title: tourt-affy-arabi-307860

status: Public on Aug 24 2006

first: Winnie

last: Liang

summary:
The Early Growth Response (Egr) family of transcription factors consists of 4 members (Egr1-4) that are expressed in a wide variety of cell types. A large body of evidence point to a role for Egr transcription factors in growth, survival, and differentiation. A major unanswered question is whether Egr transcription factors serve similar functions in diverse cell types by activating a common set of target genes. Signal transduction cascades in neurons and lymphocytes show striking parallels. Activation of either cell type activates the Ras-MAPK pathway and, in parallel, leads to increases in intracellular calcium stimulating the calcineurin-NFAT pathway. In both cell types, the strength of the activation signal affects the cellular outcomes and very strong stimuli lead to cell death.  Notably both these pathways converge on the induction of Egr genes. We believe that downstream targets of Egr transcription factors in lymphocytes may also be activated by Egr factors in activated neurons. There is precedence for common target gene activation in these two cell types: apoptosis in both activated T cells and methamphetamine stimulated neurons occurs via FasL induction by NFAT transcription factors.  We propose to use developing T lymphocytes (thymocytes) as a model system for discovery of Egr-dependent target genes for several reasons. First, we have observed a prominent survival defect in thymocytes from mice deficient in both Egr1 and Egr3 (1/3 DKO) and a partial differention block in the immature double negative (DN) stage. In addition, thymocytes are an easily manipulatable cell type, and the DN subpopulation affected in 1/3 DKO mice can be isolated to very high purity. We anticipate that 1/3 DKO thymocytes will provide an excellent experimental system that will provide insight into Egr-dependent transcription in neuronal development, activation, and death.
To identify Egr transcription factor target genes in developing thymocytes. Egr1/3 DKO mice show a profound defect in thymocyte survival and differentiation, but the target genes downstream of Egr1 and Egr3 are unknown. Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from DN thymocytes  to identify genes that are deregulated in Egr1/3 DKO cells.
Egr1 and Egr3 expression is necessary for the proper survival and differentiation of developing thymocytes.  We hypothesize that we will be able to identify crucial target genes of Egr1 and Egr3 by comparing gene expression profiles from wild type and 1/3 DKO thymocytes.
Egr1 and Egr3 are first expressed in the immature double negative (DN) thymocytes. We will isolate DN thymocytes by FACS sorting which allows isolation of cell populations of >99% purity.  We will perform microarray analysis using the Mouse 430 2.0 gene array with thymocyte RNA samples from 3 wild type and 3 1/3 DKO mice (6 arrays total).  Differentially regulated genes (up-regulated and down-regulated) will be identified from the list of genes with significantly altered expression greater than or equal to 2-fold by paired T test.  Interesting genes will be validated by real-time PCR in wild type and 1/3 DKO thymocytes.  Recognizing that both direct and indirect target genes may be identified in the list of differentially expressed genes, real-time PCR validated target genes will be further screened using chromatin immunoprecipitation coupled with PCR (ChIP-PCR) to determine whether Egr1 and/or Egr3 are bound to potential regulatory regions of the putative target genes.
Keywords: other

first: Warren

last: Tourtellotte


title: Influenza virus infection-induced gene expression changes of regional B cells are mediated in part through type I IFN

status: Public on Aug 26 2006

city: San Francisco

first: Michael

middle: E.

last: Salazar

state: CA

address: 1550 Fourth Street, RM 545

country: USA

institute: University of California, San Francisco

summary:
Influenza virus infection-induced gene expression changes of regional B cells are mediated at least in part through type I Interferon:
Our objective is to determine whether the influenza virus-infection induced gene expression changes in regional lymph node B cells are facilitated at least in part through type I interferon.  Our specific aim is to compare the gene expression profile of highly FACS-purified B cells in the regional lymph nodes of wildtype and IFNR-/- mice prior to and 48h following infection with influenza virus infection and to contrast this expression profile with that of FACS-purified wildtype B cells activated in vitro with IFN-beta +/- anti-CD86 for 12h.
Keywords: Infection-induced responses

overall_design: We analyzed gene expression from mouse lymph node B cells purified by flow cytometric sorting using single channel oligonucleotide microarrays. There were 4 groups: 1) wild type uninfected mice (control group), 2) wild type mice infected with influenza (flu) for 2 days, 3) IFNR-/- mice infected with flu for 2 days, 4) cells stimulated with IFN-b in vitro for 17 h. Each group contained 4 biological replicates obtained from independent experiments. There were 16 total samples and each was measured on a separate array.

first: Elizabeth

middle: S

last: Coro

first: W

middle: L

last: Chang

first: Virginia

middle: P

last: Doucett

first: Yuanyuan

last: Xiao

first: David

middle: J

last: Erle

first: Nicole

last: Baumgarth


title: Hoxb1 gene expressing Hox-A1 protein: Altered transcriptional profile in rhombomere 4 at E10.5

status: Public on Aug 28 2006

city: Salt Lake City

first: Petr

last: Tvrdik

state: UT

department: Human Genetics

institute: University of Utah

summary:
This study analyzed mRNA profiles in rhombomere 4 of E10.5 mouse knock-in embryos expressing either normal endogenous Hox-B1 protein or the paralogous Hox-A1 protein from the Hoxb1 locus. The Hox-A1 protein was found to be detectably less efficacious than Hox-B1 in promoting neurogenesis in the basal plate of rhombomere 4 and its transcriptional profile shared several similarities with the Hoxb1 mutant.
Keywords: gene swap, knock-in, hindbrain development, rhombomere 4

overall_design: GFP-positive cells were FACS-sorted from dissected hindbrains of entire litters of E10.5 mouse embryos expressing either normal endogenous Hox-B1 protein or the paralogous Hox-A1 protein from the Hoxb1 locus, either one tagged with IRES-tauGFP. Three independent biological replicates of each genotype were analyzed. Total RNA was isolated, amplified and hybridized to Affymetrix Mouse Genome 430 2.0 Arrays.

first: Diane

middle: M

last: Dunn

first: Robert

middle: B

last: Weiss

first: Mario

middle: R

last: Capecchi


title: Gene expression profiling in small intestine of mice administered lactoperoxidase

status: Public on Sep 01 2006

city: Zama

first: Hiroyuki

last: Wakabayashi

state: Kanagawa

address: 5-1-83 Higashihara

department: Nutritional Science Laboratory

country: Japan

institute: Morinaga Milk Industry Co., Ltd.

summary:
Lactoperoxidase (LPO) is a component of milk and other external secretions. Recently, we found antiinflammatory effect of orally administered LPO in mouse model of influenza virus-induced pneumonia. In order to study influence of bovine LPO on the digestive tract as its possible target organ, we performed comprehensive gene expression analysis of the mouse small intestine using GeneChip microarray to compare the effect of LPO with that of water or bovine serum albumin (BSA). We observed that 3 h after single ingestion of LPO the levels of 78 gene expression are upregulated and those of 9 gene expressions are downregulated. Within most changed genes, 5 upregulated and 1 downregulated genes such as FK506 binding protein 5 (FKBP5) and serum/glucocorticoid regulated kinase (SGK) are known to be transcriptionally modulated by glucocorticoid. This result suggests that ingested LPO modulates gene expressions in the small intestine in a glucocorticoid-like manner and this activity may link to its systemic antiinflammatory effects.
Keywords: treatment comparison

overall_design: Mice were orally administered 200 microL of water (n=3), bovine serum albumin (BSA, 2.5 g/kg body weight, n=3), or bovine lactoperoxidase (LPO, 2.5 g/kg body weight, n=3). Three hours after administration, comprehensive gene expressions of jejunum detached payer's patch were analysed for comparison of LPO administration with water or BSA administration.

first: Hirofumi

last: Miyauchi

first: Kouichirou

last: Shin

first: Koji

last: Yamauchi

first: Ichiro

last: Matsumoto

first: Keiko

last: Abe

first: Mitsunori

last: Takase


title: Searching for Brca1 regulated X-linked genes

status: Public on Sep 19 2006

city: Bethesda

first: Cuiying

state: MD

address: 10 Center Dr.

department: NIDDK

institute: NIH

summary:
We compared the expression levels of X-linked genes in the mammary glands of Brca1 D11/D11;p53+/- mutant and control (p53+/-) mice at three different stages of the mammary cycle:  virgin, pregnant day 16.5, and lactation day 1, using a cDNA microarray. In about 690 X-linked genes that are expressed at these three stages of mammary cycle of development, we found 16 X-linked genes showed altered expression levels in Brca1 D11/D11;p53+/- mammary glands in comparison with controls at all three time points. Among them, 9 genes were up-regulated and 7 were down-regulated. This result indicates that mutation of Brca1 could affect expression of a few X-linked genes in mammary tissues. However, this was unlikely caused by failure of X chromosome inactivation, as seven of them were down-regulated, and Xist RNA was expressed in all the Brca1 mutant mammary tissues.

Keywords: BRCA1 mutation analysis

overall_design: Mutations in breast cancer associated gene-1 (BRCA1) are associated with hereditary breast and ovarian cancers. BRCA1 encodes a tumor suppressor protein that functions in DNA repair, transcriptional regulation, chromatin remodeling, centrosome duplication and checkpoint control, etc.. We were interested in fishing out the genes regulated by Brca1. Three pairs of mammary glands from Brca1 D11/D11;p53+/- mutant and control (p53+/-) mice at three different stages of the mammary cycle were analyzed by microarray for gene expression levels.

first: Xiaoling

last: Xu

first: Ruihong

last: Wang

first: Wenmei

last: Li

first: Chuxia

last: Deng


title: Pyruvate induces mitochondrial biogenesis by a PGC-1alpha independent mechanism

status: Public on Aug 12 2006

city: Cambridge

first: Reza

last: Halse

state: MA

address: 100 Technology Square

institute: Novartis Institutes for BioMedical Research

summary:
The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells.  C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 hr.  Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate and maximal oxidative capacity.  The increase in mitochondrial biogenesis was independent of increases in PGC-1alpha and PGC-1alpha mRNA expression.  To further assess whether PGC-1alpha expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1alpha prior to treatment with pyruvate.  Despite a 70% reduction in PGC-1alpha mRNA the effect of pyruvate was preserved.  Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1alpha null mice.  These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1alpha, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity.

