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Gene Expression Profile of 293T and 293T-Aire-expressing cells

ABSTRACT: To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection

ORGANISM(S): Homo sapiens

PROVIDER: GSE24733 | GEO | 2010/10/16

SECONDARY ACCESSION(S): PRJNA132421

REPOSITORIES: GEO

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Gene Expression Profile of 293T and 293T-Aire-expressing cells

Project description:To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection 293T cells were seeded on 10cm plates and were grown in DMEM containing 10% FBS under standard TC conditions. Next day, the cells were transfected either by pCMV-Aire (6ug/plate) or empty pCMV (6ug/plate) using Mirus reagent, according to manufacturer's instructions. Total RNA was extracted 48 hrs post transfection by Trizol protocol according to the manufacturer's instructions. 3 biological replicates were used for Aire transfection and 2 replicates for transfection with a control plasmid.

2010-10-16 | E-GEOD-24733 | biostudies-arrayexpress

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GIM mTEC Aire silencing

Project description:We used the TriFectaTM (IDT, Integrated DNA Technologies, Coralville, IA, USA) anti-Aire RNAi sequence (GGAUUCUCUUUAAGGACUACAAUCTAGAUUGUAGUCCUUAAAGAGAAUCCUC) in order to silencing Aire mRNA. Confluent cultures of 3.10 mTEC cell line were transfected with 10nM of anti-Aire siRNA using Hiperfect reagent (Qiagen, GmbH, Hilden, Germany) following manufacturerM-^Rs instructions. After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kitM-. (Ambion, Austin, TX, USA), which served as template for cDNA synthesis. Gene knockdown was confirmed by quantitative reverse transcription PCR (qRT-PCR) using the primers 5_-GCAACTCTGGCCTCAAAGAG-3_ forward and 5_-GGTCTGAATTCCGTTTCCAA-3_ reverse, which allowed amplification of a 120 bp PCR product corresponding to a segment of the Aire cDNA. The cDNA samples were prepared using Superscript II reverse transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) enzyme, as recommended. Expression of the above mentioned genes was quantified using a 7500 Real Time PCR System (Applied Biosystems)

2011-08-31 | E-MEXP-3213 | biostudies-arrayexpress

Project title: Endogenous AIRE-immunoprecipitation from primary human neutrophils

Project description:Autoimmune regulator (AIRE) is a transcriptional regulator. We here assess the role for AIRE in primary human neutrophils. We found that the functional role for AIRE in neutrophils is to regulate the extrinsic apoptotic pathway involving the Fas/TNFR death receptors and Cathepsin G and the expression of unique immune mediators.

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The effect of the autoimmune regulator (Aire) gene on the transcriptional expression profile of 3.10 mTEC cell line.

Project description:Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of peripheral tissue antigens (PTAs) in the thymus. PTA expression in mTECs is largely dependent on the autoimmune regulator (Aire) gene. Here we used a Mus musculus mTEC cell line (3.10 mTEC line, which constitutively express Aire in culture) to knockdown Aire gene by means of siRNA transfection. Aire knockdown was confirmed by means of qRT-PCR and RNA-FISH (for Aire mRNA levels), immunofluorescence and western blot (for AIRE protein levels).The Agilent oligo microarrays were used to determine the large scale transcriptional expression profiles of control or Aire-knockdown 3.10 mTECs.

2015-12-31 | E-MTAB-3879 | biostudies-arrayexpress

Genome-wide map of BACH1 binding in HEK 293T cells

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2011-05-13 | E-GEOD-28051 | biostudies-arrayexpress

RNAseq profiling of recirculating thymic Tregs in Aire-KO and WT mice

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The effect of the Autoimmune regulator (Aire) gene on microRNA transcriptional profile of 3.10 mTEC cell line

Project description:Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of peripheral tissue antigens (PTAs) in the thymus. Autoimmune regulator (Aire) is to date the best characterized transcriptional regulator known to at least partially coordinate PTA expression in mTECs. Furthermore the expression of Aire-dependent and -independent genes is also modulated by microRNAs. Given the transcriptional regulation exerted by Aire in mTEC cells and the role of miRNAs in post-transcriptional control, we used a Mus musculus mTEC cell line (3.10) which constitutively express Aire in culture) to knockdown Aire gene by means of siRNA transfection and then observe the effect of Aire knockdown in the transcriptional profile of miRNAs by means of oligomicroarrays. The Agilent oligomicroarrays were used to determine the large scale the miRNA transcriptional profiles of control or Aire-knockdown 3.10 mTECs.

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