title: Candidate Genes for Expansion and Transformation of Hematopoietic Stem Cells by NUP98-HOX Fusion Genes

status: Public on Oct 01 2007

city: Gothenburg

first: Lars

last: Palmqvist

address: Sahlgrenska University Hospital

department: Institute of Biomedicine

country: Sweden

institute: University of Gothenburg

summary:
BACKGROUND: Hox genes are implicated in hematopoietic stem cell (HSC) regulation as well as in leukemia development through translocation with the nucleoporin gene NUP98. Interestingly, an engineered NUP98-HOXA10 (NA10) fusion can induce a several hundred-fold expansion of HSCs in vitro and NA10 and the AML-associated fusion gene NUP98-HOXD13 (ND13) have a virtually indistinguishable ability to transform myeloid progenitor cells in vitro and to induce leukemia in collaboration with MEIS1 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: These findings provided a potentially powerful approach to identify key pathways mediating Hox-induced expansion and transformation of HSCs by identifying gene expression changes commonly induced by ND13 and NA10 but not by a NUP98-Hox fusion with a non-DNA binding homedomain mutation (N51S). The gene expression repertoire of purified murine bone marrow Sca-1+Lin- cells transduced with retroviral vectors encoding for these genes was established using the Affymetrix GeneChip MOE430A. Approximately seventy genes were differentially expressed in ND13 and NA10 cells that were significantly changed by both compared to the ND13(N51S) mutant. Intriguingly, several of these potential Hox target genes have been implicated in HSC expansion and self-renewal, including the tyrosine kinase receptor Flt3, the prion protein, Prnp, hepatic leukemia factor, Hlf and Jagged-2, Jag2. CONCLUSIONS: In conclusion this study has identified several novel Hox downstream target genes and provides important new leads to key regulators of the expansion and transformation of hematopoietic stem cells by Hox.
Keywords: Leukemic vs. non-leukemic NUP98-HOX fusion genes

overall_design: Adult murine bone marrow cells transduced with vectors carrying ND13, NA10, ND13(N51S) or an empty GFP control vector (MIG) were isolated on the basis of GFP expression by FACS 24 hours post-transduction. Viable transduced cells were further enriched for primitive hematopoietic cells by exclusion of cells expressing linage markers (Gr-1, B220, Ter-119, CD4, CD5 and CD8) and selection for cells expressing the stem cell antigen-1 (Sca-1). Three independent experiments were performed for each of the four different conditions included in the study.

first: Keith

last: Humphries


title: Gene expression profile of the age-related hearing loss cochlea in DBA/2J mice

status: Public on Oct 16 2007

city: Madison

first: Shinichi

last: Someya

state: WI

address: 425 Henry Mall Dr

department: Medical Genetics

country: USA

institute: University of Wisconsin-Madison

summary:
Age-related hearing loss (AHL) is the progressive loss of auditory function with aging. The DBA/2J (DBA) mice have been used as a model of AHL and undergoes progressive, age-related hearing loss by 12 weeks of age. Here we analyzed cochlear gene expression of 7-week-old and 36-week-old DBA mice using microarrays. Auditory brainstem response (ABR) analysis confrimed that severe age-related hearing loss occured in 36-week-old mice, whereas moderate hearing loss occured in 7-week-old mice. Comprehensive gene expression analysis identified genes correlated with AHL and revealeed that 15 mitochondrial process categories, including “mitochondrial electron transport chain”, “oxidative phosphorylation”, “respiratory chain complex I”, “respiratory chain complex IV”, and “respiratory chain complex V”, were statistically associated with AHL-correlated genes in the cochlea of 36-week-old DBA mice, and that 25 genes encoding components of the mitochondrial respiratory chain (respiratory chain complex I, IV, and V) were significantly down-regulated in the cochlea. These observations provide evidence that AHL is associated with down-regulation of genes involved in the mitochondrial respiratory chain in the cochlea of DBA mice, and suggest that mitochondrial respiratory chain dysfunction may be a key feature of AHL in mammalian cochlea.
Keywords: Disease state analysis, Time course analysis

overall_design: To determine the effects of age-related hearing loss, each 7-week-old sample (n = 3) was compared to each 36-week-old sample (n = 3), generating a total of nine pairwise comparisons. Using DAVIS and EASE, the identified genes were assign to “GO: Biological Process” categories of Gene Ontology Consortium. Furthermore, we used EASE to determine the total number of genes that were assigned to each biological process category, and to perform Fisher exact test. Quality control measures were not used. No replicates were done. Dye swap was not used.

first: Tatsuya

last: Yamasoba

first: Richard

last: Weindruch

first: Tomas

middle: A

last: Prolla


title: Gene Expression after Cochlear Removal in Cochlear Nucleus at P7 and P21

status: Public on Dec 26 2007

city: Seattle

first: Julie

last: Harris

state: WA

department: Otolaryngology-HNS

institute: University of Washington

summary:
Deprivation of peripheral nerve input by cochlear removal in young mice results in dramatic neuron death in the cochlear nucleus (CN).  The same manipulation in older mice does not result in significant loss.  The molecular basis of this critical period of vulnerability remains largely unknown.  Here we identified genes regulated at early time points after cochlear removal at ages when neurons are vulnerable (postnatal day (P)7) or invulnerable (P21) to this challenge.  Afferent deprivation regulated very different sets of genes at P7 and P21.  These genes showed a variety of functions at both ages, but surprisingly there was no net increase in pro-apoptotic genes at P7.   A large set of upregulated immune-related genes was identified at P21.
Keywords: Time Course after Cochlear Removal, Age Comparison

overall_design: Mice received unilateral cochlear removals.  At 6, 12, 24, and 48 hours after surgery, the CN ipsilateral and contralateral were removed, and RNA isolated from separate pools of tissue for each replicate.  Deafferented CN were compared to age-matched and time-matched contralateral, control CN to identify genes regulated by cochlear removal at age P7 and P21.

first: Edwin

middle: W

last: Rubel


title: Analysis of gene expression during neurite outgrowth and regeneration (430A and 430A 2.0 array)

status: Public on Dec 04 2007

city: Princeton

first: Moriah

last: Szpara

state: NJ

address: 301 Schultz

department: Molecular Biology

institute: Princeton University

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

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

middle: L

first: Karen

last: Vranizan

first: Yu

middle: C

last: Tai

first: Corey

middle: S

last: Goodman

first: Terence

middle: P

last: Speed

first: John

last: Ngai


title: Expression Profiling of Retinal Detachment and Accelerated Re-attachment

status: Public on Nov 10 2007

city: Oklahoma City

first: Rafal

last: Farjo

state: OK

address: 940 Stanton L. Young Blvd.

department: Cell Biology

institute: The University of Oklahoma Health Sciences Center

summary:
Retinal detachment is a major cause of blindness due to penetrating trauma and ocular inflammation, and is often observed in many patients following cataract extraction surgery.  When the retinal photoreceptors detach from their epithelium, stress signals and apoptotic pathways are initiated that will lead to loss of vision, however accelerating the reattachment of these cells can prevent photoreceptor death and subsequent vision loss.  To determine the genes involved in this process, we performed a microarray screen using a mouse model or retinal detachment in conjunction with a P2Y2 agonist previously demonstrated to hasten retinal reattachment.
Keywords: disease state analysis and therapeutic analysis

overall_design: We conducted a microarray screen to identify genes involved in promoting faster resolution of retinal detachment.  Subretinal detachment was induced in Balb/cJ mice by subretinal injection of 1 uL saline or delivery of 1 uL of 10uM INS37217/Saline to cause detachment and expedite the rate of recovery.  We performed this study at three timepoints: 2 hrs post-injection to identify early response genes; 24 hrs post-detachment when the retina has reattached, but still grossly misfolded; and 7 days post-detachment when the misfolding has been resolved, but retinal function is merely 50% of wild-type function.  We used each RNA pool (each containing >5 retinas) for GeneChip hybridization giving a total of 3 biological replicates for each treatment at each timepoint.  For each GeneChip, we labeled 7 ug of total RNA according to the manufacturer’s specifications (Affymetrix Inc.).

first: Muna

middle: I

last: Naash


title: Molecular mechanisms of liver carcinogenesis in the Mdr2-knockout mice

status: Public on Oct 10 2007

city: Ramat-Gan

first: Mark

last: Katzenellenbogen

address: Ramat-Gan 52900, Israel

department: THE FACULTY OF LIFE SCIENCE, BIOINFORMATICS AND MICROARRAY UNIT

country: Israel

institute: Bar Ilan University

summary:
Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-KO mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling demonstrated that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC datasets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time RT-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We demonstrate that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of b-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for b-catenin-negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes.
Keywords: Hepatocellular carcinoma, Mouse model, Mdr2-knockout.

overall_design: The liver RNA samples from six Mdr2-KO tumors, six non-tumorous liver tissues (four matched and two unmatched), as well as from three control heterozygous mice at 16 months of age were subjected to gene expression profiling using the genome scale Affymetrix Mouse Genome 430 2.0 Array.


