Measurement of Sfpi1^lox allele deletion frequency. (A) Schematic of the Sfpi1^lox allele encoding PU.1. Arrowheads indicate loxP or lox66 sequences. The thick line extending from the 5′ loxP sequence indicates unique DNA sequence derived from the targeting vector. Arrows indicate locations recognized by PCR primers. Names of primers are indicated. DNA sequence of the Sfpi1^lox allele was determined by cloning and sequencing using primers listed in supplemental Table 1. (B) Enrichment of mature B cells by cell sorting. CD93⁻CD19⁺B220⁺ B cells were enriched from the spleen of 8-week-old wild-type (WT), CD19^+/+Sfpi1^lox/loxSpib^−/−, or CD19^+/CreSfpi1^lox/loxSpib^−/− mice by flow cytometric cell sorting. Genomic DNA was prepared from sorted cells and used as template for qPCR analysis. (C) Measurement of Sfpi1^lox allele frequencies using qPCR. Primers lox-exon4 fwd and lox-exon4 rev (supplemental Table 1) were used to determine relative frequencies of intact Sfpi1^lox alleles in B cells enriched from mice of the indicated genotypes. The Sfpi1^lox allele frequency in CD19^+/+Sfpi1^lox/loxSpib^−/− B cells was set to 1.00 (left bar), and the relative Sfpi1^lox allele frequency in CD19^+/CreSfpi1^lox/loxSpib^−/− B cells was expressed as a decimal (right bar). qPCR also was performed on C57Bl/6 B cell genomic DNA (center bar) as an additional negative control to demonstrate specificity of the qPCR reaction.