title: Complete embryonic E11.5 metanephric kidney analysis of wild type and Hoxa11, Hoxd11 null targeted mice

city: Cincinnati

first: Kristopher

middle: Robert

last: Schwab

state: OH

address: 3333 Burnett Ave

department: Developmental Biology

institute: Cincinnati Children's Hospital Research Foundation

summary:
E11.5 metanephric mesenchyme and ureteric bud were dissected from the E11.5 kidney rudiment using fine manual microdissection (ureteric bud only) or both fine manual microdissection and laser capture microdissection (metanephric mesenchyme) to define the gene expression profiles of these structures.  Additionally, HoxA11, HoxD11 compound null E11.5 metanephric mesenchyme was obtained through laser capture microdissection allowing analysis of possible Hox targets in kidney development.  Targets from multiple biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430_v2 arrays.
Keywords: embryonic metanephric kidney, kidney development, Hoxa11, Hoxd11, compound null targeted mice

overall_design: Using microdissection techniques, ureteric bud and metanephric mesenchyme were dissected from E11.5 kidney rudiments allowing the identificated genes specifically regulated in either structure. In addition, Hoxa11, Hoxd11 compound null E11.5 metanephric mesenchyme were normalized to wild type embryonic controls allowing the identification of potential Hox targets in normal kidney development.  Each structure/genotype were represented in biological (seperate embryo) replicate.

first: S

last: Potter


title: Changed gene profile in inv cystic kidney

status: Public on May 01 2006

city: Kyoto

first: Noriyuki

last: Sugiyama

state: Kyoto

department: Anatomy and Developmental Biology

country: Japan

institute: Kyoto Prefectural University of Medicine

summary:
Nephronophthisis (NPHP) is a group of progressive renal disorders characterized by a variable number of cysts associated with cortical tubular atrophy.  Recently, mutations in INV have been identified to be responsible for NPHP2. The inv/inv,inv-deltaC::GFP mouse that was created by introduction of inv gene lacking the C-terminus (inv-deltaC) into inv/inv mice develops multiple renal cysts without situs abnormality.  Renal cyst progression was dependent on expression level of inv-deltaC transgene, suggesting that inv protein functions in a dose-dependent manner. Cell proliferation and apoptosis were increased in the inv-deltaC cystic kidneys.  We searched up- and down-regulated genes during cyst development and progression using cDNA microarray.

See appended table for comparison analysis of inv/inv,inv-deltaC::GFP vs +/+,inv-deltaC::GFP (GSM100334 vs GSM100335).
Keywords: apoptosis, cell proliferation, gene expression, inv, inversin, polycystic kidney disease

overall_design: Comparison of gene expression in each two kidneys in 4-week-old inv-deltaC and control mice was carried out by the KURABO Industries, Biomedical Department


title: tourt-affy-mouse-243760

status: Public on May 02 2006

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

country: USA

institute: Translational Genomics

summary:
The early growth response (Egr) family of transcriptional regulators consists of four closely related molecules (Egr1-4) that regulate target genes involved in cellular growth and differentiation.  In the brain, Egr transcription factors have a critical role in learning and memory processing, presumably by regulating effector target genes that alter synaptic efficacy or mediate structural changes in neurons.  Previous work suggests that Egr1 and Egr3 are the most important synaptic activity induced Egr molecules in the brain and they appear to have redundant regulatory function.  How Egr transcriptional regulators influence learning and memory processing in the brain is unknown because target genes regulated by them have not been identified.  Using Affymetrix microarray analysis and Egr loss-of-function mice, we will begin to characterize the gene regulatory networks modulated by Egr transcription factors in the brain.  We anticipate that basic mechanisms related to transcriptional control of learning and memory related plasticity and the identification of plasticity effector molecules that may be involved in synaptic dysfunction associated with degenerative diseases or brain injury will result from these studies.
To identify Egr transcription factor target gene regulation in brain:  Target genes regulated by Egr transcription factors have not been identified in the brain, yet the transcription factors are essential for normal learning and memory processes.  Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from somatosensory cortex to identify genes that are deregulated in Egr1/3 dKO brains.
Egr1 and Egr3 gene expression is coupled to synaptic N-methyl D-aspartate (NMDA) receptor activation, mitogen activated protein kinase (MAPK) signaling engaged by NMDA receptor activation and long term synaptic potentiation (LTP).  Previous studies have demonstrated defects in late phase LTP, long-term memory in hippocampal dependent tasks and reconsolidation of memories in Egr1-deficient mice, but the target effector molecules regulated by Egr transcription factors are not known.  We hypothesize that it will be possible to identify Egr dependent target genes by using Affymetrix microarray analysis to compare gene expression from wild type cerebral cortex that has high levels of Egr protein expression with gene expression in cortex from Egr1/3 double knockout mice.
Egr1 and Egr3 are highly expressed in mouse cortex and hippocampus twenty one days after birth because of the large amount of maternal stimulation they receive prior to weaning.  We will compare the gene expression profile in somatosensory cortex from P21 wild type mice to that of P21 Egr1/3 dKO mice.  We will perform microarray analysis using the Mouse 430 2.0 gene array with RNA samples from 3 wild type and 3 1/3 dKO brains (6 arrays total).  Differentially regulated genes (up-regulated and down-regulated) will be identified from the list of genes with significantly altered expression greater than or equal to 2-fold by paired T test.  Interesting genes will be validated by real-time PCR in wild type and 1/3 dKO brains.  Our main goal is to identify genes that are directly regulated by Egr3.  Recognizing that both direct and indirect target genes may be identified in the list of differentially expressed genes, real-time PCR validated target genes will be further screened using chromatin immunoprecipitation coupled with PCR (ChIP-PCR) to determine whether Egr1 and/or Egr3 are bound to potential regulatory regions of the putative target genes.
Keywords: other

first: Warren

middle: G

last: Tourtellotte


title: Expression data from single cells from ICMs of mouse blastocysts at E3.5

status: Public on Apr 26 2006

city: Nara

first: Kazuki

last: Kurimoto

address: 840 Shijo-Cho, Kashihara

department: School of medicine

institute: Nara Medical University

summary:
The inner cell mass (ICM) of the early blastocyst at E3.5, a source of ES cell derivation, is a morphologically homogeneous population of undifferentiated pluripotent cells that give rise to all embryonic lineages. The immediate application of the newly developed V1V3 method to single cells in this stage of mouse embryos revealed the presence of two populations of cells, one with primitive endoderm expression and the other with pluripotent epiblast-like gene expression.  The genes expressed differentially between these two populations were well preserved in morphologically differentiated primitive endoderm and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations.  This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell, and developmental biology, where small numbers of distinctive or diseased cells play critical roles.
Keywords: Single cell analysis

overall_design: We isolated blastocysts at E3.5 and dissociated the ICM into single cells by trypsin-EDTA treatment.  To prepare cDNA samples, we then randomly picked a total of 55 single cells.  cDNAs were synthesized and amplified by the V1V3 method, and screened by gene-specific PCR using Oct4 and Cdx2 to remove trophectoderm cells, and 50 cells were identified as Oct4-positive and Cdx2-negative, ICM cells.

first: Yukihiro

last: Yabuta

first: Yasuhide

last: Ohinata

first: Yukiko

last: Ono

first: Kenichiro

middle: D

last: Uno

first: Rikuhiro

last: Yamada

first: Hiroki

middle: R

last: Ueda

first: Mitinori

last: Saitou


title: Changes in gene expression in dermal fibroblasts following exposure to Et1 peptide

status: Public on Apr 25 2006

city: Chicago

first: Tammy

last: Vallender

state: IL

department: Human Genetics

institute: University of Chicago

summary:
To determine if aberrant activation of  endothelin-1 (Et1) could lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. Mouse dermal fibroblasts were treated with exogenous Et1 peptide for 24 hours.  ET1 treatment resulted in significant expression changes — primarily downregulation — of a number of genes. In particular, Tgfβ2 and Tgfβ3 were among the downregulated genes, which in turn alter the expression status of their many target genes. These data suggest that the stable silencing of Et1 is important for the phenotypic stability of dermal fibroblasts, and perhaps many other cell types as well.
Keywords: endothelin-1; Et1; dermal fibroblast

overall_design: Three separate biological replicates were derived for both control and treated samples.  The primary dermal fibroblasts were derived by explant procedure from the skin of mouse pups aged 0-3 days.  By passage 5, cells were split to two separate cultures-- one with 100nM synthetic Et1 peptide added to the medium (treated) and the other with nothing added (control).  Cells were exposed to Et1 for 24 hrs, then treated and control populations were harvested for total RNA.

middle: W

first: Bruce

middle: T

last: Lahn


title: Comparison between classically activated and type II activated macrophages to identify novel markers

status: Public on May 17 2006

city: College Park

first: Justin

last: Edwards

state: MD

department: CBMG

institute: University of Maryland

summary:
The purpose of this study is to identify novel markers for the type II activated macrophage, which is generated by classical stimulation in the presence if IgG immune complexes. These cells gererally produce high levels of IL-10 and low levels of IL-12, in comparison to classically activated macrophages. We wish to identify gene expression which is enriched in Type II activated macrophages in comparison to classically activated macrophages.
Keywords: Stimulation comparison

overall_design: The design of this experiment is to simulateously stimulate two popultions of macrophages and compare their gene expression. In this case, macrophages are primed overnight with IFN-gamma, washed, then stimulated with LPS (Classically) or LPS+Immune complexes (Type II).

