title: HCV tumor promoting effect is dependent on host genetic background

status: Public on Mar 22 2009

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:
The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo.

We crossed the HCV-Tg mice which do not produce HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and on gene expression in the liver of produced mice.

This SuperSeries is composed of the SubSeries listed below.

overall_design:
Refer to individual Series.
The expression profile of HCV-Tg and not HCV-Tg mice on the MDR2-KO and control MDR2-heterozygous background had been tested. The non-tumorous liver tissue from 4 non-HCV-Tg Mdr2-heterozygous female mice, 5 HCV-Tg Mdr2-heterozygous female mice, 5 non-HCV-Tg Mdr2-KO female mice, 5 HCV-Tg Mdr2-KO female mice and  6 tumors developed by  HCV-Tg MDR2-KO mice were submitted to the gene expression profiling. The non tumorous match of tumorous samples number 1-4 are HCV-Tg MDR2-KO samples 1-4, of tumorous sample 5,6 - HCV-Tg MDR2-KO sample 5.


title: Transcript analysis of HCV tumor promoting effect

summary:
The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo.
We crossed the HCV-Tg mice which do not produce HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and on gene expression in the liver of produced mice.

overall_design: The expression profile of HCV-Tg and not HCV-Tg mice on the MDR2-KO and control MDR2-heterozygous background had been tested. The non-tumorous liver tissue from 4 non-HCV-Tg Mdr2-heterozygous female mice, 5 HCV-Tg Mdr2-heterozygous female mice, 5 non-HCV-Tg Mdr2-KO female mice, 5 HCV-Tg Mdr2-KO female mice and  6 tumors developed by  HCV-Tg MDR2-KO mice were submitted to the gene expression profiling. The non tumorous match of tumorous samples number 1-4 are HCV-Tg MDR2-KO samples 1-4, of tumorous sample 5,6 - HCV-Tg MDR2-KO sample 5.

first: Naama

last: Klopstock

first: Orit

last: Pappo

first: Miriam

last: Sklair-Levy

first: Devorah

last: Olam

first: Lina

last: Mizrahi

first: Eithan

last: Galun

first: Daniel

last: Goldenberg


title: P13 cortex RNA

status: Public on May 30 2009

city: New York

first: Sung Wook

last: Chi

state: NY

address: 1230 York Ave,

country: USA

institute: Rockefeller Univ

summary:
To estimate transcripts levels in P13 mouse neocortex.

MicroRNAs (miRNAs) play critical roles in the regulation of gene expression. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein-RNA interaction sites.  We used HITS-CLIP to covalently crosslink native Argonaute (Ago) protein-RNA complexes in mouse brain.  This produced two simultaneous datasets—Ago-miRNA and Ago-mRNA binding sites—that were combined with bioinformatic analysis to identify miRNA-target mRNA interaction sites.  We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain.  Here we include the expression data obtained from dissected P13 mouse neocortex. These data are used for in silico CLIP simulation for normalization of Ago HITS-CLIP tags and also for selecting transcripts expressed in P13 mouse cortex.

overall_design: RNA from the neocortex of 3 wild type P13 CD1 mice.  One array per biological replicate.

first: Sung

middle: W

first: Robert

middle: B

last: Darnell


title: Differential Streptococcus agalactiae gene expression in human blood according to the blood donors

status: Public on Jun 01 2009

city: Warsaw

first: Izabela

last: Sitkiewicz

address: Chełmska 30/34

department: Epidemiology and Clinical Microbiology

country: Poland

institute: National Medicines Institute

summary: In the present investigation, we conducted a bacterial transcriptome dynamics analysis during GBS incubation with human blood. We observed large modification in the expression of numerous genes, but GBS transcriptome was different with the blood of one donor. More genes were up-regulated and less down-regulated in respect to the other donors.

overall_design:
The serotype III GBS reference strain NEM316 was cultivated in Todd Hewitt broth supplemented with 0.2% yeast extract (THY) in 5% CO2 at 37°C until OD reached 0.7. Culture was harvested by centrifugation 8 minutes at 4000 x g at 37°C, then suspended in phosphate-buffered saline (PBS).
Fresh heparinized human blood was obtained from one healthy volunteer in accordance  with a protocol approved by the Institutional Review Board of the Methodist Hospital Research Institute. The blood was maintained at 37°C no more than one hour prior to use. Bacterial suspension was then incubated with theblood at 37°C and 40°C under slight rotation to avoid sedimentation of blood and bacterial cells. Samples were removed for microarrays analysis immediately after adding bacteria, and after 30 and 90 minutes of incubation.
Thus, we obtained results for five time points: immediately after mixing the bacterium with the blood (time 0), after 30 minutes of contact with blood (time 1) at 37°C and 40°C, and after after 90 minutes of contact with blood (time 1) at 37°C and 40°C.

first: Laurent

last: Mereghetti

first: Nicole

middle: M

last: Green

first: James

last: Musser


title: Expression profiling in Wild-type and C-JUN knockout MEFs treated with IFNgamma and cycloheximide

status: Public on Mar 20 2012

city: Parkville

first: Geoff

last: Macintyre

state: Victoria

address: University of Melbourne

department: Department of Computer Science and Software Engineering

country: Australia

institute: University of Melbourne

summary:
STAT1 is the major transcription factor (TF) driving response to exposure to IFNg. C-JUN is a TF which has been shown to bind with STAT1 and act as a partner TF. We have expression profiled WT and CJUN-/- MEFs in order to determine the set of IFNg genes regulated by STAT1 and C-JUN.
This study was designed primarily to test the accuracy of in silico cis-regulatory module prediction algorithm called SCRM. The set of genes differentially expressed after IFNg are likely to be targets of the TF STAT1. A subset of these genes will also be targeted by partner TFs of STAT1 (e.g. C-JUN). By measuring the expression of the WT IFNg responsive genes in a C-JUN-/- model, the subset of genes that are regulated by C-JUN can be ascertained. This set of genes can then be compared against those predicted to be regulated by C-JUN using the in silico approach SCRM, and therefore the accuracy of the in silico predictions can be determined.

overall_design: Cells were treated with both IFNg and cycloheximide (CHX) to determine direct target genes of the TF STAT1. CHX was added to reduce the downstream response of IFNg treatment (via inhibition of protein synthesis) , resulting in a high quality list of genes directly targeted by STAT1. CHX treatment also allowed for a longer (3hr rather than 1hr) IFNg treatment time, resulting in a more robust IFNg gene signature. Microarrays were analysed using the aroma package in R and the differentially expressed genes were determined through analysis using the limma package. Genes were ranked based on the FDR adjusted p-value calculated using a t-test between IFNg + CHX treated cells and untreated cells (in both WT and C-JUN-/- MEFs). Significantly upregulated genes (P<0.05) were considered direct targets of STAT1.

middle: J

last: Gough

first: Christopher

last: Clark

first: Ricky

last: Johnstone

last: Bailey

first: Adam

last: Kowalczyk

first: Izhak

last: Haviv


title: Memory T cell RNA rearrangement by hn RNP LL

status: Public on Dec 31 2008

city: Canberra

first: Zuopeng

last: Wu

state: ACT

address: Garren Rd

department: Dept. of Immunology & Genetics

institute: John Curtin School of Medical Research

summary:
Immunological Memory is characterized by a heightened recall response to a previously encountered antigen, resulting from clonal expansion of antigen-specific lymphocytes and differentiation into specialized memory cells. Differentiation of memory B cells involves irreversible encountered antigen, resulting from clonal expansion of antigen-specific lymphocytes and differentiation into specialised memory cells. Differentiation of memory B cells involves irrversible DNA rearrangements and mutations, but differentiation of memory T cells is less understood. In an ENU screen for mouse mutations affecting the proportion of T cells with known memory cell markers, such as the alternative spliced Ptprc isoform CD45RO, we identified an inducible RNA-binding protein of previously unknown function, hnRNPLL (gene symbol Hnrpll), whose mutation abolishes the regulated silencing of CD45RABC exons in T cells and other leukocytes. In the mutant, a single amino acid substitution destabilizes an RNA-recognition domain that binds with micromolar affinity to RNA containing the Ptprc ARS exon silencing sequence. Hnrpll mutations selectively diminishes T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon array analysis of Hnrpll mutant naive and memory T cells reveals an extensive program of alternative mRNA splicing in memory T cells coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy of RNA rearrangement for achieving immunological memory in T cells.

Keywords: Memory; T cell; Immunological memory; Cell differentiation; Alternative splicing; T cell survival; Neuron; Brain; RNA-binding; Exon silencing sequence; Mouse mutation; Exon Microarray

overall_design: Single cell suspensions of spleen and lymph node cells were prepared from either wildtype or thu/thu mice, stained with anti-CD4, anti-CD8, and anti-CD44 antibodies, and sorted into sterile ice-cold RPMI+10%FCS. Four independent pools of mRNA from naïve CD4 (CD44lo), memory CD4 (CD44hi), naive CD8 (CD44lo), and memory CD8 (CD44hi) T cells, and from flow sorted CD4 and CD8 single positive thymocytes, were prepared from wildtype and thu/thu mice. For each pool, total RNA was extracted from 2-5 million sorted cells using a RNeasy plus Mini kit (Qiagen), purity analyzed using an Agilent Bioanalyzer Chip, 1ug RNA in vitro transcribed to cRNA, and after the first round amplification 10ug cRNA was used in a second round of amplification. A hybridization cocktail that included 5.5ug of fragmented end labelled ssDNA was applied to Affymetrix GeneChip Mouse Exon 1.0 ST Array chips. Hybridization was performed using F450-001 fluidics was and stain script using the Affymetrics Gene Chip Fluidics station 450. Array were scanned using the Affymetrix GCS3000 7G and Gene Chip Operating software to produce CEL files. The probeset intensities were calculated from the CEL files of the 48 samples in Partek Genomics Suite 6.3 using the core probe sets as defined by Affymetrix. 15,788 informative genes were included in the analysis, defining an informative gene as a unique transcript ID with mean signals for individual probesets greater than 3 in the T cell samples analyzed. Alternative splicing was analysed in each T cell subset using two way (genoytpe versus probeset ID) ANOVA using Partek algorithms with no normalisation. False discovery rates for each T cell subset 2-way ANOVA comparison were calcualted by the Benjamini & Hochger step-up method in Parteck. Gene vies were created with Partek using known trancripts in the UCSC Mouse Genome database (version July 2007).

first: Xinying

last: Jia

first: Xun-Cheng

last: Su

first: Bruz

last: Marzolf

first: Pamela

last: Troisch

last: Zak

last: Hamilton

first: Belinda

last: Whittle

first: Di

last: Yu

last: Sheahan

first: Edward

last: Bertram

first: Alan

last: Aderem

first: Gottfried

last: Otting

first: Gerard

middle: F

last: Hoyne

first: Chris

middle: C

last: Goodnow


title: Transcripts regulated by elevated DKF-2 (WT background)

status: Public on Jan 12 2009

city: Bronx

first: Charles

middle: S

last: Rubin

address: 1300 Morris Park Avenue

department: Molecular Pharmacology

institute: Albert Einstein College of Medicine

summary:
Transcriptional profiling was used to determine why DKF-2 (protein kinase D) deficiency and surfeit caused hypersensitivity and resistance, respectively, to a bacterial pathogen (P. aeruginosa 14). The studies show that DKF-2 contributes to C. elegans innate immunity by inducing expression of >75 genes that protect against pathogenic bacteria.

Keywords: Induced imune response

overall_design: Three independent biological replicates were isolated for each strain. The strains for each replicate were grown and harvested in parallel.

first: Hui

last: Feng

first: Min

last: Ren


title: Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages

status: Public on Aug 01 2009

city: Edinburgh

first: Paul

middle: A

last: Lacaze

address: 49 Little France Crescent

department: School of Medicine

country: United Kingdom

institute: University of Edinburgh

summary:
Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to activation. Genome-wide changes in transcript abundance caused by siRNA activity were measured using Affymetrix exon-arrays in the presence or absence of IFNγ stimulation. Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. The 11 genes targeted were Ifnb1, Irf3, Stat1, Stat2, Nkfb2, Irf5, Casp4, Ifi47, Lyn, Sod2, and Traf1. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays.

Keywords: RNAi, stress response, macrophage, IFN, interferon, siRNA, lipofectamine, cytokine

overall_design:
Transfection of murine bone-marrow derived macrophages (BMDMs) with non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays.

