VDJ recombination is impaired in early pro-B cells with FLT3/ITD mutation. (A) Early pro-B cells from FLT3/ITD mice demonstrate significantly decreased D-J_H rearrangement. Densitometry analysis shows that recombination of D-J_H1, D-J_H2, and D-J_H3 in FLT3/ITD⁺ early pro-B cells was less than 0.05-, 0.1-, and 0.1-fold, respectively, of that in wild-type cells. Completed D-J_H rearrangements were assayed by PCR of sorted early pro-B cells from FLT3/ITD or wild-type littermate controls. (B) Quantitative RT-PCR analysis shows changes in relative expression levels of Rag1 and Rag2 in CLPs and early pro-B cells (n = 3). (C) Western blotting analysis shows expression of RAG1 and RAG2 in early pro-B cells. Values below the gel image indicate relative fold changes of protein levels normalized to lamin B. (D) Broken signal ends at the J_H locus accumulate in early pro-B cells from FLT3/ITD mice. Densitometry analysis shows that recombination intermediates for J_H1 and J_H2 in FLT3/ITD⁺ early pro-B cells were 20- and 1.8-fold of that in wild-type cells, respectively. Sorted early pro-B cells were subjected to LM-PCR assays for J_H-associated broken ends. The expected gene arrangements (DJ_H1-3 in panel A) or broken J_H fragments (J_H1-3 in panel D) are indicated. PCR amplification with primers for a nonrearranging locus (CD14) was used as a quantity control for the linker-ligated DNA samples. Data are representative of 3 independent experiments.