Gene expression analysis of thymocyte subsets. Mus musculus

Mouse thymocytes can be classified into four major subsets based on expression of CD4 and CD8 co-receptors. CD4-CD8- (double negative, DN) cells become CD4+CD8+ (double positive, DP) cells following productive T cell receptor (TCR) beta chain rearrangement. A small proportion of DP cells are selected through interaction of clonal TCRalpha/beta and MHC self peptide complex expressed on thymic stromal cells. DP cell expressing MHC class I-restricted TCR become CD4-CD8+ cells, which will finally differentiate into cytotoxic T cells, while MHC class II restricted selection generates CD4+CD8- helper lineage T cells. We used microarrays to identify genes important for thymocyte differentiation and lineage determination by profiling gene expression in different thymocyte subsets. Overall design: Mouse thymocytes were divided into four subsets based on CD4, CD8a, and TCRb expression and purified by flw cytometry. FACS purified DN (CD4-CD8a-TCRb-), DP (CD4+CD8a+), CD4SP (CD4+CD8a-TCRbhi) and CD8SP (CD4-CD8a+TCRbhi) populations were lysed in Trizol, and provided to the Genomics Core Facility of the Memorial Sloan-Kettering Cancer Center (MSKCC) for quality control, quantification, reverse transcription, labeling and hybridization to MOE430A 2.0 microarray chips (Affymetrix). Arrays were scanned per the manufacturer’s specifications for the Affymetrix MOE430v2 chip.