. et al. Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148. J. Cell Biol. 161, 793-804 (2003)]. By Affymetrix gene expression analysis we found several genes up-regulated by VE-cadherin, among which claudin-5 reached remarkably high levels. The up-regulation of these genes required not only VE-cadherin expression but also cell confluence suggesting that VE-cadherin clustering at junctions was needed.

overall_design: VEC null or positive cells in the sparse and confluent conditions have been starved in MCDB 131 medium 1% BSA for 36 h. Next, RNA has been extracted with standard guanidinium isothiocyanate lysis buffer and cesium chloride ultracentrifugation. Synthesis of biotinylated cRNA targets, array hybridization (GeneChips MG_U74Av2 and MG_U74Bv2), staining and scanning were performed according to the Affymetrix standard protocols, starting from 15 μg of total RNA. Two copies of the GeneChips were hybridized with each cRNA sample. The MAS5 algorithm was used to determine the expression levels of mRNAs; the absolute analysis was performed using default parameters and scaling factor 500. Report files were extracted for each chip, and performance of labeled target was evaluated on the basis of several values (scaling factor, background and noise values, % present calls, average signal value, etc). Gene expression levels were normalized on the median over all samples. Annotation of Probe Sets is according to Affymetrix Annotation Tables (release May 31, 2007) [Vecchi,M. et al. Gene expression analysis of early and advanced gastric cancers. Oncogene 26, 4284-4294 (2007)]. We selected the genes up-regulated by VE-cadherin expression and clustering at junctions in confluent VEC positive cells.

first: Andrea

last: Taddei

first: Annarita

last: Conti

first: Fabrizio

last: Orsenigo

first: Ferruccio

last: Breviario


title: Identifying alterations of gene expression induced by two teratogenic agents which induce a similar phenotype

status: Public on Jun 19 2008

city: Cincinnati

first: William

last: Scott

state: OH

address: 3333 Burnet Ave

department: Developmental Biology

institute: Cincinnati Children's Hospital Medical Center

summary:
Samples used for hybridization consisted of non-pooled (NP) RNA extracts from 8 groups in each of two time periods after drug administration: oil vehicle treated control embryonic limb bud mesoderm and ectoderm, phosphate buffered saline vehicle control embryonic limb bud mesoderm and ectoderm, acetazolamide treated embryonic limb bud mesoderm and ectoderm, and cadmium sulfate treated embryonic limb bud mesoderm and ectoderm. Forty-eight hybridization experiments were on non-pooled (NP) individual RNA extracts.

Keywords: Cadmium or Acetazolamide drug course over time in embryonic limb mesoderm and ectoderm.

first: Claire

last: Schreiner

first: Sheila

last: Bell


title: 2-Deoxyglucose induced transcription in control and MondoA knockdown cells

status: Public on Apr 24 2008

city: Salt Lake City

first: Donald

last: Ayer

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: University of Utah, Huntsman Cancer Institute

type: Expression profiling by genome tiling array

summary:
To determine the contibution of the transcription factor MondoA to 2-deoxyglucose-induced transcription expression analysis was carried out in control and MondoA knockdown cells
HA1ER cells are human embryonic kidney cells transformed with hTERT, SV40 Early region and activated H-Ras
Keywords: cell type comparision, genetic modification

overall_design: HA1ER cells, Control (NS) or MondoA knockdown (M2), were starved for glucose overnight and then treated with 2-deoxyglucose for 3 hours

first: Chris

last: Peterson


title: Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3.

city: Zurich

first: Paolo

last: Cinelli

address: Winterthurerstrasse 190

department: Institute of Laboratory Animal Science

country: Switzerland

institute: University of Zurich

summary:
To investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells.
Keywords: Comparative expression analysis, STAT3 pathway related genes

overall_design: FVB/N derived embryonic stem cells cultivated in the presence of LIF were compared with FVB/N ES cells overexpressing STAT3-MER and cultivated in the presence of 4-hydroxytamoxifen and absence of LIF

first: Elisa

last: Casanova

first: Syndi

last: Uhlig

first: Priska

last: Lochmatter

first: Takahiko

last: Matsuda

first: Takashi

last: Yokota

last: Rülicke

first: Birgit

last: Ledermann

first: Kurt

last: Bürki


title: Identification of Stat5 Target Genes by siRNA-mediated knockdown

status: Public on Jun 23 2008

city: Regensburg

first: Anne

last: Rascle

address: Universitaetsstrasse

institute: University of Regensburg

summary:
STAT5A and STAT5B proteins belong to the family of signal transducers and activators of transcription. They are encoded by 2 separate genes with 91% identity in their amino acid sequences. Despite their high degree of conservation, STAT5A and STAT5B exert non-redundant functions, resulting at least in part from differences in target gene activation. To better characterize the differential contribution of STAT5A and STAT5B in gene regulation, we performed single or double knock-down of STAT5A and STAT5B using small interfering RNA. Subsequent gene expression profiling and RT-qPCR analyses of IL-3-stimulated Ba/F3-beta cells led to the identification of putative novel STAT5 target genes. Chromatin immunoprecipitation assays analyzing the corresponding gene loci identified unusual STAT5 binding sites compared to conventional STAT5 responsive elements. Some of the STAT5 targets identified are upregulated in several human cancers, suggesting that they might represent potential oncogenes in STAT5-associated malignancies.
Keywords: siRNA-mediated knock-down

overall_design:
Ba/F3-beta cells transfected with either control (scramble I) or Stat5A and Stat5B siRNAs were stimulated with IL-3 for either 30 minutes or 2 hours.  Unstimulated Ba/F3-beta cells transfected with scramble I siRNA were used as a control.

Total RNA was isolated using the RNeasy Maxi kit, including an on-column DNase I treatment to eliminate genomic DNA contamination, according to the manufacturer’s protocol (Qiagen, Valencia, CA). DNase-treated total RNA (5 µg) was synthesized into biotinylated cRNA probe using one-cycle target labeling and IVT labeling (Affymetrix, Inc., Santa Clara, CA) according to manufacturer’s instructions. Fifteen µg of biotinylated cRNA probe from each sample was hybridized onto murine U74A, U74B, U74C Affymetrix Microarray chips. The hybridized chips were then washed using the Affymetrix GeneChip Fluidics 400 station and scanned in the Affymetrix GeneChip Scanner 3000 (Affymetrix, Inc., Santa Clara, CA) according to manufacturer’s instructions.  The quality of cRNA probe synthesis and efficiency of hybridization was analyzed in the GeneChip Operating System for each Affymetrix chip once scanning was complete. Microarray data were normalized using MAS5 and then analyzed using the Genespring (Agilent, Palo Alto, CA).