title: Expression data from transgenic mice (6 wk) inducibly expressing human alpha1-antitrypsin in the liver

status: Public on Jul 26 2007

city: Pittsburgh

first: Tunda

last: Hidvegi

state: PA

address: 3460 Fifth Ave

institute: University of Pittsburgh

summary:
In the classical form of α1antitrypsin deficiency a mutant protein accumulates in a polymerized form in the ER of liver cells causing liver damage and carcinogenesis by a  gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant α1antitrypsin Z in the ER specifically activates the autophagic response but not the  unfolded protein response and that autophagy plays a critical role in disposal of insoluble α1antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse  model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein.
Keywords: genomic analysis

overall_design:
Liver RNA from juvenile (6 wk old) male mice inducibly expressing human alpha1-antitrypsin wild type (M) or mutant (Z) form exclusively in the liver was subjected to genomic analysis. Groups: mutant AAT (Z), wild type AAT (M), expressing (4), non-expressing (1); 3 biological replicates/each group.
Groups: mutant AAT (Z), wild type AAT (M), expressing (4), non-expressing (1); 3 biological replicates/each group, all males, 6 weeks of age

first: Michael

last: Ewing

first: Karoly

last: Mirnics


title: Identification of cancer modifiers using parental strain expression mapping

status: Public on Oct 12 2007

city: boston

first: qing

last: xu

state: MA

address: 44 binney st

department: Medical Oncology

institute: dana farber cancer institute

summary:
Inherited genetic risk factors play an important role in cancer. However, other than cancer susceptibility genes found in familial cancer syndromes and inherited in a Mendelian fashion, little is known about modifier genes (germline variants that interact with each other and with environmental factors) that contribute to individual susceptibility. Here we develop a strategy “parental strain expression mapping” (PSEM), which utilizes the homogeneity of inbred mice and genome-wide mRNA expression analyses, to directly identify candidate germline modifier genes and pathways underlying phenotypic differences among murine strains exposed to transgenic activation of AKT1. We identified multiple candidate modifier pathways and specifically, the glycolysis pathway as a candidate negative modulator of AKT1-induced proliferation. In keeping with findings in murine models, the expression of the glycolysis pathway was strongly enriched in the non-cancer prostate tissue from patients with prostate cancer who did not recur after surgical resection. Together these data suggest that PSEM can directly identify germline modifier pathways of relevance to human disease.
Keywords: genetic modifier

overall_design: mRNA expression profiling was carried out with 24 samples of VP from 6 inbred strains.

first: Qing

last: Xu

first: Kenneth

last: Ross

first: William

last: Sellers


title: Comprehensive analysis of PPARα-dependent regulation of hepatic lipid metabolism by expression profiling - 1

status: Public on Jul 24 2007

city: Wageningen

first: Guido

last: Hooiveld

address: HELIX, Stippeneng 4

department: Div. Human Nutrition & Health

country: Netherlands

institute: Wageningen University

summary:
PPARα is a ligand-activated transcription factor involved in the regulation of nutrient metabolism and inflammation. Although much is already known about the function of PPARα in hepatic lipid metabolism, many PPARα-dependent pathways and genes have yet to be discovered.  In order to obtain an overview of PPARα-regulated genes relevant to lipid metabolism, and to probe for novel candidate PPARα target genes, livers from several animal studies in which PPARα was activated and/or disabled were analyzed by Affymetrix GeneChips. Numerous novel PPARα-regulated genes relevant to lipid metabolism were identified. Out of this set of genes, eight genes were singled out for study of PPARα-dependent regulation in mouse liver and in mouse, rat, and human primary hepatocytes, including thioredoxin interacting protein (Txnip), electron-transferring-flavoprotein β polypeptide (Etfb), electron-transferring-flavoprotein dehydrogenase (Etfdh), phosphatidylcholine transfer protein (Pctp), endothelial lipase (EL, Lipg), adipose triglyceride lipase (Pnpla2), hormone-sensitive lipase (Lipe), and monoglyceride lipase (Mgll). Using an in silico screening approach, one or more PPAR response elements (PPREs) were identified in each of these genes.  Since Pnpla2, Lipe, and Mgll contribute to hepatic triglyceride hydrolysis, gene regulation was studied under conditions of elevated hepatic lipids. In wild-type mice fed a high fat diet, the decrease in hepatic lipids following treatment with the PPARα agonist Wy14643 was paralleled by significant up-regulation of Pnpla2, Lipe, and Mgll, suggesting that induction of triglyceride hydrolysis may contribute to the anti-steatotic role of PPARα. Our study illustrates the power of transcriptional profiling to uncover novel PPARα-regulated genes and pathways in liver.
Keywords: identification of target genes

overall_design:
3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARα-null (129S4/SvJae) mice were used.

Fed mice were killed at the end of the dark cycle (wild-type and PPARα-null mice; n=5 per group), mice were fasted for 24h before sacrifice (wild-type and PPARα-null mice; n=5 per group). Liver total RNA of pooled samples (within groups) was hybridized onto Affymetrix MOE430A GeneChip arrays.

Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

first: Maryam

last: Rakhshandehroo

first: Linda

middle: M

last: Sanderson

first: Merja

last: Matilainen

first: Rinke

last: Stienstra

first: Carsten

last: Carlberg

first: Philip

middle: J

last: de Groot

last: Muller

first: Sander

last: Kersten


title: Potency of Testicular Somatic Environment to Support Spermatogenesis in XX/Sry Transgenic Male Mice

status: Public on Jul 15 2007

city: bunkyo-ku

first: Anshin

last: Hoshino

state: tokyo

address: 1-1-1 yayoi

department: agriculture

country: Japan

institute: toukyo-univ.

summary:
Sry is sufficient to induce testis formation and subsequent male development of internal and external genitalia in chromosomally female mice and humans.  In XX sex-reversed males such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth due to germ cell autonomous defects.  Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body.  Here we demonstrated that the testicular somatic environment of XX/Sry males is defective in the later phases of spermatogenesis.  Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, entering meiosis and differentiating into the round spermatid stage.  XY donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, thereby preventing further progress beyond the elongated spermatid stage.  In contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY donor tubules.  Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed missing expression of several Y-linked genes and alterations in the expression profile of genes associated with spermatogenesis.  Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, supporting our hypothesis that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice.
Keywords: comparative genomic hybridization

overall_design: Whole testes and seminiferous tubules of XX/Sry and W/Wv males were used for microarray expression analysis using the Affymetrix GeneChip system (Affymetrix, CA).  In order to isolate the seminiferous tubules, the tunica was carefully removed from the testes which were then incubated in the medium with 5 mg/ml collagnease at 37oC for 40 min.  The remaining seminiferous tubules were washed several times with PBS using a 70-ºm cell strainer to remove interstitial cells.  After total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD), double-stranded cDNA and biotin-labeled cRNA were synthesized using One-Cycle cDNA Synthesis and IVT Labeling kits (Affymetrix, CA), respectively. Twenty micrograms of fragmented biotin-labeled cRNA was hybridized to the Affymetrix Mouse Expression Array MOE 430A for 16 hr at 45oC.  The chips were washed, stained, and then scanned with the GeneArray Scanner (Hewlett Packard, CA) in accordance with the manufacturer's standard protocols. Finally, the microarray data were analyzed using Microarray Suite ver. 5.0 (Affymetrix).  Differential expression was defined as a difference of 2-fold or more in both whole testis and seminiferous tubule samples between two recipient males. Mouse 430A Affymetrix Genome Array IDs were used to query the NetAffx data mining tool for gene annotations.


title: Differential regulation of intestinal lipid metabolism in A/J versus C57BL/6J mice during diet-induced obesity

status: Public on Aug 27 2007

city: Haga-Gun, Tochigi

first: Hidehiko

last: Kondo

address: 2606 Akabane, Ichikai-Machi

department: Biological Science Laboratories

institute: Kao Corporation

summary:
The effects of high-fat (HF) feeding on the gene expression in the small intestine were examined using obesity-resistant A/J mice and obesity-sensitive C57BL/6J (B6) mice. Both strains of mice were maintained on low-fat (LF, 5% fat) or HF (30% fat) diets for two weeks. Oligonucleotide microarray and semi-quantitative RT-PCR analysis revealed that lipid metabolism-related genes including mitochondrial acyl-CoA thioesterase 1, pyruvate dehydrogenase kinase 4, and NADP+-dependent cytosolic malic enzyme were up-regulated by more than 2-fold in both strains of mice. The up-regulated gene expression levels were significantly higher in A/J mice than in B6 mice, suggesting the active lipid metabolism in the small intestine of A/J mice.
Keywords: diet response, strain difference

overall_design: Mice were maintained on the LF or HF diet for two weeks. For each group (n=6), pooled total RNA from two mice of average body weight was used for the analysis.

first: Yoshihiko

last: Minegishi

first: Yumiko

last: Komine

first: Takuya

last: Mori

first: Ichiro

last: Matsumoto

first: Keiko

last: Abe

last: Tokimitsu

first: Tadashi

last: Hase

first: Takatoshi

last: Murase


title: PERK role in the exocrine pancreas

status: Public on Aug 01 2007

city: University Park

first: Douglas

last: Cavener

address: 301 Life Sciences Building

department: Biology

institute: Penn State University

summary:
Molecular characterization of PERK's role in the mouse exocrine pancreas.  Postnatal days 16, 19 and 32 were examined.  Postnatal day 16 represents a stage immediately prior to severe cytological changes in the exPKO mice.  Postnatal day 19 represents the initial stage of severe phenotypes in the exPKO mice.  Postnatal day 32 represents the middle stage of severe phenotypes (about 2 wks from the initiation) in the exPKO mice.
Keywords: transgenic mouse

overall_design: Comparison between the two genotypes (Perk mutant vs. wild-type) at postnatal days 16, 19 and 32.


