In vivo validation of the in vitro culture system reveals that loss of Sox4 impairs the development of pro-B cells into mature B cells. (A) Schematic representation of the transplantation model. Fetal liver pro-B cells were transduced with a dual reporter carrying luciferase (Luc), which permitted the tracing of cell development in vivo by bioimaging, and mCherry, which allowed analysis of transplanted cells by flow cytometry. Cells were transplanted into 250-rad-irradiated NOD-SCID mice 9 days after SE-Cre introduction. (B) Development of transplanted pro-B cells in NOD-SCID mice (Xenogen IVIS live imaging) at the indicated posttransplantation time points. Note the increase in the signal at the bone marrow position at 1 week and the shift of the signal to the spleen position at 8 weeks. Scale bars indicate signal intensity. (C) Analysis of transplanted pro-B cells from the bone marrow and spleen of the recipient mice. Bone marrow and spleen lymphocytes were analyzed by flow cytometry for eYFP expression 10 days posttransplantation. (D) Flow cytometry analysis of B cells from the bone marrow of recipient mice 10 days posttransplantation. mCherry⁺ B220⁺CD19⁺ cells were examined for expression of IgM and IgD. eYFP⁻ cells (without Sox4 deletion) served as an internal control. Numbers indicate percent of cells (C-D). Data are representative of 4 independent experiments (B-D; n = 18 mice).