Out of the 36,767 genes, 14,553 passed our noise and P/M/A call filters. Genes were further selected for being stimulated at least 2-fold by IL-3 after 30 minutes (164 genes) and 2 hours (553 genes) stimulation compared to the unstimulated cells (ScI-transfected cells). Then, IL-3 induced genes that were down-regulated at least 2-fold in the STAT5A/B siRNA-transfected cells at 30 minutes and 2 hours (12 and 29 respectively) were identified as putative STAT5 target genes and further investigated.


title: Normal Calorie Restricted

status: Public on Mar 19 2008

city: Riverside

first: Stephen

middle: R.

last: Spindler

state: CA

department: Biochemistry

institute: University of California, Riverside

summary:
Normal female Ames mouse (+/+ or +/df)  with ad libitum access to water and fed 30% less of the standard pelleted diet with respect to the amount consumed by normal female ad libitum fed +/+ and +/df mice (LabDiet, PMI Feeds, Inc., St Louis, MO) caged with microisolator filter tops.  Mice were killed between 24 to 26 months of age, tissues removed, rapidly frozen on dry ice, and stored in liquid nitrogen. Total liver RNA was isolated from frozen tissue as described (T. Tsuchiya, J.M. Dhahbi, X. Cui, P.L. Mote, A. Bartke, S.R. Spindler, Physiological Genomics, Submitted). mRNA levels were measured using the Affymetrix mouse U74Av2 array according to standard protocols. After hybridization, arrays were scanned using a Hewlett-Packard GeneArray Scanner. Image analysis was performed as described (Cao SX, Dhahbi JM, Mote PL, and Spindler SR. Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice. Proc Natl Acad Sci U S A 98: 10630-10635, 2001). A more detailed description of the methods can be found in (T. Tsuchiya, J.M. Dhahbi, X. Cui, P.L. Mote, A. Bartke, S.R. Spindler, Physiological Genomics, Submitted).
Keywords = Ames dwarf
Keywords = mouse
Keywords = Affymetrix
Keywords = caloric restriction
Keywords: parallel sample

first: Tomoshi

last: Tsuchiya

first: Joseph

last: Dhahbi

first: Xinping

last: Cui

first: Patricia

middle: L

last: Mote

first: Andrzej

last: Bartke


title: microRNA-155 is an Epstein-Barr Virus induced gene that modulates EBV-regulated gene expression (microRNA-155 infection)

status: Public on Mar 18 2008

city: New Orleans

first: Erik

middle: K

last: Flemington

state: LA

address: 1430 Tulane Ave., SL79

institute: Tulane Health Sciences Center

summary:
The cellular microRNA, miR-155 has been shown to be involved in lymphocyte activation and is expressed in EBV infected cells displaying type III latency gene expression but not type I latency gene expression.  We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested and we show that expression in EBV infected cells requires a conserved AP-1 element in the miR-155 promoter.  Gene expression analysis was carried out in a type I latency cell line transduced with a miR-155 expressing retrovirus.  This analysis identified both miR-155 suppressed and induced cellular mRNAs and suggested that in addition to direct targeting of 3’ UTRs, miR-155 alters gene expression in part through the alteration of signal transduction pathways.  3’ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes, BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets.  Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus.  Inspection of the promoters from genes regulated in EBV infected cells and in cells infected with a miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3.  Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV mediated signaling in part through the modulation of transcriptional regulatory factors.
Keywords: Differential expression of miR-155 vs cntl expressing cells

overall_design: The EBV positive Burkitt's lymphoma cell line, Akata was infected with a control (pEhyg-miRCntl) or a microRNA-155 expressing (pEhyg-miR-155) retrovirus.  Duplicate infections with the control retrovirus and with the miR-155 retrovirus were carried out.  Control and miR-155 infection pair 1 were run on an array as well as a dye swap.  Control and miR-155 infection pair 2 were similarly run on an array as well as a second array containing a dye swap.

first: Qinyan

last: Yin

first: Jane

last: McBride

first: Claire

last: Fewell

first: Michelle

last: Lacey

first: Xia

last: Wang

first: Zhen

last: Lin

first: Jennifer

last: Cameron


title: siNTSR1-treated HNSCC cell lines: Control vs. siNTSR1-treated HSC2 or HSC3

status: Public on Mar 29 2008

city: Chiba

first: Satoya

last: Shimizu

address: 1-8-1 Inohana, Chuo-ku

institute: Chiba university

summary:
NTSR1 siRNA was used to abolish NTS signaling pathway in HSC2 and HSC3, and analyzed differentially gene expression with microarray.
Keywords: Genetic modification

overall_design: Four-condition experiment, control vs. NTSR1 siRNA-treatment in HSC2 and HSC3 cells

first: Naohiko

last: Seki


title: Meta-analysis of Clear Cell Renal Cell Carcinoma Gene Expression Defines a Variant Subgroup and Identifies Gender Influences on Tumor Biology

status: Public on Nov 05 2011

city: Nashville

first: Kimryn

last: Rathmell

state: TN

address: 2220 Pierce Ave

department: Medicine

institute: Vanderbilt

type:
Expression profiling by array
Third-party reanalysis

summary: Clear cell renal cell carcinoma comprises two dominant subtypes, ccA and ccB, with gender disparity providing additional disease information. A third minor subgroup has distinct expression profiles consistent with von Hippel-Lindau wild type status and displays variant histology features.

overall_design: 44 new tumor samples and six large, publicly available, ccRCC gene expression databases were identified that cumulatively provided data for 480 tumors for metaanalysis via meta-array compilation.

first: A

last: Brannon

first: Scott

last: Haake

first: Kathryn

middle: E

last: Hacker

first: Raj

last: Pruthi

first: Eric

last: Wallen

first: Matthew

last: Nielsen

first: W


title: MyoD-/-, Time Course

status: Public on Mar 01 2008

city: Ottawa

first: Theodore

last: Perkins

state: ON

address: 501 Smyth Rd.

department: Cellular and Molecular Medicine

country: Canada

institute: Ottawa Hospital Research Institute

summary:
Myogenic Primary Myoblast Time Course in vitro Differentiation into Myotubes for Ontario Genome Project 2004-05.
The Differentiation is leaded by removing the Proliferation Medium (Ham's F10 Medium + growth factors) and feeding with Differentiation Medium (DMEM hg + 2.5% Horse serum).
The expected data are: % Viability of cells; Total RNA extraction; BioAnalysis and MicroArray of Undifferentiated and Differentiated cells.
Time course: 0 hr, 6 hr, 12 hr, 18 hr, 24 hr, 36 hr, 48 hr, 3 days, 4 days, 7 days.
Note that the Triplicate1 sample = MyoD-/-1999; Triplicate 2 sample = MyoD-/-p6; Triplicate 3 sample = MyoD-/-p10.
Keywords: other

overall_design: this experiment include 10 samples and 90 replicates


title: microRNA-155 is an Epstein-Barr Virus induced gene that modulates Epstein Barr virus regulated gene expression pathways

first: Erik

last: Flemington

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Expression data from AJ mouse control lung tissue

status: Public on Feb 20 2008

city: Denver

first: Robert

middle: S

last: Stearman

state: CO

address: 4200 E Ninth Ave

department: Medicine/Pulmonary

institute: UCHSC

summary:
AJ mouse is susceptible to lung carcinogenesis from urethane treatment and is a good model for human adenocarcinoma.  We completed a study using microarray analysis to identify a unique macrophage expression signature in the lung tumor microenvironment.
Keywords: Urethane treatment time course

overall_design: RNA from lung tissues of age matched untreated controls (Normal) from AJ mice were from two time points 24 to 26 weeks (N) or 42 weeks (N42) after saline injection. Datasets were compared to previously published adjacent to tumor lung tissues in Stearman et al Am J Path 167 1763 (2005).

first: Lori

last: Nield

first: Michael

last: Grady

first: Alvin

last: Malkinson

first: Mark

last: Geraci


title: Comparison of gene expression in conventional and germ-free intestine

status: Public on May 19 2008

city: Lausanne

first: James

last: Holzwarth

state: Vaud

address: vers chez les blanc

department: Bioanalytical Sciences

institute: Nestle Research Center

summary:
Whole tissues corresponding to the ileum, colon and rectum were dissected from adult mice and used for RNA preparation. The aim of experiment was to study the impact of gut microbiota on gene expression in different gut regions.
Keywords: comparative genomic hybridization

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Three biological replicates were used for each condition. Each RNA preparation was independently hybridized to one chip.

first: Elena

last: Comelli


title: Airway Epithelial Cell Response to Sendai virus infection

status: Public on Jan 19 2008

city: St. Louis

first: Anand

middle: Champak

last: Patel

state: MO

address: Campus Box 8116, 660 S. Euclid Ave.

