title: Nucleosome occupancy

status: Public on May 16 2007

city: Jerusalem

first: Tommy

last: Kaplan

address: Givat Ram Campus

department: School of Computer Science and Engineering

institute: Hebrew University

summary:
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Keywords: MNase, Nucleosomes

overall_design: Nucleosomal vs genomic DNA.

last: Dion

first: Minkyu

first: Stephen

last: Buratowski

first: Nir

last: Friedman

first: Oliver

last: Rando


title: Isolated adipocytes and stromo-vascular fraction (SVF) of subcutaneous and intraabdominal adipose tissue in mice

status: Public on Jul 17 2007

city: Boston

first: Stephane

last: Gesta

state: MA

address: One Joslin Place

country: USA

institute: Joslin Diabetes Center

summary:
Obesity is an epidemic health problem worldwide that impacts the risk and prognosis of many diseases. However, not all obese patients have the same risk of developing these disorders. Individuals with peripheral obesity, i.e., fat distributed subcutaneously, are at little or no risk of the common medical complications of obesity, whereas individuals with central obesity, i.e., fat accumulated in visceral depots, are prone to these complications.
In the present study, using microaarays gene expression, we have explored the hypothesis that patterns of fat distribution and, perhaps, to some degree, obesity itself may have a developmental genetic origin.
Keywords: Comparative hybridization, cell type comparison

overall_design: Isolated adipocytes and stromo-vascular fraction (SVF) of subcutaneous and intraabdominal adipose tissue of mice were used for RNA extraction and hybridization on Affymetrix microarrays. Three samples from each categories were used for the analyse.

first: Matthias

last: Blüher

first: Yuji

last: Yamamoto

first: Andrew

middle: W

last: Norris

first: Janin

last: Berndt

first: Susan

last: Kralisch

first: Jeremie

last: Boucher

first: Choy

last: Lewis

first: C

middle: R

last: Kahn


title: Dynamics of replication-independent histone turnover in budding yeast

country: Israel

type: Genome binding/occupancy profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Role of DNA Topoisomerase IIbeta in Gene Expression

status: Public on May 02 2007

city: Piscataway

first: Yi Lisa

last: Lyu

state: NJ

address: 675 Hoes Lane

department: Pharmacology

institute: UMDNJ-Robert Wood Johnson Medical School

summary:
Mice lacking topoisomerase IIβ (Top IIβ) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild type and top2β knockout mouse embryos were generated. Surprisingly, only a small number (1-4%) of genes were affected in top2β knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated.
By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIβ is not required for early differentiation programming, but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIβ and HDAC2, a known TopIIβ-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIβ binding to the 5’ region of a number of TopIIβ-sensitive genes. Further
studies of a TopIIβ-sensitive gene, Kcnd2, revealed the presence of TopIIβ in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIβ in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.
Keywords: Gene expression in genetically modified mouse strain, top2b-/-.

overall_design: Total RNAs were isolated from the brains of E14.5, E16.5 and E18.5 Top2b+/+ and top2b-/- embryos. Corresponding biotin-labeled cRNAs were then hybridized to Affymetrix GeneChip MG_U74Av2 microarrays. Data were then uploaded and analyzed by the Rosetta Resolver® system for gene expression data analysis (Rosetta Inpharmatics®, Inc.)

first: YiLisa

first: James

middle: C

last: Wang

first: Leroy

middle: F

last: Liu


title: Prediction and Testing of Novel Networks Regulating Embryonic Stem Cell Self-Renewal and Commitment

status: Public on Apr 13 2007

city: Toronto

first: William

middle: L

last: Stanford

state: Ontario

address: 164 College St

department: IBBME

country: Canada

institute: University of Toronto

summary:
Stem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks.  To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs following the initiation of commitment and integrated these data with known genome-wide transcription factor binding.  Effects of forced under- or over-expression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells.  By using this integrative approach we have, for the first time, formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.
Keywords: time course

overall_design: 1, 3, and 5 days LIF differentiated ESCs, and 1 and 2 days RA differentiated ESCs

first: Emily

last: Walker

first: Minako

last: Ohishi

first: Ryan

middle: E

last: Davey

first: Wen

last: Zhang

first: Paul

middle: A

last: Cassar

first: Tetsuya

middle: S

last: Tanaka

first: Sandy

middle: D

last: Der

first: Quaid

last: Morris

first: Timothy

last: Hughes

first: Peter

last: Zandstra


title: Gene expression profile after doxycycline treatment in keratin5 knockout epidermis

status: Public on May 01 2007

city: Bonn

first: Hong

last: Lu

state: NRW

address: Nussallee 11

country: Germany

institute: University of Bonn

summary:
Epidermolysis bullosa simplex (EBS) is a skin disorder caused by mutations in keratin (K) 5 or K14 genes. Little effect therapy is available.Following clinical reports, we applied the small molecule doxycycline to K5-/- mice. We demonstrate that doxycycline extended the survival of neonatal K5-/- mice from less than 1 to up to 8 hours. Microarray and TaqMan real-time PCR showed a downregulation of MMP-13 and IL-1beta, indicating an effect of doxycycline on transcription. Our data offer a novel small molecule based therapy approach for EBS.
Keywords: drug treatment, global gene expression comparative profile

overall_design: doxycycline (50 µg/mlwith 5 % sucrose) was administered by oral route to pregnant K5+/- females (mated to K5+/- males) starting from E13.5.  5 % sucrose solution was administered to the control group.

first: Jian

last: Chen

first: Laura

last: Planko

first: Paola

last: Zigrino

first: Ludger

last: Klein-Hitpass

first: Thomas

middle: M

last: Magin


title: Induction of inflammatory cytokines by a keratin mutation  in a mouse model   for EBS

summary:
Epidermolysis bullosa simplex (EBS) is a skin disorder caused by mutations in keratin (K) 5 or K14 genes. It is widely regarded as a mechanobullous disease, resulting from a weakened cytoskeleton, causing extensive cytolysis. It was postulated by others that certain K14 mutations induce TNF-alfa and increase apoptosis. Here, we report that in K5-/- mice , the mRNA and protein levels of TNF-alfa remain unaltered. Transcriptome analysis of K5-/- mice revealed however, that the pro-inflammatory cytokines interleukin-6 and interleukin-1beta were significantly upregulated at the mRNA level in K5-/- mouse skin. These results were confirmed by TaqMan real-time PCR and ELISA assays. We hypothesize that keratin mutations contribute to EBS in a mouse model by inducing local inflammation that mediates a stress response.
Keywords: comparative gene expression profile

overall_design: Two groups were  K5-/- skin  and wildtype . Six animals were included in each group. All the total RNA samples were isolated from tissues taken immediately after birth, and were pooled for later microarray experiments. Using realtime PCR and ELISA analysis confirmed our microarray result.


title: Gnotobiotic mouse ileum; Listeria infection series

status: Public on Apr 23 2007

city: Saint Louis

first: Justin

middle: L.

last: Sonnenburg

state: MO

address: 4444 Forest Park

department: Center for Genome Sciences

institute: Washington University

summary:
Ileal profiles from gnotobiotic mice mono-associated with Listeria species or B. thetaiotaomicron.   Samples were derived from 72h colonizations of Fabpi-hEcad transgenic B6  mice fed a standard-chow polysaccharide rich (PR) diet.
Keywords: Germ-free vs. Mono-associations

overall_design: Total RNA was prepared from ileum of 72 hour mono-associated gnotobiotic male mice, biotin labeled, and hybridized to 430 2.0 GeneChips.

first: Marc

last: Lecuit

first: Pascale

last: Cossart

first: Jeffrey

middle: I

last: Gordon


title: Altered gene expression in the neocortices and hippocampi of the adult S1P2-deficient and S1P3-deficient mice

status: Public on Mar 09 2007

city: Maebashi

first: Noriyuki

last: Akahoshi

state: Gunma

address: 3-39-22 Showa-machi

department: Molecular and Cellular Neurobiology

country: Japan

institute: Gunma University Graduate School of Medicine

summary:
Altered gene expression in the sphingosine 1-phosphate receptor 2 (S1P2)-deficient or sphingosine 1-phosphate receptor 3 (S1P3)-deficient brain.
Keywords: genetic modification

overall_design: The S1P2-deficient mice suffer from spontaneous/sporadic seizures during 4-7 weeks of age. The S1P3 deficient mice do not show such seizures. The 8-week-old mice were sacrificed for sample preparation. Neocortices and hippocampi were isolated from wild-type, S1P2-deficient, and S1P2-deficient mice (n=10, 5, and 10, respectively).

first: Isao

last: Ishii


title: Mice with and without enteric flora

status: Public on Jun 06 2007

city: Shinjuku

first: Masahiro

last: Yamamoto

state: Tokyo

address: 35 Shinano-machi

department: Department of Kampo Medicine

institute: Keio University School of Medicine

summary:
Although microbiota plays a critical role for the normal development and function of host immune systems, the detail of the influence, especially on those in the large intestine (LI), remains unknown.
Here, we performed transcriptome analysis using Affymetrix GeneChip on LI of germ-free (GF)  and specific pathogen free (SPF) mice of IQI, an inbred strain established from ICR.
Keywords: comparison of the mice with or without enteric flora

overall_design:
Male germ-free (GF) and specific pathogen-free (SPF) IQI/Jic mice were bred and maintained in the laboratory of Central Institute for Experimental Animals (CIEA, Kawasaki, Kanagwa, Japan). GF mice were housed in a Trexler-type flexible film isolator in a standard germ free state and screened on a weekly basis for germ-free status by sampling feces sterilely and culturing on MRS-agar plates under aerobic and anaerobic conditions. All the GF, SPF, ex-GF mice were kept in a 12: 12-h light/dark cycle and at a temperature of 22+-2 ºC. Nine week old mice (n=3) were sacrificed by cervical dislocation and the colon was dissected (the colon was not divided into proximal and distal sections and was treated as a single intestinal section).

