Competitive transplantation assay is used to determine HSC development and functionality in vivo by measuring multilineage reconstitution of hematopoiesis in irradiated transplanted mice. Competitive transplantation Ly5.2/Ly2.1 was used to assess whether in vivo reexpression of Gata3 could rescue T cell development in the thymus. Total 52,500 Ly5.2 Tcf1 (WT or −/−) transduced cells (mixed LSK and LK progenitors cells) were transplanted into lethally irradiated (8.07 Gy) Ly5.1 recipient mice (8 to 12 weeks), together with 300,000 splenocytes (Ly5.1) as support cells. Chimerism and peripheral T cells were analyzed at week 6 after transplantation in peripheral blood. Mice were sacrificed for analysis 7 weeks after transplantation to evaluate hematopoietic system repopulation. Mice were considered repopulated when ≥1% multilineage Ly5.2 Tcf1 cells could be detected. Single-cell suspension from the thymus, spleen, and bone marrow, as well as lysate blood, was analyzed by flow cytometry as described previously.

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