Lymphoid-specific gene expressions in purified 11- and 13-dpc liver cell populations from normal and Pax5-deficient mice.

(A) The 3-color mAb stainings were done with FITC anti–c-kit, biotinylated anti-AA4.1 and Cy5 anti-CD19 mAbs. The selected cell populations shown in the figure were electronically gated and positively sorted. There were 2 sorting windows set up for c-kit⁺AA4.1⁻ (R1) and c-kit⁺AA4.1⁺ cell populations, as shown in the representative dot plot of the figure. These cell populations were separated on the basis of CD19 Ag signals in c-kit⁺AA4.1⁺CD19⁻ (R2) and c-kit⁺AA4.1⁺CD19⁺ (R3) cells (right histograms). The R1 cell population lacked CD19⁺ cells. (B) Gene expression obtained in purified 11-dpc (R1, R2, R3, R1+R2) and 13-dpc (R3) cell preparations, unseparated 11-, 13-, and 19-dpc liver cells, and adult thymocytes (Thy). The data are representative of 3 independent analyses/sample, and are displayed as in Figure 1. First and second round VDJCμ (V_H7183 and V_HJ558) PCR reactions (rows 1 and 2, respectively) and β-actin are shown at the right as ethidium bromide gels. (C) Cytoplasmic μHC staining (cμHC) of 11-dpc liver and BM cells. CD19⁺ surface IgM⁻ (sIgM⁻) BM cells and CD19⁺11-dpc liver cells were electronically gated (left histograms) and analyzed for cμHC (right histograms). White and shaded right histograms represent negative and specific signals, respectively. (D) Detection of rag-2, VpreB, and CD19 gene transcripts in representative samples of isolated 11-dpc liver cells from wild-type (+/+), heterozygous (+/−), and homozygous Pax5-deficient (−/−) mouse embryos. The Pax5 genetic background of each embryo was identified with a genomic PCR. Bottom and top bands correspond to the wild-type and mutant Pax5 genes, respectively.41 α-Actin was used as a positive control of genomic DNA content. Rag-2, VpreB, and CD19 gene expressions were obtained with a nested RT-PCR, as described in “Materials and methods.”