Keywords: basal state versus treatment at one time point

overall_design: C2C12 myoblasts were incubated with either basal media or basal media supplemented with 50 mM sodium pyruvate for 72 hr.  Total RNA was extracted using TRIzol and GeneChip experiment was conducted using Gene Chip Mouse Genome 430 2.0 Expression Array.  A Gene Chip analysis for each condition was performed in triplicate.

first: Leanne

last: Wilson

first: Qing

last: Yang

first: P.

last: Gullicksen


title: Gene Expression Biomarkers for Predicting Lung Tumors in Two-Year Rodent Bioassays

city: Research Triangle Park

first: Russell

middle: Scott

last: Thomas

state: NC

address: 6 Davis Drive

institute: The Hamner Institutes for Health Sciences

summary:
Two-year rodent bioassays play a central role in evaluating both the carcinogenic potential of a chemical and generating quantitative information on the dose-response behavior for chemical risk assessments. The bioassays involved are expensive and time-consuming, requiring nearly lifetime exposures (two years) in mice and rats and costing $2 to $4 million per chemical. Since there are approximately 80,000 chemicals registered for commercial use in the United States and 2,000 more are added each year, applying animal bioassays to all chemicals of concern is clearly impossible. To efficiently and economically identify carcinogens prior to widespread use and human exposure, alternatives to the two-year rodent bioassay must be developed. In this study, animals were exposed for 13 weeks to two chemicals that were positive for lung tumors in the two-year rodent bioassay, two chemicals that were negative for tumors, and two vehicle controls.  Gene expression analysis was performed on the lungs of the animals to assess the potential for identifying gene expression biomarkers that can predict tumor formation in a two-year bioassay following a 13 week exposure.
Keywords: toxicology, chemical carcinogenesis, lung

overall_design: Five week old female B6C3F1 mice were exposed for 13 weeks to the following treatments: 1) 1,5-Naphthalenediamine, CAS No. 2243-62-1, feed, 2000 ppm, positive lung carcinogen; 2) 2,3-Benzofuran, CAS No. 271-89-6, gavage, 240 mg/kg, positive lung carcinogen; 3) N-(1-naphthyl)ethylenediamine dihydrochloride, CAS No. 1465-25-4, feed, 2000 ppm, negative lung carcinogen; 4) Pentachloronitrobenzene, CAS No. 82-68-8, feed, 8187 ppm, negative lung carcinogen; 5) Feed control; 6) Corn oil gavage control.  Feed animals were exposed 7 days/week and gavage animals were exposed 5 days/week (5 ml/kg).  Microarray analysis was performed on the lungs of three mice per treatment group.

first: Linda

last: Pluta

first: Todd

last: Page

first: Longlong


title: Gene Expression Biomarkers for Predicting Liver Tumors in Two-Year Rodent Bioassays

summary:
Two-year rodent bioassays play a central role in evaluating both the carcinogenic potential of a chemical and generating quantitative information on the dose-response behavior for chemical risk assessments. The bioassays involved are expensive and time-consuming, requiring nearly lifetime exposures (two years) in mice and rats and costing $2 to $4 million per chemical. Since there are approximately 80,000 chemicals registered for commercial use in the United States and 2,000 more are added each year, applying animal bioassays to all chemicals of concern is clearly impossible. To efficiently and economically identify carcinogens prior to widespread use and human exposure, alternatives to the two-year rodent bioassay must be developed. In this study, animals were exposed for 13 weeks to two chemicals that were positive for liver tumors in the two-year rodent bioassay, two chemicals that were negative for liver tumors, and two vehicle controls.  Gene expression analysis was performed on the livers of the animals to assess the potential for identifying gene expression biomarkers that can predict tumor formation in a two-year bioassay following a 13 week exposure.
Keywords: toxicology, chemical carcinogenesis, liver

overall_design: Five week old female B6C3F1 mice were exposed for 13 weeks to the following treatments: 1) 1,5-Naphthalenediamine, CAS No. 2243-62-1, feed, 2000 ppm, positive liver carcinogen; 2) 2,3-Benzofuran, CAS No. 271-89-6, gavage, 240 mg/kg, positive liver carcinogen; 3) N-(1-naphthyl)ethylenediamine dihydrochloride, CAS No. 1465-25-4, feed, 2000 ppm, negative liver carcinogen; 4) Pentachloronitrobenzene, CAS No. 82-68-8, feed, 8187 ppm, negative liver carcinogen; 5) Feed control; 6) Corn oil gavage control.  Feed animals were exposed 7 days/week and gavage animals were exposed 5 days/week (5 ml/kg).  Microarray analysis was performed on the livers of three mice per treatment group.


title: Edema Toxin-treated Macrophage Study

status: Public on Oct 24 2006

city: Galveston

first: Ashok

middle: K

last: Chopra

state: TX

address: 301 University Blvd.

department: Microbiology & Immunology

institute: University of Texas Medical Branch

summary:
Edema toxin (EdTx), which is a combination of edema factor and a binding moiety (protective antigen), is produced by Bacillus anthracis, the etiological agent of anthrax. EdTx is an adenylyl cyclase enzyme that converts adenosine triphosphate to adenosine-3’,5’-monophosphate, resulting in interstitial edema seen in anthrax patients. We used GeneChip analysis to examine global transcriptional profiles of EdTx-treated RAW 264.7 murine macrophage-like cells at 3 and 6 hr.
Keywords: Toxin response

overall_design: RAW 264.7 cells were treated with EdTx (2.5 µg/ml of protective antigen and 0.625 µg/ml of Edema factor), PA (2.5 µg/ml), or LPS (1 ng/mL) for 0, 3, and 6 hr. Each experiment was performed in triplicate, generating a total of 21 arrays (biological replciates).

first: Jason

middle: E

last: Comer

first: Cristi

last: Galindo

first: Fan

last: Zhang

first: Autumn

last: Wenglikowski

first: Katie

last: Bush

first: Johnny

middle: W

last: Peterson


title: Spinal cord and dorsal root ganglion gene expression

status: Public on Aug 02 2006

city: Memphis

first: Ramin

last: Homayouni

state: TN

address: 3774 Walker avenue

department: Bioinformatics

institute: University of memphis

summary:
We used microarrays to test the fundamental quantitative differences between central and peripheral nervous system transcriptomes.
Keywords: repeat

overall_design: 3 independent SC and 3 independent DRG  samples were analyzed. Each sample contained material from 4 age-matched male C57B/6 mice.

first: Mark

last: LeDoux

first: Lijing

last: Xu

first: Jianfeng

last: Xiao

first: Brett

last: Ferrell

first: Daniel

last: Menkes


title: Wildtype and Per2Cry1 double mutant mice: temporal gene expression profiling of adrenal gland

status: Public on Aug 08 2006

city: Lübeck

first: Henrik

last: Oster

address: Marie Curie Street

department: CBBM

country: Germany

institute: University of Lübeck

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

overall_design: Refer to individual Series


title: Gene expression induced by flagellin stimulation in CD11c+ LPCs

status: Public on Aug 01 2006

city: Suita

first: Satoshi

last: Uematsu

state: Osaka

address: 3-1, Yamada-oka

department: Host Defense

institute: Research Institue for Micrbial Diseases Osaka University

summary:
Microarray analysis of IECs from Tlr5+/+ and Tlr5-/- mice stimulated with either medium alone or flagellin (1 µg/ml); to elucidate TLR5-mediated immune responses in CD11c+ LPCs
Keywords: ordered

overall_design: CD11c+ LPCs from Tlr5+/+ and Tlr5-/- mice were treated with or without flagellin (1 ug/ml) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), and purified using an Oligotex mRNA Kit (Pharmacia). Fragmented and biotin-labeled cDNA was synthesized from 100 ng purified mRNA using the Ovation Biotin System (Nugen), according to the manufacturer's protocol. cDNA was hybridized to Affymetrix Murine Genome 430 2.0 microarray chips (Affymetrix) according to the manufacturer's instructions. Hybridized chips were stained, washed and scanned using a GeneArray Scanner (Affymetrix). Data analysis was performed with Microarray Suite software (Version 5.0, Affymetrix) and GeneSpring software (Silicon Genetics).


title: TTP mRNA targets identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts

status: Public on Oct 17 2006

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary:
Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice, and cells derived from them, showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target
mRNAs for TTP include tumor necrosis factor alpha (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 2 beta (IL2 beta). Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained conserved TTP binding sites; 10 of these were shown by secondary analyses to be stabilized. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly, and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.
Keywords: mRNA degradation, time course, knockout experiment

overall_design:
Cell lines were derived from mouse embryonic fibroblast (MEF) cultures from littermate E14.5 embryos (for Zfp36 +/+ and -/- cells). MEFs were maintained at 37o C (5% CO2) in DMEM containing 10% FBS, 100
U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. When the cells had reached approximately 70-90 % confluence, they were trypsinized and diluted 2-50 fold in the same medium, based on morphology and growth rate, such that they would reach near confluence in 3-4 days when subcultured.

