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Gene expression profiling of perinatal or adult medullary thymic epithelial cells (MECs)

ABSTRACT: A comparative analysis of gene expression of perinate or adult Aire-GFP+ and GFP- MECs in WT or Aire-KO thymus Aire+ and Aire- MECs were sorted from adults (5 weeks old) or perinates (0-3 days old) of Aire-driven Igrp-GFP (Adig) mice in Aire-WT and -KO background. RNA from whole samples was amplified, labeled, and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.

ORGANISM(S): Mus musculus

SUBMITTER: CBDM Lab

PROVIDER: E-GEOD-66402 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression profiling of perinatal or adult medullary thymic epithelial cells (MECs)

Project description:A comparative analysis of gene expression of perinate or adult Aire-GFP+ and GFP- MECs in WT or Aire-KO thymus

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Expression data from WT and Aire KO thymic medullary epithelial cells (MECs)

Project description:The aim of this study is to analyze the transcriptional effects of Aire deficiency in the thymus, using the Affymetrix MoGene platform to analyze variation in exon usage MECs were isolated from 4-6 wk-old WT or Aire KO ((B6xNOD)F1 background) mice. Three WT and three Aire-KO mice taken individually were used.

2011-11-23 | E-GEOD-33878 | biostudies-arrayexpress

Gene expression profiling of perinatal or adult regulatory T cells (Tregs)

Project description:A comparative analysis of gene expression of 3 different experiments; 1. Perinate or adult-tagged GFP+YFP+ and bulk GFP+YFP- Tregs, 2. FL or BM-derived Tregs 3. Perinate or adult thymic Tregs. 1. Foxp3-eGFP-Cre-ERT2 x Rosa26-YFP lineage-reporter mice were injected ip with tamoxifen (4 injections, every third day) between 0-10 (perinate) or 35-45 (adult) days of age, and keep until 8 weeks of age. Perinate or adult-tagged GFP+YFP+ cells and bulk GFP+YFP- were double-sorted. 2. Hematopoietic progenitor cells were isolated from E18.5 fetal liver of CD45.1 congenic mice or from bone marrow of 5 weeks old CD45.2 B6 mice via negative selection of Thy1.2+ cells. The two populations were mixed at a 1:1 ratio, and were iv-transferred into irradiated (1000 rad) Rag-1-KO mice. FL or BM-derived Tregs were double sorted after reconstitution. 3. Foxp3-GFP+ Tregs were double-sorted from perinate (4 days old) or adult (5 weeks old) thymi. RNA from whole samples was amplified, labeled, and hybridized to Affymetrix Mouse Gene 1.0 ST Arrays.

2015-02-27 | E-GEOD-66332 | biostudies-arrayexpress

Gene Expression Profile of Medullary Epithelial Cells isolated from thymus of Bone Marrow (BM) reconstituted RAG1 null (control) and SCID NOD mouse

Project description:We identified DNAPK as one of the major proteins that physically interact with Autoimmune regulator (Aire). To establish physiological significance of DNAPK in Aire-driven expression of PTA genes in MECs, we utilized BM-reconstituted SCID mice (which express non functional DNAPK in their MECs) and RAG1 null mouse as a control. MECs from these mutated/reconstituted mice were sorted by Flow cytomety and their expression profile was analyzed using Affymetrix chips. The analysis shows that Aire-dependent PTA genes are significantly underexpressed in reconstituted SCIDS vs. RAGs (Controls), suggesting the involvement of DNAPK in Aire-mediated gene expression. Keywords: gene mutation

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Gene Expression Profile of Medullary Epithelial Cells isolated from thymus of Bone Marrow (BM) reconstituted RAG1 null (control) and SCID NOD mouse

2010-10-16 | E-GEOD-24734 | biostudies-arrayexpress

Crispr-Cas9-mediated Aire gene editing in medullary thymic epithelial (mTEC) cells shows its role as a gene expression modulator during thymocyte adhesion

Project description:The aim of this study is to evaluate the effect of Autoimmune regulator (Aire) gene disruption in a murine medullary thymic epithelial cells (mTEC 3.10 cell line) on the transcriptome of these cells during its adhesion with thymocytes. The mTEC-thymocyte adhesion is a crucial step for the negative selection of autoreactive thymocytes and prevention of autoimmune diseases. To generate Aire mutant cell clones, a total of 5x10^5 mTEC 3.10 cells were electro-transfected (Lonza Nucleofector) with CRISPR-Cas9 plasmid targeting the Aire Exon 3 (plasmid "all in one" encoding Aire Exon 3 gRNA + Cas9 + GFP, from Sigma-Aldrich). The GFP positive mTEC single cells were sorted by using a FACS Aria III cytometer and cells were cloned by expansion in culture. Sanger sequencing of PCR products from the Aire Exon 3 of these clones was used in order to evaluate the occurrence of indel mutations within the targeted Exon 3. The mTEC 3.10 clone E6 was identified and validated as a compound heterozygous Aire KO (Aire +/-). This clone features the Aire allele 1 that encodes a mutant Aire protein carring a neutral aminoacid substitution (A118P) and allele 2 encoding a truncated Aire protein. Wild type (WT) mTEC 3.10 cells or mTEC 3.10 clone E6 were cultured in the presence (or not) of thymocytes in order to establish cell adhesion. The total RNA preparations from WT or clone E6 mTEC cells (before or after mTEC- thymocyte co-cultures) were then sequenced through RNA-sequencing using a Illumina HiSeq 2500 instrument and the TruSeq Stranded mRNA Library Preparation kit resulting in about 50 million paired-end stranded specific 100 bp reads per sample. Sequencing reads were mapped to Mus musculus reference genome (mm10) using STAR v.2.5.0a. Read counts over transcripts were calculated using HTSeq v.0.6.1p2 based on a current UCSC annotation file for GRCm38/mm10 (Dec. 2011).

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WT and Aire-KO mouse MEChi RNAseq profiling

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Expression Data of Treg and Tconv Cells from FoxP3-GFP Chimeric and FoxP3-ires-GFP B6 and NOD Mice

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