middle: P

first: Xia

last: Zhang

first: Kenneth

middle: A

last: Frauwirth

first: David

middle: M

last: Mosser


title: Gene expression profiling of whole testes in embryonic (gestational day 11, 12, 14, 16 & 18) and postnatal day 2 mice. (GUDMAP Series ID: 3)

status: Public on May 12 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

country: United Kingdom

institute: IGMM MRC Human Genetics Unit

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

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

first: Kevin

last: Gaido

first: Kim

last: Lehmann


title: RNAi screen for increased resistance to oxidative stress

status: Public on Apr 18 2006

city: Suita

first: Rie

last: Nagaoka-Yasuda

state: Osaka

address: 1-3 Yamadaoka

department: Frontier Bioscience

institute: Osaka University

summary:
Specificity of a short hairpin RNA is veryfied by microarray.
Keywords: RNA interference off-target effect

overall_design: Comparisons between NIH3T3 expressing a shRNA and empty vector

first: Mark

last: Mayford


title: An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis

status: Public on Mar 20 2006

first: Kazuki

last: Kurimoto

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

overall_design: Refer to individual Series

first: Yukihiro

last: Yabuta

first: Yasuhide

last: Ohinata


title: Gene expression changes following demethylation by 5-aza-2'-deoxycytidine in primary mouse dermal fibroblasts

type:
Expression profiling by array
Methylation profiling by array

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

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


title: Cell intrinsic alterations underlie hematopoietic stem cell aging

status: Public on Mar 15 2006

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly.
Groups of assays that are related as part of a time series.
Keywords: time_series_design

overall_design: 5 old mice and 3 young mice

first: Rebecca

last: Sonu


title: katz-affy-mouse-212743

status: Public on Mar 08 2006

first: Winnie

last: Liang

summary:
There are only a few DRG specific genes which have been cloned, but almost no study about level specific gene expression.
Clone different expressed genes in different level of DRGs
There are genes specifically expressed in one level of DRG but not the other.
1. dissect different level of DRGs2. extract total RNA
Keywords: other

first: Lawrence

middle: C

last: Katz


title: Expression Studies of Melanotransferrin in Mouse Brain

status: Public on Mar 29 2006

city: Sydney

first: Des

middle: Raymond

last: Richardson

state: New South Wales

address: 92-94 Parramatta Rd, Sydney

department: Bosch Institute

country: Australia

institute: University of Sydney

summary:
Melanotransferrin (MTf) or melanoma tumor antigen p97 is an iron (Fe)-binding transferrin homolog expressed highly on melanomas and at lower levels on normal tissues. It has been suggested that MTf is involved in a variety of processes such as Fe metabolism and cellular differentiation. Considering the crucial role of Fe in many metabolic pathways e.g., DNA synthesis, it is important to understand the function of MTf. To define the roles of MTf, a MTf knockout (MTf -/-) mouse model was developed. Examination of the MTf -/- mice demonstrated no phenotypic differences compared to wild-type littermates. However, microarray analysis showed differential expression of molecules involved in proliferation such as Mef2a, Tcf4, Gls and Apod in MTf -/- mice compared to MTf +/+ littermates, suggesting a role for MTf in proliferation and tumorigenesis.
Keywords: Melanotransferrin, knockout mouse, comparative genomic hybridization

overall_design: Six 17.5 week old female mice comprising four MTf -/- and two MTf +/+ from the same litter were used. Total RNA was isolated from brain tissue and processed as per standard Affymetrix protocol to obtain final cRNA products that were hybridized to the mouse GeneChip® 430 2.0. The mouse GeneChip® 430 2.0 consists of greater than 39,000 transcripts and variants from over 34,000 well characterized mouse genes. On completion of hybridization and washing, microarray chips were scanned with the Affymetrix GeneChip® Scanner 3000 (Millenium Sciences).

first: Yohan

last: Suryo Rahmanto

first: Louise

middle: L

last: Dunn

first: Eric

middle: O

last: Sekyere


title: Distinct hematopoietic cell fates regulated by graded expression of HOXA10

city: Lund

first: Mattias

last: Magnusson

address: BMC A12

department: Molecular Medicine and Gene Therapy

country: Sweden

institute: Lund university

summary:
The homeobox (Hox) transcription factor HOXA10 has been implicated in regulation of hematopoietic cell fate. Here, using a transgenic mouse model where expression of HOXA10 is tightly regulated in a graded, doxycyclin-dependent manner we demonstrate that several key commitment steps in hematopoietic differentiation  have distinctly different outcomes depending on the expression level of HOXA10. Similarly, HOXA10 regulates hematopoietic stem cell (HSC) proliferation in a dose dependent manner, since intermediate levels of HOXA10 generated a 4.5-fold increase in long-term repopulating capacity after 13 days of liquid culture, whereas high levels reduced proliferation of HSCs. Interestingly, the effects on HSC proliferation were associated with altered expression of several known regulators of stem cell self-renewal. Taken together, our findings reveal entirely novel functional and molecular aspects of HOXA10 in regulation of hematopoiesis and emphasize the need for tightly regulated production of HOX proteins in possible future applications of stem cell expansion.
Keywords: Affymetrix

overall_design: LSK cells were sorted from rtTA-HA10 and cultured for 72 hours in complete serum free medium in three different concentrations of doxycycline (0, 0.1 and 0.5 µg/ml doxycycline). RNA was extracted using RNeasy Mini kit, Qiagen, labeled and amplified according to AffymetrixTM Two Cycle Target Labeling Protocol. Hybridization and washing was performed according to AffymetrixTM GeneChip Expression protocol. Chips were scanned using an AffymetrixTM GeneChip Scanner 3000. The Affymetrix software GCOS was used to generate cell intensity data files (CEL) from two independent experiments. The CEL file data was then imported into the GeneSpring® software version 7.2 (Silicon Genetics, Redwood City, CA). The GC-RMA method was used for normalization and data was processed as follows: values below 0.01 were set to 0.01.  All of the genes in each sample were divided by the median of the specified list of 100 positive control genes present on the MOE430 v2 chip. All samples were then normalized against the median of the untreated control samples. Each measurement for each gene in the treated samples was divided by the median of that gene's measurements in the corresponding control samples. We judged genes to be differentially expressed when the difference in expression between two conditions was at least 2-fold.

first: Ann

last: Brun

first: Noriko

last: Miyake

first: Jonas

last: Larsson

first: Mats

last: Ehinger

first: JonMar

last: Bjornsson

first: Mikael

last: Sigvardsson

first: Stefan

last: Karlsson


title: Temporal control of gene expression by ERK MAP kinase during cell cycle progression from G0/G1 to S phase

status: Public on Jun 15 2006

first: Eisuke

last: Nishida

address: Kitashirakawa, Sakyo-ku

department: Department of Cell and Developmental Biology

institute: Graduate School of Biostudies, Kyoto University

summary:
The ERK family of MAP kinase plays a critical role in growth factor-stimulated cell cycle progression from G0/G1 to S phase. But, how sustained activation of ERK promotes G1 progression has remained unclear. Here, our systematic analysis on the temporal program of ERK-dependent gene expression shows that sustained activation of ERK is required for induction and maintenance of the decreased expression levels of a set of genes. Moreover, our cell biological analysis reveals that these ERK-dependent downregulated genes have the ability to block S phase entry. Cessation of ERK activation at mid or late G1 leads to a rapid increase of these anti-proliferative genes and results in the inhibition of S phase entry. These findings uncover an important mechanism by which the duration of ERK activation regulates cell cycle progression through dynamic changes in gene expression, and identify novel ERK target genes crucial for the regulation of cell cycle progression.
Keywords: time course

overall_design: time points: 0h, 1h, 4h, 7h, 12h

first: Takuya

last: Yamamoto

first: Miki

last: Ebisuya

first: Fumito

last: Ashida


title: Cumulus-oocyte complex temporal expression

status: Public on Feb 17 2006

city: Houston

first: JoAnne

middle: S

last: Richards

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary:
Cumulus-oocyte complexes were isolated a seperate time-points to generate temporal complexes.  Targets from two biological replicates at each time point (0h, 8h, 16h post-hCG treatment) were generated and the expression profiles were determined using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays.  Comparisons between the sample groups allow the identification of genes with temporal expression patterns.
Keywords: time course

overall_design: 2 eCG-primed (48h) pooled cumulus-oocyte complexes, 2 eCG-primed (48h) hCG-treated (8h) pooled cumulus-oocyte complexes, and 2 eCG-primed (48h) hCG-treated (16h) pooled cumulus-oocyte complexes replicates were analyzed