title: Expression data from transgenic mice (3 mo) inducibly expressing human alpha1-antitrypsin in the liver

first: Tunda

last: Hidvegi

summary:
In the classical form of α1antitrypsin deficiency a mutant protein accumulates in a polymerized form in the ER of liver cells causing liver damage and carcinogenesis by a  gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant α1antitrypsin Z in the ER specifically activates the autophagic response but not the  unfolded protein response and that autophagy plays a critical role in disposal of insoluble α1antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse  model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein.
Keywords: genomic analysis, alpha1-antitrypsin deficiency, comparative study, adult animals, inducible expression, liver specific

overall_design: Liver RNA from adult (3 mo old) male mice inducibly expressing human alpha1-antitrypsin wild type (M) or mutant (Z) form exclusively in the liver was subjected to genomic analysis. Groups: mutant AAT (Z), wild type AAT (M), expressing (4), non-expressing (1), wild type littermates (WT); 3 biological replicates/each group

first: Michael

last: Ewing

first: Karoly

last: Mirnics


title: Expression data of untreated or cisplatin-treated wildtype or Rb-/- MEFs

status: Public on Sep 01 2007

city: Chicago

first: Kay

last: Macleod

state: IL

address: 929 E 57th Str, CIS W338

department: Ben May Institute for Cancer Research

institute: The University of Chicago

summary:
The absence of the Rb tumor suppressor gene changes levels/activities of transcription factors (e.g., E2F and p53) which alter gene expression patterns, related to cell cycle control and cellular response to DNA damage.  Cisplatin is a genotoxic chemotherapeutic agent and wildtype or Rb null cells have different sensitivities to cisplatin-induced cytotoxicity.
We used microarrays to compare global profiles of gene expression and distinct responses of wildtype and Rb null MEFs in response to cisplatin.
Keywords: genotype and cisplatin treatment

overall_design: Primary wildtype or Rb null MEFs were generated from embryos at embryonic day 13.5 and cultured to passage 2.  Wildtype or Rb null MEFs were either untreated or treated with 16 microM cisplatin for 24 hours prior to harvest and RNA extraction.  Four different RNA samples (wildtype or Rb-/- MEFs, untreated or cisplatin-treated) were used for hybridization in triplate to Affimatrix Chips. Then, the total of 12 hybridizations were divided into 4 subseries: UT-Rb, UT-WT, CP-Rb, CP-Wt, and each subseries contained three samples (hybridization 1-3 or a-c).

first: Huiping

last: Liu


title: Effect PPARb/d knockout in white adipose tissue

status: Public on Jul 04 2007

first: Guido

last: Hooiveld

summary:
Analysis of white adipose tissue of PPARb/d knockout mice. Data may point towards putative target genes of PPARb/d and thus the function of PPARb/d in white adipose tissue. Datasets were used to identify glycogen synthase 2 as novel PPAR target.
Keywords: gene expression array-based, count

overall_design: Three month old male PPARb/d knock-out mice were on a mixed background (Sv129/C57BL/6). Wild-type littermates served as control animals. Animals received standard chow. Epididymal white adipose tissue (WAT) was removed and total RNA was isolated and pooled afterwards (five animals per group). Pooled RNA was hybridized to Affymetrix mouse genome 430 2.0 arrays arrays. Five microgram total RNA was labeled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix protocols.

first: Stephane

last: Mandard

first: Walter

last: Wahli

last: Muller

first: Beatrice

last: Desvergne

last: Kersten


title: Atrioventricular junction gene expression at embryonic day 10.5-11.0 in the mouse

status: Public on Sep 12 2007

city: Charleston

first: Andy

last: Wessels

state: SC

address: 173 Ashley Ave. BSB 648

department: Cell Biology and Anatomy

institute: Medical University of South Carolina

summary:
Goals of this study were to identify new candidates involved in the development of the Atrioventricular cushions in the mouse heart.

Keywords = Heart development
Keywords = Atrioventricular cushions
Keywords = Atrioventricular junction
Keywords = Atrioventricular myocardium
Keywords = embryonic day ED10.5-11.0
Keywords = ventricles
Keywords: development analysis

overall_design: The atrioventricular junction and ventricular region were dissected from 80 embryonic day 10.5-11.0, wild type, C57BL/6 mouse hearts.  RNA was isolated from the respective regions, amplified using the Arcturus RiboAmp HS RNA amplification kit, and hybridized to Affymetrix MOE430A expression chips in duplicate.  Comparison between atrioventricular gene expression and ventricular gene expression will allow for the identification of genes necessary in the development of the AV cushions.

first: Elaine

middle: E

last: Wirrig

first: Jeremy

last: Barth

first: Aimee

last: Phelps

first: Victor

last: Fresco

first: Stanley

last: Hoffman

first: Bryan

last: Toole

first: W

last: Argraves

first: Arno


title: Comprehensive analysis of PPARα-dependent regulation of hepatic lipid metabolism by expression profiling - 2

overall_design:
3-5 months old male pure bred wild-type (129S1/SvImJ) and PPARα-null (129S4/SvJae) mice were used.
Wild-type and PPARα-null mice were treated with the synthetic PPARα ligand WY14643 (0.1% w/w) mixed in the food, or normal food (control) for 5 days (n=5 per group). Liver total RNA of pooled samples (within groups) was hybridized onto Affymetrix MOE430A GeneChip arrays.
Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

first: Maryam

last: Rakhshandehro

first: Linda

last: Sanderson

first: Merja

last: Matilainen

first: Rinke

last: Stienstra

first: Carsten

last: Carlberg

first: Philip

last: de Groot

first: Michael

first: Sander


first: Guido

last: Hooiveld

summary:
Gene expression regulation of transporters and phase I/II metabolic enzymes in murine small intestine during fasting
Keywords: metabolic state analysis

overall_design:
Pure bred wild-type (129S1/SvImJ) and PPARα-null (129S4/SvJae) mice were maintained on AIN93M diet, and subsequently two sets of mice  were fasted for 24 hours. The complete intestines were then removed and total RNA was isolated.
In total 4 experimental groups were present: wild type and knockout mice that had free access to feed (AIN93M) before they were killed; wild type and knockout mice mice that were fasted for exactly 24 hours before they were killed.
RNA of 3 biological replicates was hybridized to Affymetrix MOE430A arrays. Five microgram total RNA was labelled according to the ENZO protocol, fragmented and hybridized according to Affymetrix's protocols.

first: Heleen

last: van den Bosch

first: Meike

last: Bunger

first: Jolanda

last: van der Meijde

last: Muller


title: Laser microdissection and microarray analysis of the hippocampus of Rasgrf1 knockout mice

status: Public on Jul 13 2007

city: Salamanca

first: Javier

last: De Las Rivas

address: Campus Miguel de Unamuno

department: CSIC and University of Salamanca

country: Spain

institute: Cancer Research Center (CiC-IBMCC)

summary:
We used manual macrodissection or laser capture microdissection (LCM) to isolate tissue sections of the hippocampus area of Ras-GRF1 wild type and knockout mice brains, and analyzed their transcriptional patterns using commercial oligonucleotide microarrays. Comparison between the transcriptomes of macrodissected and microdissected samples showed that the LCM samples allowed detection of significantly higher numbers of differentially expressed genes, with higher statistical rates of significance. These results validate LCM as a reliable technique for in vivo genomic studies in the brain hippocampus, where contamination by surrounding areas (not expressing Ras-GRF1) increases background noise and impairs identification of differentially expressed genes. Comparison between wild type and knockout LCM hippocampus samples revealed that Ras-GRF1 elimination caused significant gene expression changes, mostly affecting signal transduction and related neural processes. The list of 36 most differentially expressed genes included loci concerned mainly with Ras/G protein signaling and cytoskeletal organization (i.e. 14-3-3γ/ζ, Kcnj6, Clasp2) or related, cross-talking pathways (i.e. jag2, decorin, strap). Consistent with the phenotypes shown by Ras-GRF1 knockout mice, many of these differentially expressed genes play functional roles in processes such as sensory development and function (i.e. Sptlc1, antiquitin, jag2) and/or neurological development/neurodegeneration processes affecting memory and learning. Indeed, potential links to neurodegenerative diseases such as Alzheimer disease (AD) or Creutzfeldt-Jacobs disease (CJD), have been reported for a number of differentially expressed genes identified in this study (Ptma, Aebp2,Clasp2, Hebp1, 14-3-3γ/ζ, Csnk1δ, etc.). These data, together with the previously described role of IRS and insulin (known Ras-GRF1 activators) in AD, warrant further investigation of a potential functional link of Ras-GRF1 to neurodegenerative processes.
Keywords: knockout analysis

overall_design: 6 samples of tissue sections of the hippocampus area of wild type (WT) and Rasgrf1 knockout (KO) mice brains, 3 biological replicates of each.

first: A

last: Fernández-Medarde

last: Porteros

first: J

last: Nuñez

last: Fuster

first: E

last: Santos


title: miRNAs and codon usage regulate striatal gene and protein expression in two mouse models of Parkinson’s disease

status: Public on Jul 17 2007

city: Los Angeles

first: Mark

middle: H.

last: Chin

state: CA

address: 650 Charles E. Young Dr

department: Human Genetics

institute: UCLA

summary:
The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson’s disease (PD) are not completely understood.  Here we use microarrays and mass spectrometry to study the transcriptomic and proteomic changes in the striatum of two mouse models of PD induced by distinct neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and  methamphetamine (METH).  Transcripts and proteins were found with similar abundance changes in both models which may be involved in the pathophysiology of PD.  GFAP transcript and protein levels were significantly up-regulated by both neurotoxins, confirming the known astrocytic response to these drugs.  Other genes and proteins were idiosyncratic in their responses to the two toxins, suggesting specific toxicological responses.  Comparing transcript and protein levels revealed that efficiently translated genes used more commonly occurring codons than inefficiently translated genes.  Additionally, a potential role was found for miRNAs in translational control in the striatum.  The results constitute one of the largest datasets integrating transcript and protein changes for these two neurotoxin models with many similar endpoint phenotypes but distinct pathologies.  Using multiple toxins while examining proteins and transcripts can be an effective method of delineating the molecular pathology of neurodegenerative diseases.
Keywords: Pharmacological Disease Mouse models comparison

overall_design: Parkinson's disease mouse models were created by injecting male C57BL/6J mice with either Methamphetamine or MPTP and were compared to saline injected controls.  3 biological replicates were examined for each condition yielding 9 total samples.  Single channel Affymetrix mouse expression arrays 430_2 were used to analyze gene expression in the striata of each mouse.

