title: Gene expression changes in FORKO mice

first: JAYAPRAKASH

last: ARAVINDAKSHAN

address: 110 Ave des Pins Ouest

institute: Clinical Research Institute of Montreal

summary:
To understand consequences of loss of FSH receptor, we used genetically altered mouse mutants with deletion of this receptor. Follitropin Receptor Knockout (FORKO) mice present various developmental and age-dependent abnormalities such as infertility, senescence and increased tumor incidences in addition to other phenotypes. To explore why the atrophic ovaries of FORKO mice develop ovarian anomalies later in life, we used expression profiling studies to gain a more comprehensive view of genes that are misregulated.
Keywords: Comparison of  Wildtype and FSH-Receptor Knock out Ovaries at 8 months of age.

overall_design:
Mice were housed under controlled temperature and constant light (12 h of light, 12 h of darkness), with food and water provided ad libitum. The female mice used in this experiment were derived by breeding heterozygotes of the 129T2 svEmsJ background.

For RNA extraction, both the ovaries from 8 month old mice were carefully dissected and pooled. Tissues were frozen in liquid nitrogen and homogenized in TRIZOL (Invitrogen, Burlington, Ontario) and processed in accordance with the manufacturer’s protocol.

Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 Array has over 45000 probe sets which analyses 39000+ transcripts and variants from over 34000 well characterized mouse genes. A total of 1 µg of double-stranded cDNA was transcribed in vitro using the Bioarray High-Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY) according to the manufacturer's instructions using biotinylated CTP and UTP (Enzo, Farmingdale, NY). Following 5-hr incubation at 37°C, the resultant biotin-labeled cRNA was purified with RNAeasy columns (Qiagen) and eluted in 40 µl of RNAse-free water. The concentration of biotin-labeled cRNA was determined by RNA bio-analyzer. Target cRNAs corresponding to either wild type ovary or FORKO ovarian tissue were hybridized to an individual GeneChip from an identical lot of Affymetrix 39 K GeneChip® Mouse Genome 430 2.0 array for 16 hr. GeneChip arrays were washed and stained using antibody-mediated signal amplification and the Affymetrix Fluidics Station's standard Eukaryotic GE Wash 2 protocol, using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA).

first: Jayaprakash

last: Aravindakshan

first: Chen

last: XinLei

first: Ram

last: Sairam


title: Effect of Synthetic Dietary Triglycerides: a Novel Research Paradigm for Nutrigenomics

status: Public on Jan 30 2008

city: Wageningen

first: Guido

last: Hooiveld

address: HELIX, Stippeneng 4

department: Div. Human Nutrition & Health

country: Netherlands

institute: Wageningen University

summary:
Dietary fatty acids have myriads of effects on human health and disease. Many of these effects are likely achieved by altering expression of genes. Several transcription factors have been shown to be responsive to fatty acids, including SREBP-1c, NF-kB, RXRs, LXRs, FXR, HNF4α, and PPARs. However, the relative importance of these transcription factors in regulation of gene expression by dietary fatty acids remains unclear. Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the acute effects of individual dietary fatty acids on hepatic gene expression in mice. The dietary interventions were performed in parallel in wild-type and PPARα-/- mice, enabling the determination of the specific contribution of PPARα. Depending on chain length and degree of saturation, dietary fatty acids caused a statistically significant change in expression of over 400 genes. Surprisingly, the far majority of genes regulated by dietary fatty acids in wild-type mice were unaltered in mice lacking PPARα, indicating PPARα-dependent regulation. We conclude that the effects of dietary fatty acids on hepatic gene expression are almost entirely mediated by PPARα, indicating that PPARα dominates fatty acid-dependent gene regulation in liver.
Keywords: identification of target genes

overall_design:
2-6 months old male pure bred wild-type (129S1/SvImJ) and PPARα -/- (129S4/SvJae) mice were used.

Wild-type and PPARα -/- mice fasted for 4 hours received a single dose of 400 µl synthetic triglyceride (tridecanoin – C10:0, triolein – C18:1, trilinolein – C18:2, trilinolenin – C18:3, trieicosapentaenoin – C20:5 or tridocosahexaenoin – C22:6), the synthetic PPARα agonists WY14643 or fenofibrate (400 μl of 10 mg/ml in 0.5% carboxymethyl cellulose, CMC) or control (400 μl of 0.5% CMC). 6 hours after gavage, mice were killed and livers extracted.

(Please note: Experiments with C10:0, C18:2 and C18:3 were carried out a few months later than the rest of the treatments. Therefore there is a second set of control arrays – named control2 – which belong to these three treatment groups.)

Liver total RNA from biological replicates was hybridized onto Affymetrix mouse genome 430 2.0 GeneChip arrays. Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

first: Linda

middle: M

last: Sanderson

first: Philip

middle: J

last: de Groot

first: Michael

last: Muller

first: Sander

last: Kersten


title: Liver toxicity of dichloroacetyl chloride and dichloroacetic anhydride in female MRL +/+ mice

status: Public on Jan 31 2008

city: Galveston

first: Rolf

last: Konig

state: TX

address: 301 University Blvd.

department: Microbiology & Immunology

country: USA

institute: UTMB

summary:
Dichloroacetyl chloride (DCAC) is a metabolic intermediate of trichloroethene (TCE), an industrial chemical and ubiquitous environmental contaminant. TCE and its metabolites have been implicated in the induction of organ-specific and systemic autoimmunity, in the acceleration of autoimmune responses, and in the development of liver toxicity. In humans, effects of environmental toxicants are often multifactorial and detected only after long-term exposure. Therefore, we developed a small-animal model to determine mechanisms by which DCAC and related acylating agents affect liver disease. Autoimmune-prone female MRL +/+ mice were injected i.p. with 0.2 mmole/kg of DCAC or the acylating agent dichloroacetic anhydride (DCAA) in corn oil twice weekly for six weeks. We then determined changes in the liver transcriptome using microarray gene expression analysis. After exposure to DCAC or DCAA, we observed changes in liver gene expression consistent with inflammatory processes. Both toxicants up-regulated expression of acute phase response and inflammatory genes. Further, metallothionein genes were strongly up-regulated, indicating effects of the toxicants on zinc ion homeostasis and stress responses. In addition, DCAC and DCAA induced up-regulation of several genes indicative of tumorogenesis. Microarray gene expression analysis using a restricted set of genes could be a valuable tool to screen for early changes in liver function following suspected exposure to environmental toxicants.
Keywords: Response to acylating agents; inflammatory response; disease state analysis.

overall_design:
Animals: Four-week-old female MRL +/+ mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in an animal room maintained at ~22°C and 50-60% relative humidity with a 12-h light/dark cycle.  Lab chow and drinking water was provided ad libitum, and mice were acclimatized for one week before starting the treatment. Mice were injected intraperitoneally with 0.2 mmol/kg of DCAC or DCAA in 100 µl of corn oil twice weekly for six weeks. Control animals received only corn oil. Mice were sacrificed 24 h after the last treatment.
Samples: Livers from three mice per group were independently processed. The integrity of the RNA samples was tested using an Agilent 2100 Bioanalyzer and RNA 6000 LabChip® kit (Agilent Technologies, Palo Alto, CA). Only RNA samples without signs of degradation were used. Comprehensive gene expression profiles were analyzed.

last: König

first: Ping

last: Cai

first: Xin

last: Guo

last: Ansari


title: Convergent molecular pathways that induce immunoglobulin light-chain recombination

status: Public on Jan 26 2008

city: Chicago

first: Harinder

last: Singh

state: IL

department: Molecular Genetics and Cell Biology

institute: Howard Hughes Medical Institute/ University of Chicago

summary:
Productive rearrangement of the immunoglobulin heavy chain locus triggers a major developmental checkpoint that promotes limited clonal expansion of pre-B cells, culminating in cell cycle  arrest and rearrangement of the kappa (κ) or lambda (λ) light-chain loci. B lineage cells lacking the related transcription factors IRF-4 and IRF-8 undergo a developmental arrest at the cycling pre-B cell stage and are blocked for light-chain recombination. Using Irf-4,8-/- pre-B cells we demonstrate that two pathways converge to synergistically drive light-chain rearrangement, a process that is not simply activated by cell cycle exit. One pathway is directly dependent on IRF-4, whose expression is elevated by pre-BCR signaling.  IRF-4 targets the κ 3′ and λ enhancers to increase locus accessibility and positions a kappa allele away from pericentromeric heterochromatin. The other pathway is triggered by attenuation of IL-7 signaling and results in activation of the κ intronic enhancer via binding of the transcription factor, E2A. Intriguingly, IRF-4 regulates the expression of CXCR4 and promotes the migration of pre-B cells in response to the chemokine CXCL12. We propose that IRF-4 coordinates the two pathways regulating light-chain recombination by positioning pre-B cells away from IL-7 expressing stromal cells.
We used microarrys to identify the changes in gene expression under different levels of the cytokine IL-7 and after rescue of genetic defect.
Keywords: growth conditions and rescue

overall_design: IRF4,8 null pre-B cells were cultures in the indicated conditions prior to RNA isolation and hybridization to Affymetrix arrays.

