Protocol for studying in vitro mouse primary thymocyte differentiation via retroviral-mediated gene expression and stromal-free stimulation

T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes. For complete details on the use and execution of this protocol, please refer to Fan Zhao et al. 1 • Instructions for the isolation of mouse DN3 thymocytes • Guidelines for retroviral-mediated gene expression in thymocytes • Procedure for stromal-free in vitro differentiation of mouse DN3 thymocytes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes.