Met mutations in MEFs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Karl Dykema
PROVIDER: E-GEOD-11319 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Tyrosine kinase-activating mutations in Met have been observed in hereditary papillary renal carcinomas as well as in other cancers. These mutations have been examined in several in vitro systems, where they cause constitutive Met activation, focus formation, and cell motility, and are tumorigenic in xenografts. To determine the influence of these mutations on tumorigenesis in vivo, we generated mice with targeted mutations in the murine met locus. Five mouse lines with mutant Met were created: wt, D1226N, Y1228C, M1248T, and M1248T/L1193V. These mouse lines develop a wide range of sarcomas, lymphomas, and carcinomas. To determine if mutationally activated forms of Met have altered signaling specificity we utilized gene expression analysis. Mouse embryonic fibroblasts (MEFs) were isolated from the mutant Met lines and here we present data for wild type and M1248T (M833). Our results indicate that Met activating mutations have unique gene expression signatures. Keywords: mutant analysis
2011-11-20 | GSE11319 | GEO
Project description:Microarrays analysis was undertaken to compare mRNA levels in exponentially growing wild-type and sub1Δ cells, or after 1 h treatment with 4NQO. After spot quantification and normalization, only few genes were found to be differentially expressed when the two strains were exponentially grown in rich medium. In contrast, the distribution of gene expression ratios was clearly different upon 4NQO treatment showing that the transcriptional response to DNA damages was affected in the absence of Sub1. Most of the differentially expressed genes were up-regulated in sub1Δ cells, suggesting a global repression effect of Sub1. About half of them were bound by Sub1 demonstrating that Sub1 occupancy correlates with gene regulation. A GoTermFinder analysis revealed an enrichment in genes implicated in cell cycle process for the down-regulated genes. The up-regulated genes were enriched in ribosome biogenesis and cell wall structure GO categories, confirming that most of the genes bound by Sub1 were up-regulated in sub1Δ cells upon 4NQO treatment. A third GO category corresponded to genes encoding proteins involved in amino acid metabolism whose expression is under the control of the Gcn4 transcriptional activator. This GO category could reflect an indirect effect since the GCN4 gene is bound by Sub1, up-regulated in sub1Δ cells and induced at the translational level in response to DNA-damaging agents. Keywords: Transcriptomic Genomic Comparative Cell culture and RNA extraction. Wild-type (yPH500) or sub1Δ cells were grown to exponential phase (OD600 = 1) in YPD medium at 30°C and an aliquot of cells was collected to serve as the time-zero reference. After the addition of 4NQO (1 µg/µl), the cells were further incubated for 1h. The cells were harvested by centrifugation and resuspended in 50 mM Na acetate, pH 5.3, 10 mM EDTA. Total RNA was isolated by heating and freezing the cells in the presence of SDS and phenol as described (Schmitt et al., 1990). Preparation of labeled cDNAs Labeled cDNAs were synthesized from 20 to 30μg total RNA using an indirect labeling protocol adapted from P.Brown (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm) with the Superscript II reverse transcription kit (Gibco-BRL) and amino-allyl dUTP. lAmino-allyl modified DNA was purified on Microcon filters (Amicon) recovered with 20 µL of H2O and the DNA was lyophilized. The DNA pellet was resuspended in 9 µL of 100 mM sodium bicarbonate, pH 9.0. This sample DNA was used to dissolve a dry pellet of monofunctional NHS-ester Cy3 or Cy5 (1/16e of the quantity delivered from the mono-reactive dye pack PA23001 or PA25001 respectively from Amersham) and the mixture was incubated for 1 hour at room temperature in the dark. The coupling reaction of the Cy dyes to the amino-allyl dUTP was stopped by quenching with the addition of 4.5 µL of 4M hydroxylamine (Sigma) and the solution was further incubated for 15 min in the dark. Cy3-immunoprecipitated DNA and Cy5-WCE control DNA were mixed and unincorporated/quenched Cy dyes were removed using a Microcon YM-30 filter (Amicon/Millipore). DNA was recovered with 60 µL of TE and ethanol precipitated. Construction of ORF-microarrays S. cerevisiae open reading frames (ORF), reamplified using a pair of universal primers from 6144 full-length ORFs (1 ng) amplified from genomic DNA by Research Genetics. The PCR products were purified by ethanol precipitation, and their size and concentration were measured by agarose gel electrophoresis. 