Conditional deletion of PU.1. (A) PU.1 conditional allele. The PU.1 targeting construct containing exon 5 of PU.1 surrounded by LoxP sites (▴) and a reporter cassette flanked by 2 Frt sites (•) containing both the internal ribosome entry site-green fluorescent protein (IRESGFP) and the phosphoglycerate kinase (PGK) promoter driving the selection marker Neomycin (Neo) was introduced into the PU.1 locus by homologous recombination to generate the PU.1^gfp allele. When crossed to mice harboring flp recombinase, the reporter/selection cassette is lost, generating PU.1^fl allele. Exon 5 of PU.1 is deleted by crossing with mice carrying Cre recombinase, creating the allele PU.1^Δ. (B) Expression of lineage markers in E18.5 fetal livers. Following isolation, fetal livers were treated with red cell lysis buffer and analyzed by flow cytometry for the presence of B cells (CD19⁺B220⁺) and monocytes/granulocytes (Gr-1⁺Mac-1⁺). (C) Deletion efficiency of PU.1^fl/fl alleles in the B-cell fractions of PU.1^Δ/ΔCD19 mice. Isolated B-cell fractions from the fetal liver (left panel), bone marrow, and spleen (right panel) were analyzed by competitive PCR for the presence of targeted (fl) and deleted (Δ) PU.1 alleles. The antibody combinations used and the rate of deletion calculated by densitometry are as follows: fetal liver (CD19⁺B220⁺, 73%); bone marrow: pre-BI (B220⁺ckit⁺, 59%), pre-BII large (B220⁺c-Kit^-CD25⁺, forward scatter [FSC] large, 86%), pre-BII small (B220⁺c-Kit^-CD25⁺, FSC small, 86%), immature B (B220^loIgM⁺, 90%), and mature recirculating (B220^hiIgM⁺, 98%); spleen: transitional 1 (CD23^-IgM⁺-CD21^lo, 98%), transitional 2 (CD23⁺IgM^hiCD21^hi, 96%), follicular (CD23⁺IgM^loCD21^lo, 92%), and marginal zone (CD23^-IgM⁺CD21^hi, 100%). (D) Loss of PU.1 in B cells from PU.1^Δ/ΔCD19 mice. Total protein extracts were made from IgM⁺B220⁺ cells sorted by flow cytometry from the spleens of PU.1^+/+CD19 and PU.1^Δ/ΔCD19 mice. The presence of PU.1 and β-actin was assessed by Western hybridization.