first: Kristen

last: Johnson

first: Tamar

last: Hashimshony


title: Inducible repositioning of genes to the inner nuclear membrane

status: Public on Jan 16 2008

summary:
Nuclear compartmentalization appears to play an important role in regulating metazoan genes. While studies on immunoglobulin (Ig) and other loci have correlated positioning at the nuclear lamina with gene repression, the functional consequences of this compartmentalization remain untested. We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM) and demonstrate with 3D DNA-ImmunoFISH, repositioning of chromosomal regions to the nuclear lamina. Relocalization requires mitotic nuclear envelope breakdown and reformation. Tethering leads to the accumulation of lamin and INM proteins but not to association with pericentromeric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Using DamID we show that as is the case for our model system, inactive Ig loci at the nuclear periphery are contacted by INM and lamina components. We propose that such molecular interactions are used to compartmentalize and limit the accessibility of Ig loci.
Keywords: Inducible

overall_design: We used microarray to analyze the transcription status of genomic regions are inducibly tethered to the INM

first: Karen

middle: L

last: Reddy


title: Functional and Genomic Profiling of Effector CD8 T Cell Subsets

status: Public on Jan 23 2008

city: Atlanta

first: Shruti

last: Subramaniam

state: GA

address: G211 RRC, Emory University School of Medicine

department: Emory University School of Medicine

institute: Emory University

summary:
Using killer cell lectin-like receptor G1 as a marker to distinguish terminal effector cells from memory precursors, we found that despite their diverse cell fates both subsets possessed remarkably similar gene expression profiles and functioned as equally potent killer cells. However, only the memory precursors were capable of making IL-2 thus defining a novel effector cell that was cytotoxic, expressed granzyme B, and produced inflammatory cytokines in addition to IL-2. This effector population then differentiated into long-lived protective memory T cells capable of self-renewal and rapid re-call responses. Mechanistic studies showed that cells that continued to receive antigenic stimulation during the later stages of infection were more likely to become terminal effectors. Importantly, curtailing antigenic stimulation towards the tail-end of the acute infection enhanced the generation of memory cells. These studies support the decreasing potential model of memory differentiation and show that the duration of antigenic stimulation is a critical regulator of memory formation
Keywords: Infection Type

overall_design: An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study we provide a comprehensive phenotypic, functional and genomic profiling of terminal effectors and memory precursors, towards better understanding the generation of these subsets. The two effector subsets were FACS purified during the early expansion phase (Days 4-5 post-infection) and extensively analyzed for their phenotypic (eg. CD127, CD62L, CD27, Bcl-2, Granzyme B, etc.) and functional properties (cytokine production, cytotoxicity, homeostatic proliferation, recall proliferation), and gene expression profiles (by genome-wide microarray analyses). Mechanistic studies involving the extent of proliferation and duration of antigen stimulation on memory differentiation potential of effectors were also performed using adoptive transfer techniques.


title: Striatal gene expression data from 22-month-old CHL2 mice and control mice.

status: Public on Jan 25 2008

city: Bethesda

first: Alexandre

last: Kuhn

state: MD

address: 35 Convent Dr

department: NIA

institute: NIH

summary:
Achieving a mechanistic understanding of disease and initiating preclinical therapeutic trials necessitate the study of huntingtin toxicity and its remedy in model systems. To allow the engagement of appropriate experimental paradigms, Huntington’s disease (HD) models need to be validated in terms of how they recapitulate a particular aspect of human disease. In order to examine transcriptome-related effects of mutant huntingtin, we compared striatal mRNA profiles from seven genetic mouse models of disease to that of postmortem human HD caudate using microarray analysis. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in models of HD took longer to appear, 15-month and 22-month CHL2Q150/Q150, 18-month HdhQ92/Q92 and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. When the affected genes were compared across models, a robust concordance was observed. Importantly, changes concordant across multiple lines mice were also in excellent agreement with the mRNA changes seen in human HD caudate. Although it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared to those caused by expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. There was, however, an overall concordance between transcriptomic signature and disease stage. We thus conclude that the transcriptional changes of HD can be modelled in several available lines of transgenic mice, comprising lines expressing both N-terminal and full-length mutant huntingtin proteins. The combined analysis of mouse and human HD transcriptomes provides an important chronology of mutant huntingtin's gene expression effects.
Keywords: genetic modification

overall_design: Striatal samples from 4 CHL2 Q150/Q150 mutant mice and 4 age-matched wild-type mice.

first: Kirupa

last: Sathasivam

first: Gillian

middle: P

last: Bates

first: Ruth

last: Luthi-Carter


title: Mutant huntingtin's effects on striatal gene expression in mice

status: Public on Jan 24 2008

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

overall_design: Refer to individual Series


title: Expression data from Mus musculus subspecies and their F1 hybrids for 3 tissues (brain, liver, testis)

status: Public on Jan 19 2008

city: Cologne

last: Rottscheidt

address: Zuelpicher Strasse 47

department: Institute for Genetics

country: Germany

institute: University of Cologne

summary:
We have studied different subspecies of the house mouse (M. m. musculus, M. m. domesticus and M. m. castaneus) and their reciprocal F1 hybrids to estimate the within-locus mode of inheritance for subspecies differences in gene expression in three tissues (brain, liver, testis) of male mice. This study investigates mode of inheritance in crosses at a larger taxonomic distance than have been previously systematically investigated. We found the vast majority of transcripts to be additively expressed with only a few transcripts showing dominance or overdominance in expression, except for one direction of one cross, which showed large mis-expression in the testis.
Keywords: comparative whole transcripome analysis

sample_organism: Mus musculus musculus x M. m. domesticus

sample_organism: Mus musculus musculus x Mus musculus castaneus

overall_design: Wild-derived inbred lines of M. m. musculus, M. m. domesticus and M. m. castaneus were used to generate reciprocal crosses of M. m. musculus x M. m. domesticus and M. m. musculus x M. m. castaeneus. The gene expression profile of  2 males each of the hybrids and the pure parental  subspecies were analyzed by using the Affymetrix GeneChip 430 2.0 to obtain evidence about the within-locus mode of inheritance of genes. First, we studied all transcripts that are differentially expressed between the parental subspecies and assessed additivity vs. nonadditivity of expression of these transcripts in the F1 hybrids. Second, we investigated those transcripts which do not differ in expression between the parental lines, but which are expressed differently in the F1 hybrids relative to both of their parents.

first: Bettina

last: Harr


title: Molecular imaging of lymphoid organs and immune activation using PET with a new 18F-labeled 2’-deoxycytidine analog

status: Public on Jan 15 2008

city: Los Angeles

first: Jason

last: Lee

state: CA

address: 675 Charles E. Young Dr. South

institute: UCLA

summary:
Differential gene expression between naive and activated CD8+ T cells was assessed using microarray analysis to determine target genes for new positron emission tomography (PET) probe screening, in particular for molecular imaging of lymphoid organs and immune activation.
Keywords: cell activation comparison

overall_design: Total RNA was purified from tissues using the Qiagen RNeasy Mini kit.  Total RNA was extracted from purified naïve and proliferating (72 hrs post activation) CD8+ T cells from the pmel-1 TCR transgenic mice. Pooled RNA from 4 independent experiments was hybridized to Affymetrix Mouse Genome 430 2.0 arrays.  Absolute calls describing whether a probe set is present (P), marginally present (M), or absent (A) were generated using the Affymetrix GeneChip Operating Software v1.3 (GCOS) and expression values were calculated using the PM/MM difference model of DNA-Chip (dChip). Expression values across samples were normalized using dChip’s invariant set method. A gene was considered differentially expressed if the corresponding probe set fit the following criteria: absolute call was P in at least half of the samples, fold change >1.4 between baseline (naïve CD8+ T cells) and experimental (activated CD8+ T cells) using the lower 90% confidence bound of fold change as defined in dChip, and expression difference between the baseline and experimental samples was >100. Genes involved in the nucleoside de novo biosynthesis and salvage pathways were taken from the KEGG database (pathway IDs 00230 and 00240), and corresponding probe sets were manually extracted from Affymetrix’s NetAffx to ensure complete coverage of all nucleoside pathway genes (239 probe sets), plus the SLC28 and SLC29 transporters (10 probe sets).

first: Caius

middle: G

last: Radu

first: Chengyi

last: Shu

first: Evan

last: Nair-Gill

first: Stephanie

last: Shelly

middle: T

first: Jorge

middle: R

last: Barrio

first: Nagichettiar

last: Satyamurthy

middle: E

last: Phelps

first: Owen

middle: N

last: Witte


title: Microarray Analysis of Treacher Collins Syndrome

city: Kansas City

first: Gaye

last: Hattem

state: MO

address: 1000 E. 50th Street

department: Bioinformatics

institute: Stowers Institute for Medical Research

summary:
The object of this study was to identify genes transcriptionally upregulated and downregulated in response to Tcof1 haploin-sufficiency during mouse embryogensis
Keywords: mouse embryo, littermates, Tcof1, Treacher Collins sydnrome, comparative hybridisation

overall_design: Total RNA was extracted from 3 E8.5 wild-type and 3 E8.5 Tcof1+/-  littermate embryos using the RNeasy Mini Protocol for Isolation of Total RNA from Animal Tissues (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity was determined using the Bioanalyser. To generate targets for microarray analysis, total RNA (100 ng) from each wild-type and mutant embryo was amplified using Two-Cycle Target Labeling (Affymetrix) according to the manufacturer’s instructions. Biotinylated target cRNAs (20 μg) from the 3 wild-type and 3 Tcof1+/- embryos were hybridised to separate GeneChip® Mouse Genome 430 2.0 arrays (Affymetrix), following standard Affymetrix procedures.

first: Natalie

middle: C

last: Jones

first: Karin

last: Gaudenz

first: Earl

middle: F

last: Glynn

first: Paul

middle: A

last: Trainor


title: Polysomal, free mRNP, and total cellular mRNA of J774.1 before and after LPS stimulation

status: Public on Feb 05 2008

city: Ebetsu

first: Hiroshi

last: Kitamura

address: 582 Midorimachi, Bunkyodai

department: School of Veterinary Medicine

country: Japan

institute: Rakuno Gakuen University

summary:
Bacterial lipopolysaccharide(LPS) dramatically activates macrophages. So far, dozen of papers indicated that many proinflammatory molecules are transcriptionaly regulated during response. Despite of this,translational regulation is not fully elucidated especially in a comprehensive fashion. In this series, we investigated expression profiles of translation active (polysome) inactive (free mRNP) mRNAs of a typical mouse macrophage cell line, J774.1. Moreover, we also measured total cellular RNA level as a reference.
Keywords: time-course

overall_design: Triplicates for each time points are included.

