Figure 8
Rescue of P2neo/neo dependent thymic defect by T-cell specific removal of the neo cassette. (A) Specific excision of the neo cassette in P2/Lck-Cre mice. Southern blot analysis using a probe spanning the region indicated by the red bar assessed the level of neo excision in thymocytes, stomach epithelium and splenocytes of 4 week-old P2/Lck-Cre mice. The positions of XbaI cleavage sites are shown (X). The probe hybridized to a 3.4 Kb- and a 5.4 Kb XbaI genomic fragments derived from WT and P2neo/neo allele, respectively. Insertion/excision of the neo cassette by Lck-Cre eliminated the middle XbaI site (shown in WT as bold blue) generating the 4.6 Kb fragment derived from the P2/Lck-Cre allele. (B) Regeneration of thymus cellularity in P2/Lck-Cre mice. At day-1.5 the number of thymocytes in P2/Lck-Cre thymic lobes was near normal compared to WT littermates. Data shown are average ± S.E. of five independent experiments using five mice of each genotype (WT, P2neo/neo and P2/Lck-Cre). The difference between WT and P2neo/neo is significant at P < 0.001 by Student's t test. (C) Recovery of thymopoiesis and thymic organogenesis in P2/Lck-Cre embryos. Histological analysis and Runx1 expression in thymic lobes derived from E15.5 and E16.5 WT, P2neo/neo and P2/Lck-Cre embryos. Shown are H&E stained transverse sections immunostained for Runx1. (D) Runx1 expression was not rescued in P2/Lck-Cre epithelial cells. IHC analysis of Runx1 expression in esophagi derived from E16.5 WT, P2neo/neo and P2/Lck-Cre embryos. Transverse sections showing Runx1 expression in epithelial cells lining the lumen of WT (arrow), but not of P2neo/neo or P2/Lck-Cre esophagus. Several other tissues including stomach and nasal cavity were similarly analyzed and revealed no expression of Runx1 in epithelia of P2neo/neo or P2/Lck-Cre mice (not shown).