department: Pulmonary/Critical Care Medicine

institute: Washington University School of Medicine

summary:
Oligonucleotide microarrays were used to establish a profile for gene expression in wild-type airway epithelial cells after paramyxoviral infection.
Analysis was performed on mRNA isolated from SeV-infected primary-culture mouse tracheal epithelial cells that were maintained under physiologic conditions (air-liquid interface).
Keywords: Treatment Comparison

overall_design: Primary-culture mouse tracheal epithelial cells (mTECs) were established on Transwell membranes using air-liquid interface (ALI) conditions. Sendai virus (SeV), strain 52, was obtained from American Type Culture Collection and stored at -70°C. Cultures were inoculated with SeV or an equivalent amount of UV-inactivated SeV (SeV-UV) in the apical compartment for 1 h at 37 °C. Air-liquid-interface conditions were re-established by washing the membrane with PBS. Each culture well was subjected to one of two treatments (SeV, or UV-SeV) for 1 day. N = 4 SeV wells, N = 6 UV-SeV wells, with each well independently analyzed by microarray. No technical replicates were performed, but arrays were evaluated for quality control using the SimpleAffy package (Miller CJ, 2004) in Bioconductor 2.0.

first: L

middle: P

last: Shornick

first: G

last: Wells

first: Y

last: Zhang

last: Huang

first: K

last: Takami

first: M

last: Sosa

first: N

last: Shukla

first: E

last: Agapov

middle: J

last: Holtzman


title: MyoD wt, Time Course

first: Theodore

last: Perkins

summary:
Time Course in vitro Differentiation of Myogenic Primary Myoblast into Myotubes for Ontario Genome Project 2004-05.
The Differentiation is leaded by removing the Proliferation Medium (Ham's F10 Medium + growth factors) and feeding with Differentiation Medium (DMEM hg + 2.5% Horse serum).
The expected data are: % Viability of cells; Total RNA extraction; BioAnalysis and MicroArray of Undifferentiated and Differentiated cells.
Time course: 0 hr, 6 hr, 12 hr, 18 hr, 24 hr, 36 hr, 48 hr, 3 days, 4 days, 7 days.
Keywords: other


title: Chronic viral infection of naive, effector, memory and exhausted virus-specific CD8 T cells

status: Public on Nov 27 2007

city: Atlanta

first: Shruti

last: Subramaniam

state: GA

address: Clifton Road

institute: Emory University

summary:
CD8 T cells normally differentiate from resting naïve T cells into function effector and then memory CD8 T cells following acute infections.  During chronic viral infections, however, virus-specific CD8 T cells often become exhausted.  We used microarrays to examine the gene expression differences between naive, effector, memory and exhausted virus-specific CD8 T cells following lymphocytic choriomeningitis virus infection.
Keywords: infection response

overall_design: Three or four independent samples were sorted by flow cytometry for each cell type (naive, effector, memory and exhausted) virus-specific CD8 T cells.  RNA was extracted and hybridized to Affymetrix microarrays.


title: Gene expression changes associated with the anti-angiogenic effect of KLK3 (PSA) on HUVECs

status: Public on Jul 22 2008

city: Helsinki

first: Hannu

last: Koistinen

address: PO Box 63

department: Clinical Chemistry

country: Finland

institute: University of Helsinki

summary:
Kallikrein-related peptidase 3 (KLK3, also known as prostate-specific antigen, PSA) is a chymotrypsin-like kallikrein that has been shown to have anti-angiogenic properties. KLK3 expression in prostate cancer is associated with low microvessel density. We have previously shown in a human umbilical vein endothelial cell (HUVEC) model that the anti-angiogenic effect of KLK3 is related to its enzymatic activity. However, the mechanism of this effect remains to be clarified. To this end we have used a DNA microarray to study the KLK3-induced changes in gene expression associated with reduction of HUVEC tube formation. Among the 41000 genes studied, 311 were differentially expressed in control and KLK3-treated cells. These changes were enriched in several pathways, including the pathways associated with proteasome, ubiquitin-mediated proteolysis, focal adhesion and regulation of actin cytoskeleton. Furthermore, the changes were opposite to those described previously to occur during tubulogenesis. In conclusion, our results show that KLK3 induces gene expression changes in HUVECs. While these changes might be relevant for the mechanism by which KLK3 exerts its anti-angiogenic activity, it cannot be judged by the present results whether the changes are secondary to morphogenic differentiation of the cells or the primary mechanism mediating the effect of KLK3.
Keywords: KLK3 treatment, tube formation

overall_design:
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cord veins and grown in Endothelial Cell Basal Medium supplemented with an Endothelial Cell Growth Medium Supplement Pack (PromoCell). The cells were cultured on Matrigel basement membrane prepara-tion together with 500 nM enzymatically active KLK3 (PSA). Phosphate buffered saline (PBS) was used for control. HUVECs were grown on Matrigel for 18 h.

RNA was isolated from KLK3-treated and control HUVECs from three different individuals. Cell Recovery Solution (BD Biosciences) was used to detach cells from the Matrigel. Total RNA was isolated with the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. RNA con-centration and quality of the samples was determined with an Agilent 2100 bioanalyser (Agilent Technologies) by measuring the RNA integrity number (RIN). All samples contained high quality RNA (RIN 9.5-10).

DNA microarray analysis was performed with the Agilent Whole Human Genome Oligo Microar-ray 4x44K (G4112F) (Agilent Technologies Inc.) following the manufacturer’s protocol. Total RNA (1 ug) was labeled using the RNA input fluorescent linear amplification kit according to the manufacturer's recommendations (Agilent Technologies Inc.). RNA was hybridized on Agilent's Human 4x44K Oligo Microarrays containing 44000 features (41000 unique genes and transcripts). Pre-processing of the data was performed by Feature Extraction software (v 7.5.1).

first: Johanna

last: Aaltonen

first: Pirjo

last: Laakkonen

first: Sami

last: Kilpinen

first: Ulf-Håkan

last: Stenman


title: The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97

status: Public on Jan 23 2008

city: Hannover

first: Doris

last: Steinemann

state: Niedersachsen

department: Cell and Molecular Pathology, Theoretische Institute II

institute: Medical School Hannover

summary:
A possible functional role of VCP/p97 during differentiation of U937 cells was addressed by siRNA-targeting of VCP/p97. Functional changes in VCP-siRNA-transfected cells following phorbol ester-induced monocytic differentiation have been examind by DNA microarray analysis. Cells transfected with the non-targeting AllStars negative siRNA (Qiagen) served as a reference.
Keywords: cellular modification design

overall_design: Computed


title: microRNA-155 is an Epstein-Barr Virus induced gene that modulates EBV-regulated gene expression (EBV infection)

first: Erik

last: Flemington

summary:
The cellular microRNA, miR-155 has been shown to be involved in lymphocyte activation and is expressed in EBV infected cells displaying type III latency gene expression but not type I latency gene expression.  We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested and we show that expression in EBV infected cells requires a conserved AP-1 element in the miR-155 promoter.  Gene expression analysis was carried out in a type I latency cell line transduced with a miR-155 expressing retrovirus.  This analysis identified both miR-155 suppressed and induced cellular mRNAs and suggested that in addition to direct targeting of 3’ UTRs, miR-155 alters gene expression in part through the alteration of signal transduction pathways.  3’ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes, BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets.  Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus.  Inspection of the promoters from genes regulated in EBV infected cells and in cells infected with a miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3.  Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV mediated signaling in part through the modulation of transcriptional regulatory factors.
Keywords: Gene expression analysis in EBV positive vs EBV negative cells

overall_design: Two RNA preps from the EBV negative Burkitt's lymphoma cell line, BL41, and two RNA preps from EBV converted BL41 cells were prepared.  In each two color array, EBV positive BL41s were run with EBV negative BL41s.  RNA prep set 1 were run in duplicate, RNA prep set 2 were run in duplicate and all four of these combinations were run as dye swaps for a total of eight arrays.