first: Michiyo

last: Takayama

first: Tomoko

last: Nakagawa

first: Masatoshi

last: Ito

first: Atsushi

last: Hijikata

first: Osamu

last: Ohara


title: Impaired liver regeneration in Nrf2 knockout mice caused by ROS-mediated insulin/IGF-1 resistance

city: Zurich

first: Stefan

last: Zoller

address: Universitatstrasse 16

department: Genetic Diversity Centre

country: Switzerland

institute: ETH Zurich

summary:
The liver is frequently challenged by surgery-induced metabolic overload, viruses, or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signalling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. As a consequence, tissue damage was aggravated, and liver regeneration after partial hepatectomy was delayed.
Using in vitro and in vivo studies we identified oxidative stress-induced insulin/insulin-like growth factor resistance as the underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase, and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signalling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration and thus offer new avenues to improve regeneration in patients with acute or chronic liver damage.
Keywords: genetic modification, wilde type (wt) vs. knock out (k.o.) liver samples

overall_design: Livers from Nrf2 k.o. and wt mice; 3 hybridizations per genoype: RNA samples were pooled from 3 individual animals

first: Tobias

last: Beyer

first: Sabine

last: Werner


title: Gene expression profiling of a mouse model of islet dysmorphogenesis

status: Public on Mar 01 2008

city: Nashville

first: Laura

last: Crawford

state: TN

address: 715 PRB 2220 Pierce Ave

department: Diabetes, Endocrinology, & Metab Division

institute: Vanderbilt University

summary:
In the past decade, several transcription factors critical for pancreas development have been identified.  Despite this success, many of the cell surface and extracellular factors necessary for proper islet morphogenesis and function remain uncharacterized.  Previous studies have shown that transgenic over-expression of the transcription factor HNF6 specifically in the pancreatic endocrine cell lineage resulted in the disruption of islet morphogenesis, including dysfunctional endocrine cell sorting, increased islet size, and failure of islets to migrate away from the ductal epithelium.  We exploited the dysmorphic islets in pdx1PBHnf6 animals as a tool to identify factors important for islet morphogenesis.  Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal pancreas tissue from wild type and pdx1PBHnf6 animals.  We report the identification of genes with an altered expression in HNF6 Tg animals and highlight factors with potential importance in islet morphogenesis.
Keywords: disease state analysis, developmental stage analysis

overall_design: Whole genome microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal pancreas tissue from wild type (WT) animals and HNF6 transgenic animals at a time when the events of normal islet morphogenesis are occurring (e18.5 and postnatal day 1). RNA was isolated from individual pancreata at the appropriate developmental stage. Highly pure samples were pooled according to their genotype (3-5 animals per pool) in order to obtain an adequate quantity of RNA for Affymetrix GeneChip analysis.  Twelve samples were run in total, 3 replicates each of 4 groups: embryonic day 18.5, wild type mouse; embryonic day 18.5, HNF6 Transgenic mouse; post-natal day 1, wild type mouse; post-natal day 1, HNF6 Transgenic mouse.

first: Elizabeth

last: Tweedie

first: Young

last: Oh

first: Braden

last: Boone

first: Shawn

last: Levy

first: Maureen

last: Gannon


title: Inhibitor Trials

city: London

first: Claudine

last: James

email: claudinegj@hotmail.com, fbeier@uwo.ca

state: Ontario

address: 1151 Richmond Street, Suite 2

department: Physiology and Pharmacology

country: Canada

institute: University of Western Ontario

summary:
Objectives: To identify similarities and differences in gene expression data in the MEK/ERK and PI3K pathways and to determine how histone modification affects these same pathways.
Goal: To identify and functionally characterize novel targets of these signaling pathways in the context of chondrocyte differentiation.
Keywords: Teatment, signaling, one-colour array, signaling pathway comparison

overall_design:
Cartilage derived from the hind limbs of CD1 mice staged at 15.5 days of embryonic development were dissected and plated in primary chondrocyte monolayer cultures.  Cells were treated with MEK/ERK pathway inhibitors (SB202190 and UO126), a PI3K inhibitor (LY294002), a histone deacetylase inhibitor (Trichostatin A) and the vehicle control (DMSO) for two hours. Total RNA was isolated from these samples and hybridized to Affymetrix MOE 430 2.0 chips at the London Regional Genomics facility.
A total of 15 genechips were analyzed between 4 treatments and the vehicle (DMSO) control. Three biological replicates per treatment were executed.

first: Veronica

last: Ulici

first: Frank

last: Beier


title: G9a histone methyltransferase maintains genomic imprinting in the mouse placenta.

first: Reiner

last: Schulz

address: 8th floor Guy's Tower

department: Medical & Molecular Genetics

country: United Kingdom

institute: King's College London

summary:
Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic repression of imprinted genes is unclear. ‘Imprinting control regions’ (ICRs), however, are consistently marked by histone H3 K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET-domain protein G9a. We find that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression is unchanged at the domains analysed, in spite of a global loss of H3-K9 di-methylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is lost in the absence of G9, and this correlates with a loss of H3K9me2 and H3K9me3. These findings provide the first in vivo evidence for the involvement of a SET domain protein in imprinting and highlight the importance of histone lysine methylation rather than DNA methylation in the maintenance of imprinting in the trophoblast lineage.
Keywords: genetic modification

overall_design: Number of samples: four (two biologically replicate G9a-/- pooled placentae samples, and two biologically replicate wildtype pooled placentae samples). The first G9a-/- and wildtype replicates were used to hybridize Affymetrix MOE430A and MOE430B arrays (four arrays total). The second replicates were used to hybridize Affymetrix Mouse430_2 arrays (two arrays total).

first: Alexandre

last: Wagschal

first: Heidi

last: Sutherland

first: Kathryn

last: Woodfine

first: Amandine

last: Henckel

first: Karim

last: Chebli

first: Rebecca

last: Oakey

first: Wendy

last: Bickmore

first: Robert

last: Feil


title: Expression data from livers of mice fed four different diets

city: Leipzig

first: Mehmet

last: Somel

address: Deutscher Platz 6

department: Evolutionary Genetics

institute: Max Planck Institute for Evolutionary Anthropology

summary:
Beyond the DNA sequence difference between humans and closely related apes, there are large differences in the environments that these species experience. One prominent example for this is diet. The human diet diverges from those of other primates in various aspects, such as having a high calorie and protein content, as well as being cooked. Here, we used a laboratory mouse model to identify gene expression differences related to dietary differences.
Keywords: diet

overall_design: We fed four groups of six genetically identical 8-week-old female NMR1 mice one of four diets ad libidum: (1) a diet consisting of vegetables, fruit and yogurt identical to the diet fed to chimpanzees in our ape facility ('Chimpanzee'); (2) a diet consisting exclusively of McDonalds. fast food ('FastFood'); (3) a diet consisting of cooked food eaten by our staff in the Institute cafeteria ('HumanCafe'); (4) the mouse pellet diet on which they were raised ('Pellet'). At the end of a 2-week period, mice were euthanized by cervical dislocation and both liver and brain (right cerebral hemisphere) tissue were dissected. RNA was extracted from the 24 liver and brain samples as per established lab protocols and processed in two batches (containing equal numbers of individuals from all groups).

first: Somel

last: Mehmet

first: Creely

last: Hilliary

first: Franz

last: Henriette

first: Uwe

last: Mueller

first: Lachmann

last: Michael

first: Paabo

last: Svante

first: Khaitovich

last: Philipp


title: Genome-wide analysis of transcript isoform variation in humans

status: Public on Jan 13 2008

city: Montreal

first: Tony

last: Kwan

state: Quebec

address: 740 Dr. Penfield

department: Human Genetics

institute: McGill University

summary:
We have performed a genome-wide analysis of common genetic variation controlling differential expression of transcript isoforms in the CEU HapMap population using a comprehensive exon tiling microarray covering 17,897 genes. We detected 324 genes with significant associations between flanking SNPs and transcript levels. Of these, 39% reflected changes in whole gene expression and 55% reflected transcript isoform changes such as splicing variants (exon skipping, alternate splice site usage, intron retention), differential 5’ UTR (initiation of transcription) usage, and differential 3’ UTR (alternative polyadenylation) usage.
These results demonstrate that the regulatory effects of genetic variation in a normal human population are drastically more complex than previously observed. This additional layer of molecular diversity may account for natural phenotypic variation and disease susceptibility.
Keywords: Comparative genomic hybridiation within the CEU HapMap population

overall_design: We used individuals from the HapMap CEU population for our analysis of examining genetic variation in transcript isoforms. 3 biological growths each from 57 individuals (parents of CEU trios) were used.

first: Jacek

last: Majewski


title: Expression data from brains of mice fed four different diets

first: Hilliary

last: Creely

first: Henriette

last: Franz

first: Michael

last: Lachmann

first: Svante

last: Paabo

first: Philipp

last: Khaitovich


title: Gene expression in mouse 3T3-L1 adipocyte tissue culture treated with cis-9,trans-11 conjugated linoleic acid(c9t11 CLA)

status: Public on Jan 11 2008

city: Lincoln

first: Jean-Jack

middle: M.

last: Riethoven

state: NE

address: 1901 Vine Street

department: Beadle Center for Biotechnology

institute: University of Nebraska-Lincoln

summary:
Trans-10, Cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse 3T3-L1 adipocyte tissue culture; however cis-9, trans-11 CLA does not (this series). The early transcriptome changes were analyzed using high-density microarrays to better characterize the signaling pathways responding to c9t11 CLA.  Their gene expression responses between 8 to 12 hr after treatment showed no gene expression changes indicative of an integrated stress response (ISR).
Keywords: control/treatment time course

overall_design: Mouse 3T3-L1 RNA for each time point was isolated from control and treatment samples for analysis on microarrays; there are four biological reps for the controls for each timepoint, and only one rep for the treatment.