first: Chad

last: Wayne

first: Inmaculada

last: Hernandez-Gonzalez

first: Ignacio

last: Gonzalez-Robayna

first: Masayuki

last: Shimada

first: Scott

last: Ochsner

first: Lisa

last: White


title: Cystic Fibrosis Mouse Lung Profiles

status: Public on Mar 01 2006

city: Montreal

first: Christina

middle: Kathleen

last: Haston

state: QC

address: 3626 St. Urbain

department: Medicine

country: Canada

institute: McGill University

summary:
Gene expression profiling with microarrays was used to identify genes differentially expressed in the lungs of B6 and BALB CF mice compared to non-CF littermates
Keywords: disease state analysis

overall_design: BALB/c and C57BL/6 mice (male and female) were examined in CF-/- and CF+/+ genotypes. 3 arrays of each combination of strain sex and CF status were analyzed (except for B6 male CF, which only had two replicates)

middle: K


title: ruben-affy-mouse-187820

status: Public on Jun 06 2006

first: Winnie

last: Liang

summary:
The Dlx homeobox genes have central roles in controlling patterning and differentiation of the brain and craniofacial primordia. In the brain, loss of Dlx function results in defects in the production, migration and differentiation of GABAergic neurons, that can lead to epilepsy. In the branchial arches, loss of Dlx function leads to craniofacial malformations that include trigeminal axon pathfinding defects. To determine how these genes function, we wish to identify the transcriptional circuitry that lies downstream of these transcription factors by comparing gene expression in wild type with Dlx mutant CNS and craniofacial tissues.
1) Compare gene expression in the maxillay branch of the first branchial arch (BA) of E10.5 wild type and Dlx2 -/- mutants. 2) Compare gene expression in the maxillary branch of the first BA of E10.5 wild type and Dlx1/2 -/- mutants. 3) Compare gene expression in wild type maxillary and mandibular branchial arches. 4) Compare gene expressionin mandibular branch of Dlx5/6 -/- mutants with wild type mandibular branch.
The Dlx transcription factors are essential for controlling patterning of the brain and craniofacial primordia. In the brain, they control differentiation of GABAergic neurons of the basal ganglia. In the branchial arches, they control regional patterning. I hypothesize that there will be some conserved and some divergent mechanisms that the Dlx genes use in controlling brain and craniofacial development. We have already performed array analyses on Dlx function in the developing basal ganglia (with TGEN) by comparing expressed genes in wild type and Dlx1/2 mutants. Here we will compare gene expression in the brachial arches of wild type and Dlx mutant mice.
1) Generate E10.5 mouse embryos that are either wild type, Dlx2-/-, Dlx1/2 -/- or Dlx5/6 -/-. 2) Determine genotype by PCR. 3) Dissect branchial arches from the different genotypes. 4) Separate maxillary and mandibular branch of each branchial arch. 5) Prepare total RNA from the specimens. Obtain sufficient tissue to obtain 10 ug of total RNA - based on previous experience we anticipate that this will require ~ 10 branchial arches. We will pool the tissue from different embryos of the same genotype. 6) Send total RNA to TGEN for probe preparation, hybridization and array result analysis.
Keywords: other

first: John

last: Rubenstein


title: Improved DNA methylation analysis via enrichment of demethylated cells expressing an X-inactivated GFP transgene

status: Public on Apr 27 2006

first: Tammy

last: Vallender

summary:
The demethylating drug 5-aza-2’-deoxycytidine (5-aza-2dC) is frequently used to investigate the
effect of global DNA demethylation on gene expression in cultured mammalian cells. Here, we utilize a novel method that uses the reactivation of an X-inactivated GFP transgene as a marker to enrich for cells that have undergone drug-induced demethylation. By combining it with microarray gene expression profiling, we demonstrate the method’s utility in identifying genes activated by global DNA demethylation.
Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast; X-linked GFP

overall_design:
Mice used have an X-linked GFP transgene driven by the chicken β-actin promoter and CMV
intermediate early enhancer in a 129/ICR mixed genetic background obtained from The Jackson
Laboratory. Male transgenic animals were bred to wildtype BL6 female mice to obtain females that were heterozygous for the transgene. Dermal fibroblasts were obtained from explant cultures of neonatal female mice from these crosses.  The cells were then sorted using FACS to obtain only GFP-nonexpressing cells.  These cells represent the population with the GFP transgene on the inactive X chromosome.  The GFP-silent cells were treated with the demethylating drug 5-aza-2'-deoxycytidine.  This drug does not affect all cells and uses a passive form of demethylation that results in a heterogenous population of unaffected and demethylated cells.  A portion of the cells affected by demethylation activate the inactive GFP transgene by demethylating the inactive X chromosome.  The GFP-active and GFP-inactive cells were sorted by FACS and assayed by microarray to determine what genes were affected by demethylation.  Also the microarray results indicated if sorting for GFP-active cells enriches for cells affected by the drug.

first: Bruce

last: Lahn


title: burde-affy-arabi-64764

summary:
In skeletal muscle, the pattern of electrical activity regulates the expression of proteins involved in synaptic transmission, contraction and metabolism.  Disruptions in electrical activity, resulting from prolonged bed-rest, cast-immobilization or trauma, inevitably lead to muscle atrophy.  The mechanisms that regulate muscle atrophy are poorly understood, but it seems likely that changes in gene expression play a key role in initiating and maintaining a muscle atrophy program.  Previously, we found that Runx1, a transcription factor previously termed AML1, was substantially induced in muscle following denervation.  More recently, we sought to determine whether this increase in Runx1 expression may be causally related to the morphological changes in skeletal muscle that accompany muscle disuse, notably muscle atrophy.  We found that Runx1 is indeed required to sustain muscle and to minimize atrophy following denervation.  Experiments described here are designed to identify the genes that are regulated by Runx1 in skeletal muscle with the particular goal of identifying genes that regulate muscle atrophy.
We propose to use microarray analysis to identify genes, expressed in skeletal muscle, that are mis-regulated in mice lacking Runx1.
We inactivated runx1 selectively in skeletal muscle and found that denervated myofibers in mutant mice atrophy far more (90% atrophy) than in wild-type mice (30% atrophy).   We therefore reason that Runx1 activates and/or represses genes that are required to sustain muscle and to minimize atrophy.
We generated MCK::cre; runx1f/- and runx1f/- control mice.  In normal mice, an increase in runx1 expression is detected by two days after denervation and is maximal by five days after denervation.  Muscle atrophy is first evident between one and two weeks after denervation.  As we wish to avoid detecting global changes in gene expression that are associated with late stages of muscle atrophy, we plan to denervate muscle for three or five days and to compare gene expression in dissected innervated and denervated muscles from mutant and control mice.  We will generate thirty samples for comparison-5 replicates per condition:  Samples 1-3 from runx1f/- control mice. (1) innervated tibialis anterior muscles (TA); (2) 3-day-denervated TA; (3) 5-day-denervated TA. Samples 4-6 from MCK::cre; runx1f/- mice. (4) innervated TA; (5) 3-day-denervated TA; (6) 5-day-denervated TA.  We obtain sufficient total RNA (10 micrograms) from each dissected muscle to avoid pooling samples.  We will analyze adult mice of the same age (~six weeks after birth; most will be littermates) and sex-male. It is difficult to anticipate how many genes will be identified in this screen, as few target genes for Runx1 have been identified in any cell type and none in skeletal muscle.  Moreover, although we would prefer to focus our attention on genes that are strongly dependent upon Runx1 expression (e.g. more than 5-fold difference in expression in wild-type and mutant mice), we do not know the extent to which target gene expression will depend upon Runx1.  For these reasons, in these experiments, we will analyze expression from five “identical” samples, so that we can be confident that even small (e.g. three-fold) differences in expression can be reliably determined.  Importantly, in order to confirm results obtained from the microarray data, we will use other assays (RNase protection) to measure RNA expression of candidate genes in innervated and denervated muscles of wild-type and mutant mice.
Keywords: other

first: Steven

middle: J

last: Burden


title: Gene expression in murine germ cells

status: Public on Feb 09 2006

city: Pullman

first: Lizhong

last: Yang

state: WA

address: 531 Fulmer Hall/POB 644660

department: School of Molecular Biosciences

institute: Washington State University

summary:
In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active [1-8]. On the other hand, the idea that the imprinted paternal X of the early embryo may be pre-inactivated by MSCI suggests that silencing may persist longer [9-12]. To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the post-meiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this post-meiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed post-meiotically, while autosomes are largely active. We conclude that chromosome-wide X-silencing continues from meiosis to the end of spermiogenesis and discuss implications for proposed mechanisms of imprinted X-inactivation.
Keywords: Specific germ cells from murine testis

overall_design: Independent germ cell preps were used for array analysis.  Duplicates were provided for each sample.


