{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Edward Oakeley"],"organism":["Mus musculus"],"software":["MicroArraySuite 5.0"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MEXP-85"],"description":["The Lck-OBF-1 construct used to generate transgenic mice contains an N-terminally HA epitope-tagged human OBF-1 cDNA under the control of the murine proximal lck promoter (-3100 to +23 relative to the transcription start site) that was purified by electroelution. Transgenic mouse lines were obtained and bred in B6CF1 x C57BL/6 background. For the analysis, age- and sex-matched littermates were used. The mice were kept in a standard mouse facility. Mice were sacrificed, the thymi were taken, rinsed in PBS and immediately homogenized in TRIZOL reagent (Life Technologies). Total RNA was prepared using TRIZOL reagent and purified on RNeasy Miniprep columns (Qiagen) according to the manufacturers' instructions.   Array set (A1): Equal amounts of RNA from four young adult male mice (6.5 weeks old) per genotype were purified as stated above and pooled in equal amounts. Array set (A2): Equal amounts of total RNA from four young adult mice (6.5 weeks old; 2 males and 2 females each time) per genotype were purified as stated above and pooled in equal amounts. Both the WT and the transgenic pool were hybridized to Affymetrix GeneChips in duplicate. Array set (B): RNA was prepared from sorted thymic cell populations (CD4+ CD8+ CD25- and CD4+ CD8+ CD25+) from a pool of 15 young adult (mixed gender, 6 to 9 weeks of age) transgenic mice as stated above. Cell sorting was performed on a MoFlo (DakoCytomation) with a purity of at least 94%."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Hybridization - Title: Fluidics Station Protocol. Description:","Nucleic Acid Extraction - Total RNA was prepared using TRIZOL reagent (Life Technologies) and purified on RNeasy Miniprep columns (Qiagen) according to the manufacturers' instructions.","Growth Protocol - In a first set of experiments (A), total thymus RNA from young adult Lck-OBF-1 transgenic mice and their WT littermates was isolated and gene expression levels were compared as a means to detect potential OBF-1 target genes.  In a second analysis (B), RNA was prepared from two sorted thymic cell populations (CD4+ CD8+ CD25- and an abnormal CD4+ CD8+ CD25+ population that expresses higher levels of transgenic OBF-1 protein) and gene expression levels were compared between these two populations. Due to the limited amount of RNA that was obtained, only one microarray experiment per sample could be made. The findings from this experiment (B) were subsequently compared to the findings from experiment (A).","Labeling - 10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5?-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3? (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc.","Sample Processing - The Lck-OBF-1 construct used to generate transgenic mice contains an N-terminally HA epitope-tagged human OBF-1 cDNA under the control of the murine proximal lck promoter (-3100 to +23 relative to the transcription start site) that was purified by electroelution. Transgenic mouse lines were obtained and bred in B6CF1 x C57BL/6 background. For the analysis, age- and sex-matched littermates were used. The mice were kept in a standard mouse facility. Mice were sacrificed, the thymi were taken, rinsed in PBS and immediately homogenized in TRIZOL reagent (Life Technologies). Total RNA was prepared using TRIZOL reagent and purified on RNeasy Miniprep columns (Qiagen) according to the manufacturers' instructions.   Array set (A1): Equal amounts of RNA from four young adult male mice (6.5 weeks old) per genotype were purified as stated above and pooled in equal amounts. Array set (A2): Equal amounts of total RNA from four young adult mice (6.5 weeks old; 2 males and 2 females each time) per genotype were purified as stated above and pooled in equal amounts. Both the WT and the transgenic pool were hybridized to Affymetrix GeneChips in duplicate. Array set (B): RNA was prepared from sorted thymic cell populations (CD4+ CD8+ CD25- and CD4+ CD8+ CD25+) from a pool of 15 young adult (mixed gender, 6 to 9 weeks of age) transgenic mice as stated above. Cell sorting was performed on a MoFlo (DakoCytomation) with a purity of at least 94 %."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","Additional Files","MAGE-TAB Files","Array Designs"],"data_protocol":["Feature Extraction - Title: Affymetrix CEL Analysis (Percentile). Description:","Assay Data Transformation - Title: Affymetrix CHP Analysis (ExpressionStat). Description:"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"pubmed_abstract":["OBF-1 (Bob.1, OCA-B) is a lymphoid-specific transcriptional coactivator that associates with the transcription factors Oct-1 or Oct-2 on the conserved octamer element present in the promoters of several ubiquitous and lymphoid-specific genes. OBF-1-deficient mice have B cell-intrinsic defects, lack germinal centers, and have severely impaired immune responses to T cell-dependent antigens. Crucial genes that are regulated by OBF-1 and that might explain the observed phenotype of OBF-1 deficiency have remained elusive to date. Here we have generated transgenic mice expressing OBF-1 specifically in T cells and examined these together with mice lacking OBF-1 to discover transcriptional targets of this coactivator. Using microarray analysis, we have identified the Ets transcription factor Spi-B as a direct target gene critically regulated by OBF-1 that can help explain the phenotype of OBF-1-deficient mice. Spi-B has been implicated in signaling pathways downstream of the B cell receptor and is essential for germinal center formation and maintenance. The present findings establish a hierarchy between these two factors and provide a molecular link between OBF-1 and B cell receptor signaling."],"study_type":["transcription profiling by array"],"species":["Mus musculus"],"pubmed_title":["The Ets factor Spi-B is a direct critical target of the coactivator OBF-1"],"pubmed_authors":["Boris Bartholdy","Herbert Angliker","Edward Oakeley","Bartholdy B, Du Roure C, Bordon A, Emslie D, Corcoran LM, Matthias P","Patrick Matthias"],"additional_accession":[]},"is_claimable":false,"name":"Transcription profiling of mouse ck-OBF-1 transgene","description":"The Lck-OBF-1 construct used to generate transgenic mice contains an N-terminally HA epitope-tagged human OBF-1 cDNA under the control of the murine proximal lck promoter (-3100 to +23 relative to the transcription start site) that was purified by electroelution. Transgenic mouse lines were obtained and bred in B6CF1 x C57BL/6 background. For the analysis, age- and sex-matched littermates were used. The mice were kept in a standard mouse facility. Mice were sacrificed, the thymi were taken, rinsed in PBS and immediately homogenized in TRIZOL reagent (Life Technologies). Total RNA was prepared using TRIZOL reagent and purified on RNeasy Miniprep columns (Qiagen) according to the manufacturers' instructions.   Array set (A1): Equal amounts of RNA from four young adult male mice (6.5 weeks old) per genotype were purified as stated above and pooled in equal amounts. Array set (A2): Equal amounts of total RNA from four young adult mice (6.5 weeks old; 2 males and 2 females each time) per genotype were purified as stated above and pooled in equal amounts. Both the WT and the transgenic pool were hybridized to Affymetrix GeneChips in duplicate. Array set (B): RNA was prepared from sorted thymic cell populations (CD4+ CD8+ CD25- and CD4+ CD8+ CD25+) from a pool of 15 young adult (mixed gender, 6 to 9 weeks of age) transgenic mice as stated above. Cell sorting was performed on a MoFlo (DakoCytomation) with a purity of at least 94%.","dates":{"release":"2006-10-25T00:00:00Z","modification":"2022-11-21T12:21:12.363Z","creation":"2022-03-09T05:01:34.881Z"},"accession":"E-MEXP-85","cross_references":{"pubmed":["16861304"],"EFO":["EFO_0002768"],"doi":["16861304"]}}