first: Christopher

last: LaRosa

first: Han

last: Chen

first: Yuannan

last: Xia

first: You

last: Zhou

first: Mei

first: Jess

last: Miner

last: Fromm


title: Gene expression in 3T3-L1 mouse tissue (preadipocytes) treated with Trans-10,Cis-12 conjugated linoleic acid(t10c12 CLA)

summary:
Trans-10, Cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse white adipose tissue (WAT) and 3T3-L1 adipocyte tissue culture; however in preadipocyte tissue (this series) the UPS/ISR and fat loss is not detected. The early transcriptome changes in 3T3-L1 preadipocyte tissue culture were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA.  Their gene expression responses between 4 to 12 hr after treatment do not show a set of genes indicative of an integrated stress response (ISR).
Keywords: control/treatment time course

overall_design: Mouse 3T3-L1 RNA for each time point was isolated from two control (LA) and two treatment (CLA) samples for analysis on four microarrays.


title: Gene expression data from Hexose-6-phosphate dehydrogenase knockout mouse muscle at 4 weeks

status: Public on Feb 01 2008

city: Birmingham

first: Gareth

last: Lavery

address: 2nd Floor IBR

institute: University of Birmingham

summary:
Hexose-6-phosphate dehydrogenase (H6PD)is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver, H6PD is required for the 11-oxoreductase activity of 11ss-hydroxysteroid dehydrogenase type 1 (11ss-HSD1), which converts inactive 11-oxo glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in Type II (fast) fibers which have increased glycogen content. They also display a progressive vacuolar myopathy evident after 4 weeks of age.
We carried out microarray analysis on TA and soleus muscles from 4 week old WT and KO mice to determine an expression profile predicting myopathy.
Keywords: Age dependant disease state phenotype comparison

overall_design: 4 week old mice are weaned and do not display overt histological evidence of myopathy. Soleus and tibialis anterior are used as comparison groups as they have distinct fibre type content and differing metabolic properties. 3 biological replicates are used for each genotpye and sample type.

last: Turan


title: Gene expression in mouse 3T3-L1 adipocyte tissue culture treated with CLA

summary:
Trans-10, Cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse 3T3-L1 adipocyte tissue culture. The early transcriptome changes were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA.  Their gene expression responses between 4 to 24 hr after treatment showed a common set of early gene expression changes indicative of an integrated stress response (ISR).
Keywords: control/treatment time course

overall_design: Mouse 3T3-L1 RNA for each time point was isolated from control and treatment samples for analysis on microarrays with two to four biological reps.


title: Gene expression in mouse white adipose tissue

summary:
Trans-10, Cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse white adipose tissue (WAT). The early transcriptome changes in WAT were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA.  Their gene expression responses between 4 to 24 hr after treatment showed a common set of early gene expression changes indicative of an integrated stress response (ISR).
Keywords: control/treatment time course

overall_design: Mouse WAT RNA for each time point was isolated from two control and two treatment samples for analysis on four microarrays.  The two control and two treatment samples consisted of RP tissues from 5 pooled mice for each sample, requiring a total of twenty mice for the four samples taken at each time point.


title: Expression data from Murine T cell in response to IL-2 at 10 time points in 24 hours after IL-2 treatment

city: Livermore

first: Zhaoduo

last: Zhang

address: 7011 East Ave

department: Biosystems

institute: Sandia National Labs

summary:
The cytokine IL-2 determines T cell fate by controlling T cell proliferation and differentiation, but the expression files of IL-2 regulated genes are not defined
Using Affymetrix mouse genome array and mouse T cell line CTLL-2, we analyzed the expression profiles of IL-2 regulated genes over 10 time points in 24 hours after IL-2 treatment. A total of 2,813 genes were differentially expressed (?2-fold change) at least at one time point in response to IL-2 stimulation. Clustering analyses showed that these genes consist of 9 clusters, suggesting that the IL-2 regulated genes in mouse T cells have 9 expression patterns. These genes involve in many biological processes, including immune response, signal transduction, apoptosis, cell cycle, transcription, transport, biosynthesis and various metabolisms.

Two supplementary tables are linked to this series:

Table 1:  IL-2 responsive genes over the first 24 hours post-stimulation
Table 2:  Newly identified IL-2 regulated genes function in a variety of biological processes
Keywords: stress response

overall_design: Murine cytotoxic T lymphocyte cell line CTLL-2, derived from a C57BL/6 mouse, is an IL-2 dependent cell line. Cells were cultured in D10 medium in an incubator with 5% CO2 at 37oC to mid-log phase (~2 X 105 cells/mL), and collected by centrifugation, washed with phosphate buffered saline (PBS) (Invitrogen), resuspended at 1 X 106 cells/mL in the medium without IL-2 (IL-2 starvation) and cultured for 14 hours (No IL-2 stimulation, T0, 5 reps). After adding human rIL-2 (100 U/mL, Chiron, IL-2 stimulation), cells were collected at time point 0.5, 1, 2, 4, 6, 8, 10, 12, 16 and 24 hours, at least 3 biological replicates were carried out for each time point.Total RNA were extracted from the cells and was hybridized on Affymetrix GeneChip Mouse Genome 430 2.0 Array.

first: Anthony

last: Martino

first: Jean-Loup

last: Faulon


title: Conjugated linoleic acid suppresses FAS and promotes mammary tumorigenesis in PyV-MT mice

status: Public on Jan 17 2008

city: Santa Monica

first: Margaret

last: Flowers

address: 2200 Santa Monica Blvd.

department: Dept of Experimental Therapeutics

institute: John Wayne Cancer Institute

summary:
Overexpression of fatty acid synthase (FAS) has been reported in both malignant and premalignant breast lesions, and has been associated with poor outcome. FAS has gained interest as a metabolic target for the treatment of breast cancer based on evidence that blockade with the antifungal antibiotic, cerulenin or synthetic inhibitor C75 inhibits proliferation of breast cancer cells and delays tumor development. Conjugated linoleic acid (CLA), a class of fatty acids found in beef and dairy products, has been shown to inhibit FAS in bovine mammary adipose. Based on previously well-documented anti-tumor activity of CLA, we hypothesized that one mechanism of CLA’s anti-tumorigenic activity may be metabolic blockade of FAS.  We fed virgin PyV-MT transgenic mice a diet supplemented with either 1% CLA, as mixed isomers, or control chow for four weeks. Tissue histology was determined by H&E staining.  cDNA microarray and real-time quantitative PCR were performed to determine relative expression of lipogenic genes. Western blots were used to examine relative protein expression of FAS. Differences in protein densitometry were analyzed using Students 2-sided T-test.  Probability was determined using the binomial sign test. Level of significance for all tests was 0.05. H&E staining revealed a shift towards advanced mammary lesions in the CLA-fed mice compared to control animals (24/26 vs. 11/26) (p for trend < 0.001). Microarray analysis revealed a >2-fold decrease in FAS in the CLA-fed group compared to controls, and was confirmed by quantitative RT-PCR (p < 0.001) and Western blot. The decrease in FAS mRNA expression was unexpectedly associated with more advanced disease (p for trend < 0.01). Conclusions: Dietary CLA suppressed fatty acid synthase in the mammary glands of the PyV-MT mouse while promoting mammary tumor progression.
Keywords: dietary intervention to compare mammary tumorigenesis between groups

overall_design: PyV-MT and wild type FVB mice were obtained from Jackson Laboratories (Bay Harbor, ME) and housed 2–4 per cage in micro insulator rooms. Mice were genotyped according to Jackson Laboratory protocols (http://jaxmice.jax.org/pub-cgi/protocols/protocols). PyV-MT positive females were randomly selected at four weeks of age to receive either a control AIN93G chow (n=5) or 1% CLA-supplemented chow (n=6). . Food disappearance and animal weights were recorded weekly. Animals were palpated three times a week, and tumor area (length x width) was measured by caliper. Only palpable masses with an area >/= 0.5 cm2 were considered established growths. Animals were euthanized by CO2 inhalation. Eight-week-old animals were selected for tissue histology and analysis of mRNA and protein expression. Mammary gland sections were collected and processed to obtain mRNA or protein lysate, paraffin embedded for histology, or stored in liquid nitrogen until further processing.

first: Joyce

last: Schroeder

first: Cynthia

last: Thomson

first: David

last: Besselsen

first: Patricia

last: Thompson


title: Comparison of gene expression in the epidermis of Tcfap2c mutant and control skin at embryonic day 16.5

city: Logan

first: Jill

last: Guttormsen

state: UT

address: 5600 Old Main Hill

department: ADVS

institute: Utah State University

summary:
The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development.  Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.
Keywords: genetic modification

overall_design: Skin samples were collected from 3 Tcfap2c and 3 control animals at embryonic day 16.5.

first: Quinton

last: Winger

first: Jillian

first: Maranke

middle: I

last: Koster

first: John

last: Stevens

first: Dennis

last: Roop

first: Trevor

last: Williams


title: Expression data during stem cell differentiation

status: Public on Jan 07 2008

city: Rochester

first: Atta

last: Behfar

state: MN

address: 200 First Street SW

institute: Mayo Clinic

summary:
Stem cell development requires selection of specific genetic programs to direct cellular fate. Using microarray technology, we profile expression trends at selected timepoints during stem cell differentiation to characterize these changes.
Keyword(s): timecourse

overall_design: Total RNA was isolated using the Micro-to-Midi isolation kit (Invitrogen), and subjected to comparative gene expression profiling by labelled cRNA hybridization to the mouse genome 430 2.0 microarray (Affymetrix). Data acquired using the GeneChip Scanner 3000 was analyzed with the Genespring GX 7.3 microarray data software bioinformatics suite (Agilent Technologies) restricting the derived gene list to identify differentially expressed genes defined by a >1.5-fold difference and P<0.05. Data population sets were normalized to the undifferentiated phenotype, and quality filtered to eliminate background noise prior to hierarchical clustering.

first: Carmen

last: Perez-Terzic

first: Randolph

middle: S

last: Faustino

middle: K

last: Arrell

first: Denice

last: Hodgson

first: Satsuki

last: Yamada

first: Michel

last: Puceat

first: Nicolas

last: Niederlander

first: Alexey

last: Alekseev

first: Leonid

last: Zingman

first: Andre

last: Terzic


title: Comparison analysis of 2 week old C57BL6J lung exposed to 2 weeks of cigarette smoke compared to age-matched controls

status: Public on Jan 01 2008

city: Baltimore

first: Sharon

last: McGrath-Morrow

address: 200 N.Wolfe St.

department: Pediatrics

institute: Johns Hopkins

summary:
We hypothesize that gene expression in the cigarette smoke (CS) exposed neonatal lung and age-matched controls will be divergent.  CS exposed lung will have divergence of immune response genes and  structural genes.  The lungs of (6) 2 week old neonatal mice exposed to 2 weeks of CS were compared to the lung of (4) 2 week old age-matched control mice.  We utilized microarray analysis to examine transcriptional differences between smoke exposed neonatal lung and age-matched controls.
Keywords: comparative expression profiling

overall_design: This study utilizes microarray analysis to test these hypotheses. Six sets of lungs were harvested from CS exposed mice and four sets of lungs were harvested from age-matched control mice.  RNA was isolated and used for global gene expression profiling (Affymetrix Mouse 430 2.0 array). Statistically significant gene expression was determined as a minimum 6 counts of 9 pairwise comparisons, minimum 1.5-fold change, and p < 0.05. Further, Absolute | FC - FC SEM | >= 1.5.

first: Shyam

last: Biswal

first: Tirumalai

last: Rangasamy

first: Cecilia

last: Cho


title: Expression profiling of cyclin D1 splice variants cyclin D1a and D1b in mouse 3T3 cells.

city: Philadelphia

first: Mathew

last: Casimiro

state: PA

address: 1035 BLSB, 233 South 10th street

department: Cancer Biology

institute: Thomas Jefferson University

summary:
Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. Analysis was performed of mouse cyclin D1 knockout 3T3 cells infected with splice variants of cyclin D1. 3T3 cells transduced with retroviral vectors expressing each cyclin D1 isoform were processed for expression analysis.
Keywords: Cancer associated risk factor

overall_design: Three Mouse Embryonic Fibroblasts cell lines obtained from littermate cyclin D1 knockout E14 embyos were serial passaged to obtain cyclin D1 knockout (D1KO) 3T3 cells. Each of the D1KO 3T3 cell lines were infected using the Murine Stem Cell Virus (MSCV) expressing splice variants of cyclin D1; cyclin D1a/ cyclin D1b or GFP control to give triplicate sample sets.  7 days post infection total RNA from each sample was extracted using Trizol and further purified using Qiagen’s RNeasy Kit. Preparation of biotinylated cRNA and hybridization to oligonucleotide arrays (Affymetrix mouse genome genechip 430 2.0) were performed in conjuction with Pestell lab and the Nucleic Acid Core Facility at Thomas Jefferson University. Mouse 430 2.0 genechip contains 39,000 transcripts. Gene chips were scanned and analyzed using Robust Multi-array Average (RMA) algorithm.

first: Zhiping

last: Li

first: Chenguang

last: Wang

first: Richard

last: Pestell


title: NUP98-NSD1 links H3K36 methylation to Hox-A gene activation and leukaemogenesis

status: Public on Jan 04 2008

city: La Jolla

first: Mark

middle: Philip

last: Kamps

address: 9500 Gilman Drive

department: Pathology

institute: University of California, San Diego

summary:
Nuclear receptor-binding SET domain protein 1 (NSD1) prototype is a family of mammalian histone methyltransferases (NSD1, NSD2/MMSET/WHSC1, NSD3/WHSC1L1) that are essential in development and are mutated in human acute myeloid leukemia (AML), overgrowth syndromes, multiple myeloma and lung cancers. In AML, the recurring t(5;11)(q35;p15.5) translocation fuses NSD1 to nucleoporin-98 (NUP98). Here, we present the first characterization of the transforming properties and molecular mechanisms of NUP98-NSD1. We demonstrate that NUP98-NSD1 induces AML in vivo, sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HoxA7, HoxA9, HoxA10 and Meis1 proto-oncogenes. Mechanistically, NUP98-NSD1 binds genomic elements adjacent to HoxA7 and HoxA9, maintains histone H3 Lys 36 (H3K36) methylation and histone acetylation, and prevents EZH2-mediated transcriptional repression of the Hox-A locus during differentiation. Deletion of the NUP98 FG-repeat domain, or mutations in NSD1 that inactivate the H3K36 methyltransferase activity or that prevent binding of NUP98-NSD1 to the Hox-A locus precluded both Hox-A gene activation and myeloid progenitor immortalization. We propose that NUP98-NSD1 prevents EZH2-mediated repression of Hox-A locus genes by colocalizing H3K36 methylation and histone acetylation at regulatory DNA elements. This report is the first to link deregulated H3K36 methylation to tumorigenesis and to link NSD1 to transcriptional regulation of the Hox-A locus.
Keywords: expression analysis

overall_design:
Total RNA was extracted from stably transformed progenitors cultured in vitro and the expression levels of mRNA transcripts quantified using the Affymetrix GeneChip Mouse Genome 430 2.0 array, as previously described. The GEO database accession numbers: for progenitors immortalized by HoxA9 (GSM190542, GSM190546, GSM190547); for progenitors immortalized by coexpressed HoxA9 plus Meis1 (GSM190548, GSM190549, GSM190550); for progenitors immortalized by NUP98-NSD1 (GSM190551, GSM190552, GSM190553); and for progenitors immortalized by MLL-ENL (GSM190554).

NOTE: CEL files and dChip data were requested by GEO but not provided.


title: mRNA expression analysis of undifferentiated Dicer +/- (D4) and Dicer -/- (27H10) embryonic cell lines

city: Basel

first: Lasse

last: Sinkkonen

address: Maulbeerstrasse 66

institute: Friedrich Miescher Institute

summary:
We have analyzed the transcript expression levels in Dicer heterozygous and Dicer knock-out embryonic stem (ES) cells  in order to identify which transcripts are regulated by RNAi pathway in mouse ES cells.
Keywords: Cell type comparison of cell line with or without knock-out

overall_design: Two cell lines were analysed in an undifferentiated state. Triplicates of both cell lines were analyzed.

first: Petr

last: Svoboda

first: Witold

last: Filipowicz


title: Identification and characterization of genes expressed in the mouse ZPA using a novel microarray approach

city: Gainesville

first: Brian

last: Harfe

state: FL

address: 1376 Mowry Road

department: Molecular Genetics and Microbiology

institute: University of Florida

summary:
Comparing gene expression of cells from the E10.5 limb bud ZPA and the rest of the E10.5 limb bud from Shhgfpcre heterozygotes separated by FACS.
Keywords: comparison, cell-type comparison, experiemental versus control

overall_design: 8 samples, 4 ZPA and 4 rest of the limb

first: Jason

last: Rock

first: M

last: Lopez

first: Henry

middle: V

last: Baker

middle: D


title: Gene expression of Wt vs CYP26A1-/- murine ES cells treated with control or 100 nM RA for 8 or 72 hr.

status: Public on Jan 02 2008

city: New York

first: Simne

last: Langton

state: NY

address: 1300 York Ave, E405

department: Pharmacology

institute: Weill Cornell Medical College

summary:
The goal of this study was to identify genes that are differentially expressed after genetic deletion of both alleles of the Cyp26a1 gene in murine embryonic stem cells.  Cyp26a1 codes for the CYP26A1 enzyme which metabolizes RA to polar RA metabolites, such as 4-oxo-RA and 4-OH-RA.  CYP26A1-/- ES cells do not metabolize RA within 48 hours of RA treatment while in Wt ES cells, polar RA metabolites are already detectable by 8 hr.  In addition, the absence of CYP26A1 enzyme increases intracellular RA levels.  By gene microarray analysis, we wanted to identify genes that would be affected by the lack of the Cyp26a1 gene.
Keywords: cell type comparison, time course

overall_design: Wt and CYP26A1-/- ES cells were treated with control+LIF for 8 hours, and this was repeated 3 times.  Wt and CYP26A1-/- ES cells were treated with 100 nM RA +LIF for 8 hours, and this was repeated 3 times.  Wt and CYP26A1-/- ES cells were treated with 100 nM RA +LIF for 72 hours, and this was repeated 2 times.

first: Lorraine

middle: J

last: Gudas


title: Individual retinal progenitor cells display extensive heterogeneity of gene expression

status: Public on Dec 31 2007

city: Boston

first: jeffrey

last: trimarchi

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary:
The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed.  While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity.  Additionally, the expression of cell cycle related transcripts showed differences among those associated with G2 and M, versus G1 and S phase, suggesting different levels of regulation for these genes.  These data provide insights into the types of processes and genes that are fundamental to cell fate choices, proliferation decisions, and, for cells of the central nervous system, the underpinnings of the formation of complex circuitry.
Keywords: Single retinal cell gene expression profiling across multiple mouse developmental stages

overall_design: Single retinal cells were isolated in tubes containing lysis buffer, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles.  Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software.  A total of 42 retinal progenitors were identified post hoc and compared with ganglion, amacrine and photreceptor cells isolated from identical timepoints.