Five identical experiments involving sequential serum deprivation, serum stimulation and actinomycin D treatment were performed on five different days; each experiment involved simultaneous identical experiments with the WT and KO cell lines. In each experiment, the cells were serum deprived for 16h, and a sample was taken at that point as a serum-deprived control. The cells were then stimulated by 10% FBS for
90 min and another sample taken, after which 5μg/ml actinomycin D was added; samples for RNA analysis were then removed at 30, 60, 90 and 120 min after actinomycin D treatment. In all experiments, each sample represented three combined 100 mm dishes of cells.

first: Wi

last: Lai

first: Joel

last: Parker

first: Sherry

middle: F

last: Grissom

first: Deborah

last: Stumpo

first: Perry

last: Blackshear


title: Gene expression induced by flagellin stimulation in IECs and CD11c+ LPCs


title: Transcriptome regulation in the adrenal gland of circadian clock deficient Per2Cry1 double mutant mice

summary:
This array set was used to determine clock regulated genes in the adrenal gland that are not necessarily rhythmic but still controlled by the circadian TTL.
Keywords: comparative genomic hybridization / time series

overall_design: LD entrained animals were released into DD an whole adrenals dissected at the specified time points after "lights off"

first: Sebastian

last: Damerow

first: Silke

last: Kiessling

first: Vladimira

last: Jakubcakova

first: Tian

last: Jiong

first: Matthias

last: Hoffmann

first: Roeloff

last: Hut

first: Gregor

last: Eichele


title: Microarray Analysis of the Delipidation of White Adipose Tissue of Mice Fed Conjugated Linoleic Acid

status: Public on Jul 28 2006

city: Lincoln

first: Chris

last: LaRosa

state: NE

address: 19th & Vine

department: Center of Biotechnology

institute: University of Nebraska, Lincoln

summary:
The white adipose tissue (WAT) rapidly loses mass when mice are fed a diet containing trans-10, cis-12 conjugated linoleic acid (t10c12 CLA).  A microarray analysis of WAT due to CLA feeding was performed to better define the processes and genes involved.  WAT weight decreased by ca. 80% over 17 days of feeding a 0.5% t10c12 CLA diet.  The lipid volume decreased by 90% and the number of adipocytes and total cells were reduced by15% and 47%, respectively.  Microarray profiling of replicated pools of control and treated mice (n=140) at seven time points over the 17day feeding indicated between 2798 to 4318 genes showed mRNA changes of 2-fold or more.  Transcript levels for genes of glucose and fatty acid import or biosynthesis were significantly reduced. A prolific inflammation response was indicated by the 2 to100-fold induction of many cytokine transcripts, including those for IL-6, IL1?, TNF ligands, and CXC family members
We used microarrays to characterize transcriptome changes in mouse adipose tissue due to feeding trans-10 cis-12 linoleic acid .
Keywords: time course

overall_design: Mice were fed either diet with 0.5 % CLA or control diet, and the retroperitoneal fat pad mRNA surveyed by high density oligonucleotide array expression profiling. Microarray profiling of replicated pools of control and treated mice (n=140) at seven time points (day 1, 2, 3, 4, 10, 17) over the 17day feeding indicated between 2798 to 4318 genes showed mRNA changes of 2-fold or more.

first: Christopher

first: Jess

last: Miner

first: Yuannan

last: Xia

first: Michael

last: Fromm

first: Steven

last: Kachman


title: Gene expression profiling of whole ovary in embryonic (gestional day 11, 12, 14, 16 & 18) and postnatal day 2 mice. (GUDMAP Series ID: 5)

status: Public on Jul 22 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

country: United Kingdom

institute: IGMM MRC Human Genetics Unit

summary:
The overall objective of this proposal is to map the temporal and spatial dynamics of gene expression in the fetal mouse testis at key developmental timepoints. Urogenital tract malformations are the most common birth defects in males and their incidence together with other male reproductive health concerns such as reduced fertility and testicular cancer are reportedly on the rise in the human population. To better understand the impact of genetic factors and environmental influences on testicular development, it is important to first understand normal gene expression patterns and signaling cascades within the fetal testis during development. The goal of this study is to identify cell-specific genes that can be used as biomarkers for key differentiation events.
Keywords: developmental time course

overall_design: Day to day comparison in whole ovary throughout development with triplicates at each time point.  Each sample is a pair of ovaries from a single animal and each animal is taken from a different dam (with the exception of gd11 sample numbers 18 and 19 which come from the same dam).

first: Kevin

last: Gaido

first: Kim

last: Lehmann


title: Gata4 is required for postnatal cardiac function and protection from pressure overload-induced heart failure

city: Boston

first: Sek Won

last: Kong

address: 300 Longwood Avenue

department: Informatics Program

institute: Boston Children's Hospital

summary:
An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiac cardiomyocytes, the transcription factor Gata4 is required for agonist-induced cardiomyocyte hypertrophy. We hypothesized that in the intact organism Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed upregulation of genes associated with apoptosis and
fibrosis, including members of the TGF? pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.
Keywords: genetic modification, pressure overload stress response

overall_design:
We reasoned that if Gata4 was a crucial regulator of pathways necessary for cardiac hypertrophy, then modest reductions of Gata4 activity should result in an observable cardiac phenotype. To test this hypothesis, we used gene targeted mice that express reduced levels of Gata4. We characterized these mice at baseline and after pressure
overload.

first: Egbert

last: Bisping

first: Sadakatsu

last: Ikeda

first: Sek-Won

first: Oleg

last: Tarnavski

first: Natalya

last: Bodyak

first: Julie

last: McMullen

first: Satish

last: Rajagopal

first: Jennifer

last: Son

first: Qing

last: Ma

first: Zhangli

last: Springer

first: Peter

last: Kang

first: Seigo

last: Izumo

first: William

middle: T

last: Pu


title: Gene expression profiling of whole epididymis in embryonic (gestational day 12, 14, 16 & 18) and postnatal day 2 mice. (GUDMAP Series ID: 4)

first: GUDMAP

overall_design: Day to day comparison in whole epididymus throughout development with triplicates at each time point.  Each sample is a pair of epididymus from a single animal and each animal is taken from a different dam.


title: Maternal BRG1 regulates zygotic genome activation in the mouse

status: Public on Jul 21 2006

city: Philadelphia

first: Hua

last: Pan

state: PA

address: 415 S University Avenue

department: Department of Biology

institute: University of Pennsylvania

summary:
Embryos were collected at 2 cell stage.  cRNA from four biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430 v2.
Keywords: repeat

overall_design: 4 embryo at 2 cell stage biological replicates were analyzed

first: Scott

last: Bultman

first: Thomas

middle: C

last: Gebuhr

first: Petr

last: Svoboda

first: Richard

last: Schultz

first: Terry

last: Magnuson


title: Differential transcript abundance in BMAL-/- mouse at ZT14

status: Public on Dec 16 2006

first: Radu

middle: Daniel

last: Rudic

address: 421 Curie Boulevard

department: Center of Experimental Therapeutics

summary:
Investigation of RNA transcript abundance in aortic tissue of BMAL-/- versus BMAL+/+ control mice at ZT14
Keywords: Differential RNA abundance in genetic mutant

overall_design: One-way ANOVA design with Strain (BMAL-/- versus BMAL+/+) as the independent variable. Control group has five biological replicates, BMAL-/- group has four biological replicates.

first: Anne

last: Curtis

first: Yan

last: Cheng

last: Price

first: Dermot

last: Reilly

first: Shiv

last: Kapoor

first: Garret

middle: A

last: FitzGerald


title: Expression analysis of Day 0 versus Day 1 involuting mouse mammary gland

status: Public on Jul 18 2006

city: Winnipeg

first: Yvonne

last: Myal

state: Manitoba

address: 770 Bannatyne Ave.

department: Pathology

country: Canada

institute: University of Manitoba

summary:
Mammary gland involution represents one of the most dramatic examples of programmed cell death/apoptosis and tissue regression during development, yet large gaps still exist in the understanding of the mechanisms involved, and the key factors that trigger involution, are not yet identified. With the focus on identifying “novel” genes associated with mammary gland regression, we used microarray analysis to examine differentially expressed genes during early mammary gland involution in the mouse.  We believe that such analysis may facilitate the identification of genes that report on proliferative changes in the breast, particularly with regards to growth arrest and growth inhibition. We further reasoned that genes that inhibit mammary gland proliferation might have relevance to breast tumorigenesis since cancer is generally associated with a defect in apoptosis and cell growth arrest. Gene expression from day 0 (D0) of involution was compared to day 1 (D1, 24 hr post weaning) to identify genes exhibiting differential expression. A total of 5,826 genes were identified as being differentially expressed. Changes in gene expression were confirmed by quantitative real time PCR analysis of 21 randomly selected genes. Following the selection of a number of highly regulated mouse genes within this cohort, database searches were then employed to identify genes associated with human tumorigenesis. With an ultimate goal being the identification of genes, which under normal physiological conditions, inhibit breast proliferation, and therefore have the potential to impact on the clinical management of breast cancer, we further examined their relevance to human tumorigenesis. Among genes identified as having human homologues, and with previously established links to human tumorigenesis, were several known genes among the top 200 differentially expressed genes including EGF, EGFR, TGFß2, PTHLH, IGFBP1, that were down regulated.  Upregulated known genes included ANNEXIN A1 and A3, THYMOSIN BETA 10, IGFBP5, LIF, BID, BCL2, JUN, KIT, and CLAUDIN 1. CLAUDIN 1 was the most highly upregulated gene in our dataset. Moreover, we identified a number of genes not previously implicated or not well characterized in human breast cancer including ANGPTL4, SLC34A2, GOS2, STEAP, SOX4, TNFRSF12A and SAA2.  The expression levels of these genes in human breast cancer were confirmed in breast cancer cell lines and breast tumor tissues. For some of these genes, expression was consistently down regulated in a small panel of human breast tumor specimens. We believe that these genes now warrant further investigation to determine their relevance to human breast tumorigenesis and their potential utility as therapeutic targets for human breast cancer. This pilot study was used to demonstrate proof of principle that through the analysis of gene expression during mammary gland involution, it may be indeed possible to identify “novel” genes relevant to the growth arrest in the mammary gland and relevant to human breast cancer.