The SPF mice were provided by the Central Institute for Experimental Animals of Japan.  These mice were determined free of the following pathogens (by various techniques including culture, microscopic observation, serological test, and PCR):  Dermatophytes, Citrobacter rodentium, Pasteurella pneumotropica, Corynebacterium kutscheri, Pseudomonas aeruginosa, Salmonella spp., Staphylococcus aureus, Mycoplasma pulmonis, Corynebacterium kutscheri, Salmonella typhimurium, Clostridium piliforme, Mycoplasma pulmonis, Mouse hepatitis virus, Sendai virus, Ectromelia virus, Lymphocytic c. virus, Hantavirus, Giardia muris, Spironucleus muris, Syphacia spp., Aspiculuris tetraptera, Ectoparasits, Intestinalprotozoa, Pinworm, Pneumocystis carinii, Helicobacter hepaticus, Helicobacter bilis, EDIM virus(Rotavirus), Pneumonia virus of mice, Minute virus of mice, Mouse adenovirus, Mouse cytomegalovirus, Mouse encephalomyelitis virus, Reovirus type3, and CAR bacillus.

first: Kaori

last: Munakata


title: Expression data from female reproductive organs of adult mice treated with estrogen

status: Public on Feb 06 2007

city: Okazaki

first: Suzuki

last: Atsuko

state: Aichi

address: 5-1, Higashiyama

department: Okazaki Institute for Integrative Bioscience

institute: National Institute of Natural Science

summary:
Estrogen induce organ-specific cell proliferation and development in female reproductive organs, though the reproductive differentiation, sex maturation, implantation and lactation. However, the mechanism of organ-specific estrogen responsive genes is unknown. Thus, we examined early estrogen responsive genes in mouse uterus, vagina and mammary gland.
Keywords: organ specificity

overall_design: 70-day-old ovariectomized mice (C57BL/6J)(n=4) were treated with 17beta-estradiol (5micro g/kg) or sesame oil. Whole uterus (Ut), vagina (Vg) and mammary gland (Mg) were sacrificed 6h after the injection.


title: A study of the differentiation of Murine ES cells [Scl+/hCD4 ES cells (Chung et al., 2002)] into hemangioblasts

status: Public on Jan 25 2007

city: st louis

first: kyunghee

last: choi

address: 660 s euclid ave

institute: washington university

summary:
Molecular mechanisms that regulate the generation of hematopoietic and endothelial cells from mesoderm are poorly understood.
To define the underlying mechanisms, we compared gene expression profiles between embryonic stem (ES) cell-derived
hemangioblasts (Blast-Colony-Forming Cells, BL-CFCs) and their differentiated progeny, Blast cells. Bioinformatic analysis indicated
that BL-CFCs resembled other stem cell populations. A role for Gata2, one of the BL-CFC-enriched transcripts, was further
characterized by utilizing the in vitro model of ES cell differentiation. Our studies revealed that Gata2 was a direct target of BMP4
and that enforced GATA2 expression upregulated Bmp4, Flk1 and Scl. Conditional GATA2 induction resulted in a temporal-sensitive
increase in hemangioblast generation, precocious commitment to erythroid fate, and increased endothelial cell generation. GATA2
additionally conferred a proliferative signal to primitive erythroid progenitors. Collectively, we provide compelling evidence that
GATA2 plays specific, contextual roles in the generation of Flk-1+ mesoderm, the Flk-1+Scl+ hemangioblast, primitive erythroid and
endothelial cells.
Keywords: Comparison of induced differetiated state to original cell line.

overall_design:
Generation of Hemangioblast and Blast Colony cell populations:
Scl+/hCD4 ES cells (Chung et al., Development, 2002) were maintained on a layer of mitotically inactivated STO cells in DMEM media in the presence of LIF.  Two days before differentiation, the cells were transferred to gelatinized tissue culture flasks and the media was changed to IMDM-based.  ES cells were differentiated in liquid-differentiation media in the presence of FCS for 2.75 days.  D2.75 EBs were recovered, dissociated in Trypsin/EDTA and stained with antibodies against Flk-1 and human CD4.  Cells were then flow sorted on the basis of Flk-1 and Scl expression and the hemangioblast population was considered to be Flk-1+Scl+.
D2.75 EB cells were replated as described (Chung et al., 2002) and individual Blast colonies were picked and pooled, and RNA was generated with TRIzol according to the manufacturers protocol.  RNA (20 ug) was given to the Washington University Array core were it was reverse transcribed and cRNA synthesized.

Replicated of the two populations (hCD4, Blast) were made.  Pairwaise comparisons were performed to identify enriched genes.

first: Jesse

middle: J

last: Lugus

first: Yun

last: Chung

first: Jason

last: Mills

first: Shin-Il

last: Kim

first: Jeffery

last: Grass

first: Michael

last: Kyba

last: Doherty

first: Emery

middle: H

last: Bresnick

first: Kyunghee

last: Choi


title: Brown and white adipocyte differentiation

status: Public on May 31 2007

city: Stockholm

first: Ola

last: Larsson

department: Department of oncology-pathology

country: Sweden

institute: Karolinska Institutet

summary:
Attainment of a brown adipocyte cell phenotype in white adipocytes, with their abundant mitochondria and increased energy expenditure potential, is a legitimate strategy for combating obesity. The unique transcriptional regulators of the primary brown adipocyte phenotype are unknown, limiting our ability to promote brown adipogenesis over white. In the present work, we used microarray analysis strategies to study primary preadipocytes, and we made the striking discovery that brown preadipocytes demonstrate a myogenic transcriptional signature, whereas both brown and white primary preadipocytes demonstrate signatures distinct from those found in immortalized adipogenic models. We found a plausible SIRT1-related transcriptional signature during brown adipocyte differentiation that may contribute to silencing the myogenic signature. In contrast to brown preadipocytes or skeletal muscle cells, white preadipocytes express Tcf21, a transcription factor that has been shown to suppress myogenesis and nuclear receptor activity. In addition, we identified a number of developmental genes that are differentially expressed between brown and white preadipocytes and that have recently been implicated in human obesity. The interlinkage between the myocyte and the brown preadipocyte confirms the distinct origin for brown versus white adipose tissue and also represents a plausible explanation as to why brown adipocytes ultimately specialize in lipid catabolism rather than storage, much like oxidative skeletal muscle tissue.
Keywords: In vitro differentiation

overall_design: Comparisons of white and brown pre-  and mature-adiposytes

first: James

last: Timmons


title: Effect of IgG cytoplasmic tail on BCR-respose genes

status: Public on Mar 13 2007

city: Acton

first: Keisuke

last: Horikawa

state: ACT

address: Garran Rd

department: DIG

country: Australia

institute: JCSMR, ANU

summary:
IgG cytoplasmic tail interferes with the induction of antigen-response genes
Keywords: Comparison of two transgenic mice

overall_design: Comparing antigen-induced genes between B cells expressing anti-HEL IgM BCR and IgMG BCR (chimeric receptor where the extracellular spacer, transmembrane, and cytoplasmic domain of IgM are replaced with those of IgG1)

first: Stephen

last: Martin

first: Sarah

last: Pogue

first: Kaiman

last: Peng

first: Kiyoshi

last: Takatsu

first: Christopher

last: Goodnow


title: Expression data from spec. transcriptional activity of primary mouse embryonic fibroblasts (MEF) in response to serum.

status: Public on Jan 16 2007

city: Bern

first: Marc

last: Kenzelmann

address: Friedbuhlstrasse 51

department: Molecular Genome Analysis

country: Switzerland

institute: Institute of Medical Microbiology, University of Bern

summary:
Current methods to analyze gene expression measure steady-state levels of mRNA. In order to specifically analyze mRNA transcription, a technique has been developed that can be applied in-vivo. The technique is referred with the acronym NIAC-NTR (Non Invasive Application and Capture of Newly Transcribed RNA). This method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in-vivo. The method has been applied to a model of serum response of cultured primary mouse embryonic fibroblasts.
Affymetrix GeneChip microarrays were used to detail regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.
Keywords: treatment experiment

overall_design: The gene expression and regulation program at the RNA level early (2h) in response to serum was studied. Total RNA, amplified total RNA and specifically enriched newly transcribed RNA was isolated from serum starved and 2h serum treated MEF cells. This experiment allowed detailed regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level of MEF cells early in response to serum.

first: Sabine

last: Maertens

first: Manfred

last: Hergenhahn

first: Stefan

last: Küffer

first: Agnes

last: Hotz-Wagenblatt

first: Li

last: Li

first: Shijun

last: Wang

first: Carina

last: Ittrich

first: Thomas

last: Lemberger

first: Rosa

last: Arribas

first: Sunitha

last: Jonnakuty

first: Monica

last: Hollstein

first: Wolfgang

last: Schmid

first: Norbert

last: Gretz

first: Hermann-Josef

last: Gröne

first: Günther

last: Schütz


title: Mechanisms of Aging in Senescence-Accelerated Mice

status: Public on Jan 24 2007

city: La Jolla

first: Jennifer

last: Greenhall

state: CA

address: 10010 N. Torrey Pines Rd.

department: MNL-H

institute: The Salk Institute

summary:
Background: Progressive neurological dysfunction is a key aspect of human aging. Because of underlying differences in the aging of mice and humans, useful mouse models have been difficult to obtain and study. We have used gene-expression analysis and polymorphism screening to study molecular senescence of the retina and hippocampus in two rare inbred mouse models of accelerated neurological senescence (SAMP8 and SAMP10) that closely mimic human neurological aging, and in a related normal strain (SAMR1) and an unrelated normal strain (C57BL/6J).
Results: The majority of age-related gene expression changes were strain-specific, with only a few common pathways found for normal and accelerated neurological aging. Polymorphism screening led to the identification of mutations that could have a direct impact on important disease processes, including a mutation in a fibroblast growth factor gene, Fgf1, and a mutation in and ectopic expression of the gene for the chemokine CCL19, which is involved in the inflammatory response.
Conclusions:  We show that combining the study of inbred mouse strains with interesting traits and gene-expression profiling can lead to the discovery of genes important for complex phenotypes. Furthermore, full-genome polymorphism detection, sequencing and gene-expression profiling of inbred mouse strains with interesting phenotypic differences may provide unique insights into the molecular genetics of late-manifesting complex diseases.
Keywords: disease state analysis

overall_design: Gene-expression profiling was performed on 3 month-old (young), 16 month-old (old) S8, S10 and SR1 mice. Two independent samples for each time point were used in gene-expression profiling for each strain. Because of greater replicate variability, three samples were used for hippocampus of 16-month SAM mice. A 10.0 ug sample of total RNA was used to generate labeled cRNA for each sample according to recommended protocols (Affymetrix). RNA from multiple animals was not pooled, except in the case of retina, where the retinas of two mice were combined to generate sufficient total RNA.