title: A function for interleukin 2 in Foxp3-expressing regulatory T cells

city: Seattle

first: Jeffrey

last: Rasmussen

department: Immunology

institute: University of Washington

summary:
Regulatory T cells (Treg cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most Treg cells constitutively express the high-affinity interleukin 2 (IL-2) receptor alpha-chain (CD25); however, the precise function of IL-2 in Treg cell biology has remained controversial. To directly assess the effect of IL-2 signaling on Treg cell development and function, we analyzed mice containing the Foxp3gfp knock-in allele that were genetically deficient in either IL-2 (Il2-/-) or CD25 (Il2ra-/-). We found that IL-2 signaling was dispensable for the induction of Foxp3 expression in thymocytes from these mice, which indicated that IL-2 signaling does not have a nonredundant function in the development of Treg cells. Unexpectedly, Il2-/- and Il2ra-/- Treg cells were fully able to suppress T cell proliferation in vitro. In contrast, Foxp3 was not expressed in thymocytes or peripheral T cells from Il2rg-/- mice. Gene expression analysis showed that IL-2 signaling was required for maintenance of the expression of genes involved in the regulation of cell growth and metabolism. Thus, IL-2 signaling seems to be critically required for maintaining the homeostasis and competitive fitness of Treg cells in vivo.
Keywords: cell type comparison

overall_design: We isolated Foxp3+ CD4+ T cells from Il2-/- mice treated with PBS or recombinant IL-2 for gene expression analysis using whole-genome oligonucleotide microarrays. To identify gene expression changes resulting from IL-2 deficiency and subsequent IL-2 stimulation in Treg cells, we directly compared expression profiles with each other and with expression profiles of Foxp3+ CD4+ T cells isolated from wild-type mice.

first: Jason

last: Fontenot

first: Marc

last: Gavin

first: Alexander

middle: Y

last: Rudensky


title: Analysis of RPE65 loss of function in mouse retina

status: Public on Feb 01 2006

city: Basel

first: Lydia

last: Michaut

address: Klingelbergstrasse 70

country: Switzerland

institute: Biozentrum, Basel University

summary:
To characterize gene response in RPE65-/- mouse model of Leber’s congenital amaurosis during progression of the disease, we analyzed differential gene expression in retinae early in the development of the disease, namely before and at the onset of photoreceptor cell death in knock-out mice of 2, 4 and 6 months of age.
Keywords: knock out response over time

overall_design: We compared gene expression in wild type and RPE65-/- retinae at 2, 4 and 6 months of age. Three biological replicates were performed for each of the six conditions analyzed; this series hence contains 18 samples.

first: Sandra

last: Cottet

first: Walter

last: Gehring

first: Daniel

last: Schorderet


title: Mouse limbal epithelial basal cells vs. corneal epithelial basal cell gene expression profile

status: Public on Jan 27 2006

first: Mingyuan

last: Zhou

address: 303 E. Chicago Ave.

department: Dermatology

summary:
Limbal vs. corneal epithelial basal cell gene expression patterns were identified and compared
Keywords: repeat

overall_design: 8 limbal and 8 corneal epithelial basal cells samples from 8 mice were dissected and mRNAs were isolated and amplified for microarray analysis

first: Robert

last: Lavker


title: Selection for contextual fear conditioning affects anxiety-like behaviors and gene expression (palme-affy-mouse-84746)

status: Public on Jan 13 2006

summary: These data are from the brains (amygdala and hippocampus) of mice originally derived from a cross between C57BL/6J and DBA/2J inbred strains. We used short-term selection to produce outbred mouse lines with differences in contextual fear conditioning, which is a measure of fear learning. We selected for a total of 4 generations. Fear learning differed in the selected lines and this difference was stronger with each successive generation of selection. These mice also showed differences for measures of anxiety-like behavior, but were not different for tests of non-fear motivated learning, suggesting that selection altered alleles that are specifically involved in emotional behaviors. We identified several QTLs for the selection response. We used Affymetrix microarrays to identify differentially expressed genes in the amygdala and hippocampus of mice from the final generation of selection. Amygdala and hippocampus samples were rapidly dissected out of experimentally naïve mice f  rom each selected line. Three samples were pooled and hybridized to each array. Experimentally naïve mice were used because the behavior of the mice can be reliably anticipated due to their lineage. Thus, these gene expression differences are not due to the response to human handling, foot shock or fear-inducing conditioned stimuli. We have a second similar study that focuses on a different selected population that was based on C57BL/6J and A/J mice (see GES4034).

first: Abraham

last: Palmer


title: Expression Profile of Day 5 5cc7 T cell blasts

status: Public on Jan 18 2006

first: Morgan

last: Huse

address: 279 Campus Dr

department: Microbiology and Immunology

institute: Stanford

summary:
Our aim was to get a general sense of which proteins are expressed in 5cc7 T cell blasts.  We use these cells for a number of different experiments in our lab
Keywords: MCC peptide stimulated cells

overall_design: RNA was isolated from day 5 5cc7 T cell blasts.  This RNA was converted to cRNA and used to hybridize 2 affymetrix mouse 430 2.0 arrays in parallel

first: Mark

last: Davis


title: Targeting of GFP to new-born rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors

status: Public on Feb 28 2006

city: Ann Arbor

first: Swaroop

last: Anand

state: MI

address: 1000 Wall St.

department: Ophthalmology & Visual Sciences

institute: University of Michigan

summary:
Purpose: To investigate the gene regulatory networks during photoreceptor differentiation.
Special aims: To generate gene expression profiles of purified photoreceptors at distinct developmental stages and from different genetic backgrounds.
Background: Rod photoreceptor genesis spans a broad temporal window during retinal development. It starts as early as E12.5 and peaks at P0-P2. At E16.5, there are some early born rods but the peak of rod genesis does not occur. At P2, the majority of rod photoreceptors are born. At P6, rod specific structural/functional genes begin to express. At P10, Outer segments morphogenesis is taking place. At 4 weeks, retinal development is complete and retina is functional. Nrl is a rod specific transcription factor and one of the earliest markers of rod photoreceptors. Nrl promoter drives the expression of GFP exclusively to rod photoreceptors shortly after they exit cell cycle. In the Nrl-knockout background, the expression of GFP is detected in S-opsin positive cells, which suggested a cell fate transformation from rods to cones in the absence of Nrl.
Design: GFP positive photoreceptors from the WT-Gfp or Nrl-knockout-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol reagent.  Around 50 ng of total RNA was used for linear amplification and biotin labeling followed Nugen kit protocol.  Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.
Conclusion: By comparing the gene expression profiles from different developmental stages, we can obtain novel insights into molecular events underlying photoreceptor differentiation.
Keywords: Transcription factor, development, photoreceptor, retina, neuron, differentiation, gene regulation, microarray, gene profiling, cell type comparison

overall_design: Postmitotic rod precursors and mature rod photoreceptors are tagged by GFP under the control of an Nrl promoter in the wild type background (Wt-Gfp mice). When cross-bred into the Nrl-knockout background (Nrl-ko-Gfp mice), the transformed “S-cones” are tagged by GFP. GFP positive photoreceptors from the Wt-Gfp or Nrl-ko-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol regent and around 50 ng of total RNA was used for linear amplification and biotin labeling following Nugene kit protocol.  Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.

first: Masayuki

last: Akimoto

first: Hong

last: Cheng

first: Dongxiao

last: Zhu

first: Joseph

last: Brzezinski

first: Ritu

last: Khanna

first: Elena

last: Filippova

first: Edwin

last: CT Oh

first: Yuezhou

last: Jing

first: Jose-Luis

last: Linares

first: Matthew

last: Brooks

first: Sepideh

last: Zareparsi

first: Alan

last: Mears

first: Alfred

last: Hero

first: Tom

last: Glaser

first: Anand

last: Swaroop


title: sikes-affy-mouse-84486

first: Winnie

last: Liang

institute: Translational Genomics

summary:
Reversible phosphorylation is a critical step in the control of cellular signaling events. The protein phosphatase 1 (PP1) regulates neuronal proteins by mediating their de-phosphorylation. PP1 selectivity and subcellular localization is conferred via association with a panel of interacting proteins. We have extensively characterized the association of PP1 with inhibitor-1, an interacting protein widely expressed in brain and heart. Inhibitor-1 deletion results in a persistently active PP1 phosphatase in vitro. Inhibitor-1 overexpression results in increased transformation of cultured cells and enhances the rate of learning and memory in mouse models through largely unknown mechanisms. Significantly, inhibitor-1 activation is cAMP-sensitive. The genes regulated by inhibitor-1/PP1 signals have not been characterized.  Identification of these genes is expected to provide key insights into the role of inhibitor-1/PP1 escape from cellular homeostasis and regulation of neuronal learning and memory.
We will determine gene expression patterns in hippocampus of wild-type and inhibitor-1 knock-out mice. The results of these studies of constitutive expression will be used as a baseline for future studies aimed to determine the role cAMP-mediated gene expression in I-1 knock-out mice.
We hypothesize that the genes regulated by the inhibitor-1/PP1 holoenzyme are critical to cellular growth commitment including resistance to growth arrest and neuronal learning and memory
We will examine the role of inhibitor-1 in regulating hippocampal gene expression using total RNA isolated from whole hippocampi of inhibitor-1 knockout (I-1-/-) animals and parental C57Bl6 controls under non-stimulated conditions. Animals will be sacrificed by a standard protocol. Hippocampal tissue will be rapidly dissected from several animals, flash frozen within 1 minute of extraction, pooled, and total RNA will be processed immediately using TriZol.
Keywords: dose response

first: Suzanne

last: Sikes


title: zhang-affy-mouse-200705

summary:
In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have used the facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice.  We would like to compare these changes with that opccured in SOD1 mice, a mouse model of motor neuron diseases.  Analysis of these genes will help to identify changes in networks and pathways that may cause the death of NR3B-null mice. These studies will further help to elucidate the functional role of NR3B in motor neurons.
We will compare samples from motor neurons of wild type and SOD1 mice to identify genes that show abnormal expression patterns, which may be implicated in the death of SOD1 mice and shared with the same changes in NR3B-null mice.
We hypothesize that genes with their transcription level changing significantly by ablation of NR3B will be associated with the molecular mechanism underlying the death of motor neurons in NR3B null mice.
As NR3B is expressed primarily in the motor neurons of hindbrain and spinal cord, we have first collected and analyzed the spinal cord samples from NR3B null mice and wild-type controls in P4, an age of disease onset.  We like to compare motor neuron smaples from SOD1 mice at the age around the disease onset.  Total RNA from total 6 samples will be purified from ~200 motor neurons obtained by Laser Capture Microdissection.  Extracted RNAs will be subjected to two rounds of amplification and the obtained cRNA will be biotinylated. The purified cRNA will be sent to the NINDS/NIMH Microarray Consortium be used to hybridize the GeneChip Mouse Genome 430 2.0 Array. The hybridization, scanning, and initial data analysis of these GeneChips will be conducted by the Consortium staff. We will analyze the collected data further after data collection. We will  first identify genes that show significant changes between wild-type and SOD1 mice and then compare that with the result from NR3B null mice.
Keywords: other

first: Dongxian

last: Zhang


title: Transcriptome profiling of Transforming Growth Factor B1-Induced Mesangial Gene Expression

status: Public on Jan 01 2006

city: Detroit

first: Lalit

last: Singh

address: 4646 John R.

department: Internal Medicine/Nephrology & Physiology

institute: Wayne State University School of Medicine

summary:
TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood.  In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease.

Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs.

Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 °C for 5 min, equilibrated at 45 °C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice.

Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david).

Keywords = TGFBeta1
Keywords = mesangial cells
Keywords = diabetic glomerulopathy
Keywords: repeat sample

first: Davis

last: Cheng

first: Yan

last: Jiang

first: Errol

last: Crook


title: Transcriptome Profiling of High Glucose and Glucosamine-Induced Mouse Mesangial Cells

summary:
The renal mesangial cells play an important role in the development of diabetic glomerulosclerosis and renal failure. We have previously demonstrated some of the effects of high glucose are mediated via the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 chips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on the transcriptomes of a mouse mesangial cell line (MES-13). Of the 34,000 genes on the chip, ~55-60% genes are detected in MES-13 cells. HG induces the expression of ~369 genes at >2-fold where 263 genes are up and 106 genes down regulated. Similarly, GlcN increases the expression of 120 genes and decreases 94 genes. Seventy-two genes are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The differential expressions of several genes found in the microarray are also confirmed by quantitative PCR. Significant pathways co-modulated by HG and GlcN are the thioredoxin system (a 20-fold increase in thioredoxin interacting protein expression), endoplasmic reticulum (ER) stress, extracellular matrix and interferon-inducible genes. Furthermore, HG and GlcN target various intracellular pathways including the mitogen-activated protein kinase, TOLL-like receptor, fructose and mannose metabolism, and the biosynthesis of steroids and N-glycans. We conclude from this microarray data and other experimental results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes ER stress, oxidative stress and the interferon-inducible gene expression to cause cell cycle arrest, ECM gene expression and apoptosis.

Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with the desired concentrations of glucose and GlcN for 48h (LG, 5.5 mM; HG, 25 mM and GlcN, 1.5 mM + LG).

Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix U430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA (from LG, HG and GlcN treated MES-13 cells) was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled-cRNAs were purified in RNeasy spin columns (Qiagen, Valencia, CA). Fifteen mg of each cRNA was fragmented at 94 °C for 35 min in a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). These cRNA fragments were then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100). The solution was equilibrated at 45 °C for 5 min and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the mater mix) were hybridized to GeneChip Mouse Genome U430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm. After the overnight hybridization, the chips were washed with a non-stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by a stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween-20) at 50 °C. The GeneChips were then stained with streptavidin-phycoerythrin (Molecular Probe), washed with 6x saline/sodium phosphate/EDTA, incubated with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin. Finally, a third wash with 6x saline/sodium phosphate/EDTA was performed using the GeneChip Fluidics Station 450. The arrays were then scanned in a GeneChip Scanner 3000 (Affymetrix).  Each experiment was repeated twice.

Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (http://appls.niaid.nih.gov/david).

Keywords = glucose
Keywords = glucosamine
Keywords = mesangial cells
Keywords = hexosamine
Keywords: repeat sample


title: Gene profiling analysis of Src chemical rescue

status: Public on Mar 07 2006

city: Baltimore

first: Philip

last: Cole

address: 725 N Wolfe St

department: Pharmacology and Molecular Sciences

institute: Johns Hopkins Medical School

summary:
The restoration of catalytic activity to mutant enzymes by small molecules is well-established for in vitro systems.  Here we show that the protein tyrosine kinase Src R388A mutant can be rescued in live cells using the small molecule imidazole.  Cellular rescue of a v-Src homolog was rapid and reversible and conferred predicted oncogenic properties.  Using chemical rescue in combination with mass spectrometry, six known Src kinase substrates were confirmed, and several new protein targets identified.  Chemical rescue data suggests that c-Src is active under basal conditions.  Rescue of R388A c-Src also allowed contributions of Src to the MAP kinase pathway to be clarified.  This chemical rescue approach is likely to be of broad utility in cell signaling.
We were also interested in examining the impact of Src rescue on the kinetics of gene expression.  Chronic gene expression changes in v-Src transformed colon cancer and NIH3T3 cells have been reported, but the chemical rescue method permits insights into rapid kinetic changes.  We used gene microarray analysis of imidazole activated Src, 1 h after imidazole treatment of 8A7F cells as well as 6N7F control cells, a set of several genes show increases (>1.7-fold) at 1 h in the 8A7F cells and another set show decreases (>1.7-fold) at 1 h with minimal changes in 6N7F control cells.  Thirteen of these genes were further analyzed using real-time RT-PCR and most of the genes tested showed similar changes using both techniques.  These gene changes were not reported in cells chronically transformed with v-Src or rapidly stimulated with growth factors suggesting that rapid initiation of Src-mediated tyrosine phosphorylation may induce a specialized pattern of gene expression changes.  However, these earlier experiments were done under different conditions which may also contribute to gene effects.
Keywords: time course, cell type comparison

overall_design:
SrcR388A/Y527F or SrcD386N/Y527F stably transfected SYF cells were serum-starved overnight and then treated with imidazole 5 mM for 1 h or 12 h. RNeasy Miniprep column (Qiagen) was used to prepare total RNA from each treatment group. All samples were run in commercial arrays from Affymetrix,
using Murine Genome MOE430 2.0 GeneChips as described in the Affymetrix website.

first: Yingfeng

last: Qiao

first: Francisco

last: Murillo

first: Chunfa

last: Jie


title: Targeted disruption of glycerol kinase gene in mice: expression analysis in liver

status: Public on Dec 22 2005

city: Los Angeles

first: Nicole

middle: Kim

last: MacLennan

address: 10833 LeConte Ave

department: Pediatrics

institute: University of California, Los Angeles

summary:
Glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologic crises. Liver shows the highest level of glycerol kinase (GK) activity in humans and mice.  Absence of genotype-phenotype correlations in patients with GKD indicate the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal glycerol kinase (Gyk) knockout (KO) and wild type (WT) mice. Unsupervised learning revealed the overall gene expression profile of the KO mice was different from that of WT. Real time PCR confirmed differences for selected genes.  Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories.  Pathway Assist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors SREBP-1c, ChREBP, HNF-4alpha, and PPAR-alpha, had increased activity in the Gyk KO mice compared with WT mice; while SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in gene expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse knockout model of a human disease.
Keywords: Glycerol kinase (Gyk) knockout mouse; mouse model of human Glycerol Kinase Deficiency; Gyk KO versus WT liver expression analysis; Affymetrix mus 430 2.0 GeneChip

overall_design: Male WT and KO mouse pups were sacrificed on day of life (dol) 3 and each liver was harvested.  Total RNA from 4 KO and 4 WT livers was isolated individually.  cDNA was synthesized from the poly(A)+ mRNA in the total RNA, Biotin-tagged and fragmented to an average strand length of 100 bases (range 35-200 bases).  Ten µg of each cRNA was hybridized onto an Affymetrix mus 430 2.0 GeneChip to analyze differences in liver gene expression between KO and WT mice.  Day of life three was chosen because the mice are phenotypically symptomatic with statistically different parameters for hypoglycemia, acidosis; low bicarbonate and decreased base excess.  On day of life 2 they are not significantly different from wild type in all of these important clinical phenotypes.

first: Lola

last: Rahib

first: Cynthia

last: Shin

first: Zixing

last: Fang

first: Steve

last: Horvath

first: Jason

last: Dean

first: James

last: Liao

first: Edward

last: McCabe

first: Katrina

last: Dipple


title: Rapid selection response for contextual fear conditioning in a cross between C57BL/6J and A/J (palme-affy-mouse-198967)

city: Phoenix

first: Winnie

last: Liang

address: 445 N. Fifth Street

summary: These data are from the brains (amygdala and hippocampus) of mice originally derived from a cross between C57BL/6J and A/J inbred strains. We used short-term selection to produce outbred mouse lines with differences in contextual fear conditioning, which is a measure of fear learning. We selected for a total of 4 generations. Fear learning differed in the selected lines and this difference was stronger with each successive generation of selection. We identified several QTLs for the selection response, including a highly significant QTL at the tyr locus (p < 9.6(-10)). We used Affymetrix microarrays to identify many differentially expressed genes in the amygdala and hippocampus of mice from the final generation of selection. Amygdala and hippocampus samples were rapidly dissected out of experimentally naïve mice from each selected line. Three samples were pooled and hybridized to each array. Experimentally naïve mice were used because the behavior of the mice can be reliably a  nticipated due to their lineage. Thus these gene expression differences are not due to the response to human handling, foot shock or fear-inducing conditioned stimuli. We have a second similar study that focuses on a different selected population that was based on C57BL/6J and DBA/2J mice (see GES4035).

first: Abraham

last: Palmer


title: Scd1 Knockout Mice on very low fat diet

status: Public on Dec 23 2005

city: Madison

first: Alan

middle: D

last: Attie

state: WI

address: 433 Babcock Drive

department: Biochemistry

institute: University of Wisconsin-Madison

summary:
Stearoyl-CoA desaturase 1-deficient (SCD1-/-) mice have impaired monounsaturated fatty acid (MUFA) synthesis.  When maintained on a very low-fat, high-carbohydrate (VLF-HC) diet, SCD1-/- mice develop severe hypercholesterolemia characterized by an increase in apolipoprotein B-containing lipoproteins and the appearance of lipoprotein-X.  Additionally, high-density lipoprotein cholesterol is dramatically reduced in VLF-HC SCD1-/- mice.  The concomitant presence of elevated plasma bile acids, bilirubin and aminotransferases in the VLF-HC SCD1-/- mouse are indicative of hepatic dysfunction.  Supplementation of the VLF-HC diet with unsaturated fat (canola oil), but not saturated fat (coconut oil), prevents these plasma phenotypes.  However, dietary oleate was not as effective as canola oil in reducing low-density lipoprotein cholesterol, signifying an additional role for dietary polyunsaturated fatty acid deficiency in the development of this phenotype.  These results indicate that lack of SCD1 results in an increased requirement for dietary unsaturated fat to compensate for impaired MUFA synthesis and to prevent hypercholesterolemia and hepatic dysfunction.
Keywords: repeat (genotype and diet)

overall_design: We used Affymetrix Mouse430v2.0 microarray chips to search for gene expression changes unique to the VLF-HC SCD1-/- group.  These chips contain 45,101 probe sets representing over 39,000 transcripts and variants from over 34,000 mouse genes.  We studied SCD1+/+ and SCD1-/- mice on chow or the VLF-HC diet, analyzing five individual livers from each subgroup for a total of twenty microarrays.

first: Matthew

last: Flowers

first: Angie

last: Oler

first: Kathryn

last: Schueler

first: Mark

last: Gray-Keller

first: Oliver

last: Richards

first: Hong

last: Lan

first: YounJeong

last: Choi

first: Folkert

last: Kuipers

first: Albert

last: Groen

last: Ntambi


title: Leptin Regulated Mouse Gallbladder Genes

status: Public on Dec 31 2005

city: Indianapolis

first: Henry

last: Pitt

state: IN

address: 535 Barnhill Dr. RT130D

institute: Indiana University School of Medicine

summary:
Gallstone disease is a major contributor to health care costs in the United States.  Approximately 12 % of the U.S. population has gallstones.  As a result, more than 700,000 cholecystectomies are performed in this country each year.  Many of these patients are obese and have a positive family history; but surprisingly, little is known about the link between obesity, genetics and gallstone formation.   Obese individuals have been shown to have supersaturated bile, larger gallbladder fasting volumes and impaired gallbladder emptying. We have recently demonstrated that leptin plays a role in gallbladder motility.  In an effort to understand the genetic basis for these observations, we tested the hypothesis that leptin would alter gallbladder gene expression.  Methods:  Affymetrix oligonucleotide microarrays 430 2.0 were used to compare gallbladder gene expression profiles from 12 week old control saline-treated leptin-deficient (Lep ob) and from leptin-treated Lep ob female mice.  Analyses were performed on pooled RNA (n=4) from the gallbladders of 12 saline-treated Lep ob mice and from 12 Lep ob mice which were administered daily IP 5 ug/g of recombinant murine leptin for 4 weeks.  Resulting data were analyzed utilizing Gene Chip Operating Software or MAS 5.0.  Results:  Of the genes analyzed 314 were upregulated and 108 were downregulated by leptin administration.  Numerous genes related to gallstone pathogenesis, gallbladder absorption/secretion, inflammatory cytokines, and insulin resistance were altered by leptin.
Keywords: comparitive response to leptin

overall_design: Female B6.V-lepob obese mice (n=24) aged 7 weeks were obtained from The Jackson Laboratory (Bar Harbor, ME).  Upon arrival, mice were placed on a standard chow diet and were allowed to acclimate for 1 week before starting the experiment.  For leptin treatments, mice received daily intraperitoneal injections of either recombinant mouse leptin (R&D Systems, Minneapolis, MN) (n=12) at a dose of 5 µg/g body weight or with saline as a control (n=12) for 4 weeks.  At 12 weeks of age, mice were fasted overnight with free access to water.  The following morning the mice underwent cholecystectomy. Three gallbladders were pooled to create 4 pools in each treatment and total RNA was isolated. Affymetrix murine 430 2.0 arrays were utilized to examine altered gallbladder gene expression as a result of leptin administration.  The resulting data was analyzed utlizing Gene Chip Operating Software or MAS 5.0.

first: Deborah

last: Swartz-Basile

first: Shannon

last: Graewin

first: Hayder

last: Al-Azzawi

first: Attila

last: Nakeeb


title: Profiling of visceral sensory neurons in dorsal root and nodose ganglia in BALB/c mice.

status: Public on Dec 21 2005

city: Beerse

first: Pieter

middle: Johan

last: Peeters

address: Turnhoutseweg 30

department: Internal Medicine

country: Belgium

institute: J&J Pharmaceutical R&D

summary:
Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central  nervous system. In order to elucidate molecular identities for these different  properties, we compared gene expression profiles of neurons located in nodose  ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of  Alexa Fluor-488 conjugated Cholera toxin B allowed identification of neurons  projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG  were isolated using laser capture microdissection. Gene expression profiles of visceral  afferent neurons, obtained by microarray hybridization, were analysed using  multivariate spectral map analysis, SAM algorithm (Significance Analysis of  Microarray data) and fold-difference filtering. A total of 1996 genes were found to be  differentially expressed in DRG versus NG, including 41 G-protein coupled receptors  and 60 ion channels. Expression profiles obtained on laser-captured neurons were  contrasted to those obtained on whole ganglia demonstrating striking differences and  the need for microdissection when studying visceral sensory neurons because of  dilution of the signal by somatic sensory neurons. Furthermore, a detailed catalogue  of all adrenergic and cholinergic, GABA, glutamate, serotonin and dopamine  receptors, voltage-gated potassium, sodium and calcium channels and transient  receptor potential cation channels present in visceral afferents is provided. Our  genome-wide expression profiling data provide novel insight into molecular  signatures that underlie both functional differences and similarities between NG and  DRG visceral sensory neurons. Moreover, these findings will offer novel insight into  mode of action of pharmacologic agents modulating visceral sensation.
Keywords: Cell type comparison

overall_design: Three separate experiments were performed. First, 5 whole dorsal root ganglia were compared to 7 whole nodose ganglia. Second, Laser captured visceral neurons derived from 5 dorsal root ganglia and 5 nodose ganglia were compared on MG-U74Av2. Third, Laser captured visceral neurons derived from 9 dorsal root ganglia and 11 nodose ganglia were compared on Mouse430_2.

middle: J.

first: Jeroen

last: Aerssens

first: Ronald

last: de Hoogt

first: Andrzej

last: Stanisz

first: Hinrich

middle: W.

last: Göhlmann

first: Kirk

last: Hillsley

first: Ann

last: Meulemans

first: David

last: Grundy

middle: H.

last: Stead

first: Bernard

last: Coulie


title: Identification of tumor immune evasion mechanism using P0 and P3 cell lines

status: Public on Dec 06 2005

first: Ken-Yu

last: Lin

address: 720 Rutland Ave, Ross 512

department: Pathology

institute: Johns Hopkins University School of Medicine

summary:
The emergence of immune resistance variants during immunotherapy is poorly understood.  We generated a highly immune resistant cell line (P3) from a susceptible cell line (P0) by subjecting it to 3 rounds of in vivo immune selection.  Subsequently, microarray analysis of P0 and P3 was performed to identify genes that may contribute to the increase in immune resistance.
Keywords: cell type comparison

overall_design: Four experimental replicates were prepared for each cell line.  P0 is the control cell line, and P3 is the experimental/resistant cell line.

