Project description:Cortical 1.4C18 and medullary 3.10 TEC lines (mTEC) were cultured in 10% fetal bovine serum-supplemented RPMI 1640 medium at 37 ?C in a 5% CO2 atmosphere. Confluent cultures of 3.10 mTEC cell line were cultured during 72 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kit® (Ambion, Austin, TX, USA).
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 manufacturers instructions. After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kit® (Ambion, Austin, TX, USA), which served as template for cDNA synthesis.<br><br>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<br><br>of a 120 bp PCR product corresponding to a segment of the Aire cDNA. The cDNA samples were prepared using Superscript II<br><br>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)
Project description:We used the TriFectaTM (IDT, Integrated DNA Technologies,<br><br>Coralville, IA, USA) anti-Dicer RNAi sequence (AGAACGAAAUGCAAGGAAUGGACTCGAGUCCAUUCCUUGCAUUUCGUUCUUC, ACAAGAAACGGAAUCACAUCACACTAGUGUGAUGUGAUUCCGUUUCUUGUCG, GCAGUUGUCCUAAACAGAUUGAUAAUUAUCAAUCUGUUUAGGACAACUGCUG) to silencing Dicer mRNA. Confluent cultures of 3.10 mTEC cell line were transfected with 20 nM of each anti-Dicer siRNA using Hiperfect reagent (Qiagen) following manufacturers instructions. After transfection, cells were cultured during 24 h in RPMI medium as above mentioned and total RNA was extracted using the mirVana kit (Ambion), which served as template for cDNA synthesis. Gene knockdown was confirmed by quantitative PCR (qRT-PCR) using the primers 5CCCAAATGTAGAACCCGAGA 3 forward and 5CAACCGACACTGTCCATCG 3 reverse, which allowed amplification of a 119 bp PCR product corresponding to a segment of the Dicer mRNA (cDNA). Transcriptional expression levels were determined using a StepOne Real-Time PCR System (Applied Biosystems, USA).
Project description:Due to high sequence identity between human and M. musculus microRNAs, we used in this study human microRNA microarrays to determine microRNA expression profiles of medullary thymic epithelial cells (mTECs) from thymus of pre-diabetic NOD mice.
Project description:Small interfering RNA (siRNA) was used to knockdown Autoimmune regulator(Aire) gene by in vivo electroporation of the thymus of BALB-c mice. In This set of data we include control and Aire-silenced mTEC cells isolated from thymus of BALB-c mice.
Project description:Relative H3K27me3 enrichment, assessed by ChIP-on-chip, in two TLX positive and two TLX negative T Acute Lymphoblastique Leukemia (T-ALL) samples. Acute lymphoblastic leukemias (ALLs) are characterized by multi-step oncogenic processes leading to cell differentiation arrest and proliferation. Specific abrogation of maturation blockage constitutes a promising therapeutic option in cancer, which requires precise understanding of the underlying molecular mechanisms. We show that the cortical thymic maturation arrest in T-lineage ALL that over-express TLX1 or TLX3 is due to binding of TLX1/TLX3 to ETS1, leading to repression of the T cell receptor (TCR) alpha enhanceosome activity and blocked TCR-Jalpha rearrangement. TLX1/TLX3 abrogation or enforced TCRalpha/beta expression leads to TCRalpha rearrangement and apoptosis. Importantly, the auto-extinction of clones carrying TCRalpha-driven TLX1 expression supports TLX 'addiction' in TLX-positive leukemias and provides further rationale for targeted therapy based on disruption of TLX1/TLX3.