first: Qinyan

last: Yin

first: Jane

last: McBride

first: Claire

last: Fewell

first: Michelle

last: Lacey

first: Xia

last: Wang

first: Zhen

last: Lin

first: Jennifer

last: Cameron


title: The Epstein-Barr Virus latent membrane protein 1 (LMP1) induces cellular microRNA146a

status: Public on Jan 04 2008

summary:
The Epstein Barr virus (EBV) encoded latent membrane protein-1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of Nuclear Factor-kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis, and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative RT-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an OCT-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.
Keywords: Analysis of gene expression changes altered by the microRNA, miR-146a

overall_design: EBV positive Burkitt's lymphoma cell line, Akata, was infected with the control retrovirus, pEhyg, or the miR-146a expressing retrovirus, pEhyg-miR-146a.  Two infections were carried out for control retrovirus and two infections were carried out with the pEhyg-miR-146a retrovirus.  Total RNA was prepared from each of the 4 infections.  Control and miR-146a infection set 1 was subjected to dual color array analysis on chip 1.  Control and miR-146a infection set 2 was subjected to dual color array analysis on second array of chip 1.  Dye swaps of each of these were carried out on two arrays from a second chip (chip 2).

first: Brian

last: Schaefer


title: human induced pluripotent stem cell

status: Public on Nov 22 2007

city: Kyoto

first: Shinya

last: Yamanaka

address: 53 Kawahara-machi, Shogoin, Sakyo-ku

institute: Center for iPS Cell Research and Applicaton (CiRA), Kyoto University

summary:
Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were expandable and similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, DNA methylation status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.
Keywords: cell type comparison

overall_design: Total RNA from HDF and hiPS cells (clone 201B) was labeled with Cy3. Samples were hybridized with Whole Human Genome Microarray 4 x 44K (G4112F, Agilent), with the one color protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent).  Data analyzed by using GeneSpring GX7.3.1 software (Agilent). Array data of hES H9 cells (GSM194390) was retrieved from GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM194390). Genes with "present" flag value in all three samples were used for analyses (32266 genes).

first: Kazutoshi

last: Takahashi

first: Megumi

last: Narita


title: Gene expression profiles in TNBS-induced murine colitis

status: Public on Dec 10 2007

city: Baltimore

first: Shukti

last: Chakravarti

address: 720 Rutland Ave

institute: Johns Hopkins University School of Medicine

summary:
We investigated the relationship between chronic inflammation and fibrosis in a mouse model of chronic colitis.  Six weekly Trinitrobenzenesulphonic acid (TNBS) enemas were given to establish colitis and temporal changes in gene expression was elucidated over the next six weeks.
Keywords: disease state analysis

overall_design: After the six TNBS enemas, mice were sacrificed at 3 days (termed as TNBS-w6, n=6), 2 weeks (TNBS-w8, n=4), 4 weeks (TNBS-w10, n=5), or 6 weeks (TNBS-w12, n=4). The saline control groups were also sacrificed at week 6 (Saline-w6, n=5), week 8 (n=2) and week 10 (n=2). The last two sets were pooled as Saline-w10 (n=4). Two replicates of saline-w6 were analyzed. We compared Saline-w6 with TNBS-w6, and Saline-w10 with TNBS-w8, TNBS-w10, TNBS-w12, respectively.

first: Feng

last: Wu


title: The EBV latent membrane protein 1 (LMP1) induces cellular microRNA-146a, a modulator of lymphocyte signaling pathways

status: Public on Jan 09 2008

type:
Expression profiling by array
Non-coding RNA profiling by array


title: Comparison of the prostate cancer cell line LNCaP and its androgen insensitive derivative C4-2B

status: Public on Aug 07 2007

city: Ann Arbor

first: Scott

last: Tomlins

address: 1400 E. Medical Center Dr., 5410 CCGC

institute: University of Michigan

summary:
LNCaP and its androgen insensitive derivative were profiled in order to identify genes differentially expressed during the conversion to androgen insensitivity.
This experiment was performed due to the presence of an ins(7;14) localizing the entire ETV1 locus to chr 14 in LNCaP and C4-2B prostate cancer cells.
Keywords: cell type comparison

overall_design: LNCaP and C4-2B were both hybridized to Agilent Whole Human Genome Oligonucleotide Microarrays, with a commercially obtained pool of benign prostate RNA serving as the reference for both cell lines. Dye flips for both cell lines were also performed

middle: A

first: Arul

last: Chinnaiyan


title: Expression profiling of the developing and mature Nrl -/- mouse retina (Yoshida et al. 2004, HMG)

status: Public on Sep 08 2007

first: Swaroop

last: Anand

address: 1000 Wall St.

department: Ophthalmology & Visual Sciences

summary:
The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed.
Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation.
Keywords: time course

overall_design: Both the Nrl-/- mice and wild type controls were of a matched mixed genetic background (R1 and C57BL/6 strains).

first: Shigeo

last: Yoshida

first: Alan

last: Mears

first: James

last: Friedman

first: Todd

last: Carter

first: Shirley

last: He

first: Edwin

last: Oh

first: Yuezhou

last: Jing

first: Rafal

last: Farjo

first: Gilles

last: Fleury

first: Carrolee

last: Barlow

first: Alfred

middle: O

last: Hero

first: Anand

last: Swaroop


title: Profiling of RWPE cells stably over-expressing ETV1

department: Pathology

summary:
RWPE benign prostatic epithelial cells were infected with a lentivirus expressing ETV1 or GUS (control), and stable clones were isolated by puromycin selection.
ETV1 over-expression, recapitulating ETS gene rearrangements observed in vivo, confers invasiveness in the benign prostate cell line RWPE.
Keywords: genetic modification

overall_design: Four hybridizations in total were performed using RWPE cells stably expressing ETV1 or GUS (control) on Agilent Whole Human Genome Oligonucleotide Microarrays. RWPE-ETV1 (Cy5) / RWPE-GUS (Cy3) and a dye flip were performed in duplicate

middle: M


title: Whole genome recruitment of Rpb3 and Rpb4 in S.cerevesiae

status: Public on Nov 15 2007

city: Bangalore

first: madavan

last: vasudevan

state: Karnataka

address: Kasturi Nagar

country: India

institute: Theomics International Private Limited

type: Genome binding/occupancy profiling by genome tiling array

summary:
ChIP on chip microarray experiment to decipher Rpb3 and Rpb4 recruitment in S.cerevesiae genome.
Keywords: ChIP-chip

overall_design: TAP- tagged Rpb3 and Rpb4 strains were cultured in 100 ml YPD medium at 25°C till log phase (O. D. 600 ~ 0.8). Cells were crosslinked with formaldehyde to a final concentration of 1% for 20 minutes. Cells were collected by centrifugation, washed three times with 40 ml of ice-cold Tris-buffered saline buffer (20 mM Tris HCl [pH 7.5], 150 mM NaCl), and resuspended in 800ul of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail supplied by Roche). Yeast cells were disrupted by shaking for 30 sec for 20 times in the presence of glass beads (diameter, 0.5 mm) using vortexer. Glass beads were removed, and samples were sonicated five times for 15 sec at 20% duty cycle on a sonicator in order to shear chromatin DNA into fragments of average size ~400- 500 bp. Samples were centrifuged 10 min at maximum speed in a micro centrifuge, and the supernatant (from now on referred to as whole-cell extract) was used as input material for immunoprecipitation. Two hundred microliters of whole-cell extract was incubated with Rabbit IgG agarose beads (Sigma) for 3 hrs at 4°C with agitation. Beads were washed twice with 1 ml of lysis buffer, twice with 1 ml of lysis buffer plus 500 mM NaCl, twice with 1 ml of wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA) and once with 1 ml of Tris-EDTA (TE; 10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Bound material was eluted from the beads by resuspending beads in 100ul of elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, and 1% SDS) and incubating 10 min at 65°C with occasional agitation. Samples were centrifuged briefly and the cross-linking was reversed by incubating overnight at 65°C. Samples were treated with proteinase K and RNase A, extracted twice with phenol, extracted once with chloroform, precipitated with ethanol, and resuspended in 30ul of TE. These samples were further purified with Qiagen PCR product purification Kit.

