Global gene expression profiling of the avian B-lymphoma DT40 cell line was used as a model to differentiate among Btk KO and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell line, subclones are selected, which have multiple changes in their gene expression pattern (p<0.01).
Expression profiling of chicken DT40 lymphoma cells indicates clonal selection of knockout and gene reconstituted cells.
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View SamplesSplenic Transitional Type-1 B-cells from CBA wild-type mice, X-linked immunodeficiency mice and Bruton's tyrosine kinase knock-out mice. Two replicates where run on Affymetrix 420 2.0 arrays for CBA wild-type, Xid samples and the Btk KO samples.
Distinct gene expression signature in Btk-defective T1 B-cells.
Specimen part
View SamplesBruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (KO) and immunocompetent CBA mice, were used in microarrays containing more than 12,000 genes and expressed sequence tags (ESTs). We found 4515 transcripts expressed in duplicate experiments in all three strains. Out of these, 38 were differentially expressed genes (21 up-regulated >2 fold and 17 down-regulated <-2 fold) between CBA and Btk defective mice. Ten out of these genes were selected and quantitative Real-Time PCR was conducted for validation and further investigation. Real-Time experiments correlated nicely with the microarray data.
Gene expression profile of B cells from Xid mice and Btk knockout mice.
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Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.
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View SamplesThe Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.
Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.
No sample metadata fields
View SamplesThe Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.
Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.
No sample metadata fields
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.
Specimen part, Treatment
View SamplesDuchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here,we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNAtarget interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.
Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.
Specimen part, Treatment
View SamplesCells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of these extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. Whilst exosomes are assumed to be a homogenous population of EVs, they have a wide range of putative functions. Therefore, we hypothesized that cells release subpopulations of exosomes with distinct molecular compositions and functional properties. Exosomes were isolated from conditioned medium by differential ultracentrifugation. Sucrose density gradient centrifugation of the resulting exosome pellet revealed the presence of two distinct subpopulations, one smaller, slow migrating population (HD-Exo), and one fast migrating, larger population (LD-Exo).
Cells release subpopulations of exosomes with distinct molecular and biological properties.
Specimen part
View SamplesRecent studies demonstrated that tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We established glioblastoma stem-like (GS-) cell cultures from 9 different glioblastomas, 8 of which generated stably expandable cell lines. Analyzing GS-cell cultures, we discovered two clearly discernable phenotypes.
Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria.
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