middle: H

first: Wei-Jun

last: Qian

first: Haixing

last: Wang

first: Vladislav

last: Petyuk

first: Joshua

last: Bloom

first: Daniel

last: Sforza

first: Goran

last: Lacan

first: Dahai

last: Liu

first: Arshad

last: Khan

first: Rita

last: Cantor

first: Diana

last: Bigelow

first: William

last: Melega

first: David

middle: G

last: Camp II

first: Richard

middle: D

last: Smith

first: Desmond


title: Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers (430A)

status: Public on Jul 19 2007

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary:
The ability to purify to homogeneity a population of hepatic progenitor cells from adult liver is critical for their characterization prior to any therapeutic application.  As a step in this direction, we have utilized gene profiling of a bipotential liver cell line from dpc 14 mouse embryonic liver to catalog genes expressed by liver progenitor cells.  These cells, known as Bipotential Mouse Embryonic Liver (BMEL) cells, proliferate in an undifferentiated state and are capable of differentiating into hepatocyte-like and cholangiocyte-like cells in vitro.  Upon transplantation, BMEL cells are capable of differentiating into hepatocytes and cholangiocytes in vivo.  Microarray analysis of gene expression in the 9A1 and 14B3 BMEL cell lines grown under proliferating and differentiating conditions was used to identify cell surface markers preferentially expressed in the bipotential undifferentiated state.  This analysis revealed that proliferating BMEL cells express many genes involved in cell cycle regulation whereas differentiation of BMEL cells by cell aggregation causes a switch in gene expression to functions characteristic of mature hepatocytes.  In addition, microarray data and protein analysis indicated that the Notch signaling pathway could be involved in maintaining BMEL cells in an undifferentiated stem cell state.  Using GO annotation, a list of cell surface markers preferentially expressed on undifferentiated BMEL cells was generated.  One marker, Cd24a, is specifically expressed on progenitor oval cells in livers of DDC treated animals.  We therefore consider Cd24a expression a candidate molecule for purification of hepatic progenitor cells.
Keywords: cell type comparison

overall_design: RNA was extracted from two independently isolated BMEL cell lines (9A1 and 14B3) after culture under three conditions (basal, aggregate 5 days, and Matrigel).  Duplicate biological replicates were collected for each cell line:culture condition combination for a total of 12 samples.  Samples were biotin-labeled, hybridized to mouse 430a chips, and scanned according to established Affymetrix protocols.

first: S

first: H

last: Strick-Marchand

last: Venable

first: A

last: Dean

first: M

last: Wilde

last: Weiss

first: G

last: Darlington


title: Expression data of MB114 Endothelial cells during in vitro angiogenesis on Matrigel

status: Public on May 20 2007

city: Aurora

first: Allan

middle: Ray

last: Albig

state: CO

department: Pharmacology

institute: University of Colorado Health Sciences

summary:
MB114 cells cultured on Matrigel for 1, 5, 15, 25 hours
Keywords: time course experiment

overall_design: Total RNA was extracted from capillary-like matricies and used to synthesize biotin labeled probes.

first: Bill

last: Schiemann


title: Expression data from spleens of mice immunized using LPS vs MPL as adjuvant.

status: Public on Jun 09 2007

city: Louisville

first: Thomas

middle: Clark

last: Mitchell

state: KY

address: 570 S. Preston St.

department: Microbiology and Immunology

institute: University of Louisville

summary:
An unresolved issue in immunology is the extent to which inflammatory effects are needed for robust T cell responses.  In this study, mice were immunized by iv injection using either high toxicity lipopolysaccharide (LPS) or low toxicity monophosphoryl lipid A (MPL) as adjuvant.  Six hours after iv immunization, whole spleens were harvested and gene expression was measured in unfractionated splenic populations of cells.  The analysis indicated that the low toxicity adjuvanticity of MPL was associated with TLR4-mediated signaling that was biased to the TRIF branch of TLR4, while LPS generated balanced MyD88 and TRIF-associated outcomes.
Keywords: Treatment comparison

overall_design: B6.SJL mice adoptively transferred with T cells from B6.OTI and OTII transgenic TCR transgenic were immunized via intravenous injection with ova peptides alone ("Ova"), or with LPS ("OvaLPS") or MPL as adjuvant ("OvaMPL").   Six hours after immunization, the mice were euthanized by cervical dislocation, the spleens were removed and immediately lysed in guanidium chloride, and kept frozen until being used to extract total cellular RNA.  Three mice each were given the indicated treatments, with independent processing and analysis of nine samples total.

first: Sabine

last: Waigel


title: Fundic mucosa gene expression in the presence/absence of gastrin and in gastrin-CCK double knockout mice.

status: Public on May 03 2007

city: ANN ARBOR

first: Linda

last: Samuelson

state: MI

address: MED SCI II, # 7761, 1150 W. MEDICAL CENTER DRIVE

department: Molecular and Integrative Physiology

institute: University of Michigan

summary:
Fundic mucosa gene expression in wildtype vs gastrin knockout, gastrin replaced gastrin KO and gastrin-CCK double knockout mice
Fundic mucosal scraping RNA were isolated from WT, Gastrin Knockout, Gastrin replaced KO and Gastrin-CCK double knockout mice.  Targets from three biological replicates of each were generated and the expression profiles were determined using Affymetrix murine 430A arrays.  Comparisons between the sample groups allow the identification of genes with gastrin responsiveness and Gastrin-CCK double effect .
Keywords: repeat

overall_design: 3 WT, 3 Gastrin KO, 3 Gastrin replaced KO and 3 Gastrin-CCK double Knockout fundic RNA replicates were analyzed

first: Renu

last: Jain


title: Effects of ischemia reperfusion injury or nephrectomy on mouse lung

status: Public on May 30 2007

city: Baltimore

first: Dmitry

middle: N

last: Grigoryev

state: MD

institute: JHU

summary:
Experiments in rodents have shown that kidney ischemia/reperfusion injury (IRI) facilitates lung injury and inflammation. To identify potential ischemia-specific lung molecular pathways involved, we conducted global gene expression profiling of lung 6 or 36 hours following 1) bilateral kidney IRI, 2) bilateral nephrectomy (BNx), and 3) sham laparotomy in C57BL/6J mice. Total RNA from whole lung was isolated and hybridized to 430MOEA (22,626 genes) GeneChips (n=3/group).
Keywords: different time points after different treatments

overall_design:
All procedures were approved by the Johns Hopkins Animal Care and Use Committee, and were consistent with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male 6-8 week-old mice (C57BL6/J), weighing approximately 25-30 grams were obtained from Jackson Laboratory (Bar Harbor, ME) and housed under pathogen-free conditions according to NIH guidelines at least five days prior to operative procedures.
Animals were placed on a heating blanket and underwent midline laparotomy with isolation of bilateral renal pedicles. For mice assigned to experimental ischemia-reperfusion injury (IRI), a non-traumatic microvascular clamp was applied across both renal pedicles for 60 minutes. After the allotted ischemia time, the clamps were gently removed, the animals administered 1 ml of sterile saline intraperitoneally, and the incision closed in two layers with 4-0 silk suture. The animals were then allowed to recover with free access to food and water. Sham animals underwent the identical procedure without placement of the vascular clamps. The mice assigned to bilateral nephrectomy (BNx) underwent similar procedures except both renal pedicles were ligated with 5-0 silk suture and the kidneys removed. At 6 or 36 hours following the experimental procedure, the mice were euthanized by exsanguination under pentobarbital anesthesia and lung tissues collected for analysis.


title: Genome-wide expression analysis of satellite cells

status: Public on Apr 01 2007

city: Chiyoda-ku

first: Naoki

last: Tanimura

state: Tokyo

address: 2-3 Kanda-Nishikicho

department: Science Solutions Division

institute: Mizuho Information & Research Institute, Inc.

summary:
(Abstract of publication submitted currently) To clarify molecular regulation of satellite cells, we performed genome-wide gene expression analysis of quiescent satellite cells isolated from mouse skeletal muscle by flow cytometry.  We identified 53 novel quiescent satellite cell-specific genes whose expressions are sharply down-regulated upon activation.  The gene list contains a number of cell surface molecules, transcriptional factors, and cytokines and other signal transduction molecules.  We further confirmed that Odz4 and calcitonin receptor proteins were expressed by quiescent but not by activated satellite cells in vivo.  Importantly, we found that Pax7+/calcitonin receptor+ satellite cells reappear in close association with regenerating myofibers 7 days after muscle damage, often outside the basal lamina.  Moreover, an agonist of calcitonin receptor suppressed the activation of quiescent satellite cells on myofibers in in vitro culture, suggesting that calcitonin receptor signaling plays an important role in renewal and maintenance of satellite cells.  Our results show the gene expression profile of quiescent satellite cells for the first time and reveal the temporal and spatial reappearance of a satellite cell pool.
Keywords: cell type comparison

overall_design: Satellite cells and non-satellite cells were examined.  Totally three types of cells (groups), the satellite cells in quiescent and activated states and the non-satellite cells, were compared. Each has 4 replicates.

first: So-ichiro

last: Fukada


title: Gene Profile of RU486 effect on LPS induced gene expression in CNS.

