Identification of an abnormal pre-B cell population in 11- to 18-week-old CD19^+/CreSfpi1^lox/loxSpib^−/− mice. (A) B-cell frequencies are severely reduced in 11- to 18-week-old CD19^+/CreSfpi1^lox/loxSpib^−/− mice. Flow cytometric analysis was performed to determine the frequency of splenic B cells expressing CD19 and B220 in the spleen of mice of 11- to 18-week-old mice of the indicated genotypes. Seven mice per group were analyzed. (B) Measurement of deleted (Sfpi1Δ) allele frequencies in B cells from 6- to 10-week-old and 11- to 18-week-old CD19^+/CreSfpi1^lox/loxSpib^−/− mice. Using qPCR, primers cko1 and cko2 (supplemental Table 1) were used to determine relative frequencies of Sfpi1Δ alleles in B cells enriched from mice of the indicated genotype and age. The Sfpi1Δ allele frequency in an 8-week-old CD19^+/CreSfpi1^lox/loxSpib^−/− B cells was set to 1.00 (left bar), and the Sfpi1Δ allele frequency in a 17-week-old CD19^+/CreSfpi1^lox/loxSpib^−/− B cells is expressed as fold increase relative to this value (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. (C) Abnormal pre-B cell frequency in bone marrow of a 13-week-old CD19^+/CreSfpi1^lox/lox Spib^−/− mouse. Flow cytometric analysis was used to determine the bone marrow pre-B cell frequencies expressing BP-1 on their cell surface from 13-week-old mice of the indicated genotypes. The histograms shown are gated on B220⁺CD43⁺ bone marrow cells. (D) Appearance of an abnormal B cell population expressing low levels of B220. Flow cytometric analysis was performed on splenocytes from 18-week-old mice of the indicated genotypes using antibodies recognizing CD19 or B220. CD19^+/CreSfpi1^lox/loxSpib^−/− mice had a CD19⁺ B220^low population in addition to the CD19⁺B220⁺ B-cell population (right panel, dashed box). (E-F) Identification of abnormal CD19⁺B220^low cells as pre-B cells. Flow cytometric analysis using antibodies recognizing the indicated cell surface markers was used to determine the identity of gated CD19⁺B220⁺ B cells in CD19^+/+Sfpi1^lox/loxSpib^−/− mice (E) or of gated CD19⁺B220^low B cells in CD19^+/CreSfpi1^lox/loxSpib^−/− mice (F). Gates are shown as dashed boxes in panel A. Results shown are representative of 9 mice analyzed.