Keywords: comparative expression analysis of involuting mouse mammary gland

overall_design: Pooled RNA from D0 mice and from D1 mice, (five mice per time point), was compared for differential gene expression by microarray analysis conducted by Paradigm Array Labs (Research Triangle Park, North Carolina, USA). The Affymetrix GeneChip Mouse Genome 430 Plus 2.0 Array (Affymetrix, Santa Clara CA. USA) was used.  The array contained 45,100 probe sets (The Affymetrix probe-set identificator) and represented 39,000 transcripts. The Affymetrix Statistical Algorithms, implemented in the Affymetrix Microarray Suite 5.0 software, were used in the expression analysis of GeneChip probe arrays.

last: Blanchard

first: Robert

last: Shiu

first: Stephanie

last: Booth

first: Garrett

last: Sorensen

first: Nicole

last: DeCorby

first: Andreea

last: Nistor

first: Paul

last: Wong

first: Etienne

last: Leygue


title: Gene expression induced by flagellin stimulation in IECs

first: Satoshi

last: Uematsu

summary:
Microarray analysis of IECs from Tlr5+/+ and Tlr5–/– mice stimulated with either medium alone or flagellin (1 µg/ml); to elucidate TLR5-mediated immune responses in IECs.
Keywords: ordered

overall_design: IECs from Tlr5+/+ and Tlr5–/– mice were treated with or without flagellin (1 ug/ml) for 4 h. Total RNA was extracted with an RNeasy kit (Qiagen), and purified using an Oligotex mRNA Kit (Pharmacia). Fragmented and biotin-labeled cDNA was synthesized from 100 ng purified mRNA using the Ovation Biotin System (Nugen), according to the manufacturer’s protocol. cDNA was hybridized to Affymetrix Murine Genome 430 2.0 microarray chips (Affymetrix) according to the manufacturer’s instructions. Hybridized chips were stained, washed and scanned using a GeneArray Scanner (Affymetrix). Data analysis was performed with Microarray Suite software (Version 5.0, Affymetrix) and GeneSpring software (Silicon Genetics).


title: In vivo function of NR2E3 in establishing photoreceptor identity during mammalian retinal development

status: Public on Aug 18 2006

city: Ann Arbor

first: Swaroop

last: Anand

state: MI

address: 1000 Wall St.

department: Ophthalmology & Visual Sciences

institute: University of Michigan

summary:
Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors.  NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation.  Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans.  To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease.  Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional.  Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo.  Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells.  The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression.  Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis.
Keywords: genetic modification

overall_design:
We mated the Crx::Nr2e3/Nrlko mice with the Nrl::GFP transgenic mice, in which the expression of GFP is driven by an Nrl promoter. Mouse retinas were dissected at 4 wk.  GFP+ photoreceptors were enriched by FACS (FACSAria, BD Biosciences, Franklin Lakes, NJ).  RNA was extracted from 1~5x105 flow-sorted cells using Trizol (Invitrogen).  Total RNA (40-60 ng) was used for linear amplification with Ovation Biotin labeling system (Nugen), and 2.75 ug of biotin-labeled fragmented cDNA was hybridized to mouse GeneChips MOE430.2.0 (Affymetrix) having 45,101 probesets (corresponding to over 39,000 transcripts, and 34,000 annotated mouse genes).
Four independent samples were used at 4 weeks. We normalized
Crx::Nr2e3/Nrl-ko-Gfp data along with Nrl-ko-Gfp 4 weeks samples ( 4 replicates, refer to Series submission GSE4051). The normalized data was then subjected to two stage analysis based on False Discovery Rate Confidence Interval (FDR-CI) for screening differentially expressed genes (24, 27) with a minimum fold change of 4.

first: Hong

first: Ritu

last: Khanna

first: Anand

last: Swaroop


title: Development of heart valves requires Gata4 expression in endothelial-derived cells

status: Public on Jul 14 2006

first: Sek Won

last: Kong

summary:
Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in formation of portions of the atrial and ventricular septae, and generation of thin, pliable valves. The transcription factor Gata4 is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.
Keywords: genetic modification

overall_design:
To further investigate the role of Gata4 in EC development, we specifically inactivated Gata4 in endothelium and endothelium-derived cushion mesenchyme. All mice were maintained in a mixed C57BL6/129 genetic background. Gata4wt/flox; T2Cre+ mice were crossed with Gata4flox/flox mice to yield Gata4flox/flox (CONTROL); T2Cre+ (Gata4T2del) (MUTANT) mice.
For RNA analysis, four mutant and four control RNA samples were prepared (Pico-Pure RNA Isolation Kit, Arcturus), each consisting of 10 microdissected AVCs. Probe was prepared from 50 ng total RNA using an isothermal amplification protocol (NuGen), and hybridized to Affymetrix Mouse 430 2.0 microarrays. Two control samples were excluded because of excessive noise.

first: Jos�

last: Rivera-Feliciano

first: Kyu-Ho

last: Lee

first: Sek-Won

first: Satish

last: Rajagopal

first: Qing

last: Ma

first: Zhangli

last: Springer

first: Seigo

last: Izumo

first: Clifford

last: Tabin

first: William

last: Pu


title: Loss of BMPR2 Signaling in Smooth Muscle in Mouse Lung

status: Public on Jul 08 2006

city: Nashville

first: James

last: West

address: 4200 E 9TH Ave, Box B133

department: Medicine

institute: Vanderbilt University

summary:
Mice expressing a doxycycline-inducible dominant negative BMPR2 transgene expressed only in smooth muscle are activated for one or eight weeks, and compared to transactivator-only mice also fed doxycycline. All mice are 12 weeks of old at sacrifice.
Keywords: time course with SM22-rtTA x tetO7-BMPR2 transgene

overall_design: Each chip is a pool of three individual mice, to reduce variability. The transactivator-only mice are controls. The other mice all had elevated RV pressures at time of sacrifice. RNA was prepared from whole lung.

first: Yuji

last: Tada

first: Susan

last: Majka

first: Michelle

last: Carr

first: Julie

last: Harral

first: Daniel

last: Crona

first: Takayuki

last: Kuriyama


title: Transcriptional Profiling of Mammary Gland Side Population Cells

status: Public on Jul 15 2006

city: Houston

first: Fariba

last: Behbod

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary:
Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst-effluxing side population cells, MG-SPs.  To better characterize MG-SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (MG-NSPs). For analysis, gene ontology (GO) that describes genes in terms of biological processes and ontology traverser (OT) that performs enrichment analysis were utilized.  OT showed that MG-SP specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multi-drug resistant transporters, organogenesis, and vasculogenesis.  The MG-NSP upregulated genes were enriched in the GO category of cellular organization and biogenesis which includes basal epithelial markers, p63, smooth muscle actin (SMA), myosin, alpha-6 integrin, cytokeratin (CK) 14, as well as luminal markers, CK8 and CD24.  Additional studies showed that a higher percentage of MG-SPs exist in the G1 phase of the cell cycle compared to the MG-NSPs.  G1 cell cycle block of MG-SPs may be explained by higher expression of cell cycle negative regulatory genes such as TGF-beta2 (transforming growth factor-beta2), IGFBP-5 (insulin like growth factor binding protein-5), P18 INK4C and Wnt-5a (wingless-5a). Accordingly, a smaller percentage of MG-SPs expressed nuclear b-catenin, possibly as a consequence of the higher expression of Wnt-5a.  In conclusion, microarray gene profiling suggests that MG-SPs are a lineage deficient mammary gland sub-population expressing key genes involved in cell cycle regulation, development and angiogenesis.

Supplemental File Descriptions:

Table 1 is a list of 1632 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 1.2-fold difference in expression levels, false discovery rate (FDR) 9.4%, P value less than 0.05.

Table 2 is a list of 771 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 1.5-fold difference in expression levels, FDR 3.4%, P value less than 0.05.

Table 3 is a list of 335 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 2-fold difference in expression levels, FDR 0%, P value less than 0.05.

Table 4 is a list of 90 Genes differentially expressed by MG-SP and MG-NSP; Criteria for comparison included 2-fold difference in expression levels, FDR 0%, P value less than 0.01.

Keywords: Normal C57BL/6 Mammary Gland Epithelial Cells

overall_design: Gene expression profiles were obtained by hybridizing amplified RNA from four replicate MG-SP and MG-NSP samples to Affymetrix 430 2.0 microarray chips. To isolate MG-SP and MG-NSPs, mammary gland cells were stained using the Hoechst dye 33342 and fluorescence displayed at two wavelength emissions, blue and red.  The MG-SP and MG-NSP regions were indicated by trapezoids on the left (R1) and right (R2), respectively.  We and others have previously shown that the R1 region is composed of side population cells since verapamil blocks their appearance.  The cells in each region (R1 and R2) were sorted, their RNA isolated and amplified by two rounds of in vitro amplification and applied to Affymetrix chips.  Hybridization, scanning, and production of raw data files were performed according to the Affymetrix standard protocols. Normalization and model-based expression measurements were performed with dChip. Two of the chips were eliminated from further analysis due to the high percentage of probe sets called an array outlier after model based expression analysis by dChip (Not included).  The genes were filtered to eliminate those with very low expression values in most samples.  From 45,000 probe sets, 16,744 probe sets were retained and used for further analysis.  For normalization purposes and to allow for data comparison with other centers, universal mouse reference RNA (Stratagene) was hybridized to two chips. All other arrays were normalized to one of the universal mouse reference RNA arrays expressing average intensity closest to the median intensity of all chips (Stratagene Ref 2).

