Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse ES cells expressing fosBd/d alleles which encode exclusively deltaFosB, and vs. phenotypes with fosB-null and wild-type ES cell lines

ABSTRACT: Among fos family genes modulating the cell fates such as cell proliferation, differentiation and cell death, only the fosB gene produces two forms of mature mRNA for FosB and deltaFosB proteins by alternative splicing of an exonic intron in the exon 4. FosB dramatically enhances the transcription regulation of AP-1 dependent promoters by Jun, while deltaFosB, a truncated form of FosB lacking its C-terminal transactivation domain, suppresses the function of Jun. We and other have shown that FosB and deltaFosB have a distinct function to control the cell fate as well as neuronal functions based on their exogenous expression. To elucidate the authentic function of each protein, it is essential to control each expression separately. We established a mutant mouse embryonic stem (ES) cell line carrying homozygous fosBd/d alleles which encode exclusively deltaFosB, and compared its gene expression profile and phenotypes with fosB-null and wild-type ES cell lines. Both mutant ES cells are devoid of FosB, therefore the common phenotypes between the two mutant ES cells in comparison to wild type depict the effects of FosB deficiency. Opposite phenotypes between the two are considered to be determined by deltaFosB itself. We analyzed the gene expression and cellular function among these two mutant and wild-type ES cells. Experiment Overall Design: Total RNA was purified from ES cells grown in the absence of feeder cells using the Isogen kit (Nippon Gene), according to the manufacturerâs instructions. For DNA microarray experiments, Total RNA (10 microg) were labeled using the Agilent Linear Amplification/Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. One microgram of each Cy3-labeled wild-type and Cy5-labeled mutant cDNA or each Cy3-labeled mutant and Cy5-labeled wild-type cDNA, respectively, were mixed, then hybridized to Agilent Mouse cDNA Microarrays (G4104A, design file number: 000522R000679, Agilent Technologies) with 8500 unique clones from Incyteâs mouse UniGene 1 clone set, according to the manufacturer's hybridization protocol. After the washing step, the microarray slides were analyzed with an Agilent G2565AA microarray scanner system. These experiments were carried out in duplicate using exchanged dye-labeled cDNA probes (i.e., Cy3 and Cy5 dye-swapping experiments).

ORGANISM(S): Mus musculus

SUBMITTER: Yoshinori Ohnishi

PROVIDER: E-GEOD-5963 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:The control of amino acid synthesis and transport in bacteria has been well-investigated at the transcriptional level. The discovery of a small Hfq-dependent regulatory RNA, GcvB, added another layer of gene expression control at the post-transcriptional level. GcvB RNA has been shown to directly regulate multiple ABC transporters for amino acids in E. coli and Salmonella using a highly conserved G/U-rich domain, R1. To identify additional GcvB targets, we have combined a sRNA pulse-expression and microarray analysis of whole transcriptome changes with biocomputational target searches for C/A- rich target sites in Salmonella. Moreover, we have included GcvB mutant RNAs in our microarray approach providing a new target search approach by inactivating conserved domains or target interaction sites. This dual approach revealed further amino acid transporters and, in addition, genes involved in amino acid metabolism as consensus R1-dependent GcvB targets. Moreover, GcvB RNA seems to bind with at least two binding sites to an R1-independent target, the glycine transporter cycA. Using GFP reporter gene fusions we have now validated 21 GcvB targets which is best to our knowledge with ~1% of all Salmonella protein coding genes, the largest bacterial sRNA-controlled regulon. Intriguingly, GcvB rewires many primary control circuits and, thus, constitutes an important metabolic knot. To predict direct GcvB targets with better confidence, we expanded the sRNA pulse-expression approach to assay effects of several versions of GcvB sRNA. We cloned GcvB wild-type and mutants RNAs deleted for the conserved regions R1 or R2 (Fig. 1A) under control of an arabinose-inducible PBAD promoter, yielding plasmid pBAD-GcvB (pKP1-1), pBAD-GcvB delta R1 (pKP2-6) and pBAD-GcvB delta R2 (pKP30-1). For confirmation of inducible expression, Salmonella wild-type carrying pBAD control vector (pKP8-35), and Salmonella ΔgcvB carrying either control vector (Ctr) or the above GcvB expression plasmids were grown to mid-exponential phase (OD600 of 1) and treated with L-arabinose for up to 15 min. We used whole-genome S. typhimurium microarrays to determine relative mRNA expression changes at 10 min of induction, comparing the mRNA profiles of the pBAD-GcvB, pBAD-GcvB delta R1 or pBAD-GcvB delta R2 strains to that of the delta gcvB deletion mutant strain carrying the control vector. Microarrays used in this study were produced by in-situ synthesis as 8x15k multipack format from Agilent Technologies. Each microarray comprises 13268 60-mer S. typhimurium strain SL1344 specific oligonucleotides supplemented with 319 60-mer S. enterica subsp. serovar Typhimurium 14028S specific oligonucleotides, 360 60-mer S. typhimurium LT2 specific oligonucleotides and 360 60-mer oligonucleotides specific for 149 Salmonella sRNAs. The experimental design involves the use of Salmonella enterica serovar Typhimurium genomic DNA as the co-hybridized control for one channel on all microarrays. Two independent biological eperiments were analyzed.