first: Jeffrey

middle: M

last: Trimarchi

first: Constance

middle: L

last: Cepko


title: Molecular heterogeneity of developing retinal ganglion and amacrine cells

summary:
During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types.  As these different cell types are born, they progress from newly specified cells to fully differentiated neurons and glia.  In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina.  Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. These experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types.  In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type.
Keywords: Single retinal cell gene expression profiling across multiple mouse developmental stages

overall_design: Single retinal cells were isolated in tubes containing lysis buffer, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software.  Cells were identified post hoc as either developing retinal ganglion cells, amacrine cells or  rod photoreceptor cells.

first: Ben

last: Sun


title: mRNA expression analysis of undifferentiated Dicer -/- (27H10) embryonic stem cells after miRNA transfection

summary:
We have analyzed the transcript expression levels in Dicer knock-out embryonic stem (ES) cells 24 hours after transfection with either control siRNA agains Renilla luciferase or miRNA Mimics (Dharmacon) of mmu-miR-290 cluster members in order to identify primary targets of miR-290 cluster miRNAs.
Keywords: Comparison of effect of two different transfections on transcriptome of Dicer-KO ES cells

overall_design: Cell were analysed 24h after transfections in an undifferentiated state. Triplicates of both transfections were analyzed.


city: Stanford

first: John

last: Cahoy

address: 299 Campus Drive

department: Department of Neurobiology

institute: Stanford University

summary:
A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate astrocytes, neurons, and oligodendrocytes.  Here we describe the first method for the isolation and purification of developing and mature astrocytes from mouse forebrain.  This method takes advantage of the expression of S100β by astrocytes.  We used fluorescent activated cell sorting (FACS) to isolate EGFP positive cells from transgenic mice that express EGFP under the control of an S100β promoter.  By depletion of astrocytes and oligodendrocytes we obtained purified populations of neurons, while by panning with oligodendrocyte-specific antibodies we obtained purified populations of oligodendrocytes.  Using GeneChip Arrays we then created a transcriptome database of the expression levels of over 20,000 genes by gene profiling these three main CNS neural cell types at postnatal ages day 1 to 30.  This database provides the first global characterization of the genes expressed by mammalian astrocytes in vivo and is the first direct comparison between the astrocyte, neuron, and oligodendrocyte transcriptomes.  We demonstrate that Aldh1L1, a highly expressed astrocyte gene, is a highly specific antigenic marker for astrocytes with a substantially broader, and therefore potentially more useful, pattern of astrocyte expression than the traditional astrocyte marker GFAP.  This transcriptome database of acutely isolated and highly pure populations of astrocytes, neurons and oligodendrocytes provides a resource to the neuroscience community by providing improved cell type specific markers and for better understanding of neural development, function, and disease.
We acutely purified mouse astrocytes from early postnatal ages (P1) to later postnatal ages (P30), when astrocyte differentiation is morphologically complete (Bushong et al., 2004), and acutely purified mouse OL-lineage cells from stages ranging from OPCs to newly differentiated OLs to myelinating OLs.  We extracted RNA from each of these highly purified, acutely isolated cell types and used GeneChip Arrays to determine the expression levels of over 20,000 genes and construct a comprehensive database of cell type specific gene expression in the mouse forebrain.  Analysis of this database confirms cell type specific expression of many well characterized and functionally important genes.  In addition, we have identified thousands of new cell type enriched genes, thereby providing important new information about astrocyte, OL, and neuron interactions, metabolism, development, and function.  This database provides a comparison of the genome-wide transcriptional profiles of the main CNS cell types and is a resource to the neuroscience community for better understanding the development, physiology, and pathology of the CNS.
Keywords: Developmental CNS Cell type comparision

overall_design: FACS purification of astrocytes: Dissociated forebrains from S100β-EGFP mice were resuspended in panning buffer (DBPS containing 0.02% BSA and 12.5 U/ml DNase) and sequentially incubated on the following panning plates: secondary antibody only plate to deplete microglia, O4 plate to deplete OLs, PDGFRα plate to deplete OPCs, and a second O4 plate to deplete any remaining OLs.  This procedure was sufficient to deplete all OL-lineage cells from animals P8 and younger, however, in older animals that had begun to myelinate, additional depletion of OLs and myelin debris was accomplished as follows.  The nonadherent cells from the last O4 dish were harvested by centrifugation, and the cells were resuspended in panning buffer containing GalC, MOG, and O1 supernatant and incubated for 15 minutes at room temperature.  The cell suspension was washed and then resuspended in panning buffer containing 20 μg donkey anti-mouse APC for 15 minutes.  The cells were washed and resuspended in panning buffer containing propidium iodide (PI).  EGFP+ astrocytes were then purified by fluorescence activated cell sorting (FACS).  Dead cells were gated out using high PI staining and forward light scatter.  Astrocytes were identified based on high EGFP fluorescence and negative APC fluorescence from indirect immunostaining for OL markers GalC, MOG, and O1.  Cells were sorted twice and routinely yielded >99.5% purity based on reanalysis of double sorted cells.; FACS purification of neurons: EGFP- cells were the remaining forebrain cells after microglia, OLs, and astrocytes had been removed, and were primarily composed of neurons, and to a lesser extent, endothelial cells (we estimate < 4% endothelial cells at P7 and < 20% endothelial cells at P17).  EGFP- cells from S100β-EGFP dissociated forebrain were FACS purified in parallel with astrocyte purification and were sorted based on their negative EGFP fluorescence immunofluorescence.  Cells were sorted twice and routinely yielded >99.9% purity.  In independent preparations, the EGFP- cell population was additionally depleted of endothelial cells and pericytes by sequentially labeling with biotin-BSL1 lectin and streptavidin-APC while also labeling for OL markers as described above.  Cells were sorted twice and routinely yielded >99.9% purity.; Panning purification of oligodendrocyte lineage cells: Dissociated mouse forebrains were resuspended in panning buffer.  In order to deplete microglia, the single-cell suspension was sequentially panned on four BSL1 panning plates.  The cell suspension was then sequentially incubated on two PDGFRα plates (to purify and deplete OPCs), one A2B5 plate (to deplete any remaining OPCs), two MOG plates (to purify and deplete myelinating OLs), and one GalC plate (to purify the remaining PDGFRα-, MOG-, OLs).  The adherent cells on the first PDGFRα, MOG, and GalC plates were washed to remove all antigen-negative nonadherent cells.  The cells were then lysed while still attached to the panning plate with Qiagen RLT lysis buffer, and total RNA was purified.  Purified OPCs were >95% NG2 positive and 0% MOG positive.  Purified Myelin OLs were 100% MOG positive, >95% MBP positive, and 0% NG2 positive.  Purified GalC OLs depleted of OPCs and Myelin OLs were <10% MOG positive and ~50% weakly NG2 positive, a reflection of their recent development as early OLs.; Data normalization and analysis: Raw image files were processed using Affymetrix GCOS and the MAS 5.0 algorithm.  Intensity data was normalized per chip to a target intensity TGT value of 500, and expression data and absent/present calls for individual probe sets were determined.  Gene expression values were normalized and modeled across arrays using the dChip software package with invariant-set normalization and a PM model. (www.dchip.org, Li and Wong, 2001).  The 29 samples were grouped into 9 sample types: Astros P7-P8, Astros P17, Astros P17-gray matter (P17g), Neurons P7, Neurons P17, Neurons-endothelial cell depleted (P7n, P17n), OPCs, GalC-OLs, and MOG-OLs.  Gene filtering was performed to select probe sets that were consistently expressed in at least one cell type, where consistently expressed was defined as being called present and having a MAS 5.0 intensity level greater than 200 in at least two-thirds of the samples in the cell type.  We identified 20,932 of the 45,037 probe sets that were consistently expressed in at least one of the nine cell types.  The Significance Analysis of Microarrays (SAM) method (Tusher et al., 2001) was used to determine genes that were significantly differentially expressed between different cell types (see Supplemental Table S2 for SAM cell type groupings).  Clustering was performed using the hclust method with complete linkage in R.  Expression values were transformed for clustering by computing a mean expression value for the gene using those samples in the corresponding SAM statistical analysis, and then subtracting the mean from expression intensities.  In order to preserve the log2 scale of the data, unless otherwise indicated, no normalization by variance was performed.  Plots were created using the gplots package in R.  The Bioconductor software package (Gentleman et al., 2004) was used throughout the expression analyses.  Functional analyses were performed through the use of Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com).

last: Emery

first: Yi

last: Xing

first: Amit

last: Kaushal


title: Gene expression in mouse 3T3-L1 adipocyte tissue culture treated with tunicamycin

first: Jean-Jack

last: Riethoven

summary:
Tunycamcin induces UPR/ISR and Inflammation in mouse 3T3-L1 adipocyte tissue culture. The early transcriptome changes were analyzed using high-density microarrays to better characterize the signaling pathways responding to tunicamycin, to be compared with similar experiments with CLA as the treatment.  Their gene expression responses between 4 to 12 hr after treatment showed a common set of early gene expression changes indicative of a UPR/Inflammation stress response.
Keywords: control/treatment time course

overall_design: Mouse 3T3-L1 RNA for each time point was isolated from control and treatment samples for analysis on microarrays with two biological reps.

first: Christopher

last: LaRosa

first: Han

last: Chen

first: Yuannan

last: Xia

first: You

last: Zhou

first: Mei

first: Jess

last: Miner

first: Michael

last: Fromm


title: IgA impact on distal small intestine after colonization with B. thetaiotaomicron

status: Public on Dec 27 2007

city: St Louis

first: Daniel

last: Peterson

address: 4444 Forest Park

department: Molecular Biology and Pharmacology

institute: Center for Genome Sciences

summary:
Wildtype B6, Rag1-/- B6 and Rag1-/- B6 mice harboring the 225.4 IgA producing hybridoma were colonized for 10 days with Bacteroides thetaiotaomicron
Keywords: RNA Expression Array

overall_design: Distal small intestine segments were snap frozen immediately after the mice were sacrificed, and small intestine divided into 16 equal segments, segments 10, 11, 12 and 14 were pooled and RNA extracted.