first: Wa

last: Xian

first: Chad

last: Shaw

middle: G

last: Hilsenbeck

first: Tsimelzon

last: Anna

first: Jeffrey

last: Rosen


title: Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

status: Public on Jul 06 2006

first: Marc

last: Uldry

address: 1 Jimmy Fund Way

institute: Dana Farber Cancer Institute

summary:
Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1α. We could then efficiently knockdown PGC-1β expression by shRNA expression. Loss of PGC-1α did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1α or PGC-1β coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1α and PGC-1β were deficient. These data show that PGC-1α is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis. Affymetrix microarray analysis of total RNA from wt, PGC-1α KO and PGC-1α KO
cells expressing an RNAi specific for PGC-1β knockdown was performed. Of the 461
mitochondrial genes analyzed, 181 were found to be at least 20% different between wt
and defective PGC-1α and β adipocytes (p < 0.05). More than 85% of these genes were downregulated in cells deficient for PGC-1alpha and PGC-1beta.
Keywords: Analysis of mitochondrial gene expression

overall_design: Brown preadipocytes that were either WT, KO for PGC-1alpha, or KO for PGC-1alpha and deficient for PGC-1beta (knockdown through siRNA expression) were differentiated for seven days. RNA was made from biological replicates of the three different types of brown adipocytes (WT, KO expressing a control siRNA, KO expressing a siRNA specific for PGC-1beta knockdown).

first: Wenli

last: Yang

last: St-Pierre

first: Jiandie

last: Lin

first: Patrick

last: Seale

first: Bruce

last: Spiegelman


title: Expression of the brown fat thermogenic gene program requires PGC-1alpha

summary:
To investigate the specific role of PGC-1 coactivators in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severly reduced the induction of thermogenic genes. In order to assess the specific requirement for PGC-1α in the global transcriptional response to cAMP, we used Affymetrix arrays to compare the sets of genes induced in response to a 4 hr dbcAMP treatment in differentiated wt and KO cells. This analysis revealed that 88 genes were induced more than 3-fold in the wt cells; of these, 54 (61% of total) were similarly increased in both wt and KO. However, 28 genes (32% of total) were decreased by at least 50% in the KO cells compared to wt cells. These data were confirmed by quantitative PCR for a subset of genes. These data indicate that PGC-1α is required for proper expression of approximately one third of the genes induced in response to cAMP in brown fat cells, but this set of sensitive genes is enriched in those involved in adaptative thermogenesis.

Keywords: thermogenic gene program

overall_design: WT and PGC-1alpha KO brown preadipocytes were differentiated into mature brown adipocytes for seven days. Cells were then treated with dibutyryl cAMP for four hours. Two replicates were made for each condition: WT non treated, WT treated with cAMP, KO non treated, KO treated with cAMP.


title: Expression data from Lbeta T2 cells upon activin treatment

status: Public on Jul 05 2006

city: Evanston

first: Ying

last: Peng

state: IL

address: 2205 Tech Drive

institute: Northwestern University

summary:
The primary response of Lbeta T 2cells upon activin treatment at 1hr, 2hr, and 6hr is the significant up-regulation of FSHbeta subunit. Affymetrix genearray missed this important information due to cross-hybridization of its probes with other genes and chromosome segments.
Activin modulates a network of genes and signaling pathways to exert a variety of functions.
Keywords: time course

overall_design: Subconfluent Lbeta T2 cells were seeded in cell culture flasks for 16 hours in phenol-free, serum-free medium, and then treated with either vehicle or activin A (30ng/ml). At 1hr, 2hr, or 6hr after treatment, total RNA was isolated using RNeasy Mini Kit. Integrity of RNA was evaluated with an Agilent 2100 Bioanalyzer, and purity/concentration was determined using a GeneSpec III before target preparationi and hybridizations.

first: Xinmin

last: Li

first: Teresa

last: Woodruff


title: Telomere dysfunction

status: Public on Jul 01 2006

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary:
We used microarrays to analyze the prevalence of signalling induced by acute telomere dysfunction (as induced by TRF2DBDM expression = Dataset 1) and in TERC- compared to TERC+ HCC (Dataset 2).
Keywords: acute telomere dysfunction, hepatocellular cancer

overall_design:
Presented are gene expression data from:
Dataset 1:  Mouse liver 48h after i.v. infection of p53-/- mice with Ad TRF2DBDM compared to Ad GFP (2x10^10 PFU) (GSM103546, GSM103547) and
Dataset 2:  p53-/- heptaocellular carcinoma (HCC) from mTERC+ HBs (GSM103548, GSM103549, GSM103553) compared to mTERC-, HBs  (GSM103550, GSM103551, GSM103552) transgenic mice.
All mice were in a C57BL/6J background.
Dataset 1: Liver RNA was extracted 48h after adenoviral infection (n=3 mice/group), samples were pooled for hybridization on Affymetrix microarrays.
Dataset 2: RNA was extracted from HCC of 12-15month old mTERC+ and mTERC- mice (n=3 mice/group). Individual tumors were hybridized on Affymetrix microarrays.
Differentially expressed gene clusters were identified using Array Assist 4.0 (Stratagene).

first: A

last: Lechel

first: K

last: Rudolph

first: R


title: Transcriptional profiling of mouse ileum in response to colonization with a zebrafish or mouse gut microbiota

status: Public on Jul 07 2006

city: Chapel Hill

first: John

last: Rawls

address: 111 Mason Farm Rd.

department: Department of Cell and Molecular Physiology

institute: University of North Carolina at Chapel Hill

summary:
We compared gene expression in the small intestine (ileum) of mice that were either (i) germ-free, (ii) colonized with a conventional mouse cecal microbiota, (iii) colonized with a conventional zebrafish gut microbiota, or (iv) colonized with Pseudomonas aeruginosa PAO1.
Keywords: response to microbial colonization

overall_design: Adult germ-free NMRI mice were colonized with either (i) a conventional mouse cecal microbiota harvested from adult Swiss-Webster mice (5 biological replicates), (ii) a conventional zebrafish intestinal microbiota harvested from adult C32 zebrafish (3 biological replicates), or (iii) a culture of Pseudomonas aeruginosa PAO1 (5 biological replicates). 14 days after colonization, total RNA was prepared from the ileum of each animal, with total RNA prepared from adult germ-free NMRI mouse ileum serving as negative controls (5 biological replicates). RNA was used as template to generate cRNA for hybridization to Affymetrix 430 v2 Mouse GeneChips.

first: Michael

last: Mahowald

first: Ruth

last: Ley

middle: I

last: Gordon


title: Impact of intestinal colonization on Paneth cell gene expression

status: Public on Jun 28 2006

city: Dallas

first: Lora

middle: V

last: Hooper

address: 5323 Harry Hines Blvd.

department: Immunology

institute: The University of Texas Southwestern Medical Center at Dallas

summary:
This study delineated how small intestinal resident microflora impact gene expression in Paneth cells.
Keywords: functional genomics; transcriptional profiling

overall_design: Paneth cells were isolated by laser capture microdissection from the small intestines of germ-free and conventionalized (10 day) mice. RNAs from 3 mice per group were pooled, and duplicate RNAs from each group were amplified and hybridized to Affymetrix arrays.

first: Heather

middle: L

last: Cash

first: Cecilia

last: Whitham

first: Cassie

last: Behrendt


title: Immunodomination leads to selective expansion of the fittest CD8 T cells

status: Public on Jul 12 2006

city: Montreal

first: Etienne

last: Caron

state: Quebec

address: 5415 Boul. L'Assomption

department: Recherche

institute: Hopital Maisonneuve Rosemont

summary:
During the course of infection, T cells are confronted with a multitude of non self epitopes but only respond to a few epitopes and neglect other potentially immunogenic peptides. Restriction of T cell responses to a small number of selected epitopes (immunodominance) is a central feature of immune responses. Immunodominance is considered to be disadvantageous to the host because it allows pathogens to escape by selectively mutating the relevant epitope(s). Using Affymetrix microarrays, we compared the gene expression profile of unprimed CD8 T cells to that of effector CD8 T cells specific for a dominant (H7a) and a nondominant (HY) Ag. Our key finding is that T cells specific for the dominant and nondominant Ags displayed similar gene expression profiles except for a few gene transcripts, such as Gzma, Sell, Il7r and Klrg1, that contribute to the fitness of effector CD8 T cells. The differences between HY- and H7a-specific CD8 T cells were validated by real-time PCR and flow cytometry analyses. We propose that, by leading to selective expansion of the fittest CD8 effector T cells, immunodominance may be beneficial to the host. Inhibition of T cell response to nondominant Ags would ensure that host resources (APCs, cytokines) for which T cells compete are devoted to T cells that have the best effector potential. One implication is that, in general, favouring expansion of the fittest effector T cells may be more important that increasing the diversity of the T cell repertoire.
Keywords: comparative gene profile, cell type comparaison, H7a, HY

overall_design: B10.H7b female mice were primed by i.p. injection of a cell mixture containing 2 x 107 B10 male splenocytes and 2 x 107 B10.H7b male splenocytes. On day 14 after priming, splenocytes were labeled with anti-CD8 Ab as well as H7a- and HY-Tet labeled with different fluorochromes. Then, three populations of splenocytes were purified using a FACS cell sorting: HYTet+ (n=4), H7aTet+ (n=4), and Tet- (n=5) CD8 T cells. RNA of sorted T cells was extracted and linearly amplified, cRNA was prepared, and Affymetrix Mouse Genome 430 2.0 oligonucleotide arrays were used to analyze gene expression.