status: Public on Mar 22 2007

city: Buenos Aires

first: Ariel

last: Chernomoretz

address: Ciudad Universitaria Pab I

department: Physics Department

country: Argentina

institute: University of Buenos Aires

summary:
The study was designed in order to identify genes differentially expressed when glucocorticoid signaling is blocked by a glucocorticoid-receptor antagonist (RU486 – mifepristone) in the context of brain inflammation induced by bacterial lipopolysaccharide (LPS). LPS is only able to cause murine brain damage in our experimental conditions upon RU486 pre-treatment. Hence, the study may reveal potential candidate genes to mediate neuroprotection or neurotoxicity. Due to the factorial design of the experiment, RU486 main-effect could be dissociated from the effects resultant of RU486/inflammation interaction. In addition, brain dissection was conducted to verify the effects in the brain side ipsilateral or contralateral to the site of intracerebral LPS infusion.
Keywords: Brain, CNS, LPS, Glucocorticoid, RU486, Mifrespitone, Innate immune response, Inflammation, Affymetrix, Gene profile, Neuroprotection, Neurodegeneration, Factorial design.

overall_design: C57Bl/6 mice received an i.p. injection of vehicle (DMSO - 50 microliters) or RU486 (50 mg/kg) and were submitted to surgery 4 h later. The mice receiving intraparenchymal injections were anesthetized and the right caudate putamen was reached, using a small cannula at the coordinates 0.0 mm anteroposterior, -2.0 mm lateral, and -3.0 mm dorsoventral according to a mouse brain atlas. The animals received an infusion of sterile pyrogen-free saline (1 microliter) or LPS (from Eschericia coli; serotype O55:B5; Sigma L2880 - 2.5 micrograms) over 2 min by means of a microinjection 18 pump. Animals were killed 12 h after the intracerebral infusion. The mice were anesthetized under isofluorane and blood was drawn via cardiac puncture before head decapitation. Brains were removed rapidly from the skulls and placed in cold phosphate buffered saline (PBS) solution. A brain region limited at plane anteroposterior +1.5 to -1.5 and dorsoventral -4.0 was dissected, separated in ipsilateral side and quickly immersed in liquid nitrogen. The tissue was stored at -80 oC until RNA extraction was performed. A total of 37 chips (MOE430A – Affymetrix, Santa Clara, CA) were used for oligonucleotide array analysis [one chip per biological sample; 8 groups (contralateral dmso/saline, dmso/LPS, RU486/saline, RU486/LPS and ipsilateral dmso/saline, dmso/LPS, RU486/saline, RU486/LPS) with 4 – 6 biological replicates each]. Expression values from the CEL files generated from scanning were obtained using RMA algorithm, available at http://www.bioconductor.org. The expression values were also inspected with GeneSpring software (Silicon Genetics). Statistical analysis was performed considering a factorial linear model according to the methods implemented in Limma package (R project packages are available at http://www.cran.r-project.org).

first: Isaias

last: Glezer

first: Serge

last: Rivest


title: Genome-wide analysis of PPARα activation in murine small intestine

status: Public on Jun 28 2007

first: Guido

last: Hooiveld

summary:
The peroxisome proliferator-activated receptor alpha (PPARα) is a fatty acid-activated transcription factor that governs a variety of biological processes. Little is known about the role of PPARα in the small intestine. Since this organ is frequently exposed to high levels of PPARα ligands via the diet, we set out to characterize the function of PPARα in small intestine using functional genomics experiments and bioinformatics tools. PPARα was expressed at high levels in both human and murine small intestine. Detailed analyses showed that PPARα was expressed highest in villus cells of proximal jejunum. Microarray analyses of total tissue samples revealed, that in addition to genes involved in fatty acid and triacylglycerol metabolism, transcription factors and enzymes connected to sterol and bile acid metabolism, including FXR and SREBP1, were specifically induced. In contrast, genes involved in cell cycle and differentiation, apoptosis, and host defense were repressed by PPARα activation. Additional analyses showed that intestinal PPARα dependent gene regulation occurred in villus cells. Functional implications of array results were corroborated by morphometric data. The repression of genes involved in proliferation and apoptosis was accompanied by a 22% increase in villus height, and a 34% increase in villus area of wild-type animals treated with WY14643. This is the first report providing a comprehensive overview of processes under control of PPARα in the small intestine. We show that PPARα is an important transcriptional regulator in small intestine, which may be of importance for the development of novel foods and therapies for obesity and inflammatory bowel diseases.
Keywords: identification of target genes

overall_design:
Pure bred wild-type (129S1/SvImJ) and PPARα-null (129S4/SvJae) mice were treated with the synthetic PPARα ligand WY14,643 (0.1% w/w) for 5 days. The complete intestines were then removed and total RNA was isolated.
In total 4 experimental groups were present: wild type mice fed the control diet (AIN93M), wild type mice fed the control diet supplemented with 0.1% w/w WY14,643 for 5 days, PPARα knockout mice fed the control diet, PPARα knockout mice fed the control diet supplemented with 0.1% w/wWY14,643 for 5 days.
RNA of 3 biological replicates was hybridized to Affymetrix 430A arrays.
Five microgram total RNA was labelled according to the ENZO-protocol, fragmented and hybridized according to Affymetrix's protocols.

first: Meike

last: Bunger

first: Heleen

last: van den Bosch

first: Jolanda

last: van der Meijde

first: Sander

last: Kersten

first: Michael

last: Muller


title: Dll3 and Notch1 genetic interactions model axial segmental and craniofacial malformations of human birth defects

status: Public on Jul 01 2007

city: Tempe

first: Kenro

last: Kusumi

state: AZ

department: School of Life Sciences

institute: Arizona State University

summary:
Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning.  Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions.  We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions.  While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, stochastic segmental anomalies similar to human congenital vertebral defects.  Unexpectedly, double heterozygous mice also displayed statistically significant decreases in mandibular height and elongated maxillary hard palate.  Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development.  Therefore, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders.
Keywords: genotype comparison

overall_design: Given the reduced penetrance and stochastic nature of the segmental and craniofacial defects in Dll3-Notch1 double heterozygous animals, we next sought to identify candidate genes that may be down or up-regulated in Dll3 and Notch1 homozygous mutant embryos during development of these structures.  We carried out microarray analysis using Affymetrix MOE430 microarrays, comparing 9.5 dpc homozygous embryos in duplicate to littermates with wild-type alleles at both loci, and comparing with heterozygous littermate embryos.  Affymetrix analysis software (Microarray Suite 5.0) was used to determine whether gene probes were present, marginal or absent.  Probes with present flags in replicates of Dll3, Notch1, or embryos with wild-type alleles at both loci were considered present for further analysis.  Altogether, out of 22,690 probe sets on the MOE430A array, we identified 12,820 probes that were present in replicates of at least one genotypic group.  To identify genes that were increased or decreased in expression in mutant embryos, we carried out robust multichip average (RMA) normalization of microarray data sets using the RMA module of Genespring GX 7.3 (Agilent).  After RMA normalization, housekeeping genes such as Gapdh showed steady expression (probe AFFX-GapdhMur/M32599_M_at, normalized expression 1.00 ± 0.02).  As a general indicator of variability between samples, we calculated Pearson’s correlation coefficients.  We found that the correlations between duplicates were moderately high: wild-type embryos (0.623), Dll3 embryos (0.584), Notch1 embryos (0.531).

first: Kathleen

last: Loomes

first: Stacey

middle: A

last: Stevens

first: Megan

last: O'Brien

first: Dorian

last: Gonzalez

first: Matthew

last: Ryan

first: Michelle

last: Segalov

first: Nicholas

last: Dormans

first: Mizuho

last: Mimoto

first: Joshua

last: Gibson

first: William

last: Sewell

first: Alyssa

last: Schaffer

first: Hyunduck

last: Nah

first: Eric

middle: F

last: Rappaport

first: Stephen

last: Pratt

first: Sally

last: Dunwoodie


title: The influence of the deletion of Serum Response Factor in B cells

status: Public on Jun 27 2007

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

country: Germany

institute: HCI - Helmholtz Centre for Infection Research

summary:
Serum response factor (SRF), a MADS-box transcription factor, is essential for murine embryonic development and for the function of muscle cells and neurons. SRF and its transcriptional co-factors are broadly expressed. To determine the in vivo role of SRF in developing lymphocytes we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes.
Keywords: Genetic modification

overall_design: Comparison of the gene expression profile between murine wildtype IgM+IgD+ cells and srf-deficient IgM+IgD+ cells isolated from spleens

first: A

last: Fleige

first: R

last: Schippers


title: Differential gene expression in BALB/c Npc1-/- and Npc1+/+ cerebellum

city: Berlin

first: Roland

last: Distl

address: Am Augustenburger Platz 1

department: Institute of Neuropathology

institute: Charité Universitätsmedizin Berlin

summary:
Purkinje cells (PC) of the cerebellum degenerate in adult mice with mutations in the Niemann-Pick type C (NPC) disease 1 (Npc1) gene. We subjected BALB/c Npc1+/+ and Npc1-/- mouse cerebella from an early and a later time point of PC degeneration to a genome-wide microarray gene expression analysis. We found general underrepresentation of PC-specific transcripts, consistent with PC loss, and elevated markers of microglia activation at the later time point.
Keywords: Niemann-Pick type C, Purkinje cell degeneration

overall_design: 12 BALB/c Npc1 mice of the two ages P21 and P49 and the two genotypes Npc1+/+ and Npc1-/- were used, 3 replicates for each age and genotype. The animals were of the same breed and lived under identical housing conditions. All except one animal were female. The animals were not further treated, but only sacrificed at P21 or P49.