first: Parag

last: Sadhale

first: Jiyoti

last: Verma Gaur


title: Human breast cancer LM2 cell lines: control vs. MTDH knockdown

status: Public on Feb 01 2008

city: Princeton

first: Yibin

last: Kang

state: NJ

address: LTL 254, Washington Road

department: Molecular Biology

institute: Princeton University

summary:
Transcriptional profiling of human breast cancer cell line LM2, a subline of MDA-MB-231 highly metastatic to lung when injected to nude mice, to identify the genes that are regulated after the metastasis gene metadherin is knocked down.
Keywords: Genetic modification

overall_design: Empty pSuper vector control cells were compared to the cells transfected with the MTDH knockdown shRNA construct. Two cultured conditions were studied: the LM2 cancer cells were cultured alone or on top of a monolayer of human lung endothelial HMVEC-L cells. Three arrays for each sample.

first: Guohong

last: Hu


title: Gene induction profile among wild-type, NF-IL6KO and KI(LAP) PECs

status: Public on Dec 05 2007

city: Suita

first: Satoshi

last: Uematsu

state: Osaka

address: 3-1 Yamada-oka

department: Department of Host Defense

institute: Research Institute for Microbial Diseases, Osaka University

summary:
PECs from wild-type, NF-IL6KO and KI(LAP) mice were stimulated with or without LPS+IFN-gamma for 12h. We used microarrays to check the difference of gene induction among wild type, KO and KI mice.
Keywords: Wild, KO and KI(LAP)

overall_design: Peritoneal macrophages were collected for RNA extraction and hybridization on Affymetrix microarrays.

first: Shizuo

last: Akira


title: Striatal gene expression data from 12 months-old Hdh4/Q80 mice and control mice.

status: Public on Oct 22 2007

city: Bethesda

first: Alexandre

last: Kuhn

state: MD

address: 35 Convent Dr

department: NIA

institute: NIH

summary:
To test the hypotheses that mutant huntingtin protein length and wild-type huntingtin dosage have important effects on disease-related transcriptional dysfunction, we compared the changes in mRNA in seven genetic mouse models of Huntington's disease (HD) and postmortem human HD caudate. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in and full-length transgenic models of HD took longer to appear, 15- and 22-month CHL2(Q150/Q150), 18-month Hdh(Q92/Q92) and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. Whereas it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared with those caused by the expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. In addition, very high correlations between the signatures of mice expressing normal levels of wild-type huntingtin and mice in which the wild-type protein is absent suggest a limited effect of the wild-type protein to change basal gene expression or to influence the qualitative disease-related effect of mutant huntingtin. The combined analysis of mouse and human HD transcriptomes provides important temporal and mechanistic insights into the process by which mutant huntingtin kills striatal neurons. In addition, the discovery that several available lines of HD mice faithfully recapitulate the gene expression signature of the human disorder provides a novel aspect of validation with respect to their use in preclinical therapeutic trials.
Keywords: genetic modification

overall_design: Striatal samples from 3 Hdh4/Q80 mutant mice (12 months-old) and 3 age-matched wild-type littermates.

first: Peggy

middle: F

last: Shelbourne

first: Andrew

last: Strand


title: Dystrophin-deficient and dystrophin and utrophin double-deficient mice crossed with mice with full-length hDMD genes

status: Public on Dec 13 2007

city: Leiden

first: Peter

last: 't Hoen

address: PO Box 9600

department: Center for Human and Clinical Genetics

country: Netherlands

institute: Leiden University Medical Center

summary:
Crossing of hDMD mice that contain the full-length 2.3 Mb hDMD gene were crossed with dystrophin-deficient mdx mice and dystrophin and utrophin double-deficient mdx x utrn-/- mice resulted in a full rescue of the dystrophic features of these mice, as concluded from histological analysis. Analysis on Affymetrix gene chips demonstrated that also expression profiles of the dystrophic mice were normalized by crossing with transgenic hDMD mice. This confirms the full functionality of the hDMD transgene in mice.
Keywords: disease state analysis

overall_design: RNA from gastrocnemius muscle from individual mice was hybridized to Affymetrix U74Av2

first: Judith

last: Boer

first: Johan

middle: T

last: den Dunnen


title: ERRalpha wt vs. ko hearts, baseline

status: Public on Oct 16 2007

city: Duarte

first: Janice

last: Huss

address: 1450 E. Duarte Rd.

department: Gene Regulation and Drug Discovery

institute: Beckman Research Institute, City of Hope National Medical Center

summary:
We hypothesized that the estrogen-related receptor a (ERRa), which recruits PGC-1a to metabolic target genes in heart, exerts protective effects in the context of stressors known to cause heart failure. ERRa-/- mice subjected to left ventricular (LV) pressure overload developed signatures of heart failure including chamber dilatation and reduced LV fractional shortening. 31P-NMR studies revealed abnormal phosphocreatine depletion in ERRa-/- hearts subjected to hemodynamic stress, indicative of a defect in ATP reserve. Mitochondrial respiration studies demonstrated reduced maximal ATP synthesis rates in ERRa-/- hearts. Cardiac ERRa target genes involved in energy substrate oxidation, ATP synthesis, and phosphate transfer were downregulated in ERRa-/- mice at baseline or with pressure overload. These results demonstrate that ERRa, a potential therapeutic target, is indispensable for the adaptive bioenergetic response to hemodynamic stressors known to cause heart failure.
Keywords: Genetic modification, stress response

overall_design: Microarray analyses were performed with two samples each of ERRawt and ERRako to compare baseline changes in gene expression.  Validation real-time PCR (n=7) was subsequently performed to characterize expression changes of gene targets identified in microarray and ChIP-chip studies in hearts of ERRa wt and KO mice at baseline and subjected to pressure overload stress.