92.4% of the ORFs were correctly amplified and purified. The purified DNAs were spotted onto amino-silane coated glass slides (GAPS II, Corning) using an automated arrayer (MicroGrid II, BioRobotics) with a spotting success percentage higher than 96 %.. Prehybridization and hybridization. Prehybridization and hybridization conditions were those described on P. Brown's web site (http://brownlab.stanford.edu/protocols.html) with few minor modifications. The labeled precipitated DNA samples were recovered in hybridization buffer (50% formamide, 5X Denhardt’s, 0.5% SDS, 7X SSPE, 10 µg herring sperm DNA) and hybridized to the DNA microarray in a Corning slide chamber at 42°C overnight. Arrays were washed at room temperature once with 0.1X SSC + 0.1% SDS, twice with 0.1X SSC, then dried by centrifugation. Data analysis Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 6.0 software (Axon Instruments, Inc.). Array analyses were undertaken using the Limma package (Smyth, 2005) from the R/Bioconductor software (R-Development-Core-Team, 2007). Microarray spot intensities have been normalized by subtracting the background and using the LOWESS method (Cleveland, 1979) with the smooth parameter set to 0.33 as recommended (Quackenbush, 2002). Data from four technical replicates corresponding to two independent experiments were compiled. Normalized measures served to compute the ratios of Cy3/Cy5 intensity and the associated log2-transform (denoted log2-ratios) for each gene. To identify genes with a log2-ratio significantly different between the mutant and wild-type strains, p-values were calculated for each gene using a moderated t-test. The moderated t test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference.
2011-04-01 | E-GEOD-11124 | biostudies-arrayexpress
Project description:Acute physical exercise elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. Here, we investigated the impact of a single bout of running exercise until exhaustion on global transcriptional profiles in porcine skeletal muscles. Using a combined microarray and candidate gene approach, we identified a suite of genes that are differentially expressed in muscles during post-exercise recovery. Thus, several members of the heat shock protein family and proteins associated with proteolytic events were significantly up-regulated, suggesting that protein breakdown, prevention of protein aggregation and stabilization of unfolded proteins are important processes for restoring cellular homeostasis. We also detected an up-regulation of genes, which have been reported to be associated with muscle cell proliferation and differentiation, possibly reflecting an activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression of the nuclear hormone receptors, which regulates metabolic functions associated with lipid, carbohydrate and energy homeostasis. Finally, we observed an unanticipated involvement of long non-coding RNA transcripts, which have been implicated in RNA processing and nuclear retention of adenosine-to-inosine edited mRNAs. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery phase. Gene expression study of the porcine muscle Biceps femoris in regard to exercise, pigs allowed to rest for 0 hours, 1 hour and 3 hours after exercise were compared with pigs that had not been exercising, using in-house printed porcine two-colour oligonucleotide microarrays.
2012-07-12 | E-GEOD-30013 | biostudies-arrayexpress
Project description:Acute physical exercise elicits changes in gene expression in skeletal muscles to promote metabolic changes and to repair exercise-induced muscle injuries. Here, we investigated the impact of a single bout of running exercise until exhaustion on global transcriptional profiles in porcine skeletal muscles. Using a combined microarray and candidate gene approach, we identified a suite of genes that are differentially expressed in muscles during post-exercise recovery. Thus, several members of the heat shock protein family and proteins associated with proteolytic events were significantly up-regulated, suggesting that protein breakdown, prevention of protein aggregation and stabilization of unfolded proteins are important processes for restoring cellular homeostasis. We also detected an up-regulation of genes, which have been reported to be associated with muscle cell proliferation and differentiation, possibly reflecting an activation, differentiation and fusion of satellite cells to facilitate repair of muscle damage. In addition, exercise increased expression of the nuclear hormone receptors, which regulates metabolic functions associated with lipid, carbohydrate and energy homeostasis. Finally, we observed an unanticipated involvement of long non-coding RNA transcripts, which have been implicated in RNA processing and nuclear retention of adenosine-to-inosine edited mRNAs. These findings expand the complexity of pathways affected by acute contractile activity of skeletal muscle, contributing to a better understanding of the molecular processes that occur in muscle tissue in the recovery phase. Gene expression study of the porcine muscle Longissimus dorsi in regard to exercise, pigs allowed to rest for 0 hours, 1 hour and 3 hours after exercise were compared with pigs that had not been exercising, using in-house printed porcine two-colour oligonucleotide microarrays.
2012-07-12 | E-GEOD-30014 | biostudies-arrayexpress
Project description:To understand better the mechanisms controlling the balance between proliferation and and differentiation during myelopoiesis we have utilized teh bi-potent FDB1 myeloid cell line which differentiates to granulocytes and macrophages in response to GM-CSF and thus provides a dissectable model to analyse the switch between growth and differentiation. This was further studied using this cell line in which a second site mutation Y577F had been generated. A factorial time-course design between the two cell lines and over time, combined with linear modelling and geneset enrichment analysis identified the expression changes associated with the switch between granulocyte differentiation and macrophage differentiation. We observed that a single intracellular tyrosine residue (Tyr577) mediates the granulocyte fate decision. The loss of granulocyte differentiation and enhanced macrophage differentiation observed in this second site mutant is associated with accumulation of Beta-Catenin and activation of Tcf4 and other Wnt Target genes. Factorial design. First Factor was cell type with levels FDB1 expressing FI-Delta receptor mutant and FDB1 expressing FI-Delta Y577F receptor mutant. Second factor is time with levels 0 and 72 hours. Two biological replicates wre prepared for each cell line and comparions were perfomed for each cell line and directly between cell lines at 0 and 72hs. Additional dye swaps were done with FI Delta and FIDelta Y577F at 0h and the FI Delta 0-72h comparison.
2010-12-07 | E-GEOD-25857 | biostudies-arrayexpress
Project description:ChIP-chip of Pol II, H3K36me3 and CENP-A in met-1 C. elegans early embryo EXPERIMENT TYPE: CHIP-chip. BIOLOGICAL SOURCE: Strain: met-1(n4337); Developmental Stage: Early Embryo; Genotype: met-1(n4337); Sex: population predominantly Hermaphrodites perhaps with some Males; NUMBER OF REPLICATES: 2; EXPERIMENTAL FACTORS: temperature 20
2012-01-24 | E-GEOD-35079 | biostudies-arrayexpress
Project description:Parkinson's disease (PD) is defined by the degeneration of nigral dopaminergic (DA) neurons and can be caused by monogenic mutations of genes such as parkin. The lack of phenotype in parkin knockout mice suggests that human nigral DA neurons have unique vulnerabilities. Here we generate induced pluripotent stem cells from normal subjects and PD patients with parkin mutations. We demonstrate that loss of parkin in human midbrain DA neurons greatly increases the transcription of monoamine oxidases and oxidative stress, significantly reduces DA uptake and increases spontaneous DA release. Lentiviral expression of parkin, but not its PD-linked mutant, rescues these phenotypes. The results suggest that parkin controls dopamine utilization in human midbrain DA neurons by enhancing the precision of DA neurotransmission and suppressing dopamine oxidation. Thus, the study provides novel targets and a physiologically relevant screening platform for disease-modifying therapies of PD. Genomic DNA was isolated from each of the four lines of iPSCs and labeled with Cy5. Pooled sex mismatched normal human genomic DNA was labeled with Cy3. Both samples are hybridized together on RPCI 21K BAC aCGH array.
2012-01-21 | E-GEOD-35190 | biostudies-arrayexpress
Project description:acetylation of histone H4 was studied using ChIP on chip technique in breast epithelial HME1 cell lines overexpressing wt or mutant p53
2008-10-25 | E-MEXP-1494 | biostudies-arrayexpress
Project description:acetylation of histone H3 was studied using ChIP on chip technique in breast epithelial HME1 cell lines overexpressing wt or mutant p53
2008-10-25 | E-MEXP-1493 | biostudies-arrayexpress