first: Chien-Fu

last: Hung

first: Shiwen

last: Peng

first: Chunfa

last: Jie

first: Drew

last: Pardoll

last: Wu


title: Comparison of HNF4 null mouse embryonic livers with control mouse embryonic livers

city: Charleston

first: Stephen

middle: A

last: Duncan

state: SC

address: 173 Ashley Avenue, BSB 6th Floor, Room 657A

department: Regenerative Medicine and Cell Biology

institute: Medical University South Carolina

summary:
To study the role of hepatic nuclear factor alpha (HNF4a in hepatogenesis, we used loxP-Cre technology to eliminate it from developing mouse livers.
A comparison of control and experimental livers revealed that hepatocytes lacking HNF4a_failed to fully differentiate.  Specifically, HNF4a null liver cells failed to express genes that are markers of mature hepatocytes including Gys2, Pck1, and G6pc.  These cells also failed to form normal cellular junctions, which are critical for establishment of the hepatic epithelium [Parviz et al. (2003) Nat.Genet. 34, 292-96].  Because HNF4a functions as a transcription factor, we hypothesized that it regulates liver development by controlling the expression of genes whose products are essential in executing cellular differentiation and morphogenesis.  To identify these genes, we compared expression profiles between HNF4a null and wild type E18.5 fetal livers using Affymetrix gene arrays.  We identified 564 genes whose expression was decreased by at least 2.5-fold and 34 genes whose expression was increased by at least 2.5-fold when HNF4a was eliminated from hepatocytes.  These represent genes involved in diverse molecular pathways, reflecting the pleiotropic phenotype of HNF4a null livers. Our goal was to use the information from the gene arrays to define the mechanisms through which HNF4a controls the generation of the hepatic epithelium. We identified 27 genes from our list of downregulated genes with either defined or predicted roles in cellular junctions and/or adhesion.  Most striking was that loss of HNF4a altered the expression of genes encoding proteins involved in virtually all types of cellular junctions including tight junctions (Cldn1, Cldn-2, Cldn-12, Ocln, F11r/Jam1, Cxadr, Crb3), adherens junctions (Cdh1), desmosomes (Dsc2, Pkp2, Krt2-8), and gap junctions (Gjb1, Gjb2).  Using immunoblotting and immunohistochemistry, we found that the expression of proteins representing each of these junction types was reduced or absent.  Analysis of these genes for the presence of putative HNF4a binding sites revealed that 25 of 27 contain such sites.  We have used chromatin immunoprecipitation to confirm that HNF4a occupies several of these sites in vivo, including Cdh1, Cldn1, and F11r/Jam1.  From these data, we conclude that HNF4a is a central mediator of the fully differentiated hepatocyte gene expression program and that it orchestrates the expression of cell junction and adhesion proteins required for establishing the hepatic epithelium.
Keywords: E18.5 fetal hnf4 null and control mouse livers

overall_design: comparison of three hnf4 null livers to three control


title: RUNX1 (AML1) knockout in mouse embryo

status: Public on Dec 02 2005

city: Parkville

first: Ken

last: Simpson

state: Victoria

address: 1G Royal Parade

department: Genetics and Bioinformatics

institute: The Walter and Eliza Hall Institute

summary:
Series of samples studying effect of knocking out RUNX1 (a.k.a. AML1) in mouse embryos at 8.5 and 12 days.  Wildtype and knockout animals at the two time points were generated and all conditions done in duplicate.
Keywords: other

first: Joelle

last: Michaud

middle: M

first: Yoshiaki

last: Ito

first: Motomi

last: Osato

first: Gordon

last: Smyth

first: Hamish

last: Scott


title: Expression profiling of mouse gonadal somatic cells

status: Public on Dec 17 2005

city: St Lucia

first: Sean

last: Grimmond

state: QLD

address: Research Rd

department: Genomics and Bioinformatics

institute: Institute of Molecular Bioscience, Uni of QLD

summary:
Gene expression profiling of FACS sorted GFP+ve cells from sexed gonads of transgenic pSF1-eGFP mice
Keywords: Time course and comparison of sexes

overall_design: Experiment is both a time course and an assesment of sex specific expression in SF1+ve somatic gonadal cells. Each time point and sex has been profiled in triplicate

first: A

last: Beverdam

first: P

last: Koopman


title: SRF-null neonatal cardiomyocytes

city: Milwaukee

first: Ravi

middle: P.

last: Misra

address: 8701 Watertown Plank Road

institute: Medical College of Wisconsin

summary:
Serum Response Factor (SRF) is a transcriptional regulator required for mesodermal development. Numerous studies have implicated SRF as a central regulator of muscle gene expression and myogenesis. In this present study we used a loss of function approach to delineate the role of SRF in cardiac myocyte gene expression and function. In SRF null neonatal cardiomyocytes, we observe severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric proteins. Consistent with this, gene array and RT-PCR analyses show downregulation of genes encoding key cardiac transcriptional regulatory factors and proteins required for the maintenance of sarcomeric structure, function, and regulation. Chromatin IP analysis reveals that at least a subset of these proteins are likely regulated directly by SRF. Together the results presented here reveal new cellular and genetic mechanisms through which SRF exacts control over the contractile apparatus in cardiac myocytes.
Keywords: genetic modification

overall_design: There are three control replicates (cardiomyocytes transduced with Adeno-GFP virus) and three experimental replicates (cardiomyocytes transduced with Adeno-CRE to excise SRF).

first: Robert

last: Balza

middle: P


title: Comparative analysis of 10 week and 5 week Bitransgenic mice (LH mouse crossed with MMTV-neu mouse)

city: Cleveland

first: Melissa

last: Landis

state: OH

address: 2109 Adelbert

department: Pharmacology

institute: Case Western Reserve School of Medicine

summary:
To identify genes that may facilitate early steps of ErbB2/Neu-mediated mammary tumorigenesis, we performed comparative microarray analysis of 5- and 10-week bitransgenic mammary glands (LHxMMTV-neu) in triplicate.
Keywords: transgenic mouse, erbB2, MMTV-neu, HER2, mammary tumor, breast cancer

overall_design: We analyzed 3 pooled RNA samples that  represented 3 animals for each time point so that a total of 9 bitransgenic mice were analyzed per time point.

first: Darcie

last: Seachrist

first: Fadi

middle: W

last: Abdul-Karim

first: Ruth

last: Keri


title: Gene expression profiles in developing nephrons using Lim1 metanephric mesenchyme-specific conditional mutant mice

status: Public on Feb 13 2006

city: Houston

first: You-Tzung

last: Chen

address: 1515 Holcombe Blvd. Unit 1006

department: Molecular Genetics

institute: The University of Texas MD Anderson Cancer Center

summary:
BACKGROUND: Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons.
METHODS: In the present study, we used Affymetrix probe arrays to screen for nephron-specific genes by comparing the expression profiles of control and Lim1 conditional mutant kidneys. Kidneys from two developmental stages, embryonic day 14.5 (E14.5) and 18.5 (E18.5), were examined.
RESULTS: Comparison of E18.5 kidney expression profiles generated a list of 465 nephron-specific gene candidates that showed a more than 2-fold increase in their expression level in control kidney versus the Lim1 conditional mutant kidney. Computational analysis confirmed that this screen enriched for kidney-specific genes. Furthermore, at least twenty-eight of the top fifty (56%) candidates (or their vertebrate orthologs) were previously reported to have a nephron-specific expression pattern. Our analysis of E14.5 expression data yielded 41 candidate genes that are up-regulated in the control kidneys compared to the conditional mutants. Three of them are related to the Notch signaling pathway that is known to be important in cell fate determination and nephron patterning.
CONCLUSIONS: Therefore, we demonstrate that Lim1 conditional mutant kidneys serve as a novel tissue source for comprehensive expression studies and provide a means to identify nephron-specific genes.
Keywords: tissue specificity, time course, development, kidney, metanephric mesenchyme, nephron, Lim1, knockout mice, conditional knockout

overall_design:
Microarray experiment on each time point and genotype was performed in technical duplicates (ie. a single RNA preparation of pooled kidneys of one genotype used for two separate target preparations).  Data from a total of 8 independent arrays were used in this study, 2 arrays were used for RNA samples from E18.5 control kidneys, 2 for E18.5 Lim1 conditional mutant kidneys, 2 for E14.5 control kidneys, and the other 2 for E14.5 Lim1 conditional mutant kidneys.  Data generated from all arrays that satisfied the preliminary analysis were exported and loaded into DNA-Chip Analyzer (dChip2004), where statistical and comparative analyses were performed to verify the data.  The data were normalized using the default normalization method.  Briefly, an iterative procedure was used to identify an invariant set of probes, which presumably consisted of non-differentially expressed genes.  A piecewise-linear running median curve was then calculated and used as the normalization curve.  After normalization, all arrays had similar brightness.  Median intensities around 155 (between 155 to 158) were obtained after normalization.  Percent gene present (P call%) values between 55.7% and 63.6% were observed using default detection p-value cut offs (a1=0.04 and a2=0.06).  Array outlier (%) and single outlier (%) were detected at ranges from 0.016% to 0.080% and from 0.009% to 0.045%.  Expression data obtained from all arrays used are provided.
Normalized data were exported in a tab delimited text format.  Fold changes of each transcript from different samples were calculated and sorted using Microsoft Excel 5.0 software.  Signal obtained from control kidney samples were used as an experiment to compare to the signal obtained from Lim1 conditional mutant kidneys that was designated as a baseline.  A 2-fold change in the means of signal obtained from experimental duplicates and those from baseline duplicates was used as the criterion to identify differentially expressed transcripts.  To ensure the quality of the data, probe sets that showed a fold change between duplicates greater than between the experimental mean and baseline mean were removed.  To study only genes that showed consistent expression on experimental chips, probe sets that did not show consistent present calls in the experimental duplicates were removed.  To focus on genes with a significant fold change between the experiment and the baseline, only probe sets that the product of their experimental mean and fold change were more than 100 were retained.  To produce a compact differentially expressed gene list, the probe set list was sorted within Microsoft Excel based on Locus Link number and redundant entries were removed.  Our experimental design description and the data format provided in the Additional files fulfill the MIAME (minimum information about a microarray experiment) standards.

