Figure 1
Figure 1. BAF53a deficiency results in the development of lethal aplastic anemia. (A) Expression profiles of the BAF53a, BAF45a, and BAF60a genes in purified populations of mouse BM cells. Total RNA was isolated from the different populations, reverse-transcribed, and analyzed by quantitative PCR. SLAM-HSCs are CD150+ CD48− Sca1+ c-kit+ Lin−. Immunophenotypes of all cell populations analyzed are shown in supplemental Table 1. Values (RQ) are relative expression of each gene in each population over total BM cells after HPRT normalization. Error bars are based on an RQ minimum/maximum of 95% CI. (B) Generation of the BAF53a targeting vector. The genomic organization of the BAF53a locus is shown. Exon 1 contains the translational start site (ATG). LoxP sites were inserted into introns 3 and 5 of the mouse BAF53a gene. The location of the genomic probe used for Southern blot analysis is shown (green box). (C) Southern blot analysis of BAF53a-targeted ES clones using a 5′ external genomic probe (AK389). Genomic DNA was digested with EcoN1 enzyme. (D) Mendelian distribution of embryos with germline deletion of BAF53a at different embryonic stages. (E) PCR analysis of BAF53a allele in genomic DNA isolated from the BM of BAF53a+/+ (control), BAF53afl/+, and BAF53afl/fl conditional mice in the presence or absence of the Mx1-Cre transgene 17 days after pIpC treatment. The wild-type (WT), floxed (flox), knockout (deleted), and Cre alleles are identified. (F) Western blot analysis of BAF53a and BAF53b expression levels in nuclear extracts isolated from Cre-transduced BAF53a+/+ (CTRL), BAF53afl/+, and BAF53afl/fl E14.5 fetal liver cells (day 4 of infection). Note that BAF53b is undetectable in these extracts. Antibodies against RNA polymerase II were used as loading control. (G) Wright staining of blood smear preparations of BAF53afl/flCre+ mice on day 14 after pIpC-induced deletion. Representative images are shown. (H) Western blot analysis of BAF subunit expression in total cell extracts isolated from pIpC-treated BAF53afl/flCre+ and control Ter119− BM cells (day 4 after pIpC). Note that expression of other BAF subunits remained unaffected by the loss of BAF53a. (I) Kaplan-Meier survival curve of BAF53afl/+Cre+ (HET) and BAF53afl/flCre+ (KO) mice after pIpC-induced deletion. (J) Kinetic of BM aplasia development after pIpC-induced BAF53a deletion. Data are mean ± SD; n > 6 per time point. (K) Absolute number of peripheral blood cells in BAF53afl/flCre+ mice on day 12 after pIpC-induced deletion. FACS analysis confirmed the near-complete absence of B220+ and CD3+ lymphocytes in the peripheral blood of BAF53a-deficient animals at similar time points (data not shown). Ctrl, n = 2; KO, n = 2. CTRL indicates control; WBC, white blood cell; LY, lymphocyte; MO, monocyte; GR, granulocyte; RDW, red cell distribution width; RBC, red blood cell; Hgb, hemoglobin; and PLT, platelet.

BAF53a deficiency results in the development of lethal aplastic anemia. (A) Expression profiles of the BAF53a, BAF45a, and BAF60a genes in purified populations of mouse BM cells. Total RNA was isolated from the different populations, reverse-transcribed, and analyzed by quantitative PCR. SLAM-HSCs are CD150+ CD48− Sca1+ c-kit+ Lin−. Immunophenotypes of all cell populations analyzed are shown in supplemental Table 1. Values (RQ) are relative expression of each gene in each population over total BM cells after HPRT normalization. Error bars are based on an RQ minimum/maximum of 95% CI. (B) Generation of the BAF53a targeting vector. The genomic organization of the BAF53a locus is shown. Exon 1 contains the translational start site (ATG). LoxP sites were inserted into introns 3 and 5 of the mouse BAF53a gene. The location of the genomic probe used for Southern blot analysis is shown (green box). (C) Southern blot analysis of BAF53a-targeted ES clones using a 5′ external genomic probe (AK389). Genomic DNA was digested with EcoN1 enzyme. (D) Mendelian distribution of embryos with germline deletion of BAF53a at different embryonic stages. (E) PCR analysis of BAF53a allele in genomic DNA isolated from the BM of BAF53a+/+ (control), BAF53afl/+, and BAF53afl/fl conditional mice in the presence or absence of the Mx1-Cre transgene 17 days after pIpC treatment. The wild-type (WT), floxed (flox), knockout (deleted), and Cre alleles are identified. (F) Western blot analysis of BAF53a and BAF53b expression levels in nuclear extracts isolated from Cre-transduced BAF53a+/+ (CTRL), BAF53afl/+, and BAF53afl/fl E14.5 fetal liver cells (day 4 of infection). Note that BAF53b is undetectable in these extracts. Antibodies against RNA polymerase II were used as loading control. (G) Wright staining of blood smear preparations of BAF53afl/flCre+ mice on day 14 after pIpC-induced deletion. Representative images are shown. (H) Western blot analysis of BAF subunit expression in total cell extracts isolated from pIpC-treated BAF53afl/flCre+ and control Ter119− BM cells (day 4 after pIpC). Note that expression of other BAF subunits remained unaffected by the loss of BAF53a. (I) Kaplan-Meier survival curve of BAF53afl/+Cre+ (HET) and BAF53afl/flCre+ (KO) mice after pIpC-induced deletion. (J) Kinetic of BM aplasia development after pIpC-induced BAF53a deletion. Data are mean ± SD; n > 6 per time point. (K) Absolute number of peripheral blood cells in BAF53afl/flCre+ mice on day 12 after pIpC-induced deletion. FACS analysis confirmed the near-complete absence of B220+ and CD3+ lymphocytes in the peripheral blood of BAF53a-deficient animals at similar time points (data not shown). Ctrl, n = 2; KO, n = 2. CTRL indicates control; WBC, white blood cell; LY, lymphocyte; MO, monocyte; GR, granulocyte; RDW, red cell distribution width; RBC, red blood cell; Hgb, hemoglobin; and PLT, platelet.

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