title: Neocortical and hippocampal gene expression in kainate- and nicotine-injected juvenile mice

status: Public on Jan 04 2007

first: Noriyuki

last: Akahoshi

summary:
To examine irreversible changes in the developing brain following seizures, juvenile inbred mice were intraperitoneally injected with kainate and nicotine.
Keywords: seizure induction

overall_design: Kainate and nicotine were dissolved in sterile saline, and intraperitoneally injected into postnatal day 23 males at a dose of 15 mg/kg and 10 mg/kg, respectively. Control mice received a comparable injection of saline. The injection was repeated three times at 2-hour intervals in the daytime, and after one week, the P30 survivors were sacrificed for sample preparation. Neocortices and hippocampi were isolated from mice that had received the repeated injections of saline, kainate, and nicotine (n=6, 5, and 6 mice, respectively).

first: Isao

last: Ishii


title: Microarray Analysis of Murine IEL Subsets

status: Public on Apr 03 2007

first: Steve

middle: W.

last: Granger

address: 9420 Atherna Circle

department: Data Analysis

institute: The La Jolla Institute for Allergy and Immunology

summary:
Mouse small intestine intraepithelial lymphocytes (IEL) that express a ab TCR and CD8aa homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined.  To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis, combined with PCR and flow cytometry to determine the level of expression of selected genes. Using these methods, TCR ab+ CD8aa IEL were compared to their TCR ab+ CD8b+ and TCR gd+ counterparts.
Keywords: Cell type comparison

overall_design: In this study, the three IEL populations were isolated by cell sorting from a pool of IELs from 20 mice.  RNA from each IEL subset was used to probe one microarray.  A replicate experiment was performed on a seperate occasion using a new pool of 20 mice and three additional microarrays were used.

first: Timothy

last: Denning

first: Steven

first: Ryan

last: Graddy

first: Georges

last: Leclercq

first: Weiguo

last: Zhang

first: Karen

last: Honey

first: Denis

last: Heck

first: Jeffrey

middle: P

last: Rasmussen

first: Hilde

last: Cheroutre

first: Alexander

middle: y

last: Rudensky

first: Mitchell

last: Kronenberg


title: Comparison of Hybridization-based and Sequencing-based Gene Expression Technologies on Biological Replicates

status: Public on Jan 01 2007

first: Winston

middle: Patrick

last: Kuo

address: 188 Longwood Avenue

department: Genetics

institute: Harvard Medical School

summary:
High-throughput systems for gene expression profiling have been developed and matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised regarding data comparability and agreement across technologies. Within an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing). The DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained.  Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery. Based on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive, and currently, both provide indispensable tools for transcriptome profiling.
Keywords: biological replicates

overall_design:
METHODS
Biological samples
RNA samples were isolated from three sources: two pools of C57/B6 adult mouse retina (MRP1 and MRP2, n=700) and Swiss-Webster post-natal day one (P1) mouse cortex (MC) (n=19). Retinas were dissected, collected and stored in Trizol (one pair of retinas per eppendorf tube) at -80°C prior to pooling. During the RNA extraction process, two pools of adult mouse retina (MRP1, MRP2) were created (700 retinas per pool), aliquoted. All samples were stored at -80°C prior to conducting the experiments. The animal experiments were approved by the Institutional Animal Care Facility at Harvard University.
Microarray platforms, data processing and consistency assessment
Whole-genome mouse gene expression arrays (one-dye oligonucleotide microarrays) were investigated in this study, including: Affymetrix GeneChip®, Amersham (now GE Healthcare) CodeLink®, Mergen ExpressChip®, Applied Biosystems (ABI) microarrays, and Illumina BeadArray®. Microarray experiments are composed of sample preparation, hybridization, scanning and image quantitation, which are a series of integrative procedures being conducted at a laboratory, generally according to the manufacturer’s recommended protocols. To obtain sufficient statistical confidence in the data analysis, for each biological replicate (MRP1 and MRP2), five technical replicates on each platform were obtained, with an exception on Illumina, in which a single experiment for each sample was performed (included to make data available to the public). For details of the experimental protocols and laboratories, we refer to Kuo et.al.[6], except for Illumina, which can be found in the Supplementary Material A.
The raw data sets of 63 chips after image scanning and quantification in each platform were collected. For Illumina data, we set the filtering threshold as “Detections” ≥ 0.9. Filtering for the other microarray platforms are described in Kuo et.al.[6]. We also performed percentile transformation of intensities, quantiles normalization and log2ratio calculation, as described[6].
Data repeatability and reproducibility[7] are two important aspects of microarray data consistency assessment. The former evaluates the degree of data variations among technical replicates of a platform, and the latter refers to data agreement across different microarray platforms when using the same biological samples. Two popularly used metrics, coefficient of variations (CV) among replicated measurements per gene and correlation coefficient (Pearson and Spearman correlations) between any pair of replicated experiments, were adopted to measure microarray performance. For intra-platform data consistency, the mean and standard deviation of CVs or correlation coefficients were used as summations of each platform’s performance. For inter-platform data agreement, either the mean (for normalized log2ratios) or the median after percentile transformation (for intensities) of repeated measurements on each platform were used in calculating correlation coefficients.
MPSS experiment and data processing
Total RNA of MRP1 and MRP2, which were identical to those used in microarray experiments, was sent to Lynx Therapeutics, Inc. (now Solexa, Hayward, CA) for MPSS experiments. Following an RNA quality test on a Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA), cDNA libraries were generated according to the Megaclone protocol[5, 21]. Signatures adjacent to poly (A) proximal DpnII restriction sites (“GATC”) were cloned into a Megaclone vector. The resulting library was amplified and yielded about 1.6 million loaded microbeads, which were loaded onto a flow cell. Thereafter, an iterative series of enzymatic reactions decoded the signatures as 17-bp or 20-bp sequences (including DpnII recognition sites “GATC”)[22]. The abundance of each signature was converted to transcripts per million (tpm) as supplied by Lynx Therapeutics.
The mapping of signatures to genes was done based on the mouse genome sequence (Release #3, Feb 2003, UCSC Golden Path genome browser, http://hgdownload.cse.ucsc.edu/downloads.html) and the mouse UniGene sequences (ftp://ftp.ncbi.nih.gov/repository/UniGene/, Build #122). The mapping procedure include: extraction of ‘virtual’ signatures from genomic sequence, classification of ‘virtual’ signatures from genomic sequence, and matching of MPSS expressed signatures to genomic signatures[22]. In this study, we included only the reliable signatures which were located close to polyadenylation signal (“A[A/T]TAAA” at the 155 nts at the 3’-end of the cDNA sequence)  or poly(A) tail (15 base sequences containing 12 or more “A”s that occur within the 114 3’-most bases of the sequence) on a mRNA sequences with known orientation information.
Gene mapping among microarray platforms and between microarray and MPSS
Two approaches to match probes across different microarray chips, annotation-based and sequence-based probe matching were used[6]. Briefly, by the annotation-based approach, we obtained UniGene (UG) and LocusLink (LL) based matching, whereas probe matches at the RefSeq (RS) and RefSeq-exon (RSEXON) levels by utilizing actual sequence information belong to the latter.
MPSS signatures were mapped to UniGene clusters, using an in silico constructed “virtual tags” library, as described above. Thus, the gene expression data measured by microarrays and by MPSS were paired up for comparisons via UniGene clusters.

first: Fang

last: Liu

first: Tor-Kristian

last: Jenssen

first: Jeff

last: Trimarchi

first: Connie

last: Cepko

first: Lucila

last: Ohno-Machado

first: Eivind

last: Hovig


title: MEF_serum response

status: Public on Mar 27 2007

first: Marc

last: Kenzelmann

summary:
Expression data from specific translational activity and cytoplasmic localization (nuclear retention) of mRNAs of primary mouse embryonic fibroblasts (MEF) early in response to serum.

The goal of the study was to roughly discriminate between different levels of RNA regulation, namely mRNA de-novo transcription, mRNA stability and degradation, mRNA nuclear retention, mRNA translation as well as miRNA expression within a defined cellular system and time point and to link these levels together in order to get a more detailed analysis of the complex RNA regulation system, the ?RNA regulome?. To make the data more stringent the same microarray platform (Affymetrix GeneChip system) for all analyses except the miRNA expression profiling were used. Nuclear and cytoplasmic RNA fractions were separated in order to get more insight into potential nuclear retention of mRNAs. In addition the cytoplasmic mRNA pool was fractionated by its polyribosomal load using sucrose density gradients. Likewise the small RNA was specifically analyzed for miRNA precursor expression and mature miRNA expression. As study system the early serum response model of primary mouse embryonal fibroblast cells was used.
Affymetrix GeneChip microarrays were used to detail regulatory mechanisms of mRNA translation and localization in response to serum (2h).
Keywords: treatment experiment

overall_design: The gene expression and regulation program at the RNA level early (2h) in response to serum was studied. mRNAs was isolated from serum starved and 2h serum treated MEF cells. This experiment allowed detailed regulatory mechanisms of mRNA translational activity and cytoplasmic localization (nuclear retention) of mRNAs of MEF cells in early response to serum.

first: Stefan

last: Küffer

first: Sabine

last: Maertens

last: Feuerborn

first: Prashant

last: Srivastava

first: Aaron

last: Grandy

first: Minqiang

last: Chai

first: Markus

last: Seifert

first: Matthias

last: Seedorf

first: Mirco

last: Castoldi

first: Vladimir

last: Benes

first: Agnes

last: Hotz-Wagenblatt

first: Svitlana

last: Bulashevska

first: Li

last: Li

first: Xiaolei

last: Yu

first: Priyanka

last: Shai

last: Haak

first: Benedikt

last: Brors

first: Martina

last: Muckenthaler

first: Norbert

last: Gretz

first: Günther

last: Schütz

first: Hermann

last: Gröne


title: PolII occupancy

status: Public on May 16 2007

first: Tommy

last: Kaplan

summary:
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Keywords: Chip-chip, PolII

overall_design: ChIP-chip for RNA polymerase II (vs genomic DNA).

