The new in vitro Sox4-knockout system uncovers the requirement of Sox4 at the transition from fraction B to fraction C in pro-B cells. (A) Schematic illustration of the deletion of floxed Sox4 by the SE-Cre recombinase. Cre also induced deletion of the stop cassette in eYFP reporter (to allow eYFP expression as an indicator of Cre activity) as well as deletion of its own gene to terminate its expression. (B) Semiquantitative reverse transcription–PCR (RT-PCR) analysis of Sox4 expression in fivefold serial dilutions of complementary DNA (cDNA) from eYFP⁺Sox4^fl/+SE-Cre and eYFP⁺Sox4^fl/flSE-Cre pro-B cells 10 days after Cre-induced floxed Sox4 deletion. β-Actin served as a loading control. (C) Flow cytometry analysis of cultured pro-B cells in the presence of IL-7 and OP9 stromal cells for the indicated numbers of days after the introduction of SE-Cre. In eYFP⁺Sox4^fl/+SE-Cre and eYFP⁺Sox4^fl/flSE-Cre cells, 1 and 2 Sox4 alleles were deleted, respectively. eYFP⁻ cells served as an internal control. Note that the ratio of fraction B (CD24⁺BP1⁻) to fraction C (CD24⁺BP1⁺) cells was gradually increased in the eYFP⁺Sox4^fl/flSE-Cre cultures. (D) Effect of Sox4 deletion on the differentiation of fraction B into fraction C. Fraction B and fraction C cells were each sorted out 9 days after the introduction of SE-Cre (day 0) and cultured for an additional 2 days before flow cytometry analysis. The percentages of cells in each quadrant are indicated (C-D). Data are representative of 2 (C) or 3 (B,D) independent experiments.