first: Chantal

last: Baron

first: Étienne

first: Marie-Christine

last: Meunier

first: Caroline

last: Côté

first: Mark

middle: J

last: Cameron

first: David

last: Kelvin

first: Vincent

last: Rineau

first: Claude

last: Perreault


title: zhang-affy-mouse-308606

status: Public on Jun 09 2006

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have used the facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice. We would like to compare these changes with that opccured in SOD1 mice, a mouse model of motor neuron diseases. Analysis of these genes will help to identify changes in networks and pathways that may cause the death of NR3B-null mice. These studies will further help to elucidate the functional role of NR3B in motor neurons.
We will compare samples from motor neurons of wild type and SOD1 mice to identify genes that show abnormal expression patterns, which may be implicated in the death of SOD1 mice and shared with the same changes in NR3B-null mice.
We hypothesize that genes with their transcription level changing significantly in SOD1 mutant mice will be associated with the molecular mechanism underlying the death of motor neurons.
We like to compare motor neuron and spinal cord smaples from SOD1 mice at the age prior to the disease onset. Total RNA from total 9 samples will be purified, each from ~200 motor neurons obtained by Laser Capture Microdissection and the total spinal cord. Extracted RNAs will be subjected to one or two rounds of amplification and the obtained cRNA will be biotinylated. The purified cRNA will be sent to the NINDS/NIMH Microarray Consortium be used to hybridize the GeneChip Mouse Genome 430 2.0 Array. The hybridization, scanning, and initial data analysis of these GeneChips will be conducted by the Consortium staff. We will analyze the collected data further after data collection. We will first identify genes that show significant changes between wild-type and SOD1 mice and then compare that with the result from NR3B null mice.
Keywords: other

first: Dongxian

last: Zhang


title: rhythmic transcriptome in the murine wt adrenal

status: Public on Aug 08 2006

city: Lübeck

first: Henrik

last: Oster

address: Marie Curie Street

institute: University of Lübeck

summary:
This study was performed to characterize the rhythmic transcirptome of the murine adrenal gland with a focus on the corticosterone synthesis pathway.
Keywords: time course

overall_design: LD entrained male animals were released into DD and from the second day on whole adrenals were prepared from 12 time points at 4 hrs intervals.

first: Sebastian

last: Damerow

first: Silke

last: Kiessling

first: Vladimira

last: Jakubcakova

first: Tian

last: Jiong

first: Matthias

last: Hoffmann

first: Roeloff

last: Hut

first: Gregor

last: Eichele


title: zhang-affy-mouse-217286

summary:
In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have used the facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice. We would like to compare these changes with that opccured in SOD1 mice, a mouse model of motor neuron diseases. Analysis of these genes will help to identify changes in networks and pathways that may cause the death of NR3B-null mice. These studies will further help to elucidate the functional role of NR3B in motor neurons.
We will compare samples from motor neurons of wild type and SOD1 mice to identify genes that show abnormal expression patterns, which may be implicated in the death of SOD1 mice and shared with the same changes in NR3B-null mice.
We hypothesize that genes with their transcription level changing significantly by ablation of NR3B will be associated with the molecular mechanism underlying the death of motor neurons in NR3B null mice.
As NR3B is expressed primarily in the motor neurons of hindbrain and spinal cord, we have first collected and analyzed the spinal cord samples from NR3B null mice and wild-type controls in P4, an age of disease onset. We like to compare motor neuron and spinal cord smaples from SOD1 mice at the age prior to the disease onset. Total RNA from total 12 samples will be purified from ~200 motor neurons obtained by Laser Capture Microdissection and the total spinal cord. Extracted RNAs will be subjected to one or two rounds of amplification and the obtained cRNA will be biotinylated. The purified cRNA will be sent to the NINDS/NIMH Microarray Consortium be used to hybridize the GeneChip Mouse Genome 430 2.0 Array. The hybridization, scanning, and initial data analysis of these GeneChips will be conducted by the Consortium staff. We will analyze the collected data further after data collection. We will first identify genes that show significant changes between wild-type and SOD1 mice and then compare that with the result from NR3B null mice.
Keywords: dose response


title: Identification of potential KLF7 target genes in olfactory sensory neurons

status: Public on Jun 10 2006

city: New York

first: Friedrich

middle: Bernhard

last: Laub

state: NY

address: 535 East 70th Street

department: Research

institute: Hospital for Special Surgery

summary:
KLF7 null mice show profound axonal growth defects in the olfactory epithelium. The goal of this study was the identification of potential KLF7 target genes in olfactory sensory neurons.
Keywords: Molecular analysis of knockout mice.

overall_design: Olfactory epithelia were isolated from 3 wildtype and 3 mutant 1 day old pups and the RNA isolated, labeled and hybridized to one chip each.

first: Daisuke

last: Kajimura


title: zhang-affy-arabi-50806

summary:
In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function.  We have now successfully generated NR3B null mice.  Interestingly, NR3B-null mice invariably die at age P4-P8.  Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene.  We would like to use the facility provided by the NINDS/NIMH Microarray Consortium to identify true outlier genes that show abnormal expression patterns in these mice.  Analysis of these outlier genes will help to identify changes in networks and pathways that may cause the death of NR3B-null mice.  These studies will further help to elucidate the functional role of NR3B in motor neurons.
We will compare samples from the hindbrain and spinal cord of wild type and NR3B null mice to identify true outlier genes that show abnormal expression patterns, which may be implicated in the death of NR3B-null mice
We hypothesize that genes with their transcription level changing significantly by ablation of NR3B will be associated with the molecular mechanism underlying the death of motor neurons in NR3B null mice.
As NR3B is expressed primarily in the hindbrain and spinal cord, we will first collect and analyze the hindbrain and spinal cord samples from NR3B null mice and wild-type controls in triplicate at two time points (P0 and P5).  Total RNA from total 12 samples will be purified.  Extracted RNAs will be treated for with DNase I to remove contaminating genomic DNA.  The purified RNA will be sent to the NINDS/NIMH Microarray Consortium to generate biotin-labeled probe according to the Affymetrix protocol.  These probes will be used to hybridize the GeneChip Mouse Genome 430 2.0 Array.  The hybridization, scanning, and initial data analysis of these GeneChips will be conducted by the Consortium staff.  We will analyze the collected data further after data collection. We will use computer software, GeneSpring (Silicon Genetics, Redwood City, CA), to analyze data from microarray experiments. We will first identify genes that show significant changes between wild-type and NR3B null mice by applying ANOVA analysis to sample groups of different ages.  The genes identified in this manner will then be subjected to hierarchical (Gene tree) and nonhierarchical (K-means) cluster analysis, in order to group them according to similar expression patterns.   Another clustering method, the self-organizing map, will be used to determine whether one gene cluster is a variant of another.  Finally, the Principal Components Analysis (PCA) will be employed to analyze large sample groups, which may include experimental variability with respect to age, anatomical location and genotype.   We will determine from these analysis whether further experiments are needed using different array platforms, such as Agillent Oligo Array.
Keywords: time-course


title: zhang-affy-mouse-175010

summary:
In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have previously used the UCLA array facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice using whole brain samples.  Now, we would like to use the same approach to analyze pure motor neuron samples obtained by Laser Capture Microdisection (LCM).  As these samples are of small size we need to use RNA amplification method.  We would like to compare probes from two amplification methods to see which one gives the best result.
We will collect the cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. and the aRNA probe obtained from T7-based RNA amplification.  Both the hybridization intensity and the percentage of the present call of these two types of probe will be analyzed.
The cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. may have some advantage over the aRNA probe obtained from T7-based RNA amplification.
Two samples will be processed by either RNA-based single primer isothermal amplification developed by Nugen or T7-based RNA amplification to obtain cDNA or aRNA probes, respectively.  For each sample we will use 600 motor neurons obtained by LCM.   Both types of probe will be labeled by biotin and hybridize to Affymetrix GeneChip Mouse Genome 430 2.0 Array.  Data analysis will be performed by array facility.
Keywords: dose response


title: oksen-affy-mouse-212956

status: Public on Jun 06 2006

summary:
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune inflammatory disease of the CNS that is mediated by T cells and macrophages and represents the paradigmatic model for multiple sclerosis (MS). In EAE, therapeutic approaches targeting T cells have been successfully utilized leading to immunosuppression or tolerance. It has been recently shown that mesenchimal stem cells (MSC) isolated from C57BL/6J mice are an effective treatment of EAE induced by the encephalitogenic peptide pMOG 35-55. In this project we aim to track and evaluate the molecular changes the MSC acquire through several in-vitro passages that determine their therapeutic potential. Elucidation of the molecular pathways involved in this process will allow us to formulate better models of disease progression and curative strategies.
Specific aim:The specific aim of this proposal is to dissect the molecular pathways that mesenchimal stem cells undergo during in-vitro treatment and confer them the therapeutic potential to cure EAE. To accomplish this aim, we will purify and grow murine MSC under the same conditions used for therapy as described in Zappia et al (In press. Brain, 2005) and obtain samples at several stages of the culture. RNA will be purified from each sample and profiled using Affymetrix microarrays. Initial bioinformatics analysis will include data transformation, normalization and filtering. A second stage analysis will involve time-series analysis by ANOVA and dynamic events modeling using neural networks.
Stem cells are now viewed as potential source of cells for almost any tissue due to their postulated capacity to give rise to virtually any type of cell. Among them, mesenchymal stem cells (MSC), can extensively proliferate in vitro and differentiate under appropriate conditions in bone, cartilage, and other mesenchymal tissues. It has been also reported that MSC can differentiate into other cells types including neuroectodermal cells. Furthermore, upon tissue injury MSC have been shown to migrate into the damaged brain. These results suggest that MSC could provide an ideal cell source for repair of injured organs including the central nervous system (CNS). In addition to their plasticity, MSC have been recently demonstrated to suppress several T lymphocyte activities thus exerting an immunoregulatory capacity both in vitro and in vivo. The mechanisms mediating such effect are only partially understood. We hypothesize that an analysis of how MSC acquire their therapeutic potential in vitro by expression profiling in a time series fashion will allow us to dissect the involved mechanisms.
Before arriving to our lab in UCSF, samples will be processed as follows: Bone marrow from 6-8 weeks old C57BL/6J mice will be flushed out of tibias and femurs. After two washings by centrifugation at 1500 rpm for 5 min in PBS (Sigma Chemical Co., St. Louis, MO, USA), cells will be plated in 75 cm2 tissue culture flasks at the concentration of 0.3-0.4 x 106 cells/cm2 utilizing Murine Mesencult as medium (Stem Cell Technologies Inc., Vancouver, BC, Canada). Cells will be kept in a humidified 5% CO2 incubator at 37°C for about 4-5 weeks, when only adherent cells will be collected following 10 min incubation at 37°C with 0.05% trypsin solution containing 0.02% EDTA. After the first cut and for the subsequent 4 or 5 passages, cells will be plated in 25 cm2 flasks at 1.2-2.0 x 104cells/cm2 . For the following passages cells will routinely seeded at 4-10 x 103cells/cm2 reaching confluence only after 4-5 days. Cells will be collected at selected timepoints and immersed in TRizol reagent and shipped to UCSF.  For the initial stage of this project cell will be collected at the following 5 time points:
T0: Bone marrow aspirates (uncultured)
T1: pre-mesenchymal stem cells (some cut before the population completely subtracted of Hematopoietic stem cells)
T2: Mesenchymal  stem cells (in vitro assessed, functionally and phenotipically)
T3: Mesenchymal stem cells biological active in vivo
T4: Mesenchymal stem cells at the passage in vitro next to the one active in vivo
T5: Mesenchymal stem cells no more active in vitro neither in vivo
Once in our lab, RNA will be extracted using the Trizol reagent (Invitrogen) and further purified by the RNeasy kit (Qiagen). Sample labeling will be performed according to the array manufacturer (Affymetrix) instructions. Subject to initial results, we plan to add intermediate timepoints in subsequent stages.
Keywords: time-course