Project description:This experiment was conducted as part of an overall study aimed at examining effects of a dopamine receptor antagonist on reproduction, behavior, and gene expression in cyprinid fish. Relative to gene expression, we hypothesized that if haloperidol disrupted normal regulation of the HPG axis, there may be robust transcriptional alterations in the ovary of exposed females that might serve as useful markers of exposure and/or effects. For the purposes of this study, robust transcriptional alterations were defined as those detected in two separate fish species (fathead minnow and zebrafish), exposed to the same concentration of haloperidol for the same duration. There is a companion zebrafish microarray experiment (GSE14861) that these data are compared to. Transcriptional responses in the ovary of the two species are compared in terms of differentially expressed genes, enriched gene ontology categories they represent, and associated pathways. Fathead minnow and zebrafish microarray experiment Samples for microarray analysis were generated in a separate, 96 h, continuous flow-through experiment. Nominal treatment concentrations for the microarray experiment were 0 and 50 Î¼g haloperidol/L. Chemical (or control water) delivery was initiated 24 h prior to adding fish to the tanks. To start the experiment, fathead minnows (4 males, 4 females per tank) were added to each of three tanks per treatment group, while zebrafish (6 males, 6 females per tank) were added to each of two tanks per group. The time of fish addition was staggered by replicate within each treatment such that all samples from a given exposure tank could be collected within 60 min of the intended 96 h exposure duration. After 96 h, male and female zebrafish and female fathead minnows were anesthetized in buffered MS-222 and weighed. Whole gonads were removed, weighed, and preserved in RNAlater. Total RNA was isolated from fathead minnow ovary samples using RNeasy kits (Qiagen). RNA quality was assessed with a Agilent 2100 Bioanalyzer and quantity was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Aliquots of six fathead minnow ovary RNA samples per treatment group (two from each replicate per treatment) were prepared for microarray analysis.

Project description:The fathead minnow (Pimephales promelas) is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin, and gene expression data for the brain isoform of aromatase (CYP19B), vitellogenin precursors, and transferrin all provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and 7 genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as one potentially impacted pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time PCR analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology. Keywords: chemical stress response --Fadrozole exposure-- Treatments: 0, 60 ug fadrozole/L Replication: 4 replicate tanks per treatment, 2 replicate pairs (1 male, one female per pair) per tank for a total of 8 male, 8 female per treatment. Route of exposure: Waterborne, continuous flow through, without the use of carrier solvents, flow rate approx. 45 ml/min. Tanks: 20 L tanks containing 10 L of exposure water Fadrozole (purity â¥ 99%; Dr. H. Cooper Eckhardt, Summit, NJ, USA). Fish: Adult (ca. 6 month old) male (4.65Â±0.93 g) and female (1.90Â±0.44 g) Conditions: (25Â°C, 16:8 light:dark photoperiod, fed adult brine shrimp twice daily) Duration: 7 d (Â± 4 h) Sampling: Fish humanely euthanized in tricaine methanesulfonate (Finquel; Argent, Redmond, WA, USA). Liver, and brain samples transferred directly to pre-weighed vials of RNAlaterÂ® (Sigma, St. Louis, MO). Plasma samples were stored frozen at -80Â°C. Average water quality characteristics (Â±SD): temperature 25.5Â±0.1 Â°C; pH 7.21Â±0.04; dissolved oxygen 5.74Â±0.52 mg/L. --Microarray analysis-- n=3 microarrays per treatment and tissue Experimental samples: total RNA pooled from n=2 fish from the same treatment and replicate (except for pooled sample BC3 which included fish from two different replicate tanks). Reference sample: pooled liver, brain, and gonad samples from adult male and female fathead minnows Microarray design: Two color, reference design Hybridization: Experimental samples Cy5, reference sample Cy3