first: Ken-ichi

last: Imahashi

first: Catherine

last: Dufour

first: Carla

last: Weinheimer

first: Michael

last: Courtois

first: Atilla

last: Kovacs

first: Vincent

last: Giguère

first: Elizabeth

last: Murphy

first: Daniel

last: Kelly


title: Expression changes in mouse brains following nicotine-induced seizures; the modulation of transcription factor networks

status: Public on Dec 26 2007

city: Tel Aviv

first: Avi

last: Orr-Urtreger

address: 6 Weizmann St

department: Genetic Institute

country: Israel

institute: Tel Aviv Sourasky Medical Center

summary:
Nicotine, acting through the neuronal nicotinic acetylcholine receptors (nAChR), can induce seizures in mice. We aimed to study brain transcriptional response to seizure and to identify genes whose expression is altered after nicotine-induced seizures. Whole brains of untreated mice were compared to brains one hour after seizure activity, using Affymetrix U74Av2 microaarays. Experimental groups included wild-type mice and both nicotine-induced seizures sensitive and resistant nAChR mutant mice. Each genotype group received different nicotine doses to generate seizures. This approach allowed the identification of significantly changed genes whose expression was dependent on seizure activity, nicotine administration or both, but not on the type of nAChR subunit mutation or the amount of nicotine injected. Significant expression changes were detected in 62 genes (p < 0.05, FDR correction). Among them, GO functional annotation analysis determined that the most significantly over-represented categories were of genes encoding MAP kinase phosphatases, regulators of transcription and nucleosome assembly proteins. In-silico bioinformatic analysis of the promoter regions of the 62 changed genes detected the significant enrichments of 16 transcription regulatory elements (TREs), creating a network of transcriptional regulatory responses to seizures. The TREs for ATF and SRF were most significantly enriched, supporting their association with seizure activity. Our data suggest that nicotine-induced seizure in mice is a useful model to study seizure activity and its global brain transcriptional response. The differentially expressed genes detected here can help understand the molecular mechanisms underlying seizures in animal models, and may also serve as candidate genes to study epilepsy in humans.
Keywords: treatment, seizure activity, genotypes

overall_design: Whole brain expression profiles were determined in two experimental groups of mice, sixteen mice that were not treated with nicotine and twelve mice one hour after experiencing nicotine-induced seizure. The untreated group included six wild-type mice, five alpha7+/T and five beta4-/- mice. The group of mice that underwent nicotine-induced seizures included three wild-types, five alpha7+/T and four beta4-/- mice. Different doses of nicotine were injected intraperitoneally (i.p.) to each genotype group of mice in order to achieve a similar seizure score in all three genotypes.

first: Merav

last: Kedmi


title: Gene expression analysis of Kidneys from FGF23 TG and WT mice

status: Public on Nov 29 2007

city: Uppsala

first: Richard

last: Marsell

address: Sjukhusvägen

department: Dep. of Surgical Sciences

country: Sweden

institute: Uppsala University

summary:
Fibroblast growth factor-23 (FGF23), a circulating protein produced in bone, causes renal inorganic phosphate (Pi) wasting by down-regulation of sodium phosphate co-transporter 2a (Npt2a). The mechanism behind this action is unknown. We have previously generated transgenic mice (TG) expressing human wild-type FGF23 under the control of the α1 (I) collagen promoter. In this study we performed a large scale gene expression study of kidneys from TG mice and wild-type littermates. Several genes that play a role in Pi regulation had decreased expression levels, such as Npt2a, but also Pdzk1 which is a scaffolding protein known to interact with NPT2a. Importantly, the Klotho gene, a suggested crucial co-factor for FGF23 receptor binding and activation, was the most affected decreased gene. However, other genes proposed to regulate Pi levels, such as secreted Frizzled Related Protein 4 (sFRP4), Na+/H+ exchanger regulatory factor 1 (NHERF1) and the FGF-receptors 1-4, revealed no changes. Interestingly, expression levels of inflammatory response genes were increased and histological analysis revealed tubular nephropathy in the TG mice kidneys. In conclusion, FGF23 TG mice have altered kidney gene expression levels of several genes thought to be part of Pi homeostasis and an increase in inflammatory response genes, data supported by histological analysis. These findings may lead to further understanding of how FGF23 mediates its actions on renal Pi regulation.
Keywords: Genetic Modification

overall_design: Five kidneys from FGF23 TG mice and five kidneys from WT littermates was used for Affymetrix Genechip analysis. One Genechip was used/animal. Animals were 8 weeks old when kidneys were collected.


title: Gene expression analysis of Peyers patches after infection of C57BL/6 mice with Yersinia enterocolitica

status: Public on Nov 10 2007

city: Tuebingen

first: Erwin

last: Bohn

address: Elfriede-Aulhorn-Strasse 6

department: Institutefor Medical Microbiology and Hygiene

institute: University of Tuebingen

summary:
Orogastral infection of mice with Yersinia enterocolitical leads to HIF-1 alpha activation.To elucidate whether this HIF-1 alpha activation also results in a HIF-1 dependent gene programming, the transcriptomes from Peyers Patches of uninfected and Yersinia enterocolitica infected mice were analyzed by means of of microarray analyses using Affymetrix GeneChip probe arrays (MG-U74Av2). In total, 288 genes were differentially regulated three day after infection in PP compared with the expression of uninfected control mice. Of these 288 genes, 217 were found to be differentially upregulated and from these, 14 genes ( 6.5% of all upregulated genes) are well described to be regulated via HIF-1. These data indicate that orogatral infection with Y. enterocolitica results in HIF-1 dependent gene programmning
Keywords: Time course

overall_design: Per group five C57BL/6 mice were infected orogastrally with 500 Million Yersinia enterocolitica. 1 and 3 days after infection, Peyers Patches were removed and total RNA was prepared. In parallel, RNA was isolated from uninfected mice.  The generation of fragmented cRNA was performed following the manufacturers instructions and used for hybridization onto GeneChip arrays MG-U74Avs2. Analysis of microarray data was performed using the Affymetrix Microarray Suite 5.0, Affymetrix Mining Tool 3.0. A median signal log2 ratio (SLR) grater than 1.5 or less than -1.5 was considered a significant change.

first: Gianluca

last: Matteoli

first: Hanna

last: Hartmann

first: Ingo

middle: B

last: Autenrieth

first: Volkhard

last: Kempf


title: Expression profiling of K14-DN-Clim mouse backskin

status: Public on Jun 23 2007

city: Irvine

first: Kevin

last: Lin

address: 250 Sprague Hall

department: Biological Chemistry & Medicine

institute: University of California, Irvine

summary:
The homeostasis of both cornea and hair follicles depends on a constant supply of progeny cells produced by populations of keratin (K) 14-expressing stem cells localized in specific niches. To investigate the potential role of Co-factors of LIM domains (Clims) in such tissues, we generated transgenic mice expressing a dominant-negative Clim molecule (DN-Clim) under the control of the K14 promoter. As expected, the K14 promoter directed high level expression of the transgene to the basal cells of cornea and epidermis, as well as the outer root sheath of hair follicles. In corneal epithelium, the transgene expression causes decreased expression of adhesion molecule BP180 and defective hemidesmosomes, leading to detachment of corneal epithelium from the underlying stroma, which in turn causes blisters, wounds and an inflammatory response. After a period of epithelial thinning, the corneal epithelium undergoes differentiation to an epidermis-like structure. The K14-DN-Clim mice also develop progressive hair loss due to dysfunctional hair follicles that fail to generate hair shafts. The number of hair follicle stem cells is decreased by at least 50% in K14-DN-Clim mice, indicating that Clims are required for hair follicle stem cell maintenance. We hypothesize that Clim2 is an essential co-factor for the LIM homeodomain factor Lhx2, which was previously shown to play a role in hair follicle stem cell maintenance. Together, these data indicate that Clim proteins play important roles in the homeostasis of corneal epithelium and hair follicles.
Keywords: Comparison of wild-type and transgenic skin tissue

overall_design: We profiled mRNA expression in mouse back skin from 3 time points, representing the initial hair follicle morphogenesis (P6 and P14) and the first telogen (P23); hair growth is synchronized during these time points. For each time point, RNA was isolated and analyzed from 3 to 5 transgenic mice and same number of wild-type littermates.

first: Xiaoman

last: Xu

first: Jaana

last: Mannik

first: Elena

last: Kudryavteseva

first: Lisa

last: Flanagan

first: Joel

last: Spencer

first: Amelia

last: Soto

first: Ning

last: Wang

first: Zhongxian

last: Lu

first: Zhengquan

last: Yu

first: Edwin

last: Monuki

first: Bogi

last: Andersen


title: Analysis of gene expression during neurite outgrowth and regeneration (MG-U74A)

status: Public on Dec 04 2007

first: Moriah

last: Szpara

address: 301 Schultz

summary:
We have undertaken a genome-wide study of transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root ganglia (DRG) during a time course of neurite outgrowth in vitro.  Gene expression observed in these models likely includes both developmental gene expression patterns and regenerative responses to axotomy, which occurs as the result of tissue dissection.  Comparison across both models revealed many genes with similar gene expression patterns during neurite outgrowth.  These patterns were minimally affected by exposure to the potent inhibitory cue Semaphorin3A, indicating that this extrinsic cue does not exert major effects at the level of nuclear transcription.  We also compared our data to several published studies of DRG and SCG gene expression in animal models of regeneration, and found the expression of a large number of genes in common between neurite outgrowth in vitro and regeneration in vivo.
Keywords: time course

overall_design: We wished to determine the transcriptional profiles of neurons undergoing neurite outgrowth in vitro. We were particularly interested in finding genes whose expression is generally associated with the process of neurite outgrowth, rather than with cell type-specific effects. Thus, in order to avoid focusing on transcripts unique to one tissue type versus another, we used a comparative strategy to look for effects that were common to two tissue types and therefore more likely to be involved in the general process of neurite outgrowth.  While these explants contain multiple cell types, we felt this was preferable to the more disruptive conditions required to dissociate neurons or obtain a pure neuron population.  To this end, we monitored gene expression in cultured explants from SCG and DRG using DNA microarrays.  We initiated our studies by culturing embryonic day 13 (E13) mouse SCG in vitro and harvesting tissue for RNA isolation at time points from 2 to 65 hours.  Time points were selected to detect both fast, short-term responses (2, 5 and 12 hours), as well as sustained, long-term changes (24, 40, and 65 hours).  Samples were hybridized to Affymetrix MG-U74v2 A and B microarrays, with RNA from acutely dissected explants serving as a baseline reference.  We followed these experiments with a parallel analysis of a more heterogeneous tissue type, the DRG, which is more frequently used than SCG for in vivo studies of neurite regeneration.  Cervical and upper thoracic DRG from E12 embryos were cultured with NGF (the same trophic support as in SCG cultures), harvested at time points from 2 to 40 hours, and hybridized to Affymetrix MOE 430A microarrays.

first: Karen

last: Vranizan

first: Yu

middle: C

last: Tai

first: Corey

last: Goodman

first: Terence

last: Speed

first: John

last: Ngai


title: Genome-wide Replication-independent H3 Exchange Occurs Predominantly at Promoters and Implicates H3K56ac and Asf1

status: Public on Aug 02 2007

city: Montréal

first: François

last: Robert

state: Québec

address: 110, avenue des Pins Ouest

department: Chromatin and Genomic Expression

institute: Institut de recherches cliniques de Montréal (IRCM)

summary:
In yeast, histone H3/H4 exchange independent of replication is poorly understood. Here, we analyzed the deposition of histone H3 molecules, synthesized during G1, using a high-density microarray histone exchange assay. While we found that H3 exchange in coding regions requires high levels of transcription, promoters exchange H3 molecules in absence of transcription. In inactive promoters, H3 is deposited predominantly in well-positioned nucleosomes surrounding nucleosome free regions, indicating that some nucleosomes in promoters are dynamic. This could facilitate induction of repressed genes. Importantly, we show that histone H3 K56 acetylation, a replication-associated mark, is also present in replication-independent newly assembled nucleosomes and correlates perfectly with the deposition of new H3. Finally, we found that transcription-dependent incorporation of H3 at promoters is highly dependent on Asf1. Taken together our data underline the dynamic nature of replication-independent nucleosome assembly/disassembly, specify a link to transcription and implicate Asf1 and H3 K56 acetylation.
Keywords: ChIP-chip

overall_design:
To analyze histone H3 dynamics we used an experimental system based on the method developed to study chromatin reassembly at the PHO5 promoter upon repression (Schermer et al., 2005) and coupled it to high-resolution microarrays. In this system there are two different sources of histone H3 in the cell, the endogenous histone tagged with the Myc epitope and a galactose-inducible form fused to the Flag tag coexpressed with histone H4. In order to eliminate the contribution of DNA replication-dependant histone deposition, exponentially growing cells containing the double tag system are blocked in G1 with alpha-factor. After incubation with alpha-factor, cells are either fixed or induced to express Flag-H3 prior to formaldehyde treatment. Next, the level of Myc-H3, Flag-H3, and RNAP II are assayed either by standard ChIP-QPCR or by ChIP-chip using tiling path DNA microarrays (Guillemette et al., 2005; Pokholok et al., 2005).

The raw data were corrected (foreground-background) then normalized using the limma's loess function (Yang et al., 2002) in BioConductor (from the ArrayPipe Analysis Pipeline (Hokamp et al., 2004)) and replicates were combined using a weighted average method as described previously (Ren et al., 2000, Pokholok et al., 2005).

Histone H3 exchange in WT and in the asf1Δ mutant, RNAPII occupancy, and H3 K56 acetylation experiments were done in duplicate, the Asf1-dependence of H3 exchange was assayed in triplicate, and the MNase ChIP-chip experiment once. The combined datasets are available in the supplemental files of the original publication.

REFERENCES
Guillemette, B., Bataille, A. R., Gevry, N., Adam, M., Blanchette, M., Robert, F., and Gaudreau, L. (2005). Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol 3, e384.
Hokamp, K., Roche, F. M., Acab, M., Rousseau, M. E., Kuo, B., Goode, D., Aeschliman, D., Bryan, J., Babiuk, L. A., Hancock, R. E., and Brinkman, F. S. (2004). ArrayPipe: flexible processing pipeline for microarray data. Nucleic Acids Res 32, W457-459.
Pokholok, D. K., Harbison, C. T., Levine, S., Cole, M., Hannett, N. M., Lee, T. I., Bell, G. W., Walker, K., Rolfe, P. A., Herbolsheimer, E., et al. (2005). Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517-527.
Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306-2309.
Schermer, U. J., Korber, P., and Horz, W. (2005). Histones are incorporated in trans during reassembly of the yeast PHO5 promoter. Mol Cell 19, 279-285.
Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15.

first: Anne

last: Rufiange*

first: Pierre-Étienne

last: Jacques*

first: Wajid

last: Bhat

first: Amine

last: Nourani


title: Gene expression in undifferentiated human ES cells - Agilent

status: Public on Jun 27 2007

city: Cleveland

first: Paul

last: Tesar

state: Ohio

address: 10900 Euclid Ave.

department: Genetics & Genome Sciences

institute: Case Western Reserve University

summary:
The application of human embryonic stem (ES) cells in medicine
and biology has an inherent reliance on understanding the starting
cell population. Human ES cells differ from mouse ES cells and the
specific embryonic origin of both cell types is unclear. Previous
work suggested that mouse ES cells could only be obtained from
the embryo before implantation in the uterus1–5. Here we show
that cell lines can be derived from the epiblast, a tissue of the postimplantation
embryo that generates the embryo proper. These
cells, which we refer to as EpiSCs (post-implantation epiblastderived
ES cells), express transcription factors known to regulate
pluripotency, maintain their genomic integrity, and robustly differentiate
into the major somatic cell types as well as primordial
germ cells. The EpiSC lines are distinct from mouse ES cells in
their epigenetic state and the signals controlling their differentiation.
Furthermore, EpiSC and human ES cells share patterns of
gene expression and signalling responses that normally function
in the epiblast. These results show that epiblast cells can be maintained
as stable cell lines and interrogated to understand how
pluripotent cells generate distinct fates during early development.
*Note: EpiSCs were previously referred to as post-ES cells
Keywords: cell type comparison

overall_design: 3 biological replicates of human ES cells were compared to mouse ES cells and EpiSCs.

