Abnormal thymocyte development in the absence of Bax and Bak occurs without affecting proliferation and TCRβ rearrangement. (A) To determine the proliferation of premalignant thymocytes, Control and LckCre⁺BaxBak DKO mice were injected with BrDU intraperitoneally. Mice were killed after 3 hours and the DN CD44⁺ population of thymocytes was evaluated by immunostaining followed by flow cytometry. (B) Surface TCR-β staining in DN, DP, and SP CD4⁺ and CD8⁺ cells. (C) To define the status of β chain rearrangement in the subpopulation of DN thymocyte cells DN1, DN2, DN3, and DN4 (CD44⁻CD25⁻) populations were isolated by FACSAria (BD BioSciences) cell sorting. Genomic DNA was isolated from these subpopulations and analyzed by PCR for rearrangements at the TCRβ locus using Dβ2 and Jβ2 region specific primers. PCR products were analyzed on 1% agarose gels. Shown are images of 2 agarose gels with DNA markers on each gel. (D) DN1 and DN3 populations were further specified with CD44⁺c-kit^high and CD25⁺c-kit^low gates, respectively and the presence of surface TCR-β was evaluated by flow cytometry.