Project description:The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal, but is occasionally associated with inflammatory diseases. Recent work has indicated that evolutionary distinct lineages of P. acnes play etiologic roles in disease while others are associated with health. To shed light on the molecular basis for differential strain properties, we carried out genomic and transcriptomic analysis of distinct P. acnes strains. We sequenced the genome of the P. acnes strain 266, a type I-1a sequence type (ST) 18 strain. Comparative genome analysis of strain 266 and four other P. acnes strains revealed that overall genome plasticity is relatively low; however, a number of island-like genomic regions, encoding a variety of putative virulence-associated and fitness traits, differ between phylotypes. Comparative transcriptome analysis revealed that 225 genes of strain KPA171202 (type I-2, ST34) were differentially transcribed in strain 266 during exponential growth. 47% of these genes belong to the strain-specific gene content of strain KPA171202, indicating that strain-specific functions are utilized. Next, we studied differential expression during exponential and stationary growth phases. Genes encoding components of the energy-conserving respiratory chain as well as secreted and virulence-associated factors were transcribed during the exponential phase, while the stationary growth phase was characterized by up-regulation of genes involved in the stress response and amino acid metabolism. Taken together, our data highlight the genomic basis for strain diversity and identify, for the first time, the transcribed part of the genome, underling the important role active growth plays in the inflammatory activity of P. acnes. We argue that the disease-causing potential of different P. acnes strains is not only determined by variable genome content but also, and to a greater degree, by variable transcriptomes. Microarray experiments were performed as dual-color hybridizations. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color-swap dye-reversal was performed. Two different labeling methods were applied. RNA labeling was performed with the two color Quick Amp Labeling Kit (Agilent Technologies) using FullSpectrum MultiStart Primer for T7 IVT RNA Amplification (BioCat GmbH, Heidelberg, Germany) as random T7 labeling. Alternatively, the total RNA samples were amplified with the TransPlex Whole Transcriptome Amplification Kit (Sigma-Aldrich, Munich, Germany) and labeled with BioPrime Plus Array CGH Indirect Genomic Labeling System (Invitrogen, Karlsruhe, Germany) as WTA BioPrime indirect. Both methods were compared and combined for final data analysis.

Project description:Whirling disease, caused by the pathogen Myxobolus cerebralis, afflicts several salmonid species. Rainbow trout are particularly susceptible and suffer high mortality rates. The disease is persistent and spreading in hatcheries and natural waters of several countries, including the U.S., and the economic losses associated with whirling disease have been extremely large. In this study, gene expression profiling was conducted for resistant and susceptible rainbow trout strains following pathogen exposure with the primary objective of identifying specific genes associated with whirling disease resistance. Several genes were significantly up-regulated in skin following pathogen exposure for both the resistant Hofer and susceptible Trout Lodge rainbow trout strains. For both strains, response to infection appears to be associated with the interferon system. Metallothionein is differentially expressed between the resistant and susceptible strains and is a good candidate for future studies due to its diverse functional roles in inflammation and immune response. The present study has provided the first examination into the genetic basis underlying rainbow trout’s immune response and resistance to the whirling disease pathogen. The identified genes have allowed us to gain initial insight into the molecular mechanisms associated with a salmonid host’s immune response and resistance to whirling disease infection. Keywords: Disease state comparison. 1st comparison: exposed vs control. 2nd comparison: resistant vs susceptible strains Resistant (Hofer) and susceptible (Trout Lodge) rainbow trout strains were exposed to 2,000 triactinomyxons (whirling disease pathogen) per fish. Unexposed controls for each strain were treated identically to exposed other than their lack of pathogen exposure. After twenty-four hours, caudal fin tissue was collected for RNA extraction. Amplified RNA was competitively hybridized (exposed versus control) for each strain. Four biological replicates were used for each experimental condition and a balanced block design was incorporated.

Dataset Information

Transcription profiling of mouse ES cells expressing fosBd/d alleles which encode exclusively deltaFosB, and vs. phenotypes with fosB-null and wild-type ES cell lines

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets