Transcription profiling of mouse thymic deletion in B10 430B strains

Microarray analysis of thymic deletion in the B10 mouse strain; The gene expression patterns of thymocytes at the pre-selection stage and undergoing positive selection or negative selection were compared in the C57BL/10-H2k (B10k) strain. In order to sort thymocytes homogenously undergoing positive selection the 3A9 TCRHEL transgenic was used, which directs thymocyte development towards a MHC class II-restricted HEL-reactive lineage. To sort thymocytes homogenously undergoing negative selection, the 3A9 TCRHEL transgenic was crossed to the insHEL transgenic, which expresses HEL under the control of the insulin promoter in the thymic stroma, leading to efficient negative selection at the early single positive (CD4+CD8low) stage. To prepare the samples, thymic cell suspensions from female 6-8 week old B10k mice, either 3A9 TCRHEL or 3A9 TCRHEL x insHEL transgenics, were stained with CD4-FITC, CD69-PE, CD8-PerCP, and 1G12 supernatant (which recognises the 3A9 TCRHEL complex) followed by anti-IgG1-APC, and sorted. Pre-selection cells (from both transgenics) were defined as CD4+CD8+CD69-1G12low. Cells undergoing positive selection (from 3A9 TCRHEL transgenics) or negative selection (from 3A9 TCRHEL x insHEL transgenics) were defined as CD4+CD8lowCD69+1G12high. Experiment Overall Design: Single positive or double positive thymocytes from B10 mice were compared to those from B10 TCRHEL transgenic mice. 3 biological replicaes were used for each of the 4 different groups of thymocytes.