first: Josh

middle: G

last: Chenoweth

first: Ron

last: McKay


title: Expression profiling of pancreas development

status: Public on Jun 09 2007

city: Maaloev

first: Andreas

last: Petri

address: Novo Nordisk Park D6 2.154

department: Molecualr Genetics

country: Denmark

institute: Novo Nordisk A/S

summary:
Development of the pancreas from the endoderm is initiated at embryonic day 9 of mouse development and over the following days several different cell types develop from pancreas progenitor cells. A distinct phase of pancreas development, known as the secondary transition, is initiated at day 13 of development and one of the key features of this transition is a massive increase in the number of mature endocrine cells. To study gene expression in pancreas during the secondary transition we performed high-density oligonucleotide microarray experiments on dorsal pancreas tissue isolated from NMRI embryos on consecutive days from e12.5 to e16.5.
Keywords: time course

overall_design: Dorsal pancreata were isolated from embryos at embryonic day 12.5, 13.5, 14.5, 15.5, and 16.5 and pooled litter-wise prior to total RNA extraction. From each pool, two independent labelling reactions were made, and each sample was hybridized to the entire Murine Genome U74 version 2 chip set (A, B, and C).


title: Comparative gene expression profiles of T-dependent and T-independent germinal centre B cells in mice

status: Public on Sep 05 2007

city: Canberra

first: Diego

last: Silva

state: ACT

address: Mills Road

department: Division of Immunology and Genetics

country: Australia

institute: John Curtin School of Medical Research

summary:
Selection of B cells subjected to hypermutation in germinal centres (GC) during T-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and T-independent germinal centre B cells. We have now compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC.
Keywords: T-dependent and T-independent germinal center B cells

overall_design: QMxB6 F1 mice were immunised with T-independent antigens (NP-Ficoll) or T-dependent antigens (NP-CGG). T-dependent responses were analysed in carrier primed mice and non-carrier primed mice. The kinetics in carrier primed mice are comparable to those of TI-2 antigens, and thus, both of these groups were analysed 4 days after immunization with soluble antigen. The response in non-carrier primed mice peaks later, and was therefore analysed on day 10 after immunization. Germinal centre B cells were MACS enriched and then FACS sorted according to GL-7 and B220 expression. Sorted B cells from the relevant mice were pooled (4-10 mice per sample). Each sample thus contains B cells from different mice, and the replicates are biological, not technical replicates.

first: Carola

last: Vinuesa

first: Di

last: Yu

first: Chris

last: Goodnow

first: Mike

last: Cooke


title: Expression data from wildtype and Rb-/- fetal liver at e12.5

status: Public on Jun 05 2007

city: Chicago

first: Kay

last: Macleod

state: IL

address: 927 E 57th St

institute: Ben May Institute/University of Chicago

summary:
Rb null embryos exhibit defective fetal liver erythropoiesis. We used microarrays to compare  Wt and Rb null fetal livers and to analyse gene expression differences which accompany and may underlie Rb null fetal liver degeneration, erythroid failure, and erythropoietic island dissolution.
We used microarrays to compare  Wt and Rb null fetal livers and analyse gene expression changes which accompany and may underlie fetal liver.
Keywords: retinoblastoma, fetal liver, erythroblast, macrophage, cell death

overall_design: Wild type and Rb null  embryos were sacrificed at e12.5 and fetal livers were dissected for RNA extraction.    Three embryos of each genotype were analysed.

first: Benjamin

last: Dibling

last: Spike


title: Age-related transcriptional changes and the effect of dietary supplementation of vitamin E in the mouse heart

status: Public on Aug 31 2007

city: Boulder

first: Sang-Kyu

last: Park

department: IBG

institute: University of Colorado at Boulder

summary:
To investigate the global effects of vitamin E supplementation on heart aging, we used high-density oligonucleotide arrays to measure transcriptional alterations in 30-month-old B6C3F1 mice supplemented with α- and γ-tocopherol since middle age (15 months).
Keywords: expression profiling

overall_design: Gene expression profiles were obtained from 5-month-old controls, 30-month-old controls and 30-month-old mice supplemented with α-tocopherol (1g/kg), or a mixture of α- and γ-tocopherol (500mg/kg of each tocopherol).

first: Grier

last: Page

first: Kyoungmi

last: Kim

first: David

last: Allison

first: Mohsen

last: Meydani

first: Richard

last: Weindruch

first: Tomas

last: Prolla


title: Molecular profiling of tumor progression in NSCLC

status: Public on Jun 04 2007

city: Hannover

first: Astrid

last: Rohrbeck

address: Carl-Neuberg-Straße 1

department: Department of Toxicology

institute: Medizinische Hochschule Hannover

summary:
To identify genes associated with lung cancer progression, we examined gene expression profiles of tumor cells from 20 patients with primary, untreated non-small cell lung cancer (10 adenocarcinomas (AC) and 10 squamous cell carcinomas (SCC)) in comparison to lung tissue of 23 patients with stage IIIB or stage IV non-small cell lung cancer (15 AC and 8 SCC). Bronchoscopical biopsies from patient with recurrent lung tumor were taken after initial treatment. Cancer cells were isolated using laser capture microdissection in order to obtain pure samples of tumor cells. For expression analysis, microarrays covering 8793 defined genes (Human HG Focus Array, Affymetrix) were used. Array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM), respectively. Genes were considered to be up- or down-regulated when the ratio between primary and recurrent tumor samples were at least 1.5-fold differentially expressed with an estimated false discovery rate: < 5%.
Based on differentially expressed genes, primary cancer samples could be separated from recurrent tumor samples. We identified 115 and 124 significantly regulates genes in AC and SCC, respectively. For example, in recurrent AC we found increased expression of genes related to the wingless (FZD6, RYK, MYC) and calcium (CALM1, ATB2B1, S100A2) signalling pathways which might play a role in metastasis of tumor cells. Other differentially expressed genes were related to cell cycle (CCND1, CDK2), transcription factors (TTF1, TAF2, YY1), nuclear mRNA splicing and mRNA processing (SFRS1, HNRPL), protein-nucleus import (NUTF2, KPNB1, NUP50) and chromatin modification (HIST1H4C, SMARCC1). In SCC, we found an increased expression of CTNNB1, an important mediator in wingless signalling pathway. Among the down-regulated genes in SCC, the utmost fraction belonged to genes coding for ubiquitin mediated proteolysis (UCHL1, PSMA3, COPS6) and ribosomal proteins (RPS26, RPL7A, RPS15). Other down regulated genes were related to transcription factors (TCEA2, TAF10), nuclear mRNA splicing and mRNA processing (SNRPD2, HNRPM).
In conclusion, a distinct pattern of gene expression is found during the progression from primary carcinoma to recurrent NSCLC. Our microarray-based expression profiling revealed interesting novel candidate genes and pathways that may contribute to lung cancer progression.
Keywords: Lung cancer, NSCLC, gene expression, progression, Wnt signalling pathway

overall_design:
- 20 patients with primary, untreated non-small cell lung cancer (10adenocarcinomas (AC) and 10 squamous cell carcinomas (SCC)) in comparison to lung tissue of 23 patients with stage IIIB or stage IV non-small cell lung cancer (15 AC and 8 SCC)
- Human HG Focus Array, Affymetrix) were used
- Array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM)
- Genes were considered to be up- or down-regulated when the ratio between primary and recurrent tumor samples were at least 1.5-fold differentially expressed with an estimated false discovery rate: < 5%

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