first: Jorge

last: Oksenberg


title: Abundant transcripts from retrotransposons are unstable in fully-grown mouse oocytes

status: Public on Jun 07 2006

city: Basel

first: Mareike

last: Puschendorf

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary:
One physiological function proposed for RNA interference (RNAi) is to constrain expression of repetitive elements and thereby reduce the incidence of retrotransposition.  Consistent with this model is that inhibiting the RNAi pathway results in an increase in expression of repetitive elements in preimplantation mouse embryos.  Mouse oocytes are essentially transcriptionally quiescent providing a unique opportunity to assess the stability of repetitive element-derived transcripts in these cells.  We compared the transcriptome of freshly isolated fully-grown germinal vesicle (GV)-intact oocytes to that of oocytes in which meiotic maturation in vitro was inhibited for 48 h by milrinone.  Consistent with the aforementioned function for RNAi is that the abundance of only a relatively small number of transcripts decreased in the cultured oocytes, when compared to changes that occur during maturation or following fertilization, and of those, several belonged to mobile elements.
Keywords: untreated GV oocytes and GV oocytes cultured in milrinone for 48h

overall_design: untreated GV oocytes (four replicates), GV oocytes cultured in milrinome for 48h (four replicates)

first: Paula

last: Stein

first: Edward

last: Oakeley

first: Richard

middle: M

last: Schultz

first: Antoine

middle: H

last: Peters

first: Petr

last: Svoboda


title: Gene expression profile in isolated E13 gonadal somatic support cells and E13 gonads.

status: Public on May 27 2006

city: Bar Harbor

first: Gerrit

last: Bouma

state: ME

address: 600 Main St

institute: The Jackson Laboratory

summary:
Gonadal sex determining (GSD) genes that initiate fetal ovarian and testicular development and differentiation are expressed in the cells of the urogenital ridge that differentiate as somatic support cells (SSCs), i.e., granulosa cells of the ovary and Sertoli cells of the testis. To identify potential new mammalian GSD genes, we analyzed the gene expression differences between XX and XY SSCs cells isolated from the gonads of embryonic day (E) 13 mouse fetuses carrying an EGFP reporter transgene expressed specifically in SSCs. In addition, genome wide expression differences between XX and XY E13 whole gonads were examined. Newly identified differentially expressed transcripts are potential GSD genes involved in unexplained human sex reversal cases.
Keywords: microarray, mouse fetal gonadal somatic support cells, sex determination

overall_design: Two seperate RNA samples were obtained from E13 XX and XY sorted EGFP+ cells, and two seperate RNA samples were obtained from E13 XX and XY pooled gonads. Approximately 20ng of total RNA from each sample was used to generate biotin-labelled cDNA. Approximately 2.5ug biotin labelled cDNA of each sample was used for each Mouse Genome 430v2.0 GeneChip array (Affymetrix). Significantly differentially expressed transcripts were identified using R/maanova. Statistical significance was determined at a false discovery rate value of equal to or less than 1%.

first: Jason

middle: P

last: Affourtit

first: Linda

last: Washburn

first: Carol

last: Bult

first: Eva

last: Eicher


title: Gene expression profiles of the P1 bladder isolated from SMAA or SMAGA (Acta2 or Actg2) transgenic and wild type mice. (GUDMAP Series ID: 2)

status: Public on May 12 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

institute: IGMM MRC Human Genetics Unit

summary:
The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing bladder. The central thesis is straightforward. The combination of laser capture microdissection (LCM) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing bladder. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in laser capture microdissected components of the developing bladder. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. As a first step we sought to determine if expression of the reporter gene (EGFP or EYFP) used to identify smooth muscle cells altered the gene expression profile of the bladder.
Keywords: Comparison of postnatal day 1 bladders.

overall_design: Postnatal day 1 bladders were isolated from transgenic and wild-type mice to determine the effects of transgene expression on the gene expression profile in the bladder.

first: James

last: Lessard

first: John

last: Szucsik


title: Expression data from CD4+ splenocytes 6h post-sepsis

status: Public on Jun 01 2006

city: Saint Louis

first: Jonathan

last: McDunn

state: MO

address: 660 South Euclid Avenue

department: Surgery

institute: Washington University School of Medicine

summary:
Lymphocytes are adversely affected during sepsis.  Some CD4+ splenocytes undergo apoptosis while others become Th2 polarized.  The molecular determinants of these phenotypic changes are not known.  Here we compare the transcriptional response of septic CD4 splenocytes to CD4 splenocytes from sham-manipulated animals 6h after sepsis and identify an early transcriptional component to the septic CD4+ splenocyte phenotype.
We used microarrays to detail the global program of gene expression underlying the sepsis-induced changes in CD4+ splenocyte phenotype.
Keywords: disease state analysis

overall_design: CD4+ splenocytes were isolated 6h after the surgical induction of sepsis for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain a homogeneous cell population in order to reduce any effects of cellular heterogeneity on expression profiles. To that end, immunomagnetic negative selection was used to enrich CD4+ splenocyte populations to ~91%.  (n=5 biological replicates each CLP and sham)

first: Isaiah

middle: R

last: Turnbull

first: Ashoka

middle: D

last: Polpitiya

first: Alice

last: Tong

first: Sandra

middle: K

last: MacMillan

first: Dale

last: Osborne

middle: S

last: Hotchkiss

first: Marco

last: Colonna

first: Perren

last: Cobb


title: Global analysis of transcriptional changes following genomic demethylation in chondrocytes, osteoblasts, and fibroblasts

status: Public on May 06 2006

city: Chicago

first: Tammy

last: Vallender

department: Human Genetics

institute: University of Chicago

type:
Expression profiling by array
Methylation profiling by array

summary:
DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2’-deoxycytidine, and changes in gene expression monitored by microarray analysis.
Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast; chondrocyte; osteoblast

overall_design: Three biological replicates for both control and 5-aza-2'-deoxycytidine treatment were derived. Cells were primary mouse dermal fibroblasts, primary mouse chondrocytes, and primary mouse osteoblasts derived separately for each biological replicate. Treated cells were exposed to 5uM 5-aza-2'-deoxycytidine for 96 hours with recovery in normal medium for 24 hours. Control cells were untreated.

middle: F


title: GDNF-regulated gene expression in cultured mouse spermatogonial stem cells

status: Public on May 11 2006

city: University Park

first: Jon

last: Oatley

address: 324 Henning

department: Dairy and Animal Science

institute: Pennsylvania State University

summary:
GDNF-regulated gene expression was studied in cultures of actively self-renewing spermatogonial stem cells established from 6 day old male mice. GDNF is the essential growth factor regulating mouse spermatogonial stem cell self-renewal. Using a serum-free chemically defined culture system that supports mouse spermatogonial stem cell self-renewal for extended periods of time, GDNF-regulated genes were identified using microarray profiling.
Keywords: GDNF withdrawal and time-course replacement

overall_design: Established cultures of highly enriched self-renewing spermatogonial stem cells were subjeted to withrawal of GDNF and GFRalpha1 for 18-hr followed by replacement of the growth factors for 2, 4, and 8-hr. Gene expression was studied using microarray profiling prior to withdrawal, after withdrawal and at each time-point of GDNF/GFRalpha1 replacement.