first: Ralf-Juergen

last: Kuban

first: Vasily

last: Komkov

first: Knut

last: Kallwellis

first: Andreas

last: von Deimling

first: Thomas

last: Ohm


title: “Compensatory” transcriptional mechanisms:  Comparison of transcriptomes of Cx43 null and knockdown astrocytes

status: Public on Jun 20 2007

city: Prairie View

first: Dumitru

middle: Andrei

last: Iacobas

address: Ann Preston St

department: Undergraduate Medical Academy

institute: Prairie View A&M University

summary:
Our previous studies on brains and hearts of Cx null mice have revealed that expression level, control and coordination of a very large number of genes are regulated compared to wildtype littermates. This result suggests the possibility that the phenotype of the Cx null animals may include genes not only linked to the intracellular communication that gap junction channels provide but also to genes with very different functions. The question often arises, however, of the degree to which “compensation” occurs in knockouts such that gene regulation depends on pathways altered to make up for the missing gene rather than reflecting normal gene interlinkage. In order to explore this question in the Cx43 null setting, we have compared gene expression patterns in Cx43 null astrocytes with that in wildtype astrocytes acutely treated (36 hrs) with Cx43 siRNA. In these studies, Cx43 levels determined by Western blot analysis were reduced by at least 70% following siRNA treatment, comparable with the 3.24x downregulation for mRNA. For each group of astrocytes, four independent cultures were compared, using oligonucleotide microarrays printed with the mouse Qiagen library. 8060 well annotated unigenes were identifiable in all 8 arrays; of these, 8.2% were upregulated and 6.0% downregulated in Cx43 null astrocytes and 6.2% upreguated and 7.0 downregulated in siRNA treated astrocytes. Interestingly, regulation of 92.3% unigenes significantly regulated in both Cx43 deficient astrocytes had the same orientation, representing a highly significant overlap of gene expression alteration. These experiments thus indicate that the gene regulation in Cx43 null astrocytes is largely due to direct interlinkage rather than to developmental compensation for the missing gene.
Keywords: genetic modification

overall_design: Gene expression patterns in Cx43 null astrocytes and in wildtype astrocytes acutely treated (36 hrs) with Cx43 siRNA were compared with that in wildtype astrocytes.  In these studies, Cx43 levels determined by Western blot analysis were reduced by at least 70% following siRNA treatment, comparable with the 3.24x downregulation for mRNA.  For each group of astrocytes, four independent cultures were compared, using oligonucleotide microarrays printed with the mouse Qiagen library.

first: Sanda

first: Eliana

last: Scemes

first: David

middle: C

last: Spray


title: Aged hematopoietic stem cells, p53 mutants

status: Public on Jun 01 2007

first: Stuart

last: Chambers

summary:
Age-related defects in stem cells can limit proper tissue maintenance and hence contribute to a shortened life-span.  Using highly purified hematopoietic stem cells from mice aged 2 to 21 months, we demonstrate a deficit in function yet an increase in stem cell number with advancing age.  Expression analysis of more than 14,000 genes identified 1500 that were age-induced and 1600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the upregulated expression profile, while the downregulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Many chromosomal regions showed coordinate loss of transcriptional regulation, and an overall increase in transcriptional activity with aged, and inappropriate expression genes normally regulated by epigenetic mechanisms was observed. Hematopoietic stem cells from early-aging mice expressing a mutant p53 allele reveal that aging of stem cells can be uncoupled from aging at an organismal level. These studies show that HSC are not protected from aging.  Instead, loss of epigenetic regulation at the chromatin level may drive both functional attenuation of cells, as well as other manifestations of aging, including the increased propensity for neoplastic transformation.
Keywords: Aging time course

overall_design: Time course contains four time points in duplicate.  Whole bone marrow and p53 mutant arrays were used in a pairwise comparison and age estimate calculation, respectively.


title: Role of DNA methylation in stable gene repression

status: Public on Mar 06 2007

city: Jerusalem

first: Laura

last: Lande-Diner

address: Ein Kerem

department: Cell Biochemistry and Human Genetics

institute: Hebrew University Medical School

summary:
The experiment was designed to study the effects of the Dnmt1 gene knockout on gene expression, as well as to analyze the effects of TSA treatment on both WT (p53-/-) and knockout (p53-/-Dnmt1-/-) cells
The effect of TSA treatment on knockout and WT cells was  tested at two different time points (24 and 48 hs), as well as two weeks after removal of TSA
Keywords: Effect of demetyhlation and/or reacetylation on gene expression

overall_design: Seven hybridizations were performed: 1. p53-/- untreated (control), 2. p53-/- +TSA  harvested after 24hs of exposure to TSA, 3. p53-/- +TSA 48hs, 4. p53-/-Dnmt1-/- untreated, 5.p53-/-Dnmt1-/- +TSA  24hs 6.p53-/-Dnmt1-/- +TSA 48hs 7.p53-/-Dnmt1-/- two weeks after removal of TSA

first: Jianmin

last: Zhang

first: Ittai

last: Ben-Porath

first: Ninette

last: Amariglio

first: Ilana

last: Keshet

first: Veronique

last: Azuara

first: Amanda

last: Fisher

first: Gideon

last: Rechavi

first: Howard

last: Cedar


title: MyD88-dependent changes in the pulmonary transcriptome after infection with Chlamydia pneumoniae

status: Public on Mar 27 2007

city: Munich

first: Joerg

last: Mages

address: Trogerstrasse 30

department: Institute of Medical Microbiology, Immunology and Hygiene

institute: Technical University Munich

summary:
Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. Toll-like receptors and the key adaptor molecule MyD88 play a critical role in inducing immunity against this microorganism and are crucial to survive the infection. To explore the influence of MyD88 on induction of immune responses in vivo on a genome wide level, WT or MyD88-/- mice were infected with C. pneumoniae upon anesthesia and the pulmonary transcriptome was analyzed three days later by microarrays. We find that the infection induced the transcription of 360 genes and repressed 18 genes in WT mice. Of these, 221 genes were not or weakly induced in lungs of MyD88-/- mice. This cluster contains primarily genes encoding for chemokines, cytokines and other immune effector molecules. Genes induced by interferons were abundant in a cluster of 102 genes which were only partially MyD88-dependent. Interestingly, a set of 37 genes were induced more strongly in MyD88-/- mice and most of them are involved in the regulation of cellular replication. In summary, ex vivo analysis of the pulmonary transcriptome upon infection with C. pneumoniae demonstrated a major impact of MyD88 on inflammatory responses but not on interferon-type responses, and identified MyD88-independent genes involved in cellular replication
Keywords: lung, Chlamydia pneumoniae, mice, myd88, TLR, infection, knock out

overall_design: Microarray analysis was performed in biological triplicates with RNA from individual mice derived from three independent infection experiments. Samples were lungs from wt and myd88 knock out mice, three days after Chlamydia pneumoniae or mock infection. Altogther 12 samples were analyzed on Affymetrix MOE430A arrays

first: Jörg

first: Nuria

last: Rodríguez

first: Harald

last: Dietrich

first: Nina

last: Wantia

first: Hermann

last: Wagner

first: Roland

last: Lang

last: Miethke


title: Expression data from mouse liver RNA

status: Public on Feb 20 2007

city: Singapore

first: Chew-Kiat

last: Heng

address: 5, Lower Kent Ridge Road, Singapore 119074

country: Singapore

institute: National University of Singapore

summary:
Different diets and lipid-lowering treatment influence gene expression profiles in mouse liver
We used microarrays to detail the hepatic gene expression profiles and identified distinct classes of changed genes under the different treatments.
Keywords: different treatment

overall_design: Mouse livers were selected after different treatments for RNA extraction and hybridization on Affymetrix microarrays.

first: Yulan

last: Zhao

first: Mei-Yen

last: Chan

first: Shuli

last: Zhou


title: Transcripts enriched in mouse olfactory sensory neurons

status: Public on Apr 30 2007

city: Lexington

first: Neeraja

last: Sammeta

address: 800 Rose Street,

department: Physiology

institute: University of Kentucky

summary:
The overall aim of the experiment is to understand the phenotype of mature mouse olfactory sensory neurons by analyzing the transcripts expressed and enriched in them as compared to the rest of the cell types in the olfactory epithelium (consisting of immature neurons, supporting cells, progenitor cells and cells in lamina propria) and brain ( with out the olfactory bulbs). Comparision with the other cell types in the olfactory epithelium should eliminate the transcripts commonly expressed in the olfactory epithelium and comparision with brain will eliminate the transcripts common to most neurons.
Our gene chip data indicates that mature mouse olfactory sensory neurons express 10,000 genes. Mature OSNs specifically contained three clusters of over represented Gene ontology categories: smell, ion transport and cilia.  Analysis for the functionally over represented categories among the transcripts with a positive signal in the mature OSNs yielded largely broad categories common to all cells with the exception of chromatin modelling and RNA processing categories. Biological process categories of movement, development and immune response came as under represented categories.
Keywords: cell type comparison

overall_design: To purify mature olfactory neurons we took advantage of the OMP-GFP mice. OMP(olfactory marker protein) is expressed specifically in mature olfactory and vomeronasal sensory neurons. In the OMP-GFP mice the coding region of OMP is replaced by GFP. We purified OSNs from the rest of the epithelium from these mice by using FACS. . We used the Affymetrix gene chips mouse expression set 430 (consisting of 430A and 430B chips). Our gene chip data is extensively validated by insitu hybridizations.

first: Tun-Tzu

last: Yu

first: Timothy

middle: S

last: McClintock


title: Expression microarray analysis of pubertal mouse mammary gland development

status: Public on May 01 2007

city: Dublin

first: Finian

last: Martin

address: Conway Institute, Belfield

department: UCD School of Biomolecular and Biomedical Science

country: Ireland

institute: University College Dublin

summary:
The aim was to carry out global analysis of gene expression changes occurring in the normal pubertal mouse mammary gland from the appearance to the regression of terminal end buds.
Keywords: developmental time course

overall_design: 5 time points were chosen for analysis: 3wk, 4wk, 5wk, 6wk and 7wk. Each time point was analysed in triplicate with the exception of the 5wk time point which was analysed in duplicate. Total: 14 samples. Each sample was prepared from a pool of RNA from mammary glands of a minimum of three mice.

first: Jean

last: McBryan


title: Genetic alterations in mouse medulloblastomas and generation of tumors from cerebellar grunule neuron precursors

status: Public on Mar 15 2007

city: Memphis

first: Chunxu

last: Qu

state: TN

address: 262 Danny Thomas Pl

department: Pathology

institute: St Jude Children's Research Institute

summary:
Mice lacking p53 and one or two alleles of the cyclin D-dependent kinase inhibitor p18Ink4c are prone to medulloblastoma development. The tumor frequency is increased by exposing postnatal animals to ionizing radiation at a time when their cerebella are developing. In irradiated mice engineered to express a floxed p53 allele and a Nestin-Cre transgene, tumor development can be restricted to the brain. Analysis of these animals indicated that inactivation of one or both Ink4c alleles did not affect the time of medulloblastoma onset but increased tumor invasiveness. All such tumors exhibited complete loss of function of the Patched 1 (Ptc1) gene encoding the receptor for sonic hedgehog, and many exhibited other recurrent genetic alterations, including trisomy of chromosome 6, amplification of N-Myc, modest increases in copy number of the Ccnd1 gene encoding cyclin D1, and other complex chromosomal rearrangements. In contrast, medulloblastomas arising in Ptc1+/- mice lacking one or both Ink4c alleles retained p53 function and exhibited only limited genomic instability. Nonetheless, complete inactivation of the wild type Ptc1 allele was a universal event, and trisomy of chromosome 6 was again frequent. The enforced expression of N-Myc or cyclin D1 in primary cerebellar granule neuron precursors isolated from Ink4c-/-, p53-/- mice enabled the cells to initiate medulloblastomas when injected back into the brains of immunocompromised recipient animals. These engineered tumors exhibited gene expression profiles indistinguishable from those of medulloblastomas that arose spontaneously. These results underscore the functional interplay between a network of specific genes that recurrently contribute to medulloblastoma formation.
Keywords: disease state analysis

overall_design: 5 samples, 2 states

first: Frederique

last: Zindy

first: Tamar

last: Uziel

first: Olivier

last: Ayrault

first: Christoper

last: Calabrese

first: Marc

last: Valentine

first: Jerold

last: Rehg

first: Richard

last: Gilbertson

first: Charles

last: Sherr

first: Martine

last: Roussel


title: Conserved and distinct regulatory targets of Brn3a at different levels of the sensory axis

status: Public on Jan 28 2007

city: La Jolla

first: Eric

last: Turner

address: 9500 Gilman Dr., BSB 2077

department: Psychiatry

institute: University of California San Diego

summary:
General somatic sensation is conveyed to the central nervous system at cranial levels by the trigeminal ganglion (TG), and at spinal levels by the dorsal root ganglia (DRG).  Although these ganglia have similar functions, they have distinct embryological origins, in that both contain neurons originating from the neural crest, while only the TG includes cells derived from the placodal ectoderm.  Here we use microarray analysis of E13.5 embryos to demonstrate that the developing DRG and TG have very similar overall patterns of gene expression.  In mice lacking the POU-domain transcription factor Brn3a the DRG and TG exhibit many common changes in downstream gene expression, but a subset of genes show increased expression only at cranial levels.  Although silent in wild-type ganglia, the promoter regions of genes which are activated in the absence of Brn3a also exhibit increased histone H3-acetylation at levels similar to constitutively transcribed gene loci, and this H3-acetylation is tissue-specific for genes which are increased only in the TG.  These results demonstrate that one developmental role of Brn3a is to repress potential differences in gene expression between sensory neurons generated at different axial levels, and to regulate a convergent program of developmental gene expression, in which functionally similar populations of neurons are generated from different embryological substrates.
Keywords: Gene expression in the developing nervous system

overall_design: Microarrays used to compare the patterns of gene expression in the dorsal root ganglia and trigeminal ganglia of Brn3a knockout and wild-type mice. Embryonic day 13.5 (E13.5) was chosen because at this point in development mutant mice exhibit major defects in sensory axon growth, but have yet to undergo the period of extensive sensory neuron death associated with later stages.

first: Sani

middle: R

last: Eng

first: Iain

last: Dykes

first: Lanier

last: Jason

first: Natalia

last: Fedtsova


title: Expression data from 8 xenograft L2987 lung tumor tissue samples

status: Public on Apr 21 2007

city: Pennington

first: Mark

middle: David

last: Ayers

address: 311 Pennington-Rocky Hill Rd.

department: Clinical Discovery Technologies

institute: Bristol-Myers Squibb

summary:
Transcriptional profiling was conducted on RNA from 8 xenograft L2987lung tumor tissue samples to identify genes affected by treatment with a VEGFR-2 small molecule inhibitor
Keywords: Identfication of genes modulated by treatment with a small mollecule VEGFR-2 inhibitor

overall_design: Baseline and 14 days post treatement gene expression profiling was performed using 8 xenograft L2987 lung tumor tissue samples to identify genes affected by a VEGFR-2 small molecule inhibitor


title: Comparison of mock treated L929 cells with L929 cells infected with wildtype versus nt3A mutant VRP at 5hpi

status: Public on Apr 28 2007

city: Chapel Hill

first: Jennifer

last: Konopka

state: NC

address: CB 7292; 9th Floor Burnett-Womack Bldg; Manning Drive

department: Carolina Vaccine Institute

institute: University of North Carolina at Chapel Hill

summary:
This series includes arrays from two separate experiments where the gene expression profile of mock treated L929 cells was compared to that of L929 cells that had been infected with wildtype versus nt3A mutant Venezuelan equine encephalitis virus replicon particles (VRP).  Total RNA was isolated from all samples at 5 hour post-infection.
Keywords: virus infection-induced gene expression changes

overall_design: L929 cells were mock treated or infected at an MOI of 5 with either wildtype or nt3A mutant null-VRP, and total RNA was isolated at 5 hpi using the UltraSpec reagent protocol as described above.  The UNC-CH Functional Genomics Core Facility provided Affymetrix sample preparation and hybridization services, hybridizing the samples to Affymetrix Mouse Expression 430A (MOE430A) GeneChip arrays, which include analysis of ~22,600 genes.  Affymetrix GeneChip Microarray Suite 5.0 software was used for washing, scanning, and basic analysis, with sample quality assessed by examination of 3’ to 5’ intensity ratios of certain genes.  Expression analysis files created by Microarray Suite were imported into GeneSpring 6.2 (Silicon Genetics) for further gene expression analysis and filtering.  Two independent array studies were completed, and gene lists were compiled representing only those genes that were up- or downregulated by greater than 2-fold in replicate analyses.

middle: L

first: Robert

last: Johnston


title: Pac1+/+ versus Pac1-/- in macrophages and mast cells

status: Public on Jan 26 2007

city: Darlinghurst

middle: Reay

last: Mackay

state: NSW

address: 384 Victoria St

country: Australia

institute: Garvan Institute for Medical Research

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

overall_design: Refer to individual Series


title: Expression analysis of primary mouse megakaryocyte differentiation

status: Public on Jan 08 2007

city: Boston

first: Ramesh

last: Shivdasani

address: 44 Binney St.

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary:
Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate or advanced differentiation, and establish its value using mouse models of defective thrombopoiesis resulting from absence of GATA-1, NF-E2 or tubulinß1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stagespecific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis
Keywords: expression profiling differentiation megakaryocyte

overall_design: MK progenitors expand in mouse bone marrow or fetal liver cell preparations cultured with thrombopoietin, and mature over 5-6 days in vitro. After 3 days of culture, a significant fraction of cells shows features of committed MK progenitors, and numerous terminally mature, proplatelet-forming MKs appear by day 5. Because isolation of cell populations that correspond to sequential stages in MK differentiation is hindered by the lack of synchrony in primary MK cultures, we applied flow cytometry to harvest sub-populations that are substantially enriched for MKs with defined properties. High surface expression of the lineage marker CD41 identified MKs, whereas forward-scatter (FSC) properties distinguished cells on the basis of size. We collected thrombopoietic culture suspensions from days 3, 4 and 6, and sorted populations by flow cytometry. RNAs were prepared from sorted cells to probe Affymetrix MOE430A mouse oligonucleotide arrays.

first: Zhao

last: Chen

first: Mike

last: Hu


title: Earlybird mutation and knockout of Rab3a on gene expression in the cortex and hippocampus

status: Public on Dec 15 2006

city: Philadelphia

first: Maja

last: Bucan

address: 415 Curie Blvd

institute: UPENN

summary:
The cortex and hippocampus were dissected from each individual mouse.  Targets from three biological replicates of earlybird mutants and their wildtype littermates and six replicates of Rab3a knockouts and wildtype littermates were generated and the expression profiles were determined using Affymetrix MOE430 A and B arrays.  Comparisons between the sample groups allow the identification of tissue specifically expressed genes and genes that are affected by mutations of Rab3a.
Keywords: repeat

overall_design: Cortex and hippocampus from 3 biological replicates of earlybird mutants and wildtype littermates and 6 biological replicates of Rab3a knockouts and wildtype littermates were analyzed

first: Shuzhang

last: Yang

first: Michael

last: Farias

first: David

last: Kapfhamer

first: John

last: Tobias

first: Gregory

last: Grant

first: Ted

last: Abel


title: Surviving Sepsis: bcl-2 Overexpression Modulates Splenocyte Transcriptional Responses in vivo

status: Public on Feb 15 2007

city: St. Louis

first: J

middle: Perren

last: Cobb

state: MO

address: Campus Box 8109, 660 South Euclid Avenue

institute: Washington University in St. Louis

summary:
Background: We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the affect of bcl-2 on improving survival during sepsis.