first: Michael

last: Dion

first: Minkyu

last: Kim

first: Stephen

last: Buratowski

first: Nir

last: Friedman

first: Oliver

last: Rando


title: Cardiac-specific deletion of ménage-à-trois-1 (MAT1)

status: Public on Feb 08 2007

city: Houston

last: Schneider

state: TX

address: One Baylor Plaza, Room 506D

department: Center for Cardiovascular Development

institute: Baylor College of Medicine

summary:
The Cdk7/cyclin H/ménage-à-trois 1 (MAT1) heterotrimer has proposed functions in transcription as the kinase component of basal transcription factor TFIIH and is activated in adult hearts by hypertrophic pathways. Using cardiac-specific Cre, we ablated MAT1 in myocardium. Despite reduced Cdk7 activity, MAT1-deficient hearts grew normally. However, fatal heart failure ensued at 6-8 weeks. By microarray profiling, quantitative RT-PCR, and Western blotting at 4 weeks, genes for energy metabolism were found to be suppressed selectively, including targets of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1). Cardiac metabolic defects were substantiated in isolated perfused hearts and isolated mitochondria. In culture, deleting MAT1 with Cre disrupted PGC-1 function: PGC-1α failed to activate PGC-1-responsive promoters and nuclear receptors, GAL4-PGC-1α was functionally defective, and PGC-1β likewise was deficient. PGC-1 was shown to interact with MAT1 and Cdk7, in co-precipitation assays. Thus, we demonstrate an unforeseen essential role for MAT1 in operation of the PGC-1 family of co-activators.
Keywords: conditional knockout in mice

overall_design: MAT1F/F mice (Korsisaari et al., 2002) were bred with mice expressing Cre recombinase under the control of the cardiomyocyte-specific α-myosin heavy chain (αMHC) promoter (Gaussin et al., 2002a) and back-bred to MAT1F/F mice to generate the cardiac-specific knockout (αMHC-Cre+/0; MAT1F/F; CKO; Fig. 1A, B). Control mice were αMHC-Cre+/0; MAT1F/+ littermates, differing by the presence of one wild-type MAT1 allele, and excluding Cre-mediated toxicity as a basis for phenotypic disparity. Cardiac RNA samples were analyze at 2 or 4 weeks of age (N = 3-5 for each condition tested.

first: Motoaki

last: Sano


title: SOX13 regulated genes in CD4+CD8+ double positive thymocytes

status: Public on Dec 20 2006

city: Worcester

first: joonsoo

last: kang

address: 55 lake ave n.

department: Pathology

institute: University of Massachusetts Medical School

summary:
ab and gd T cells originate from a common, multi-potential precursor population in the thymus, but the molecular mechanisms regulating this lineage fate decision process are unknown.  We have identified Sox13 as a gd-specific gene in the immune system.  Using Sox13 transgenic mice, we show that SOX13 promotes gd T cell development while opposing ab T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gd T cells, but not ab T cells. One mechanism of SOX13 function is the inhibition of WNT/TCF signaling, suggesting that differential WNT/TCF activity is an essential parameter for this binary cell fate choice.
Keywords: global gene expression profile comparison

overall_design: CD4+CD8+ double positive thymocyte subsets were sorted by FACS using total pooled thymocytes from minimum of two mice and immediately lysed in Trizol. Comparison groups in each experiment were DP thymocytes from Sox13 transgenic mice and wild type B6 littermate controls. Gene expression profiling was performed according to the manufacturer’s protocol (Affymetrix). Labeled cRNA (from total RNA) was generated and applied to Affymetrix Mu11K(A and B) (expt 1) or muU74Av2 (expt 2) microarrays. Results were analyzed using Microarray Analysis Software v4 and v5 (Affymetrix).

first: Joonsoo

last: Kang

first: Sandy

last: Der

first: Heather

last: Melichar


title: Tumor suppression by Interferon regulatory factor-1 relies on down-regulation of cyclin D1

status: Public on Dec 16 2006

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary:
Interferons have been ascribed to mediate antitumor effects. IRF-1 is a major target gene of interferons. It inhibits cell proliferation and oncogenic transformation. Here we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of non-transformed cells by IRF-1. These include cell cycle regulating genes. Activation of IRF-1 decreases cyclin D1 expression and CDK4 kinase activity concomitant with dephosphorylation of pRb. These effects of IRF-1 are mediated by inhibition of the MEK-ERK pathway and a transcriptional repression of cyclin D1. IRF-1 mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Ablation of cyclin D1 by RNA interference experiments prevents transformation and tumor growth in nude mice. The data demonstrate that cyclin D1 is a key target for IRF-1 mediated tumor suppressive effects.
Keywords: NIH3T3 cells, myc, ras, IRF-1, proliferation, transformation, G1/S cell cycle ceckpoint

overall_design: 3 samples were analysed, two replicates per sample. NIH3T3 cells were compared with myc/ras transformed NIH3T3 cells and with IRF1 expressing myc/ras transformed cells.

first: R

first: A

last: Kroger


title: Expression data from mouse uteri after ovariectomy and E2 treatment.

city: Quebec

first: Ezequiel

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

institute: CRCHUL

summary:
17ß-Estradiol (E2) is well known to be associated with uterine cancer, endometriosis, and leiomyomas. Although insulin-like growth factor I (IGF-I) has been identified as a mediator of the uterotrophic effect of E2 in several studies, this mechanism is still not well understood. In the present study, identification of the genes modulated by a physiological dose of E2, in the uterus, has been done in ovariectomized mice using Affymetrix microarrays. The E2-induced genomic profile shows that multiple genes belonging to the IGF-I pathway are affected after exposure to E2. Two phases of regulation could be identified. First, from 0 to 6 h, the expression of genes involved in the cell cycle, growth factors, protein tyrosine phosphatases, and MAPK phosphatases is quickly upregulated by E2, while IGF-I receptor and several genes of the MAPK and phosphatidylinositol 3-kinase pathways are downregulated. Later, i.e., from 6 to 24 h, transporters and peptidases/proteases are stimulated, whereas defense-related genes are differentially regulated by E2. Finally, cytoarchitectural genes are modulated later. The present data show that a physiological dose of E2 induces, within 24 h, a series of transcriptional events that promote the uterotrophic effect. Among these, the E2-mediated activation of the IGF-I pathway seems to play a pivotal role in the uterotrophic effect. Furthermore, the protein tyrosine phosphatases and MAPK phosphatases are likely to modulate the estrogenic uterotrophic action by targeting, at different steps, the IGF-I pathway.
Keywords: E2 time course

overall_design: Ten 10-11 weeks old C57Bl/6 mice were gonadectomized under isoflurane-induced anesthesia and sacrified seven days after GDX.  Mice  were injected s.c with E2 (0.05ug/mice) or vehicle, at 0.5, 1, 3, 6, 12 or 24 h before sacrifice. Uteri was removed and pooled before total RNA extraction. Samples was hybridized to U74Av2 Genechips (Affymetrix).

first: Francine

last: Durocher

first: Yvan

last: Labrie

first: Mahinè

last: Ivanga

first: Fernand


title: ANA-1 macrophages infected with Chlamydia pneumoniae

city: Munich

first: Joerg

last: Mages

address: Trogerstrasse 30

department: Institute of Medical Microbiology, Immunology and Hygiene

institute: Technical University Munich

summary:
This experiment is an additional experiment to GSE6688. Mouse macrophages (ANA-1 cells) were infected in vitro with C. pneumoniae with a M.O.I. of 10. Twenty two genes were significantly upregulated. Examples of the most upregulated genes in mouse macrophages after C. pneumoniae infection are serum amyloid A3 (saa3), a protein that is mainly produced by activated macrophages during tissue injury or inflammation, MIP-2 (cxcl2) and irg1. Expression levels of all genes induced by C. pneumoniae in macrophages in vitro correlated with the results obtained from infected lungs from wild type mice (GSE6688), suggesting that this cell type participates in host defense in vivo against C. pneumoniae.
Keywords: Chlamydia pneumoniae, ANA-1 macrophages, in vitro, infection

overall_design: ANA-1 macrophages were infected with Chlamydia pneumoniae or left untreated. After 8h total RNA was extracted. Two biological replicates were performed resulting in 4 arrays in total

first: Jörg

first: Nuria

last: Rodríguez

first: Harald

last: Dietrich

first: Nina

last: Wantia

first: Hermann

last: Wagner

first: Roland

last: Lang

first: Thomas

last: Miethke


title: H3 occupancy

city: Jerusalem

first: Tommy

last: Kaplan

address: Givat Ram Campus

department: School of Computer Science and Engineering

institute: Hebrew University

summary:
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Keywords: Chip-chip, H3

overall_design: ChIP-chip for Myc-H3 vs WCE.

first: Michael

last: Dion

first: Minkyu

last: Kim

first: Stephen

last: Buratowski

first: Nir

last: Friedman

first: Oliver

last: Rando


title: gene expression profiling of NFIA deficient mice brain

status: Public on May 30 2007

city: Hamburg

first: Yong Wee

last: Wong

address: 94 Falkenried Str

institute: Center for Molecular Neurobiology

summary:
Background. Nuclear factor I-A (NFI-A), a phylogenetically conserved transcription/replication protein, plays a crucial role in mouse brain development. Previous studies showed that disruption of the Nfia gene in mice leads to perinatal lethality, corpus callosum agenesis, and hydrocephalus.
Results. To identify potential NFI-A target genes involved in the observed tissue malformations, we analyzed gene expression in brains from NFI-A-deficient and wild-type littermate control mice at the mRNA level using oligonucleotide microarrays. In young postnatal animals (P16), 356 genes were detected as differentially regulated, whereas at the late embryonic stage (E18), only 5 dysregulated genes were found. An in silico analysis identified phylogenetically conserved NFI binding sites in at least 70 of the differentially regulated genes. Moreover, assignment of gene function showed that marker genes for immature neural cells and neural precursors were expressed at elevated levels in young postnatal NFI-A mutants. By contrast, marker genes for differentiated neural cells were down-regulated at this stage. In particular, genes relevant for oligodendrocyte differentiation were affected.
Conclusions. Our results suggest that a delay in early postnatal development, especially oligodendrocyte maturation, is exhibited by NFI-A knock-out mice, and at least partly accounts for their phenotype. The identification of potential NFI-A target genes by our study should both help to elucidate NFI-A-dependent transcriptional pathways and contribute to a better understanding of this period of brain formation, especially with regard to the function of NFI-A.

