Only Lmo2, Arf^−/− DN2 cells can engraft and self-renew in the thymus. (A) Double-negative (CD4⁻/CD8⁻) thymocytes transduced with either MSCV-IRES-mCherry or MSCV-Lmo2-IRES-mCherry were cultured in vitro on OP9-DL1 stromal cells together with IL-7 and FLT-3. After 20 days in culture, thymocytes were sorted to obtain mCherry⁺ DN2-stage cells, 2 × 10⁵ of which were transplanted into lethally irradiated mice together with 2 × 10⁵ bone marrow cells. At 3 weekly intervals after transplantation, mice were killed and their thymi analyzed for the presence of transduced mCherry⁺cells. Each thymus was stained for mature (CD4 and CD8) and immature (CD44 and CD25) thymocyte markers. The lower panels illustrate results with mCherry-marked cells at week 12. (B) Sterile-sorted mCherry⁺ cells (2 × 10⁵) collected from the mouse killed on week 12 illustrated in panel A were transplanted into γ_C^−/−, Rag1^−/− mice. Nine weeks after transplantation, an animal was killed, and the thymus was analyzed for the presence of mCherry⁺ T cells expressing CD4 and CD8.