first: Mary

last: Avarbock

first: Aino

last: Telaranta

first: Douglas

middle: T

last: Fearon

first: Ralph

last: Brinster


title: conra-affy-mouse-89474

status: Public on May 02 2006

first: Winnie

last: Liang

summary:
Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are the two most common examples of synucleinopathies, i.e. human disorders that display intracellular deposition of the Alpha-synuclein (aSYN) protein. The two forms of aSYN deposits, intracellular Lewy bodies and Lewy neurites, are in the PD brain primarily found in the substantia nigra whereas in DLB brains they are diffusely spread in cortical and limbic areas. We are currently analyzing the protective effects on aSYN pathology by the molecular chaperone hsp70 in mice overexpressing wildtype human aSYN. Moreover, transgenic mice overexpressing hsp70 have been cross-bred with the aSYN mouse line. Intriguingly, the aSYN / hsp70 double transgenics display a pronounced reduction in biochemically assessed aSYN aggregation. Gene expression of these mice will not only provide insight into the disease mechanisms of aSYN pathology but its prevention by the overexpression of hsp70, leading to new potential avenues of treatment.
Project 1: To investigate and compare alterations in gene expression profiles of brains from wild-type mice and mice overexpressing wildtype human aSYN, human hsp70 and aSYN / hsp70, respectively.
The comparison of the expression profiles of the groups of mice: aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates will help to identify the genes that are involved in the mediation of aggregation toxicity of aSYN expression as well as the genes that may mediate an attenuation of aSYN pathology in hsp70-coexpressors.
Project 1: Selection of animals Mice of 4 month old (n=6) of age of each background (aSYN, Hsp70, aSYN/ Hsp70 and non-transgenic littermates) will be used for RNA-extraction for a total of 24 mice. Preparation of samples For the proposed experiments, we will use the recommended procedures, as outlined by the consortium, for extracting and purifying RNA from whole brain homogenate. Briefly, RNA will be extracted by the trizol method and purified by using the RNeasy MinElute Cleanup (QIAGEN Inc., USA).  RNA labeling and hybridization procedures will be carried out by the microarray consortium. SPECIAL NOTE: The samples will be sent upon availability.  Gene Expression Microarray Analysis Six mice will be included in each group per timepoint to generate statistically testable data and limit the possible confound of individual mouse gene expression variations. The genome expression will be evaluated through the use of the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix) for each individual mouse constituting a total of 24 arrays for the proposed study. Gene expression data interpretation will be assisted by the Microarray Consortium Center bioinformatics core.
Keywords: time-course

first: Chris

last: Conrad


title: Inducible Ngn3 Embryonic Stem Cells

city: Morristown

first: Nathan

last: Treff

state: NJ

address: 111 Madison Ave

department: Molecular Biology

institute: RMA of NJ

summary:
Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets.  In order to better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profile of differentiating murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3.  An ESC line was created that allows for the induction of Ngn3 by adding doxycycline (Dox) to the culture medium.  Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of both undifferentiated and differentiated conditions.  Characterization of pancreatic developmental markers during embryoid body (EB) formation revealed an optimum context for Ngn3 induction. Neuroendocrine genes including neurogenic differentiation 1 (NeuroD1) and single minded 1 (Sim1) were found to be significantly upregulated.  Genes regulated by Ngn3 independent of the context were analyzed using systematic gene ontology tools and revealed Notch signaling as the most significantly regulated signaling pathway (p=0.009).  This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and conversely, more sensitive to Notch signaling inhibition.  Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p<0.0001).  Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin transcription.
Keywords: Ngn3 induction, embryonic stem cell differentiation

overall_design: 16 embryonic stem cell (ESC) samples (8 groups in duplicate) were processed for microarray analysis using the Affymetrix Mouse Genome 430 2.0 GeneChip.  The samples included the parental Ainv15 ESC line (1) undifferentiated (Ainv15 ESC), (2) differentiated for 3 days as embryoid bodies (Ainv15 EB3) or (3) for 10 days as embryoid bodies (Ainv15 EB10).  The remaining samples included tetracycline inducible Ngn3 ESC line derived from the parental Ainv15 ESC line (4) after differentiation and addition of doxycycline for 3 days without embryoid body formation (Ngn3 ES3 ON), (5 and 6) after differentiation as embryoid bodies for 3 days with (Ngn3 EB3 ON) or without (Ngn3 EB3 OFF) doxycycline, and (7 and 8) after differentiation as embryoid bodies for 10 days with (Ngn3 EB10 ON) or without (Ngn3 EB10 OFF) doxycycline.

first: Robert

last: Vincent

first: Melisa

last: Budde

first: Victoria

last: Browning

first: Joseph

last: Magliocca

last: Odorico

first: Vivek

last: Kapur


title: ruiz-affy-mouse-243540

summary:
The retinal degeneration slow gene (rds/peripherin), is essential for normal photoreceptor (rods  and cones) outer segment structure  in the neurosensory retina. Our rds animal model, are stable transgenic mice that carry a null mutation (complete lost of function) in the rds gene. Those animals become blind, mimicking a human form of retinitis pigmentosa. In our experience we have observed dramatic levels of photoreceptor rescue after subretinal injection of those animals with a ciliary neurotrophic factor (CNTF) inserted into a recombinant Adeno-associate virus (rAAV) vector. However, this cell rescue is accompanied by changes in photoreceptor nuclear morphology, attenaution of the electroretinogram and evetually Apoptosis. By using Microarray analysis we hope to identify genes whose expression or lack thereof brings the cell to the brink of suicide. Identification of these genes will suggest therapeutic targets for the future.
We will determine gene expression patterns in the retina of rds mice non-injected and injected with CNTF/rAAV virus after 60 days of injection.
Our working hypothesis is that the injected CNTF/rAAV vector induces the expression of genes that somehow partially restores the photoreceptor morphology of rds mice.  Paradoxically, it also has a negative effect on the phisiology of these cells (ERG). The up or down regulation of genes in different areas of the neurosensory retina collected by Laser Capture Microdissection will give us an insight on the relevance of candidate genes responsible for this phenomenon.
We will use rds mice at age P25 and inject their rigth eye with CNTF/AVV virus (2.68x e13 particles/ml) and use the left eye as a control.  Injection is performed via subretinal. Retinas will be collected at age P90 post injection.  RNA will be extracted and processed accordingly for MIcroarray analysis.
Keywords: dose response

first: Alberto

middle: C

last: Ruiz


title: Mouse oocyte and one-cell embryo polysomal mRNA

status: Public on Jun 30 2006

city: Philadelphia

first: Keith

middle: E

last: Latham

address: 3307 N Broad St

department: The Fels Institute for Cancer Research and Molecular Biology

institute: Temple University School of Medicine

summary:
Transcriptional activation in mammalian embryos occurs in a stepwise manner. In mice, it begins at the late one-cell stage, followed by a minor wave of activation at the early two-cell stage, and then the major genome activation (MGA) at the late two-cell stage. Cellular homeostasis, metabolism, cell cycle, and developmental events are orchestrated before MGA by time-dependent changes in the array of maternal transcripts being translated (i.e., the translatome). Despite the importance of maternal mRNA and its correct recruitment for development, neither the array of recruited mRNA nor the regulatory mechanisms operating have been well cheracterized. We present the first comprehensive analysis of changes in the maternal component of the zygotic translatome during the transition from oocyte to late one-cell stage embryo, revealing global transitions in the functional classes of translated maternal mRNAs, and apparent changes in the underlying cis-regulatory mechanisms.
Keywords: Time course

overall_design: Batches of 160-500 MII oocytes or one-cell embryos. 4 independent replicates per sample kind. Lysed and processed for polysomal mRNA extraction. Two rounds of IVT.

first: Santhi

last: Potireddy

first: Rita

last: Vassena


title: Basonuclin: A novel mammalian maternal effect gene

status: Public on May 01 2006

first: Hung

last: Tseng

address: 415 Curie Boulevard Room 242B

department: Dermatology

institute: University of Pennsylvania

summary:
	Basonuclin, which is a zinc-finger protein found in abundance only in the keratinocytes of the stratified epithelium, male germ cells and oocytes, qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal.  Using a transgenic-RNAi approach, we knocked-down basonuclin specifically in mouse oocytes, which led to female sub-fertility.  Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription and oocyte morphology was affected as evidenced by cytoplasmic and cell surface abnormalities.  The affected oocytes, however, could still mature to and arrest at metaphase II and be ovulated, suggesting the impaired pathways were not essential for oocyte development and maturation.  Nevertheless, an early embryonic failure in pre-implantation development was identified and likely accounted for the sub-fertility phenotype.  These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.
Keywords: RNAi knockdown

overall_design: Basonuclin-deficient oocytes were created by a mouse transgenic RNAi approach, which knocks down basonuclin mRNA level specifically in oocytes.  Microarray analysis was performed on total RNA from four non-transgenic (control) and six transgenic (basonuclin-deficient) oocyte samples (approximately 50 oocytes per sample).

first: Jun

last: Ma

first: Fanyi

last: Zeng

first: Richard

last: Schultz


title: Expression data for validation of single cell cDNA amplification method (V1V3 method)

status: Public on Apr 26 2006

city: Nara

first: Kazuki

last: Kurimoto

address: 840 Shijo-Cho, Kashihara

department: School of medicine

institute: Nara Medical University

summary:
A systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells.  We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification.  Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy.
Keywords: Method validation

overall_design: To verify the ability of the developed method, we prepared total RNA from ES cells and diluted it to the single-cell level (10 pg).  Eight array data from the cDNA samples amplified independently from the 10 pg RNA (amplified samples) were compared to each other, and to eight array data from the undiluted 5 ug RNA (nonamplified controls).  Since all the materials were the same, all of the observed variations were attributable to the methodogoly.

first: Yukihiro

last: Yabuta

first: Yasuhide

last: Ohinata

first: Yukiko

last: Ono

first: Kenichiro

last: Uno

first: Rikuhiro

middle: G

last: Yamada

first: Hiroki

last: Ueda

first: Mitinori

last: Saitou


title: Immune Response to Nippostrongylus brasiliensis in the mouse lung

city: Baltimore

first: Joshua

last: Reece

state: MD

address: 615 N. Wolfe Street

department: MMI

institute: Johns Hopkins University BSPH

summary:
The goal of this experiment was to examine the innate immune response to helminth infection in the lung. Hookworms (like many other helminths) use an obligate migration pathway through the lung. Their infection has been characterized in the gut in detail, but early immune responses in the lung have not been fully characterized.
Keywords: Time Course

overall_design: SCID mice were used to examine the innate immune response. SCID mice have no functional B or T cells but a fully functional innate immune system. SCID and WT mice were infected with 500 L3 stage infectious Nippostrongylus brasiliensis SC and their lungs were removed at days 2,3,4,8 and 12 post infection.

first: Mark

last: Siracusa

first: Alan

last: Scott