Material and method
Animals
NFI-A-deficient mice were bred and maintained as described [20]. Animals were sacrificed by CO2 at embryonic stage E18.5 (E18) or postnatal day 16 (P16). Brains were dissected and snap frozen in liquid nitrogen.
The respective KO and WT animals used for analysis at E18 and P16 were littermates. Only male mice were used. All of the animals were F1 hybrids of C57Bl/6 and 129S6 animals. Within these matings, males had a B6 and females had a 129S6 background. In a large scale test, 38.5% of the offspring survive until P30 using this kind of mating. In purebred B6, we observed 0% survival at P1 (R.M.G., unpublished observations).
All animal use was performed under the approved protocol BCH05082N (RMG) of the University at Buffalo Institutional Animal Care and Use Committee in the UB Laboratory Animal Facility, an AAALAC licensed facility.

RNA preparation
Total RNA was extracted from the entire brain of NF-I-A-deficient and wild-type control mice (n=3 each) using TRIzol (Invitrogen) according to the manufacturer’s instructions. Further purification of RNA was performed with the RNeasy Mini Kit (Qiagen). Total RNA concentration was determined using a spectrophotometer at 260 nm and 280 nm wavelength. To check RNA integrity, total RNA was separated in a 1% agarose gel containing formaldehyde, and the intensity ratio of 28S and 18S ribosomal RNA bands was assessed after ethidium bromide staining.

Microarray hybridization and signal detection
Procedures for cDNA synthesis, labeling and hybridization were carried out according to the manufacturer's protocol (Affymetrix). All experiments were performed using Affymetrix mouse genome Genechip U74A version 2. Briefly, 15mg of total RNA were used for first strand cDNA synthesis with an HPLC-purified T7-(dT)24 primer. Synthesis of biotin-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (Affymetrix). For hybridization, 15 µg of fragmented cRNA were incubated with the chip in 200 µl of hybridization solution in Hybridization Oven 640 (Affymetrix) at 45oC for 16 hours. Genechips were washed and stained with streptavidin-phycoerythrin using the microfluidic workstation™ (Affymetrix) and scanned with a laser scanner (Agilent Technologies).

Microarray quantification and statistical analysis
Quality controls were performed using Affymetrix Microarray Suite 5.0 (MAS 5.0) software. Data and statistical analysis and data visualization were performed with GeneSpring™ (Agilent Technologies). Expression values were extracted from the CEL file, and imported to GeneSpring using Robust Multi-array Average (RMA) for further analysis [24]. Two criteria were used to select for significant changes in gene expression. First, probe sets with less than 1.2 fold change comparing the KO to WT (control) were removed in order to reduce the number of genes undergoing statistical analysis. Second, genes were then tested statistically for significant changes in all three wild-type or NFI-A-deficient samples, using Student's t-test. Finally, genes were grouped based on their biological function using Affymetrix NetAffx, and NCBI Locuslink.
Keywords: biotinylated cRNA template

overall_design: 3 knockout and 3 wildtype from each embryonic stage day 18 and postnatal day 16 were used for gene expression profiling

first: Yongwee

last: Streichert

first: Richard

last: Gronostajski

first: Melitta

last: Schachner

last: Tilling


title: Transcriptional profiling of the cellular transformation induced by Rho subfamily GTPases

status: Public on Jan 03 2007

city: Salamanca

first: Xose

middle: Garcia

last: Bustelo

state: Salamanca

address: Campus Unamuno

country: Spain

institute: Centro de Investigacion del Cancer

summary:
We have used microarray technology to identify the transcriptional targets of Rho subfamily GTPases.  This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles.  Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of biological functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are up-regulated preferentially.  Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc, and p53.  Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation.  Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells.  Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways.  Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells.  However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation.  These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells
Keywords: Rho/Rac GTPases, microarray, oncogenesis, proliferation, gene expression, transcription, Rock, c-Myc

overall_design:
In order to generate the cell clones used in this study, we took advantage of the oncogenic properties of the constitutively active versions (Q63L mutants) of Rho subfamily proteins when overexpressed in rodent fibroblasts (Schuebel et al., 1998).  Based on this property, we transfected NIH3T3 cells with plasmids encoding the indicated versions of Rho subfamily proteins to obtain foci of transformed cells. Selected foci were picked, pooled, and used for the subsequent microarray experiments.
To avoid the activation of genetic programs related to serum withdrawal or contact inhibition that may confound the detection of Rho-specific transcriptomal changes (Coller et al., 2006), we cultured the chosen cell lines and the parental NIH3T3 cells in the presence of serum and maintained them at confluency levels lower than 70% prior to RNA extraction.  In addition, we isolated total RNAs from eight (in the case of NIH3T3 cells), seven (in the case of RhoAQ63L-transformed cells) and six (in the case of RhoBQ63L- and RhoCQ63L-transformed cells) independent cell cultures in order to make it possible a robust statistical treatment of the data obtained.
three 10-cm diameter plates containing exponentially growing cultures of IMB11-1P (expressing RhoAQ63L) IMB11-2P (expressing RhoBQ63L), or IMB11-3P (expressing RhoCQ63L) cells were washed with PBS and their total cellular RNAs isolated using the RNeasy kit (Qiagen) according to the supplier’s specifications. The quantity and quality of the total RNAs obtained was determined using 6000 Nano Chips (Agilent Technologies).  Total RNA samples (4 ug) were then processed for hybridization on MGU75Av2 microarrays (Affymetrix) using standard Affymetrix protocols at the CIC Genomics and Proteomics Facility (www.cicancer.org).  Normalization, filtering and analysis of the raw data obtained from the microarrays was carried out with the Bioconductor software (www.bioconductor.com) using de ReadAffy package and the RMA application. The RMA algorithm was selected over the standard Affymetrix software because it provides a better precision in signal detection to achieve adequate normalization of multiple microarray hybridizations, especially in cases of low levels of gene expression (Bolstad et al., 2004; Gautier et al., 2004; Gentleman et al., 2004; Parrish & Spencer, 2004).  We considered a gene to be differentially expressed when exhibiting a signal ≥ 100 and met the following criteria: in the case of the characterization of the transcriptome of Rho-transformed cells, we regarded a gene as common to all GTPases when: i) It showed a fold change ≥ 1.5 in at least two of the cell lines used.  ii) The fold change in the third cell line was ≥ 1.0 and displayed a similar variation trend when compared to the other two cell lines (i.e., similar up-regulation or down-modulation in the three cell lines).  iii) The fold change values in the three cell lines had always P values ≤ 0.01.  We regarded a gene as common to only two GTPases when: i) It showed a fold change ≥ 1.5 in two the cell lines with P values ≤ 0.01.  ii) The fold change in the third cell line was non-existent or, alternatively, had P values ≥ 0.01.  We considered a gene as uniquely-regulated by a GTPase when: i) The fold change in the expression levels of is transcript in the cell line transformed by that GTPase was ≥ 1.5 with P value ≤ 0.01.  ii) The fold change, if any, obtained in the other cell lines had P values ≥ 0.01. Statistical analyses were performed using F-statistics.

first: Inmaculada

last: Berenjeno

first: Fatima

last: Nuñez

middle: R


title: Four weeks expression data of the antipsychotics Clozapine and Haloperidol in the mouse brain (Affymetrix, GCRMA).

status: Public on Dec 13 2006

city: London

first: Mie

last: Rizig

address: Windeyer Institute of Medical Sciences,46 Cleveland Street

department: Mental Health Sciences

country: United Kingdom

institute: University Collage London

summary:
Atypical antipsychotic Clozapine has a superior antipsychotic and antimanic effects compared to other antisphicotics. Its widespread use was limited by the side effects of agranulocytosis, cardiomyopathy and metabolic anomalies such as weight gain, and diabetes. Very little is known about mechanisms by which Clozapine works. The aim of this experiment is to compare the chronic gene expression profile (i.e four weeks) of the atypical antipsychotic Clozapine to the typical antipsychotic drug Haloperidol using gene expression Microarray in order to understand the intercellular mechanism behind the therapeutic and the toxic effects of Clozapine.
Keywords: drug response

overall_design: The study was designed to compare the chronic therapeutic and toxic expression profile of Clozapine to Haloperidol in the mouse brain. All experiments were performed in male C57BL mice at four weeks of age (Biological services, University Collage London). Several theoretical and practical considerations influenced the final experimental design. To avoid the effect of injections on genes’ expression and to simulate the clinical scenario in human, both drugs were applied to the animals’ drinking water using the maximum human therapeutic dose (i.e.1.6mg/kg/day for Haloperidol and 12mg/kg/day for Clozapine).Thirty animals were divided equally between three treatment groups and received either Haloperidol (10 animals), Clozapine (10 animals) or no treatment (10 animals) for 4 weeks. After four weeks,the plasma drug level for both drugs was assesed by Tandem mass Spectrometry LC- MS/MS. The total RNA from the right forebrains of nine selected animals (three from each treatment group) were extracted and hybridized to the Affymetrix U74Av2. In all the Microarray experiments we have avoided pooling and each RNA sample was an independent biological replicate. The total numbers of used arrays were 9  Affymetrix U74Av2.

first: Hugh

last: Gurling


title: Spleen and Liver

status: Public on Dec 03 2006

city: Pasadena

first: Sangdun

last: Choi

address: 1200 E. California Blvd

department: Biology

institute: California Institute of Technology

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

overall_design: Refer to individual Series


title: Transcriptional comparison between whole kidneys from E14.5 Wnt4 mutants and wildtype mice (MG_U74Av2 platform). (GUDMAP Series ID: 7)

status: Public on Nov 18 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

institute: IGMM MRC Human Genetics Unit

summary:
Our laboratory's interest is in understanding the molecular principles that underlie the regional organization of the mammalian metanephric kidney. Our goal is to generate a detailed spatial map of the cellular expression of selected regulatory genes during mammalian kidney development.  The goal of this study is to identify a population of genes that are enriched in the renal vesicle (RV) and its derivatives using Wnt4 mutants.
Keywords: mutant comparison

overall_design: Wnt4 is required for renal vesicle (RV) induction.  Therefore, RV and the derivatives (s-shaped body and eventually the mature nephron) are missing in Wnt4 mutants.  At E14.5, these structures are present in wildtype kidneys.  Transcriptional profile comparison between E14.5 wildtype and Wnt4 mutants therefore identify genes expressed in the RV and derivatives. Minimally pooled kidney samples were used as single biological replicates.  Total RNA was isolated from these pools and subjected to a single round of amplification for use on Affymetrix arrays.

