1. The development of functional B lymphocytes in conditional PU
The development of functional B lymphocytes in conditional PU.1 knock-out mice
by Matthew Polli, Aleksandar Dakic, Amanda Light, Li Wu, David M. Tarlinton, and Stephen L. Nutt
Blood
Volume 106(6):
September 15, 2005
©2005 by American Society of Hematology

2. Conditional deletion of PU.1.
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.1gfp allele. When crossed to mice harboring flp recombinase, the reporter/selection cassette is lost, generating PU.1fl allele. Exon 5 of PU.1 is deleted by crossing with mice carrying Cre recombinase, creating the allele PU.1Δ. (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.1fl/fl alleles in the B-cell fractions of PU.1Δ/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 (B220loIgM+, 90%), and mature recirculating (B220hiIgM+, 98%); spleen: transitional 1 (CD23-IgM+-CD21lo, 98%), transitional 2 (CD23+IgMhiCD21hi, 96%), follicular (CD23+IgMloCD21lo, 92%), and marginal zone (CD23-IgM+CD21hi, 100%). (D) Loss of PU.1 in B cells from PU.1Δ/ΔCD19 mice. Total protein extracts were made from IgM+B220+ cells sorted by flow cytometry from the spleens of PU.1+/+CD19 and PU.1Δ/ΔCD19 mice. The presence of PU.1 and β-actin was assessed by Western hybridization.
Matthew Polli et al. Blood 2005;106:
©2005 by American Society of Hematology

3. The effect of PU.1 deletion on IL-7Rα and EBF expression in pre-BII cells.
The effect of PU.1 deletion on IL-7Rα and EBF expression in pre-BII cells. (A) IL-7Rα expression on pre-BII cells. Pre-BII cells (B220+c-Kit-CD25+) from the bone marrow of PU.1Δ/ΔCD19 and PU.1+/+CD19 were analyzed for the expression of IL-7Rα by FACS. (B) Gene expression in pre-BII cells. Pre-BII cells were sorted by FACS, total RNA extracted, and gene expression analyzed by RT-PCR.
Matthew Polli et al. Blood 2005;106:
©2005 by American Society of Hematology

4. The effect of PU.1 deletion on B-cell function.
The effect of PU.1 deletion on B-cell function. (A) Immunoglobulin isotype levels in untreated mice. Sera from 8- to 12-week-old mice (n = 6 for each genotype) were analyzed for levels of IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, Igκ, and Igλ by ELISA. PU.1+/+CD19 and PU.1Δ/ΔCD19 are shown in black and white circles, respectively. (B-C) Number of NP-specific IgG1 ASCs in bone marrow and spleen. PU.1+/+CD19 and PU.1Δ/ΔCD19 mice were immunized with 100 μg NP-KLH precipitated in alum and boosted at day 28 with 20 μg soluble NP-KLH. On the indicated day, spleens (Sp) and bone marrow (BM) were harvested and a known number of cells were cultured overnight on ELISPOT plates with high (B) or low (C) conjugation ratio NP-specific coat. Bound antibody was detected by incubating with IgG1-HRP and spots revealed with AEC substrate. The number of PU.1+/+CD19 and PU.1Δ/ΔCD19 ASCs are shown in gray and white, respectively. Numbers represent the mean ± standard deviation. (D) Germinal center formation. Spleens from mice were collected at day 7 and 28 following immunization. Sections were cut and stained for the germinal center B cells (GL7, red/brown) and follicular B cells (IgD, blue).
Matthew Polli et al. Blood 2005;106:
©2005 by American Society of Hematology

5. Enhanced apoptosis of PU.1Δ/ΔCD19 B cells upon BCR cross-linking.
Enhanced apoptosis of PU.1Δ/ΔCD19 B cells upon BCR cross-linking. (A) Reduced proliferation of PU.1Δ/ΔCD19 B cells upon BCR cross-linking. Small resting naive B cells were isolated from the spleen and cultured in the presence of 10 μg/mL F(ab′)2 anti-IgM. Cultures were pulsed with 1 μCi (37 Bq) [3H]thymidine for 6 hours and thymidine incorporation assessed. Mean counts per minute (CPM) ± standard deviation is shown. PU.1+/+CD19 is shown in black circles and a solid line and PU.1Δ/ΔCD19 in white with a dashed line. (B) Enhanced apoptosis in PU.1Δ/ΔCD19 B cells upon BCR cross-linking. Cells were cultured in the presence of 10 μg/mL anti-IgM or IL-4 or in combination for the indicated time, the number of viable cells (annexin V negative, propidium iodide-negative) assessed by FACS, and the data converted to the percentage of the initial culture that remained viable at that time point. Numbers represent the mean ± standard deviation. (C) The expression of antiapoptotic genes in PU.1Δ/ΔCD19 and PU.1+/+CD19 BCR-stimulated B cells. Total RNA was isolated from cells cultured for 24 hours and assessed for the expression of antiapoptotic genes by RT-PCR.
Matthew Polli et al. Blood 2005;106:
©2005 by American Society of Hematology

6. PU. 1Δ/ΔCD19 B-cell response to anti-CD40 and LPS
PU.1Δ/ΔCD19 B-cell response to anti-CD40 and LPS. (A) Syndecan-1 expression in B-cell cultures.
PU.1Δ/ΔCD19 B-cell response to anti-CD40 and LPS. (A) Syndecan-1 expression in B-cell cultures. Small naive B cells were isolated from spleens of PU.1+/+CD19 and PU.1Δ/ΔCD19 mice and cultured with either anti-CD40/IL-4/IL-5 or LPS for 3 days, after which the expression of syndecan-1 was assessed. (B) Expression of immunoglobulin isotypes by anti-CD40/IL-4/IL-5- or LPS-stimulated PU.1+/+CD19 and PU.1Δ/ΔCD19 B cells. B cells from PU.1+/+CD19 and PU.1Δ/ΔCD19 mice were cultured for 3 days, washed, counted, and a known number of cells reseeded and cultured overnight in fresh media containing no stimuli. The supernatants were then harvested and the level of the indicated immunoglobulin isotypes measured by ELISA. (C) Western and RT-PCR analysis of gene expression in PU.1+/+CD19 and PU.1Δ/ΔCD19 mice. (Top) Western hybridization of PU.1 in cells following 3 days of stimulation with anti-CD40/IL-4/IL-5 or LPS; β-actin is shown as a loading control. (Bottom) RT-PCR analysis of gene expression in PU.1+/+CD19 and PU.1Δ/ΔCD19 cells after 3 days of stimulation. Hprt is shown as a loading control, and a sample of PU.1+/+CD19 minus RT was used a negative control. (D) Electrophoretic mobility shift assays. Nuclear extracts were made from PU.1+/+CD19 and PU.1Δ/ΔCD19 B cells stimulated with LPS and incubated with a radiolabeled oligonucleotide representing the high-affinity PU.1 binding site from the SV40 promoter. Binding of PU.1 was confirmed by the addition of anti-PU.1 and anti-Pax5 antibodies. FP indicates free probe; BP, bound probe; NS, nonspecific; and SS, supershift.
Matthew Polli et al. Blood 2005;106:
©2005 by American Society of Hematology