Figure 3.

Development of B cells is blocked at the early stage in the absence of IRE1α. (A) FACS analysis of cell surface markers CD43 and B220 in bone marrow cells from rag2^–/– mice after 4 weeks of reconstitution with ire1α^–/– or ire1α^+/+ fetal liver and AGM cells. (B) Genotypes of the CD43⁺B220^– cells sorted from the reconstituted bone marrow. The upper panel shows amplification of segments from the neo gene and the murine ire1α gene. The first 2 lanes show CD43⁺B220^– cells sorted from the ire1α^–/– and ire1α^+/+ reconstituted bone marrow. The third lane shows CD43⁺B220^– cells sorted from nonirradiated rag2^–/– mice. The fourth and fifth lanes show ire1α^–/– and ire1α^+/+ MEFs, which served as controls for the genotype of ire1α^–/– and ire1α^+/+ cells, respectively. The lower panel shows amplification of the rag2 gene WT (rag2^+/+) and knockout (rag2^–/–) alleles. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α^–/– and ire1α^+/+ reconstituted CD43⁺B220^– populations were found to be more than 10:1. (C) RT-PCR analysis of expression of ire1α and neo genes in the CD43⁺B220^– cells sorted from the reconstituted bone marrow. The samples are the same as described in B. (D–F) FACS analysis of cell surface markers BP-1 and HSA (D), B220 and IgM (E), and TER119 and Mac-1 (F) in ire1α^–/– and ire1α^+/+ reconstituted bone marrow cells. (G) Genotypes of TER119⁺ cells sorted from the reconstituted bone marrow. The first 2 lanes show TER119⁺ cells sorted from the ire1α^–/– and ire1α^+/+ reconstituted bone marrow. The third lane shows TER119⁺ cells from nonirradiated rag2^–/– mice. The fourth and fifth lanes show ire1α^–/– and ire1α^+/+ MEFs. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α^–/– and ire1α^+/+ reconstituted TER119⁺ populations were found to be more than 8:1. For A–G, experiments were performed at least 3 times and representative data are shown.