first: Andrew

last: McMahon

first: M

middle: T

last: Valerius


title: OVX/P4&oil/UTX 129 WT vs. Hoxa-10-/-

city: Boston

last: Maas

address: 20 Shattuck St, Room 1019A

department: Medicine/Division of Genetics

institute: Brigham and Women's Hospital/Harvard Medical School


title: OVX/P4/UTX 129/SvImJ WT


title: Comparison of gene expression for contraction- and freeze-induced skeletal muscle injuries

status: Public on Dec 31 2006

city: Morgantown

first: Petia

last: Simeonova

state: WV

address: 1095 Willowdale Rd.

institute: NIOSH, CDC

summary:
Common acute injuries to skeletal muscle can lead to significant pain and disability.  The current therapeutic approaches for treating muscle injuries are dependent on the clinical severity but not on the type of injury.  The aim of this study was to compare the molecular events accompanying the degeneration and repair phases of contraction- and trauma-induced muscle injuries by applying DNA microarray methodology to two well-characterized mouse models of skeletal muscle injury, i.e., eccentric contraction-induced injury (CI) and traumatic injury induced by freezing (FI).  Histopathological evaluation and measurements of muscle strength were accompanied by analyses of expression for 12,488 known genes at four time points ranging from 6 hours to 7 days post-injury.  Real-time RT-PCR was used to confirm some of the gene expression temporal profiles.  While both types of injury cause early induction of transcription, myogenic, and stress-responsive factors, they also induce injury type-specific gene expression profiles.  CI only activates a set of genes associated with the protection and repair of protein and structural integrity while FI activates gene sets which result in extensive inflammatory responses, tissue remodeling, angiogenesis, and myofibre and extracellular matrix synthesis.  This study identified genes that are candidates for therapeutic manipulation following two disparate types of muscle injury.
Keywords: time course, comparative genomic hybridization

overall_design: 2 types of skeletal muscle injury (eccentric contraction- and freeze-induced) x 4 time points after injury (6 hours, 1 day, 3 days, and 7 days post-injury). There were 3 samples for each of the 8 cells with the exception of the 'contraction injury, 1 day post-injury' and 'freeze injury, 7 days post-injury' cells; for each of these 2 cells, there were only 2 samples. Additionally, there were 3 control (i.e., uninjured) muscle samples.

first: Gordon

middle: L

last: Warren

first: Mukesk

last: Summan

first: Xin

last: Gao

first: Rebecca

last: Chapman

first: Tracy

last: Hulderman

first: Kurt

last: Brumbaugh


title: Murine model of decidulization and menstruation

status: Public on Dec 01 2006

city: Cambridge

first: Ching-wen

last: Cheng

address: Robinson Way

department: Obstetrics and Gynaecology

institute: University of Cambridge

summary:
A murine model that mimic the decidualization and regression observed in human was used to investigate the molecular mechanisms underlying the dynamic processes in endometrium. Ovariectomized mice were treated sequentially with steroid hormones and then, to induce decidualization, oil was injected into the uterine lumen.  A process similar to menstruation was induced by hormone-withdrawal.  The uterine tissues were collected at 4 time-points after the induction of decidualization.
Keywords: decidualization, menstruation, progesterone-withdrawal, time-course

overall_design: The uterine tissues were collected at 36 (T1), 48 (T2), 60 (T3) and 84 hours (T4) after the last progesterone injection. There are 4 experimental animals for each time-point, and the RNAs collected from animals within the same time-point group were pooled together for Affymetrix microarray analysis using U74Av2 Chips.

first: Holli

last: Bielby

first: Di

last: Licence

first: Stephen

middle: K

last: Smith

first: Cristin

middle: G

last: Print

last: Charnock-Jones


title: T cell gene expression profile

status: Public on Oct 04 2006

city: Chicago

first: yuanyuan

last: zha

state: IL

department: Department of Pathology

institute: University of Chicago

summary:
compare gene expression profiles between normal and anergic T cells and identify upregulated genes in anergic T cells
Keywords: cell type comparison

overall_design: RNA from normal Th1 T cell clone and anergic Th1 T cell clone made anergic by plate-bound anti-CD3 antibody were isolated and amplified for microarray analysis

first: Reinhard

last: Marks

first: Allen

last: Ho

first: Thomas

last: Gajewski

first: Yuanyuan

last: Zha


title: Olfactory epithelium of SR-A mutant mice following bilateral olfactory bulbectomy

status: Public on Oct 14 2006

city: Lexington

first: Marilyn

last: Getchell

state: KY

address: 800 South Limestone St.

department: Anatomy and Neurobiology

institute: University of Kentucky

summary:
Sham and bilateral OBX were performed on wild-type and SR-A mutant mice.
Tissue collected 2, 8, 16, and 48 hours following OBX. Expression profiles were determined using Affynetrix MG U74Av2 chips.
Keywords: repeat

overall_design: olfactory epithelium from 3 wild type 6 week old mice and 3 SR-A 6 week old mutant mice

middle: V


title: Gene expression profiling of CD11b purified from mouse spleen (tumor-free and tumor-bearing mice)

status: Public on Oct 06 2006

city: Padova

first: Silvio

last: Bicciato

address: via U. Bassi 59/b

department: Molecular Medicine

country: Italy

institute: University of Padova

summary:
Active suppression of tumor-specific T lymphocytes can limit the immune-surveillance and immunotherapy efficacy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+/IL-4Rα+, inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-γ released from T lymphocytes. CD11b+/IL-4Rα+ cells produce IL-13 and IFN-γ and integrate the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits are active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors has detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumorinduced immune dysfunctions.
Keywords: Analysis of Cd11b cells from tumor-free and tumor-bearing mice

overall_design: Labeled cRNA extracted from a a total of 9 samples was hybridized to the Affymetrix GeneChip MG-U74Av2 which contains 12,488 probe sets . The 3 control samples represented 3 replicates of RNA extracted from Cd11b cells purified from the spleen of tumor-free mice. The 6 samples obtained from tumor-bearing mice represented 3 replicates each of RNA extracted from Cd11b cells purified from the spleen of tumor-bearing mice. Three out of six samples were incubated for 24 hours in complete medium.

first: Giovanna

last: Gallina

first: Luigi

last: Dolcetti

first: Paola

last: Zanovello

first: Vincenzo

last: Bronte


title: Expression data from control untreated mouse melanoma B16F1 cells

status: Public on Sep 27 2006

first: Assel

last: Biyasheva

address: 303 East Chicago Ave., Ward 8-063

department: Cell and Molecular Biology

institute: Northwestern University

summary:
B16F1 cells are a good model to study cell motility and cytoskeletal organization. In our lab, a combination of microscopy and gene silencing was used to approach the problem. Having gene expression profiles for B16F1 would facilitate and support subsequent gene silencing by RNAi as well as potentially identify new molecular players.
We used microarrays to detail the expression profiles in cells cultured in regular conditions. These profile will serve as a reference point for further studies.
Keywords: control

overall_design: Three biological replicats of control untreated B16F1 mouse melanoma cells were processed on mouse GeneChip arrays. Reliability, variability, and reprodusibility of data generated by microarrays were addressed.

first: Shin-ichiro

last: Kojima

first: Spencer

middle: C

last: Huang

first: Gary

last: Borisy


title: Expression data from wild type versus HDAC knock out mouse embryonic stem cells

status: Public on Sep 19 2006

city: Vienna

first: Gordin

last: Zupkovitz

address: Dr. Bohrgasse 9

department: Medical Biochemistry

country: Austria

institute: MFPL

summary:
Histone deacetylase 1 (HDAC1) is an enzyme that promotes deacetylation of acetylated lysine residues in histones and other proteins. Histone acetylation is often associated with gene activation and expression. Los of HDAC1 leads to severe problems in development and proliferation. Moreover, it seems to be the major histone deacetylase in mouse embryonic stem cells.
We used microarrays to identify putative HDAC1 target genes.
Keywords: Fold change anaylsis between wild type nad HDAC KO ES cells

overall_design: Wild type and HDAC1 mouse embryonic stem cell, originating from same litter, were used for RNA extraction and hybridization on Affymetrix microarrays.

first: Markus

last: Posch

first: Gerda

last: Egger

first: Norbert

last: Schweifer

first: Christian

last: Seiser


title: whole brains from nicotinic acetylcholine receptor subunit mutant mice and littermate controls

status: Public on Sep 22 2006

city: Tel Aviv

first: Avi

last: Orr-Urtreger

address: 6 Weizmann St

department: Genetic Institute

institute: Tel Aviv Sourasky Medical Center

summary:
Studies using mice with beta4 nicotinic acetylcholine receptor (nAChR) subunit deficiency (beta4-/- mice) helped reveal the roles of this subunit in bradycardiac response to vagal stimulation, nicotine-induced seizure activity and anxiety. In order to identify genes that might be related to beta4-containing nAChRs activity, we compared the mRNA expression profiles of brains from beta4-/- and wild-type mice using Affymetrix U74Avr2 microarray.
Keywords: knockout mice, nicotinic acetylcholine receptor

overall_design: The experimental groups included six wild-type mice and five beta4-/- mice. All mice were 6-9 weeks old. Total RNA was extracted from whole brain tissue of the mice.

