Identification of genes regulated by RORg in mouse thymus
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE4248 | GEO | 2006/04/01
SECONDARY ACCESSION(S): PRJNA94961
REPOSITORIES: GEO
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Project description:Microarray analysis was performed in order to identify changes in gene expression in thymocytes isolated from RORg-/- mice in comparison to those of wild type mice. Experiment Overall Design: Microarray analyses were carried out by the NIEHS Microarray Group (NMG). Gene expression analysis was conducted using Agilent Mouse arrays (Agilent Technologies, Palo Alto, CA) representing about 20,000 genes. For each condition, equal amounts of total RNA from thymocytes of three individual wild type or RORg-/- mice were pooled and amplified. Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 1 mg of total RNA, Cy3- or Cy5-labeled cRNA was synthesized according to manufacturerâs protocol. For each comparison, 750 ng of each Cy3- and Cy5-labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained with the Agilent Feature Extraction software (v7.1) using defaults for all parameters.
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Project description:The DNA binding factor Tcf-1 is one of the most prominently expressed genes in thymocytes yet its global DNA binding pattern remained unknown. Here we have assessed by ChIP-seq the Tcf-1 binding pattern in murine thymocytes in WT B6 mice and in mice expressing a stabilized form of the Tcf-1 binding partner beta-Catenin (CAT mice).
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Project description:CD5 is characterized as an inhibitory co-receptor with important regulatory role during T cell development. To study the molecular mechanism by which CD5 operates, we used quantitative mass spectrometry to analyze the components of the CD5 signaling machinery in primary T cells. In a first set of experiments, CD5-containing complexes were immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice. Thymocytes were treated with pervanadate to induce widespread activation of protein tyrosine kinases. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls, to discriminate CD5-binding molecules from the background of contaminant proteins. Eight biological replicates were prepared for both conditions, and samples were analyzed in duplicate LC-MS runs. This first set of experiment is presented in TableS1 of the paper, and is associated to the MaxQuant result file “Interactome WT” in the present PX dataset. We analyzed the CD5 interactome at different time of stimulation with pervanadate (1min and 10min) using thymocytes from wild-type (WT) C57BL/6 mice. Corresponding samples prepared from thymocytes of Cd5−/− mice were used as controls. Three biological replicates were prepared for both conditions (WT and KO) and each time point (1min and 10min). This set of experiments is presented in TableS2 of the paper and is associated to the MaxQuant result file “Interactome at 1min and 10min” in the present PX dataset. We checked that the activation of CD5 (phosphorylation sites) and the formation of the signaling complex was comparable in different stimulatory conditions (comparison of WT thymocytes stimulated with either pervanadate or anti-CD3+anti-CD4 antibodies). We also analyzed the binding of CD5 interactors in thymocytes from a c-Cbl-/- mouse model (comparison of WT and c-Cbl-/-thymocytes stimulated with pervanadate). These experiments and the associated experimental design of the comparisons are summarized in TableS3 of the paper, and are based on the MaxQuant result file “Comparison of different stimulatory conditions” in this dataset. To check the phosphorylation status of the CD5 tyrosine residues at different time of stimulation, we analyzed samples immunoprecipitated from thymocytes of wild-type (WT) C57BL/6 mice, following stimulation with anti-CD3+anti-CD4 antibodies for 1min, 3min and 10min. This set of experiment is presented in TableS4 of the paper and is associated to the MaxQuant result file “CD5 pY kinetics” in the present PX dataset. To determine the role of Y429 in the context of CD5 signaling, we expressed a wild-type (CD5tgWt) or a mutated form of CD5, containing a tyrosine to phenylalanine substitution at position 429 (CD5tgY429F) in transgenic mice. We analyzed the CD5 interactome by comparing samples immunoprecipitated from thymocytes of these 2 mice models (following pervanadate stimulation, 6 and 5 biological replicates respectively) with samples prepared in the same way from Cd5−/− mice as controls (6 biological replicates). The results are shown in TableS5 of the paper, and correspond to the MaxQuant result file “interactome CD5tgWt_CD5tgY429F”.
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