last: Gordon


title: Impaired heart function in Apelin gene-deficient mice

status: Public on Dec 25 2007

city: Toronto

first: Sara

last: Arab

state: ONT

address: 610 University Avenue

department: Cardiology

institute: Toronto General Hospital

summary:
The endogenous peptide Apelin is crucial for maintaining heart function in pressure overload and aging
Keywords: knockout

overall_design: Heart samples from Apelin knockout mice with pressure overload and sham control together with the wild-type mice with pressure overload and sham were compared

first: Liyong

last: Zhang

first: Peter

last: Liu

first: Keiji

last: Kuba

first: Josef

last: Penninger


title: Alpha2-6-linked Sialic Acids on N-Glycans Modulate Carcinoma Differentiation In Vivo

first: Maria

last: Hedlund

address: 9500 Gilman Dr CMM-E #1087

institute: UCSD

summary:
Sialic acids on vertebrate cell surfaces mediate many biological roles. Altered expression of certain sialic acid types or their linkages can have prognostic significance in human cancer. A classic but unexplained example is enhanced α2-6-sialylation on N-glycans, resulting from over-expression of the Golgi enzyme β-galactoside:α2-6-sialyltransferase (ST6Gal-I). Previous data supporting a role for the resulting Siaα2-3Galβ1-4GlcNAc (Sia6LacNAc) structure in tumor biology were based on in vitro studies in transfected carcinoma cells, in which increased Sia6LacNAc on β1-integrins enhanced their binding to ligands, and stimulated cell motility. Here we examine for the first time the in vivo role of the ST6Gal-I enzyme in the growth and differentiation of spontaneous mammary cancers in mice transgenic for an MMTV-promoter-driven polyoma-middle-T antigen, a tumor in which beta1-integrin function is important for tumorigenesis, and in maintaining the proliferative state of tumor cells. Tumors induced in St6gal1 null animals were more differentiated in comparison to those in the wild-type background, both by histological analysis and by protein expression profiles. Furthermore, we show the St6gal1 null tumors have selectively altered expression of genes associated with focal adhesion signaling, and have decreased phosphorylation of FAK, a downstream target of β1-integrins.  This first in vivo evidence for a role of ST6Gal-I in tumor progression was confirmed using a novel approach, which conditionally restored St6gal1 in cell lines derived from the null tumors. These findings indicate a role for ST6Gal-I as a mediator of tumor progression, with its expression causing a less differentiated phenotype, via enhanced β1-integrin function.
Keywords: sialic acid, genetic modification, mouse mammary carcinoma

overall_design: Messenger RNA from tumors arising in wild-type or St6gal1 null littermates was used for the gene expression analysis on the Mouse Genome 430 2.0 Array (Affymetrix).  Six PyV-mT St6gal1 null and wild-type tumor pairs were used, in four independent pools of three tumors each. Each sample is a pool of three different tumors from three different animals. For the first set of microarray analysis samples GSM239969 and GSM239970 were being compared, and in the second set, samples GSM239971 and GSM239972 were compared.

first: Elisa

last: Ng

first: Ajit

last: Varki

first: Nissi


title: Eradication of Solid Human Tumors in Nude Mice with an Intravenously Injected Light-Emitting Oncolytic Vaccinia Virus

status: Public on Dec 26 2007

city: Doha

first: Francesco

middle: Maria

last: Marincola

address: Al Nasr Tower, AL Corniche Street, PO Box 26999

country: Qatar

institute: Sidra Medical and Research Center

summary:
Previously we reported that a recombinant vaccinia virus (VACV) carrying a light-emitting fusion gene enters, replicates in, and reveals the locations of tumors in mice. A new recombinant VACV, GLV-1h68, as a simultaneous diagnostic and therapeutic agent, was constructed by inserting three expression cassettes (encoding Renilla luciferase-green fluorescent protein (RUC-GFP) fusion, b-galactosidase, and b-glucuronidase) into the F14.5L, J2R (encoding thymidine kinase, TK), and A56R (encoding hemagglutinin, HA) loci of the viral genome, respectively. Intravenous (i.v.) injections of GLV-1h68 (1 × 107 pfu/mouse) into nude mice with established (500 mm3)  subcutaneous (s.c.) GI-101A human breast tumors were used to evaluate its toxicity, tumor targeting specificity and oncolytic efficacy. GLV-1h68 demonstrated an enhanced tumor targeting specificity and much reduced toxicity compared to its parental LIVP strains. The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus-mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of interferon stimulated genes (STAT-1 and IRF-7), cytokines, chemokines and innate immune effector function. These findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers.
Objective: To determine the gene expression changes induced by GLV-1h68 vaccinia virus injection in mouse carrying human breast cancer implant and leading to tumor eradication.
Methods: Gene expression was analyzed using oligonucleotide microarrays. Responsiveness to vaccina virus injection was assessed by toxicity and survival study, gene expression anaysis and tumor volume change.
Result: The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus-mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of interferon stimulated genes (STAT-1 and IRF-7), cytokines, chemokines and innate immune effector function.
Conclusion: Our findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers.
Keywords: Responsiveness to vaccina virus injection

overall_design: tumor tissues 3 and 6 weeks post virus injection


title: Gene expression analysis of SMARTA in response to LCMV or Lm-gp61 infection

city: Salt Lake City

first: Matthew

last: Williams

address: 15 N. Medical Dr. E.

institute: University of Utah

summary:
Following infection with LCMV, CD4+ SMARTA TCR transgenic cells (specific for the gp61-80 epitope of the LCMV glycoprotein) rapidly expand, become effector cells, and go on to form a long-lived memory population.  Following infection with a recombinant Listeria monocytogenes expressing the LCMV epitope gp61-80, SMARTA cells also expand but display defective effector differentiation and fail to form memory.  In an attempt to understand the signals required for CD4 T cell memory differentiation, we compared gene expression by SMARTA cells at the peak of the primary response following either Lm-gp61 or LCMV infection.
Keywords: response to LCMV or Lm-gp61 infection

overall_design: SMARTA cells were purified at day 7 post-infection with either LCMV or Lm-gp61.  SMARTA cells were sorted on the basis of Thy1.1 expression using a FACSAria.  Cells were sorted through the machine twice to enhance purity.  Two biological replicates of each group are provided.  Each replicate represents the results of SMARTA pooled from three animals.

last: Bevan


title: Endothelial Cell Line Expression Patterns

status: Public on Dec 23 2007

first: Levi

last: Ledgerwood

address: One Gustave L. Levy Place

department: Gene and Cell Medicine

institute: Mount Sinai School of Medicine

summary:
Sphingosine 1-phosphate (S1P) influences T cell migration into and out of secondary lymphoid organs; however, its mechanism of action remains uncertain. Our previous research shows that agonism of the S1P receptor S1P1 inhibits the egress of T lymphocytes from the peripheral tissues into afferent lymphatics. To better define the mechanism of inhibition, we developed an in vitro model to characterize T cell transendothelial migration across lymphatics.  Two commercially available endothelial cell lines (MS-1 and SVEC4-10) were characterized by flow cytometry, real time RT-PCR, and Affymetrix Gene Array. These cell lines were grown to confluent monolayers in transwell systems, on either the upper or lower surface of the transwell insert. T cells were isolated from the spleens of (C57BL/6 x C3H/HeJ)F1, S1P1 KO, or S1P1 KO littermate controls, and either treated with the S1P receptor modulator FTY720 or left untreated. Cells were migrated to chemokines (CCL19 or CCL21) for 4 hours, and migration quantified.  Flow cytometry, RT-PCR, and array results identified MS-1 as a blood vascular endothelial cell line, expressing high levels of CD31, CD34, and ICAM-1 as well as other endothelial cell markers; while SVEC4-10 closely resemble a lymphatic phenotype, expressing LYVE-1, VEGFR-3, and podoplanin. T cells efficiently migrate across MS-1, whether grown on the upper or lower surface; whereas migration across SVEC4-10 only occurs when cells are grown on the lower surface of the transwell (iSVEC), recapitulating basal (abluminal) to apical (luminal) migration that occurs in vivo. FTY720 inhibits T cell migration across iSVEC, but not across MS-1. Inhibition is due to drug effects only on T cells but not endothelial cells. S1P1 KO T cells treated with FTY720 are not inhibited in their migration across the iSVEC line, showing that S1P1 stimulation is required for migration inhibition.  The in vitro model developed here is the first to use endothelial cell lines to analyze the regulation of T cell migration across lymphatic endothelium. The results show there is directional control of T cell migration across lymphatic cells, such that T cells only migrate from a basal to apical direction. Agonism of S1P1 specifically inhibits migration, while absence of the receptor does not.  These findings have important implications for the use of S1P1 agonists in transplantation, as inhibition of cell entry into afferent lymphatics and lymph nodes could impede the development of graft rejection.
We used microarrays to detail the global gene expression of these two cell lines in order to beter determine their phenotype as blood vascular or lymphatic endothelial cell line for use in our newly-developed in vitro model.
Keywords: Cell type comparison

overall_design: MS-1 or SVEC4-10 endothelial cell lines were grown in culture flasks for 3-5 days in standard culture medium (see protocol below) until reaching confluence.  Cells were then gently dissociated from flask and cells were placed in Trizol reagent for RNA isolation.

first: Jonathan

last: Bromberg

middle: G


title: integrin alpha7 overexpression effects on skeletal muscle transcriptions

city: Urbana

first: Jianming

address: B107 CLSL, 601 S. Goodwin Avenue

department: Department of Cell and Developmental Biology

institute: University of Illinois at urbana-champaign

summary:
Analysis of integrin alpha7 transgenic mice skeletal muscle transcription profiles comparing to wild type controls. Integrin alpha7 is the major laminin binding integrin in muscle cells. Enhancing its expression has been demonstrated to alleviate pathology in a murine model of Duchenne muscular dystrophy. Results of this study provide insights into the effects of increasing integrin alpha7 expression on skeletal muscle transcription and physiology in vivo.  This analysis also evaluates any potential possible side effects associate with enhancing integrin alpha7 in skeletal muscle.
Keywords: transgenic mice comparision to wild type controls

overall_design: Equal amount of total RNA from six animals of each genotype were pooled to generate a biological replicate. Total of three biological replicates were used. Together, 18 mice of each genotype were used in this analysis.

first: jianming

first: Stephen

last: Kaufman


title: Microarray screening of RARgamma responsive genes in F9 teratocarcinoma cells

status: Public on Dec 18 2007

first: Dan

last: Su

address: 1300 york Ave

summary:
We compared the differentially expressed genes between the F9 Wt cells and F9 RAR gamma knock out cells before and after RA treatment. 3 replicates for each conditions.
We also identified the RA responsive genes in the F9 Wt cells.
Keywords: mutant type

overall_design: F9 wt cells and F9 RAR gamma knock out cells are treated with RA (1 uM) or ethanol for 24 hours. 3 replicates for each condition.

first: Lorraine

last: Gudas


title: Enhancing integrin alpha7 expression effect on myoblast transcription

summary:
Analysis of C2C12 myoblast induced with tetracycline to enhance integrin alpha7 expression. Integrin alpha7 is the major laminin binding integrin in muscle cells. Enhancing its expression has been demonstrated to alleviate pathology in a murine model of Duchenne muscular dystrophy. Results of this study provide insights into the effects of increasing integirn alpha7 expression on muscle cells and possible side effects associate with enhancing integrin alpha7 in muscle cells.
Keywords: comparitive of integrin lapha7 induced and non-induced c2C12 myobalst

overall_design: Triplicate biological smaples for each condition(induced and non-induced) C2C12 myoblast were used.


title: Functional analysis of exon 2 of p130Cas in fibroblasts derived from exon 2-specific knockout mice.

city: Hiroshima

first: Eiso

last: Hiyama

state: Hiroshima

address: 1-2-3, Kasumi, Minami-ku

department: Natural Science Center for Basic Research and Deve

institute: Hiroshima University

summary:
p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD), and a C-terminal Src binding domain (SBD). The SH3 domain of Cas has been shown to associate with various signaling molecules, including focal adhesion kinase (FAK), but its role in cellular function remains unclear. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking the exon 2 encoding the SH3 domain (Cas exon 2∆/∆). In comparison to wild-type (Cas exon 2+/+) cells, Cas exon 2∆/∆ primary fibroblasts showed delayed migration in wound healing and reduced spreading on fibronectin (FN), which would be due to reduced complex formation of Cas exon 2∆/∆ with FAK and CrkII and also to impaired localization of Cas exon 2∆/∆ to focal adhesions on FN. In addition, to analyze downstream signaling pathway regulated by Cas exon 2, we compared gene expression profiles between Cas exon 2+/+ and Cas exon 2∆/∆ cells by a microarray analysis. Interestingly, we found that Cas exon 2-deficiency upregulated expression of CXC Chemokine Receptor-4 (CXCR4), CC Chemokine Receptor-5 (CCR5), and thrombospondin 4, which are implicated in cell motility and adhesion. These results define the role of Cas SH3-encoding exon in cell motility, FAK/Cas/CrkII complex formation, recruitment of Cas to focal adhesions, and regulation of cell adhesion-associated gene expression in primary fibroblasts.
Keywords: SH3-encoding exon, p130Cas

overall_design: RNA samples extracted from Cas exon 2+/+, Cas exon 2+/∆, and Cas exon 2∆/∆ fibroblasts (12.5dpc, two embryos for each genotype) were labeled and hybridized on Affymetrix microarray. Gene expression patterns of Cas exon 2∆/∆ fibroblasts were compared with those of Cas exon 2+/+ and Cas exon 2+/∆ cells.

first: Tatsuya

last: Tazaki

first: Hiroaki

last: Honda


title: Expression data from early symptomatic Sca1154Q/2Q and Sca7266Q/5Q knock-in cerebellum

city: Houston

first: Chad

last: Shaw

state: TX

address: One Baylor Plaza

department: Department of Neuroscience

institute: Baylor College of Medicine

summary:
Comparative analysis of cerebellar gene expression changes occurring in Sca1154Q/2Q and Sca7266Q/5Q knock-in mice
Polyglutamine diseases are inherited neurodegenerative disorders caused by expansion of CAG repeats encoding a glutamine tract in the disease-causing proteins.  There are nine disorders each having distinct features but also clinical and pathological similarities.  In particular, spinocerebellar ataxia type 1 and type 7 (SCA1 and SCA7) patients manifest cerebellar ataxia with degeneration of Purkinje cells.  To determine whether the disorders share molecular pathogenic events, we studied two mouse models of SCA1 and SCA7 that express the glutamine-expanded protein from the respective endogenous loci.  We found common transcriptional changes, with down-regulation of Insulin-like growth factor binding protein 5 (Igfbp5) representing one of the most robust changes.  Igfbp5 down-regulation occurred in granule neurons through a non-cell autonomous mechanism and was concomitant with activation of of the Insulin-like growth factor (IGF) pathway and the type I IGF receptor on Purkinje cells.  These data define one common pathogenic response in SCA1 and SCA7 and reveal the importance of intercellular mechanisms in their pathogenesis.  Given that SCA1 and SCA7 share a cerebellar degenerative phenotype, we proposed that some shared molecular changes might occur in both diseases, and that common molecular alterations could pinpoint pathways that could be targeted to modulate or monitor the pathogenesis of more than one disease.  We focused on transcriptional changes because both ATXN1 and ATXN7 play roles in transcriptional regulation and transcriptional defects can be detected in early-symptomatic stages of both SCA1 and SCA7 mouse models.
To test our hypothesis, we examined cerebellar gene expression patterns in SCA1 and SCA7 knock-in (KI) models--Sca1154Q/2Q and Sca7266Q/5Q mice.
Keywords: comparative disesae state analysis between Sca1154Q/2Q and Sca7266Q/5Q knock-in cerebellum

overall_design: Total cerebellar RNA samples were collected from Sca1154Q/2Q knock-in and wild type mice at the early symptomatic disease stage (4 weeks, n=3 knock-in and 3 wild type; 9-12 weeks, n=3 knock-in and 3 wild type).  In parallel experiments, total cerebellar RNA samples were collected from Sca7266Q/5Q knock-in and wild type mice also at the early symptomatic disease stage (5 weeks, n=5 knock-in and 5 wild type).

first: Jennifer

middle: R

last: Gatchel

first: Kei

last: Watase

first: Christina

last: Thaller

first: James

middle: P

last: Carson

first: Paymaan

last: Jafar-Nejad

first: Tao

last: Zu

first: Harry

middle: T

last: Orr

first: Huda

middle: Y

last: Zoghbi


title: Large-scale analysis of the mouse transcriptome

status: Public on Dec 20 2007

city: Leuven-Heverlee

first: Léon-Charles

last: Tranchevent

address: Kasteelpark Arenberg 10

department: Departement of Electrical Engineering

country: Belgium

institute: Katholieke Universiteit Leuven

summary:
We used microarrays to compare gene expression across different murine tissues.
Keywords: Other

overall_design: Different tissues were dissected from 10-12 week old C57Bl6 mice for RNA extraction and hybridization on Affymetrix microarrays.

first: Leentje

last: Lommel

first: Lieven

last: Thorrez

first: Katleen

last: Lemaire

first: Frans

last: Schuit


title: Striatal gene expression data from 12 weeks-old R6/2 mice and control mice

status: Public on Dec 17 2007

city: Bethesda

first: Alexandre

last: Kuhn

address: 35 Convent Dr

department: NIA

institute: NIH

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

overall_design: Refer to individual Series


title: Striatal gene expression data from 12 weeks-old R6/2 mice and control mice (set 2)

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

overall_design: Striatal samples from 5 R6/2 mutant mice (12 weeks-old) and 4 age-matched wild-type mice.

first: Kirupa

last: Sathasivam

first: Gillian

last: Bates

first: Ruth

last: Luthi-Carter


title: Striatal gene expression data from 12 weeks-old R6/2 mice and control mice (set 1)

overall_design: Striatal samples from 4 R6/2 mutant mice (12 weeks-old) and 5 age-matched wild-type mice.


title: Gene expression analysis of Pax5-/- proB cells transduced with control or EBF retrovirus.

status: Public on Dec 15 2007

city: Chicago

first: Jian

last: Zhou

state: IL

address: 5841 S Maryland Ave

department: functional Genomics Facility

institute: University of Chicago

summary:
We have determined that sustained expression of EBF suppresses alternate lineage genes independently of Pax5.
Keywords: Transcription factor EBF restricts alternate lineage options and promotes B cell fate commitment independently of Pax5.

overall_design: Pax5-/- pro-B cells transduced with control or EBF retrovirus.  Transduced cells were FACS sorted based on GFP expression and total RNA was isolated.   RNA isolated from control or EBF transduced cells was analyzed using the Affymetrix  Rat 230A microarrays.

first: Jagan

last: M.R. Pongubala

first: Daniel

last: L. Northrup

first: David

last: W. Lancki

first: Kay

last: L. Medina

first: Thomas

last: Treiber

first: Eric

last: Bertolino

first: Matthew

last: Thomas

first: Rudolf

last: Grosschedl

last: Allman

first: Harinder

last: Singh