Ldb1 is not essential for T-cell development. (A) Expression of the Rosa26-EGFP^fCre reporter in DN subsets from Rosa26-EGFP^f;Rag1-Cre;Ldb1^fl/fl mice. Numbers are percent of gated cells that express EGFP. Similar subsets from non-Cre transgenic Rosa26-EGFP^f mice are shown as negative control. Shown is 1 representative of 12 mice analyzed from 6 independent experiments. (B) Deletion of Ldb1 in thymocytes mediated by Rag1-Cre. Total thymocyte DNA from the indicated mice was used as a template for polymerase chain reaction (PCR) with primers that amplify the Ldb1⁺ (wt), Ldb1^flox (Ldb1^fl), or Ldb1^Δ (deleted) alleles. (C) Western blot of total thymocyte lysates from the indicated mice with anti-Ldb1, anti-Lmo2, or anti-actin (loading control). (D) Phenotype of Rag1-Cre;Ldb1^fl/fl mice. Upper panels: Flow cytometry (fluorescence-activated cell sorting [FACS]) analysis of total thymocytes (Thy) from the indicated mice stained with fluorochrome-conjugated anti-CD4 and anti-CD8. Lower panels: gated lin^neg thymocytes consisting of immature DN cells stained with fluorochrome-conjugated anti-c-kit and anti-CD25 to delineate DN(1-4) subsets. (E) FACS analysis of total lymph node (LN) cells from the indicated mice stained with fluorochrome-conjugated anti-CD4 and anti-CD8. Numbers in quadrants or gates are percentage of total cells. Results shown in panels B-E are 1 representative of 3 experiments.