90 samples were analyzed in total.
1. Control experiments: 18 arrays studying the host response to Lipfectamine2000 and RiscFree non-targeting control siRNA (ThermoFisher) at 5 and 24 hours post treatment.
2. siRNA targeting experiments: 72 arrays in 12 different siRNA treatment groups, with each treatment group containing 6 arrays (3 replicates unstimulated, 3 replicates stimulated with IFNg). In each group a different targeting siRNA was used and in one group control non-targeting siRNA (RiscFree siRNA ThermoFisher) was used.

first: Sobia

last: Raza

first: Garwin

last: Sing

first: David

last: Page

first: Thorsten

last: Forster

first: Petter

last: Storm

first: Marie

last: Craigon

first: Tarif

last: Awad

first: Peter

last: Ghazal

first: Thomas

last: Freeman


title: The role of Insulinoma associated 1 protein in the development of the mammalian neocortex

status: Public on Oct 14 2008

city: Leipzig

last: Giger

address: Deutscher Platz 6

department: Evolutionary Genetics

country: Germany

institute: Max-Planck-Institute of Evolutionary Anthropology

summary:
Basal (intermediate) progenitors are the major source of neurons in the mammalian cerebral cortex. The molecular machinery governing basal progenitor biogenesis is unknown. Here we show that the zinc finger transcription factor Insm1 (insulinoma-associated 1) is expressed  specifically in progenitors undergoing neurogenic divisions and has a key role in basal progenitor formation. Mouse embryos lacking Insm1 contained half the number of basal progenitors and showed a marked reduction in cortical plate radial thickness. Forced premature expression of Insm1 in neuroepithelial cells resulted in their mitosis occurring at the basal (rather than apical) side of the ventricular zone and induced expression of the basal progenitor marker Tbr2. Remarkably,  these cells remained negative for Tis21, a marker of neurogenic progenitors, and did not generate neurons but underwent self-amplification. Our data imply that Insm1 is involved in the generation  and expansion of basal progenitors, a hallmark of cerebral cortex evolution.

Keywords: knock out, genetic modification,

overall_design: Dorsal telencephalon from four Insm1 knock out, six heterozygous , and eight wild type mouse embryos (E13.5) were isolated and genome wide gene expression was monitored in order to find target genes of Insm1.


title: MADS: a New and Improved Method for Analysis of Differential Alternative Splicing by Exon-tiling Microarrays

status: Public on May 09 2008

city: Iowa City

first: Yi

last: Xing

state: IA

address: 3294 CBRB, 285 Newton Rd

department: Internal Medicine, Biomedical Engineering

institute: University of Iowa

summary:
We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB.  From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing.
Keywords: control / knockdown comparison

overall_design: Short hairpin knockdown of PTB in mouse N2A neuroblastoma cells was performed as described before (Boutz et al., 2007, Genes Dev 21:1636-1652). The efficiency of the PTB knockdown was monitored by western blot using PTB-NT primary antibody and Cy5 labeled secondary antibody (GE Life Sciences). The blots were imaged using Typhoon 9410 (GE Life Sciences). The band intensities were measured using ImageQuant and normalized to GAPDH. In all cases the efficiency of the knockdown was close to 80% (data not shown). We conducted Exon array profiling on RNAs from three shRNA-PTB treated samples and three mock-treated controls (using empty vectors).

last: Stoilov

first: Douglas

middle: L

last: Black

first: Wing

middle: H

last: Wong


title: Expression data from Emx2 wildtype and knockout olfactory epithelium

status: Public on Oct 15 2008

city: Lexington

first: Jeremy

last: McIntyre

state: KY

address: 800 Rose St

department: Physiology

institute: University of Kentucky

summary:
Emx2 is a homeobox transcription factor that plays a critical role in development. Olfactory sensory neuron axons from Emx2 knockout mice fail to innervate their target tissue, the olfactory bulb. Homeobox transcription factors may also play an important role in olfactory receptor expression.
We used microarrays to analyze differences in mRNA abundance in the olfactory epithelium of Emx2 wildtype and knockout embryonic day 18.5 mice.

Keywords: Knockout-Wildtype comparision

overall_design: Total RNA from 9 Emx2 wildtype and 9 Emx2 knockout mice was analyzed. Equal amounts of RNA from 3 animals was pooled to create one biological sample, 3 pools per genotype, 6 chips total. RNA was hybridized to the Affymetrix Mouse Exon 1.0 ST array.

first: Timothy

last: McClintock