first: Merav

last: Kedmi


title: IL-10 or IL-6 stimulation of IL-6-/- and IL-10-/- murine bone marrow derived macrophages in the presence of LPS

status: Public on Aug 25 2006

city: Memphis

first: Peter

last: Murray

state: TN

address: 332 Nth Lauderdale

department: Infectious Diseases

institute: St. Jude Children's Research Hospital

summary:
IL-10 or IL-6 stimulation of control 129xC57BL/6 murine bone marrow derived macrophages in the presence of LPS.
We used microarrays to detail the global programme of gene expression changes in response to IL-6 or IL-10 stimulation in the presence of lipopolysaccharide.  BMDMs were isolated from control, IL-6-/-, and IL-10-/-  mice on a 129XBL/6 mixed background mice and differentiated in the presence of CSF-1 for 6-7 days.  Cells were scraped and plated in 6 well plates at 2x10e6/well.  Cells were washed with complete DMEM and rested for 1-2 hr before stimulation with combinations of IL-10 (10 ng/ml), IL-6 (2 ng/ml) or LPS (100 ng/ml) for 45 min or 180 mins.  Complete biological replicates were performed.
Keywords: time course

overall_design: Data sets from wild-type, IL-10-/- and IL-6-/- BMDMs treated with IL-6 or IL-10 in the presence of LPS over time

first: Jie

middle: D

last: Chen


title: Transcriptional profile of E14.5 micro-dissected kidney structures: renal vesicle, s-shaped body & collecting duct (MG_U74Av2 platform). (GUDMAP Series ID: 9)

first: GUDMAP

last: Developers

summary:
Our laboratory's interest is in understanding the molecular principles that underlie the regional organization of the mammalian metanephric kidney. Our goal is to generate a detailed spatial map of the cellular expression of selected regulatory genes during mammalian kidney development.  The goal of this study is to identify genes expressed during the morphogenesis of the nephron.  By profiling two specific developmental nephron structures, we expect to identify genes expressed in both, and unique to each, which suggests they may play a role in the underlying mechanism responsible for the morphogenesis.
Keywords: kidney component comparison

overall_design: Two early nephron structures, the renal vesicle and the s-shaped body, and the collecting duct system, were microdissected from E14.5 kidneys based on morphological criteria.  Antibody and lectin staining together with RT-PCR were used for identity verification and for exclusion of contamination by other tissues.  Samples are minimally pooled for amplification.  A biological replicate is a single minimally pooled sample.  RNA isolated from the collected structures was put through two rounds of T7-driven amplification to obtain the microgram quantities of cRNA needed for hybridization to Affymetrix microarrays.  Detection of genes previously shown to be expressed in these early structures, such as Wt1, Wnt4 and Pax8, validates the data from the screen.  The collecting duct sample serves as a reference sample for comparing the renal vesicle and s-shaped body comparison in this study.  We also tested amplification of single s-shaped bodies as compared to pooled samples and found the minimally pooled samples resulted in better data for this study (better sensitivity when inspected for genes known to be expressed in s-shaped bodies).

first: Andrew

last: McMahon

first: M

last: Valerius


title: Expression profiling of the mouse prostate after castration and hormone replacement

status: Public on Sep 25 2006

city: South San Francisco

first: Zemin

last: Zhang

address: 1 DNA Way, MS 93

department: Bioinformatics

institute: Genentech Inc.

summary:
In this study, we used microarray analysis to determine gene expression profile changes in the mouse prostate following castration and hormone replacement. We first identified genes with significant expression changes in each of these two processes and then generated a list of androgen responsive genes and a list of genes whose expression were inversely correlated with the presence of androgen. The analysis of this data set is described in Wang et al., Differentiation, 2006
Keywords: Time course

overall_design: C57/B6 mice of 10-12 weeks old (Charles River, Hollister, CA) were used. Mice were castrated, and at 3 or 14 days after surgery (C3 or C14), mice from these groups were sacrificed and whole prostates were collected. Prostates were also collected from sham-treated mice for control (N3). To obtain re-growing or regenerating prostate, mice that had been castrated 14 days before were implanted with testosterone (T) pellet (15 mg/pellet/mouse, Innovative Research, Sarasota, FL). Three days after pellet implantation, mice were sacrificed and prostates were collected (C14+T3). Five mice were used for each treatment/time point. Total RNA was purified with RNeasy kit.

first: Xi-De

last: Wang

first: Bu-Er

first: Robert

last: Soriano

first: Jiping

last: Zha

first: Zora

last: Modrusan

first: Gerald

last: Cunha

first: Wei-Qiang

last: Gao


title: Circadian Profiling of NIH3T3 Fibroblasts: Comparison with Rhythmic Gene Expression in SCN2.2 Cells and the Rat SCN

status: Public on Oct 31 2006

city: College Station

first: Gus

middle: John

last: Menger

institute: Texas A&M Health Science Center

summary:
To screen for specific circadian outputs that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared to that found in the rat SCN in vivo and SCN2.2 cells in vitro. Similar to the scope of circadian gene expression in SCN2.2 cells and the rat SCN, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Bmal1 (Mop3), and Cry1 and 323 functionally diverse transcripts (2.6%), many of which were involved in cell communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among cellular pathways mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase, the enzyme responsible for the production of nitric oxide, in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that this gaseous neurotransmitter may play a key role in SCN pacemaker function. Thus, this comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective identification of circadian signals involved in the coupling of SCN oscillators and the regulation of rhythmicity in downstream cells or tissues.
Keywords: Circadian time course

overall_design: Circadian profiling of the NIH/3T3 fibroblast transcriptome entailed the treatment of NIH/3T3 cells with a 15uM forskolin pulse, subsequent washout of the drug, and collection of total RNA immediately after washout and every 6 hours across two circadian cycles for each of three experiments. Timepoint values reflect the average of three samples from these biological replicates.

first: Kim

last: Lu

first: Gregg

last: Allen

first: Nichole

last: Neuendorff

first: Sang

last: Nahm

first: Terry

last: Thomas

first: Vincent

middle: M

last: Cassone

first: David

last: Earnest


title: Time series of diabetes and exercise training induced expression changes in cardiac muscle of mice

status: Public on Sep 01 2006

city: Jyväskylä

first: Mika

last: Silvennoinen

address: Rautpohjankatu 8, Viveca

country: Finland

institute: LIKES - Research Center

summary:
We investigated the effects of diabetes, physical training, and their combination on the gene expression of cardiac muscle. Mice were divided to control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed 1, 3, or 5 weeks of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 hours after the last training session. Gene expression of cardiac muscles were analyzed using Affymetrix Gene chip MG U74Av2 (Affymetrix , Inc., Santa Clara, CA).
Keywords: time course

overall_design: Experiment was performed on 10 to 15 weeks old male NMRI mice (Harlan, Holland) housed in standard conditions (temperature 22°C, humidity 60 ± 10 %, artificial light from 8.00 am to 8.00 pm, normally 5 animals per cage). Animals had free access to tap water and food pellets (R36, Labfor, Stockholm, Sweden). Animals were randomly divided into healthy and diabetic groups. The diabetic group received a single peritoneal injection of streptozotocin (STZ, Sigma-Aldrich, France, 180 mg/kg) dissolved in sodium citrate buffer solution (0.1 mol/l, pH 4.5) to induce experimental diabetes similar to type 1. The other group received injection of an equal volume of buffer. Diabetes was confirmed 72 hours after the injection by urine glucose testing (Glukotest(r), Roche, Germany), and mice were characterized diabetic when urine glucose values were greater than 200 mg/dl. Diabetic and healthy animals were randomly assigned into 12 groups (n = 5 per group), which were sedentary or trained for one, three or five weeks. Training groups performed 1 hour per day of treadmill running at 21 m/min and 2.5° incline. After one day of familiarization on a rodent treadmill, the mice ran as described above 5 days per week. Mice were sacrificed 24 hours after the last training bout (respective sedentary controls at the same time) by cervical dislocation followed by decapitation. Cardiac muscle was removed, weighed, snap frozen in liquid nitrogen and stored at -80°C for further analysis.

first: Riikka

last: Kivelä

first: Maarit

last: Lehti

first: Anna-Maria

last: Touvras

first: Jyrki

last: Komulainen

first: Veikko

last: Vihko

first: Heikki

last: Kainulainen


title: Modulation of pro- and antiapoptotic molecules in double-positive (CD4+CD8+) thymocytes following dexamethasone treatmen

status: Public on Aug 08 2006

city: Perugia

first: Carlo

last: Riccardi

state: Perugia

address: via del Giochetto

department: Clinical and Experimetal Medicine

institute: Università degli Studi di Perugia

summary:
 Glucocorticoids play a role in regulation of T lymphocytes homeostasis and development. In particular, glucocorticoid treatment induces massive apoptosis of CD4+CD8+ double positive (DP) thymocytes. This effect is due to many mechanisms, mainly driven by modulation of gene transcription. To find out which genes are modulated, we analyzed DP thymocytes treated for 3 hours with dexamethasone or medium alone, by global gene expression profiling using the Affymetrix technology (MGU74Av2 GeneChip). The data were analyzed with MAS 5.0  imposing a cut off of 1.7 fold in up regulation and 1.35 fold in down regulation. To further filter results we also used the statistical software, SAM (d 0.88 see figure 1s in supplementary data of the above cited manuscript). Results indicate modulation of 156 genes, also confirmed by either RNAse protection assay or Real Time PCR. For data mining we also used Go Miner to explore the Gene Ontology data bank (see tables 1-3 of the above cited manuscript). The overall results demonstrated that dexamethasone caused the down-regulation of genes promoting survival of DP thymocytes (e.g. Notch1, Socs1 and Id3) or the modulation of genes activating cell death through the ceramide pathway (Ugcg, Sgpp1, Degs1 and Gpr65) or through the mithocondrial machinery. Among the latter, there are Bcl-2 family members (Bim, Bfl-1, Bcl-xL and Bcl-xbeta), genes involved in the control of redox status (thioredoxin reductase, TXNIP and idh2) and genes belonging to Tis11 family which are involved in mRNA stability. Our study suggests that dexamethasone treatment of DP thymocytes modulates several genes belonging to apoptosis-related systems that can contribute to their apoptosis.
Keywords: Dexamethasone/PBS treatment comparison

overall_design: Thymi from 4 week old C3H/HeN mice were teased in culture medium, CD4+CD8+ double positive (DP) thymocytes were sorted with microbeads Miltenyi Biotech methods and were plated in 6 well plates with 3 ml of the culture medium at a concentration of 5.0E+06 cell/ml together with 1E-07 M Dexamethasone or a corresponding volume of PBS. DP thymocytes were kept at 37°C, 5% CO2 in a humidified atmosphere for 3 hours. Total RNA was extracted and  analyzed. We used 2 technical replicate-extracts.