Methods: Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2.  Whole spleens were obtained 6 hours after septic injury.  DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements.  Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene.

Results:  A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied.  At 6 hours, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., IL-6).  These sepsis-induced alterations were blocked by expression of the bcl-2 transgene.  Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1.  Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1.

Conclusion:  These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks.  In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses.
Keywords: Sepsis, bcl-2, wildtype, spleen, RNA expression, microarray

overall_design: To determine the splenic response in these lethal models of CLP and Pseudomonas pneumonia, microarray analysis was performed on each spleen harvested from wild-type animals 6 hours after CLP or tracheal instillation of bacteria.  The responses of the CLP or Pseudomonas spleens were compared concurrently to those of the wild-type controls, sham laparotomy and tracheal instillation of saline, respectively.  This study was repeated in animals overexpressing bcl-2.  Thus, the splenocyte effect of sepsis secondary to CLP (n=6) or Pseudomonas pneumonia (n=5) could be determined compared to their controls (n=6 and 5, respectively), and the effect of bcl-2 overexpression in turn also could be determined in both CLP (n=5) and pneumonia models (n=5) compared to controls (n=5 and 5, respectively).

first: Tracey

last: Wagner

first: Anne

middle: M

last: Drewry

first: Sandra

last: MacMillan

middle: W

last: Dunne

first: Katherine

last: Chang

first: Irene

middle: E

last: Karl

first: Richard

last: Hotchkiss

first: Perren

middle: J


title: Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: analysis by mRNA profiling

status: Public on Dec 30 2006

city: Bruce

first: Kristine

last: Hardy

state: ACT

address: University of Canberra

institute: University of Canberra

summary:
Background. T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non obese diabetic (NOD) mouse strain. Results. Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data we identify differentially expressed candidate genes including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. Conclusions. The data provide a molecular map of the negative selection response in vivo, and by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.
Keywords: Murine

overall_design: Analysis of two thymic populations - early single positive thymocytes (CD4+CD8lowCD69+TCRhi) and early double positive thymocytes (CD4+CD8+CD69-TCRlow). Samples purified from the 3A9 TCR transgenic (TCR) to characterise positive selection and the 3A9 TCR x insHEL double transgenic (Dbl) to characterise negative selection towards an Aire-dependent organ-specific antigen. Both the non autoimmune C57BL10.H2k (B10) and autoimmune Non obese diabetic (NOD) strains were analysed in this method. Three biological replicates were used for each of these eight different groups.

first: Adrian

last: Liston

first: Yvonne

last: Pittelkow

first: Susan

last: Wilson

first: Lydia

last: Markaroff

first: Aude

last: Fahrer

first: Christopher

last: Goodnow


title: MLL-AF9 transforms committed progenitors to leukemia stem cells by activation of a stem cell program (expt 2)

status: Public on Dec 02 2006

first: Scott

middle: A.

last: Armstrong

state: MA

address: 1 Blackfan Circle

department: Hematology/Oncology

institute: Children's Hospital

summary:
Leukemias and other cancers possess a rare population of cells capable of self-renewal, and eradication of these cancer stem cells is likely necessary for long-term cancer-free survival. Given that both normal and cancer stem cells are capable of self-renewal the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed.  We introduced the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23) found in human acute myelogenous leukemia (AML) into murine committed granulocyte-macrophage progenitors (GMP). The resultant leukemias contained cells with an immunophenotype similar to normal GMP that were highly enriched for leukemia stem cells (LSC). Detailed gene expression comparisons between normal hematopoietic stem cells (HSC), committed progenitors, and the LSC population demonstrated the LSC were globally more similar to the normal GMP than any other population. However, a subset of genes highly expressed in normal stem cells was re-activated in the LSC. These data demonstrate LSC can be generated from committed progenitors without widespread reprogramming of gene expression, and a leukemia self-renewal associated signature is activated in the process. Our findings define progression from normal hematopoietic progenitor to leukemia stem cell, and suggest that targeting a self-renewal program expressed in an abnormal context may be possible.
Keywords: MLL, stem cells, leukemia stem cells

overall_design: A normal HSC enriched population, common myeloid progenitors, granulocyte macrophage progenitors, megakaryocyte progenitors, and leukemia stem cells (leukemic-GMP) were isolated and labeled RNA was hybridzed to Affymetrix microarrays


title: Specific changes of liver transcriptome in the early stages of copper accumulation in the mouse model of wilson disease

status: Public on Dec 19 2006

city: Portland

first: Svetlana

last: Lutsenko

state: OR

address: 3181 SW Sam Jackson Pk Rd L224

institute: OHSU

summary:
Wilson disease (WD) is a severe metabolic disorder caused by genetic inactivation of copper-transporting ATPase ATP7B.  In WD, copper accumulates in several tissues, particularly in the liver, inducing marked time-dependent pathological changes.  To identify initial events in the copper-dependent development of liver pathology we utilized the Atp7b-/- mice, an animal model for WD.  Analysis of mRNA from livers of control and Atp7b-/- 6 weeks-old mice using oligonucleotide arrays revealed specific changes of the transcriptome at this stage of copper accumulation.  Few messages (29 up-regulated and 46 down-regulated) change their abundance more than 2-fold pointing to the specific effect of copper on gene expression/mRNA stability.  The gene ontology analysis revealed copper effects on distinct metabolic pathways.
Keywords: comparative genomic hybridization

overall_design: RNA isolation for microarrays:  Immediately after removal, what size [mg] the liver pieces for RNA isolation were frozen in liquid nitrogen and stored till further use.  The total RNA was isolated from frozen mouse livers using TRIZOL reagent (Invitrogen) according to manufacturer’s recommendations followed by a subsequent sample

first: Dominik

last: Huster

first: Tina

last: Purnat

first: Martina

last: Ralle

first: NErik

last: Olson

first: Franziska

last: Stuckert


title: Expression data from spec. transcrip. activity of contralateral mouse kidneys in response to Ischemia-Reperfusion-Injury

status: Public on Jan 16 2007

city: Bern

first: Marc

last: Kenzelmann

address: Friedbuhlstrasse 51

department: Molecular Genome Analysis

country: Switzerland

institute: Institute of Medical Microbiology, University of Bern

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

overall_design: The gene expression and regulation program at the RNA level of contralateral mouse kidneys of model IRI-mice was studied. Total RNA, amplified total RNA and specifically enriched newly transcribed RNA was isolated from normal untreated mouse kidneys and from contralateral mouse kidneys of IRI-mice. This experiment allowed detailed regulatory mechanisms in-vivo of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level of contralateral kidneys early (3h) after ischemia-reperfusion-injury..

first: Sabine

last: Maertens

first: Manfred

last: Hergenhahn

first: Stefan

last: Küffer

first: Agnes

last: Hotz-Wagenblatt

first: Li

last: Li

first: Shijun

last: Wang

first: Carina

last: Ittrich

first: Thomas

last: Lemberger

first: Rosa

last: Arribas

first: Sunitha

last: Jonnakuty

first: Monica

last: Hollstein

first: Wolfgang

last: Schmid

first: Norbert

last: Gretz

first: Hermann-Josef

last: Gröne

first: Günther

last: Schütz


title: Effect of photoperiod on gene expression in Peromyscus polionotus brain nuclei

status: Public on Dec 31 2006

city: Columbus

first: Brian

middle: C.

last: Trainor

state: OH

address: 1835 Neil Ave.

department: Psychology

institute: Ohio State University

summary:
The effect of photoperiod on gene expression was assessed in punch samples of the bed nucleus of the stria terminalis and medial preoptic area.  Long day mice were housed in 16L:8D and short day mice were housed in 8L:16D.
Keywords: cDNA microarray

overall_design: For the medial preoptic area, independent samples were collected from long day (n=3) and short day (n=3) mice.  For the bed nucleus of the stria terminalis independent samples were collected from long day (n=3) and short day (n=2) mice.

first: Shili

last: Lin

first: Michael

last: Rowlan

first: Sima

last: Finy

first: Randy

last: Nelson


title: Transcriptional profile of E14.5 micro-dissected kidney structures: renal vesicle, s-shaped body & collecting duct (MOE430A platform). (GUDMAP Series ID: 8)

status: Public on Nov 18 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

country: United Kingdom

institute: IGMM MRC Human Genetics Unit

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

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

first: Andrew

middle: P

last: McMahon

first: M

middle: T

last: Valerius


title: Mouse Retina P7 Rs1h KO versus Control

city: Regensburg

first: Bernhard

last: Weber

address: Franz-Josef-Strauss-Allee 11

institute: Institute of Human Genetics, University of Regensburg

summary:
Genome-wide expression profiling of the retinoschisin deficient retina in C57CL/6 mice.
Keywords: Genetic modification

overall_design: RNAs from retinae from 3 Wildtype Mice versus 3 Retinoschisin-KO mice (Rs1h-/Y) at postnatal day 7 (P7) were hybridized onto Affymetrix microarrays.

first: Andrea

last: Gehring

first: Franziska

last: Horling

first: Andreas

last: Janssen

last: Bonin

last: Walter

first: Sven

last: Poths

last: Langmann

middle: H