After electroporation, the transfected BALB/c ESC were selected for G418 resistance (0.4 mg/mL), and homologous recombination events were identified by PCR and Southern blot analysis of XbaI-digested ESC genomic DNA using a 0.52-kb PCR fragment upstream of the 5′ homologous arm as probe. To generate Hax1−/+ ESC, the positively

targeted mother clone (10/C3) was further transfected with a Cre recombinase (Cre) expression vector (pMC-CreN). The deletion of exons 2 and 3 as well as the selection cassette was verified by PCR and Southern blot analysis of HindIII-digested genomic DNA using a 0.37-kb PCR fragment as probe. Cre-transfected ESC clones were injected into C57BL/6 blastocysts. To generate Hax1−/+ mice, chimeric males were crossed with BALB/c females. White heterozygous offsprings were bred for homozygosity (Fig. 1B). Dabrafenib No HAX1 protein was detectable in bone marrow-derived cells or other tissues isolated from Hax1−/− mice (Fig. 1B). The Z-VAD-FMK supplier phenotype of Hax1−/− mice, characterized by growth retardation, decreased body size and weight and loss of motor coordination, became visible at the age of 3–4 wk. Premature death occurred at the age of 10–14 wk. Computed tomography of 7-wk-old mice showed that lack of HAX1 did not lead to abnormalities in skeletal growth and body proportions (Fig. 1C). However,

as observed during bone marrow preparations, bones (femurae and tibiae) from Hax1−/− mice were found to be more fragile than those from WT controls (unpublished observations). Nintedanib (BIBF 1120) Compared to WT mice, the total cellular mass of spleen, thymus and bone marrow was significantly reduced in Hax1−/− mice; n=8 mice; p<0.001 (Fig. 1D). We next investigated early B-cell developmental stages using Hardy's classification 21. Due to the significantly reduced cellular

mass of bone marrow, spleen and thymus in Hax1−/− mice, we decided to express the individual populations in absolute cell numbers. The expression of a decrease in the percentage of one population would inherently result in the increase of another and may not in fact represent an actual change in cell number or deficiency of that population. B220+ B cells were reduced by 62% in Hax1−/− compared to WT mice (Hax1−/−: 3.14±0.5×106 and WT: 8.17±0.96×106; p<0.0001) (Fig. 2A; primary gating history is shown in Supporting Information Fig. 2). The observed decrease distributed equally on both the CD43− and the CD43+ population. Within the CD43+B220+ population, the absolute numbers of pre-pro-B cells (Fr. A) (Hax1−/−: 0.50±0.02×106 and WT: 0.82±0.11×106 of CD43+ B220+ cells; p<0.001) as well as the pro-B cells (Fr. B) (Hax1−/−: 0.31±0.07×106 and WT: 1.30±0.23×106 of CD43+B220+ cells; p<0.001) were significantly reduced. In the CD43−B220+ population, representing the later stages of B-cell development, the percentage of small pre-B (Fr. D) and newly formed B (NF, Fr. E) cells was significantly decreased in Hax1−/− mice (Hax1−/−: 0.84±0.