first: Rodolfo

last: Bianchini

first: Giuseppe

last: Nocentini

first: Ludovic

last: Krausz

first: Katia

last: Fettuciari

first: Stefano

last: Coaccioli

first: Simona

last: Ronchetti


title: Genes enriched in secondary alveolar septae of neonatal lung tissue

status: Public on Aug 19 2006

city: Iowa City

first: Jeanne

last: Snyder

state: IA

address: 51 Newton Road

department: Anatomy and Cell Biology

institute: University of Iowa

summary:
The tips of secondary alveolar septae in day 6 neonatal mouse lung tissue were isolated using laser capture microscopy. RNA was isolated from pooled secondary alveolar tips and also from pooled neonatal day 6 whole lung tissue. The isolated RNAs were then amplified in parallel. Gene array profiling of the two RNA samples was performed. Gene expression in the secondary alveolar septal tips was compared to gene expression in the whole lung tissue. In this way, the genes that are expressed in the tip of secondary alveolar septae were identified as well as the genes that are enriched in the alveolar septal tips versus in whole lung tissue.
Keywords: Comparison of gene profiles in the tips of secondary alveolar septae versus in whole lung tissue of neonatal mice

overall_design: We performed an experiment in which we used laser capture microscopy to collect 10,000 secondary alveolar tips from frozen sections of lung tissue obtained from many different day 6 neonatal mice obtained from different litters. In the same experiment, we also isolated RNA from pooled day 6 neonate whole lung tissue. The isolated RNAs were amplified in parallel and then hybridized to microarrays in parallel in order to profile and compare gene expression in the two samples. The entire experiment was performed twice with tissue from different litters. In addition, the amplified RNAs were hybridized to two different microarrays, at different times.


title: E14 mouse molar tooth germs cultured with/without TCDD

city: Helsinki

first: Carin

last: Sahlberg

address: PL 63, Haartmaninkatu 8

department: Institute of Dentistry

institute: University of Helsinki

summary:
Dioxins are ubiquitous environmental poisons that are developmentally toxic. Exposure of mouse E14 tooth germs to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to reduced tooth size and deformation of cuspal morphology implying the induction of a variety of biological responses on both cellular and molecular levels.
To verify such responses at the gene level, mouse embryonic cap staged tooth germs were cultured for 24 h with/without 1 µM TCDD.
Keywords: tissue culture, exposure to TCDD, tooth development

overall_design:
Organ culture: Mandibular molar tooth regions from E14 mouse embryos (NMRI X NMRI; the day of vaginal plug = E0) were dissected under a stereomicroscope and transferred to an organ culture. The tissue explants were supported by polycarbonate Nuclepore filters [pore size, 0.1 µm; Corning Inc., New York] placed on a stainless steel grid. The basal culture medium was Dulbecco’s modified Eagle’s medium (DMEM) containing Glutamax-1 (Gibco BRL, Paisley, Scotland) supplemented with 10% fetal calf serum (FCS; Gibco BRL). TCDD (from the Department of Chemistry, University of Jyväskylä, Jyväskylä, Finland) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 µg/ml. TCDD at the concentration of 1 µM, or DMSO, was added to the basal culture medium from the start of culture. The culture dishes were kept in a humidified incubator at 37 ºC in an atmosphere of 5% CO2. The tissue explants were cultured for 24 hours, after which they were snap frozen in liquid nitrogen. Explants from several experiments were collected and kept in liquid nitrogen. In a single experiment, 15–29 explants were included in each experimental group.
Microarray analysis:
Three separate experiments were combined for each replicate sample. About 60 explants each were pooled and homogenized while still frozen in liquid nitrogen. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from the total RNA by using a GeneChip T7-Oligo(dT)24 Promoter Primer Kit (Affymetrix) and Superscript II Choice System (Invitrogen Life Sciences). The double stranded cDNA was cleaned-up with the GeneChip Sample Cleanup Module (Affymetrix). Biotin-labelled cRNA was synthesized by in vitro transcription usin a RNA labelling kit from Enzo Life Sciences, Inc. The labelling time was 5 h in 37ºC waterbath. All syntheses were done according to the manufacturers instructions. Unincorporated NTPs were removed  with the GeneChip Sample Cleanup kit and the concentration of the labelled cRNA was mesured by spectrophotometric analysis and quantificated. 15 or 16 µg of labelled cRNA was fragmented by incubation at 94 ºC for 35 min in the fragmentation buffer supplied in the GeneChip Sample Cleanup kit. The cRNA was hybridized to a murine U74v2A array (Affymetrix) for 16 h at 45 ºC, washed and stained with streptavidine-phycoerythrin. The arrays were scanned in a Genechip System confocal scanner (Aligent) and Affymetrix Microarray Suite 5.0 was used to scan and analyze the relative abundance of each gene. The target signal scaling was set to 100.

first: Eija

last: Peltonen

first: Pirjo-Liisa

last: Lukinmaa

first: Satu

last: Alaluusua


title: Nemaline myopathy mouse model

status: Public on Jul 21 2006

city: Athens

first: Despina

last: Sanoudou

address: Soranou Efesiou 4

department: Molecular Biology

country: Greece

institute: Foundation for Biomedical Research, Academy of Athens

summary:
The aim of this study was to investigate the molecular mechanisms implicated in this mouse model of nemaline myopathy, and to further compare the molecular disease response in different skeletal muscles. For this purpose, snap frozen skeletla muscle specimens from wild type and transgenic for alpha tropomyosin slow mice were studied. Five different muscle types were used (diaphragm, plantaris, extensor digitorum longus, tibialis anterior, gastrocnemus). Mice were sacrificed between 7 and 10 months. RNA pools from 3-5 animals were created and each pool was hybridized to a U74Av2 Affymetrix GeneChip. Datasets from 36 GeneChips were included in this study.
Keywords: disease mouse model analysis

overall_design: 36 skeletal mouse muscle RNA pools were used, from 5 different skeletal muscles, in two different conditions (wild type and transgenic)

first: Alan

last: Beggs


title: comparison of MILI+/- and MILI-/- at D10 testis

status: Public on Aug 01 2006

city: Suita

first: Satomi

middle: Kuramochi

last: Miyagawa

state: Osaka

address: Yamada-oka 2-2

institute: Osaka university

summary:
Spermatogenesis in MILI-KO mice was blocked completely at early prophase of the first meiosis during the zygotene and early pachytene stage. To analyze the molecular basis of MILI function, we examined the difference between gene expression for MILI-/- and MILI+/- testes.
Keywords: total RNAs of day10 testes

overall_design: To exclude secondary effects, we prepared mRNA from day10 mice. Because the apoptotic cells in KO testis were observed at day11 after birth.

last: Kuramochi-Miyagawa

first: Toru

last: Nakano


title: Earliest Changes in the Left Ventricular Transcriptome Post-Myocardial Infarction

status: Public on Jul 14 2006

city: Laramie

first: Mark

last: Stayton

state: WY

address: Dept. 3944, 1000 E. University Ave.

institute: University of Wyoming

summary:
We report a genome-wide survey of early responses of the mouse heart transcriptome to acute myocardial infarction (AMI). For three regions of the left ventricle (LV), namely ischemic/infarcted tissue (IF), the surviving LV free wall (FW) and the interventricular septum (IVS), 36,899 transcripts were assayed at six time points from 15 min to 48 h post-AMI in both AMI and sham surgery mice. For each transcript, temporal expression patterns were systematically compared between AMI and sham groups, which identified 515 AMI-responsive genes in IF tissue, 35 in the FW, 7 in the IVS, with three genes induced in all three regions. Using the literature, we assigned functional annotations to all 519 nonredundant AMI-induced genes and present two testable models for central signaling pathways induced early post-AMI.  First, the early induction of 15 genes involved in assembly and activation of the activator protein-1 (AP-1) family of transcription factors implicates AP-1 as a dominant regulator of earliest post-ischemic molecular events.  Second, dramatic increases in transcripts for arginase 1 (ARG1), the enzymes of polyamine biosynthesis and protein inhibitor of nitric oxide synthase (NOS) activity indicates that NO production may be regulated, in part, by inhibition of NOS and coordinate depletion of the NOS substrate, L-arginine. ARG1 was the single most highly induced transcript in the database (121-fold in IF region) and its induction in heart has not been previously reported.
Keywords: heart attack, mouse, time course, regional responses

overall_design:
We report temporal and regional changes in steady state mRNA levels in the mouse LV following a surgically-induced AMI.  Three regions of the infarcted LV were surveyed: IF, FW and IVS.  For each region, mRNA levels were assayed at t=0 and six time points after occlusion of the left anterior descending coronary artery (t = 15 min, 1 h, 4 h, 12 h, 24 h, 48 h).

As control, an identical time course was carried out in the mouse LV after sham surgery in which the suture was passed under the artery, but not ligated.  For sham surgical mice and naïve mice (t=0), the IVS and the LV free wall were harvested and assayed.  The AMI and sham time courses were repeated for a total of two independent repetitions of the experiment.  For each repetition, 96 GeneChips were hybridized.  Thus, for two repetitions, 192 GeneChips (2 x 96) were hybridized.  In addition, the naïve time point was assayed a second time during Repetition 1 for six additional GeneChips (two tissues x three chips).  The overall total number of samples is 198 GeneChips (192 + 6).

Total RNA was isolated from each individual tissue sample.  Equal weights of total RNA from 4-10 mice were combined for each LV region, time point and treatment.  Copy RNA mixtures were synthesized from each total RNA pool and hybridized to the Affymetrix GeneChip set MG U74 v2 (A, B, and C) that carries 36,899 probe sets.

The global normalization of fluorescence emissions for the 198 GeneChips used in this study and their conversion to relative transcript concentrations measured in relative fluorescence units (RFU) was conducted using Robust Multi-array Averaging (RMA; www.bioconductor.org).  Each of the 198 Accessions is titled as follows:  time point / treatment / tissue, repetition, GeneChip.  The additional assays of naïve mice in Repetition 1 are labeled “Naive2”.

middle: H

last: Harpster

first: Somnath

last: Bandyopadhyay

first: Paul

last: Thomas

first: Pavel

middle: S

last: Ivanov

first: Jacque

middle: A

last: Keele

first: Natalia

last: Pineguina

first: Bifeng

last: Gao

first: Vijay

last: Amarendran

last: Gomelsky

first: Richard

middle: J

last: McCormick