Effect of aire gene mutations induced by CRISPR-Cas9 on the transcriptional and post-transcriptional activity of mTEC cells and in its function on the thymocyte migration

• Grant number: 21/02081-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2021
• End date: January 31, 2024
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Geraldo Aleixo da Silva Passos Júnior
• Grantee: Cintia Junia Monteiro
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/10780-4 - Effect of CRISPR-Cas9-induced mutations in the Aire gene (APS1 syndrome) on protein conformation, mTEC cell transcriptome and their interaction with thymocytes, AP.TEM

Abstract

The post-doc fellow will study the effect of mutations in the Aire gene, associated with the APS1 syndrome, on two of the essential processes of mTEC cells during the induction of central immunological tolerance in the thymus, that is, the thymocyte migration - mTECs, which is necessary for adhesion to occur between these cells. Also, we will assess whether these mutations influence the transcriptome, including mRNAs and miRNAs, and the interaction between these molecules. As already discussed in the general project, the interaction between thymocytes and Thymic Epithelial Cells (TECs) is essential for developing these two cell types. To test this hypothesis, we will use the thymocyte migration model system (transwell assay). We will select the human Aire gene mutations (SAND and PHD domains) to assemble the CRISPR-Cas9 system, perform transfections of mTEC cells, and select the mutant Aire clones. The mTEC control (wt) and mTEC Aire mutant cells will serve for the transwell assay (cell migration). The mutant clone mTEC3.10E6 (mutation in the Aire exon 3, NLS domain) has already been isolated in our laboratory. We will perform extractions of total RNA from these cells, whose samples will be hybridized with microarrays to assess any transcriptome changes (mRNAs), miRnoma (miRNAs), and post-transcriptional interactions involving miRNAs-mRNAs. The results will help establish an association between Aire mutations and the gene activity of these cells and their biological function on thymocytes (migration thymocytes - mTECs). (AU)