first: Akio

last: Kobayashi

first: KinMing

last: Kwan

first: Randy

middle: L

last: Johnson

first: Richard

middle: R

last: Behringer


title: thymic mouse cells

status: Public on Nov 14 2005

city: Bolzano

first: Johannes

last: Rainer

address: Via A. Volta 21

department: Institute for Biomedicine

country: Italy

institute: Eurac Researc

summary:
Glucocorticoids (GC) are in most chemotherapy protocols for lymphoid malignancies, particularly childhood acute lymphoblastic leukaemia (ALL) for their ability to induce apoptosis in malignant blast. The underlying mechanism, however, has so far only been investigated in model systems. This study comprises Affymetrix hgu133 plus 2.0 analyses of
Peripheral blood lymphoblasts purified at three time points (0h, 6-8h, 24h after treatment initiation) from 13 children under therapy for ALL . Treated samples were compared to untreated (0h). For comparison, expression profiles were generated from an adult ALL patient, peripheral blood lymphocytes from GC-exposed healthy donors, GC-sensitive and -resistant ALL cell lines and mouse thymocytes treated with GC in vivo and in vitro. This series consists of the mouse thymocyte samples.
Findings. An essentially complete list of GC-regulated candidate genes in clinical settings experimental and was generated, enabling immediate analysis of any gene with respect to its potential significance for GC-induced apoptosis in these systems. Gene regulations previously thought responsible for cell death in experimental systems were reconfirmed in few children only. In contrast, a small number of genes, most not implicated in GC-induced apoptosis previously, were co-ordinately regulated in the majority of children.
Keywords: treatment response

overall_design: In vivo and in vitro glucocorticoid treated samples were compared to their control samples (ethanol or PBS treated).

first: Stefan

last: Schmidt

last: Riml

first: Christian

last: Ploner

first: Simone

last: Jesacher

first: Clemens

last: Achmüller

first: Elisabeth

last: Presul

first: Sergej

last: Skvortsov

first: Roman

last: Crazzolara

first: Michael

last: Fiegl

first: Taneli

last: Raivio

first: Olli

last: Jänne

first: Stephan

last: Geley

first: Bernhard

last: Meister

first: Reinhard

last: Kofler


title: Silencing of microRNAs in vivo with "antagomirs"

status: Public on Nov 02 2005

city: New York

first: Markus

last: Stoffel

state: NY

address: 1230 York Avenue

department: Laboratory of Metabolic Diseases

institute: Rockefeller University

summary:
Using a novel class of chemically-engineered oligonucleotides, termed "antagomirs", we studied the biological significance of silencing miR-122 in the liver of mice at the mRNA level
Keywords: treatment response

overall_design: Mice were injected on three subsequent days with antagomir-122 or scrambled control (mm-antagomir-122), respectively; 80mg/kg bodyweight/day. 24 hrs after the last treatment, mice were sacrificed and total RNA isolated from the livers. RNA from three mice was pooled and used for generation of biotin-labelled cRNA.

first: Jan

last: Krutzfeldt

first: Nikolaus

last: Rajewsky

first: Ravi

last: Braich

first: Kallanthottathil

middle: G

last: Rajeev

first: Thomas

last: Tuschl

first: Muthiah

last: Manoharan


title: Distinct Gene Expression Profiles in Adult Mouse Heart Following Targeted  MAP Kinase Activation

status: Public on Nov 01 2005

first: Scherise

last: Mitchell

address: 650 Charles E Young Dr, BH 569 CHS

department: Anesthesiology

institute: UCLA

summary:
Three major MAP kinase signaling cascades, ERK, p38 and JNK, play significant roles in the development of cardiac hypertrophy and heart failure in response to external stress and neural/hormonal stimuli.  In order to study the specific function of each MAP kinase branch in adult heart, we have generated three transgenic mouse models with cardiac specific and temporally regulated expression of activated mutants of Ras, MKK3 and MKK7, which are selective upstream activators for ERK, p38 and JNK, respectively.  Gene expression profiles in transgenic adult hearts were determined using cDNA microarrays at both early (4-7 days) and late (2-4 weeks) time points following transgene induction.  From this study, we revealed common changes in gene expression among the three models, particularly involving extracellular matrix remodeling.  However, distinct expression patterns characteristic for each pathway were also identified in cell signaling, growth and physiology.  In addition, genes with dynamic expression differences between early vs. late stages illustrated primary vs. secondary changes upon MAP kinase activation in adult hearts. These results provide an overview to both short term and long term effects of MAP kinase activation in heart and support some common as well as unique roles for each MAP kinase cascade in the development of heart failure.
Keywords: MAP Kinase induction comparison, time course

overall_design: αMHC-floxed-HRas-v12/MKK3bE/MKK7D transgenic mice were  bred with αMHC-Mer-Cre-Mer (MCM) mice (from Dr. J. Molkentin, Cincinnati Children’s Hospital)to generate double transgenic animals harboring both floxed transgenes and Mer-Cre-Mer transgene.  At 12 weeks of age the double transgenic mice and non-transgenic littermate controls were treated via i.p. injection of tamoxifen at a dosage of 20mg/kgBW once a day for 3 consecutive days as reported.  The hearts were harvested at an early (4-7 days post first tamoxifen injection) and a late (2-4 weeks) time point.  Left ventricles were dissected and rapidly frozen in liquid nitrogen and stored at -80˚C prior to protein and RNA analysis.

first: Asuka

last: Ota

first: William

last: Foster

first: Bin

last: Zhang

first: Zixing

last: Fang

first: Shilpa

last: Patel

first: Stanley

middle: F

last: Nelson

first: Steve

last: Horvath

first: Yibin

last: Wang


title: Two transactivation mechanisms are responsible for the bulk of HIF-1alpha-responsive gene expression

status: Public on Oct 24 2005

city: Memphis

first: Paul

middle: K.

last: Brindle

state: TN

address: 332 N Lauderdale

institute: St Jude Children's Research Hospital

summary:
The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1? and HIF-2? binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (?CH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of ~35-50% of global HIF-1?-responsive gene expression, whereas another HIF-transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSAS) accounts for ~70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription.
Keywords: genetic modification, dose response

overall_design: Three separate affymetrix experiments using mouse embryonic fibroblasts derived from embryos bearing the ?CH1 mutation in p300 and/or CBP and treated with hypoxia or combinations of dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) are described (GSE3195, GSE3196 and GSE3296).  Samples are not directly comparable between experiments because of differences in experiment design and Affymetrix chips used.

first: Lawryn

middle: H

last: Kasper

first: Fayçal

last: Boussouar

first: Kelli

last: Boyd

first: Wu

last: Xu

first: Michelle

last: Biesen

first: Jerold

last: Rehg

first: Troy

last: Baudino

first: John

last: Cleveland

middle: K


title: Gene expression in hypoxic MEFs having only p300 and CBP with deleted CH1 domains

summary:
The CH1 protein interaction domain of the transcriptional coactivators p300 and CBP is thought to interact with HIF-1alpha and this interaction is thought to be critical to the expression of HIF-1alpha target genes in response to hypoxia.  To test the requirement of the CH1 domain for gene expression in response to hypoxia, rimary mouse embryonic fibroblasts (MEFs) were generated from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300. The remaining allele of p300 or CBP was a conditional knock out allele. Control MEFs with only a single conditional knockout allele of p300 or CBP were also generated.  At passage 3 MEFs were infected with Cre Adenovirus and grown until they had expanded at least 100 fold. Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction.
Keywords: genetic modification, dose response

overall_design: 12 samples; 4 genotypes [CBP+/flox (flox1), p300 +/flox (flox2), CBP CH1/flox;p300 CH1/CH1 (triCH1flox1),CBP CH1/CH1;p300 CH1/flox (triCH1flox2)] , 2 treatments (normoxia and hypoxia).