Targeted disruption of mouse Deltex-1 gene. (A) Schematic representation of Deltex-1 protein domains, with the deleted region including the RING finger domain underlined. (B) Schematic representation of wild-type and targeted Deltex-1 loci. Dotted boxes represent noncoding sequences within exons. Positions of BamHI sites (B) and of the probes used for Southern (S) and Northern (5′ and 3′ cDNA probes) blot analyses are indicated. PGK p(A) signal, polyadenylation signal of phosphoglycerate kinase gene; TK, herpes simplex virus thymidine kinase gene. Triangles flanking the neomycin resistance cassette (Neo^r) represent LoxP sites. (C) Southern blot analysis of BamHI-digested thymic DNAs from wild-type mice (+/+) and from mice that were heterozygous (+/Δ) and homozygous (Δ/Δ) for the targeted Deltex-1 gene. (D) Northern blot analysis of Deltex-1 expression in spleens and brains of wild-type, heterozygous, and homozygous mice by use of a cDNA probe outside (probe 5′) or inside (probe 3′) the deletion. “a,” “b,” and “c” mark the three truncated forms of the Deltex-1 transcript observed in mutant mice (see the text for details). Blots were normalized with an actin probe. (E) Northern blot analysis of B cells from Deltex-1^Δ/Δ mice after excision of the neo^RloxP gene (F/F). The analysis was performed on LPS-stimulated splenic B cells of Δ/Δ mice, with B-cell-restricted expression of the Cre recombinase (CD19-CRE).

Normal lymphoid development in Deltex-1^Δ/Δ mice. (A) B-cell subpopulations in the bone marrow, spleen, peritoneal cavity, and Peyer's patches were detected by flow cytometry. (B) Thymic and splenic T-cell subpopulations were detected by flow cytometry. Mean values and standard deviations for at least five animals of each genotype are given.

Deltex-4, a new member of the deltex gene family. (A) Comparison of human and mouse Deltex-1 (accession no. NP_004407 and BAB18939), Deltex-2 (NP_065943 and BAB18940), and Deltex-4 (XP_166213 and AAH58647) proteins, performed with the Multalin program (4). Conserved amino acids are indicated with bold uppercase letters in the consensus sequence line, with amino acids conserved between two Deltex members shown in lowercase. Open boxes represent the RING finger motif. The line above the sequence indicates the region that is deleted in Deltex-1^Δ/Δ mice. (B) Two-by-two comparison of mouse Deltex-1 (BAB18939), Deltex-2 (BAB18940), Deltex-3 (BAB18942), and Deltex-4 (AAH58647) proteins, performed with the BLAST program. The number of amino acids and identity (left) and similarity (right) values are indicated for each pair of proteins.

Deltex-1^Δ/Δ mice have normal lymphoid organ structures. Sections from spleens (A and B) and mesenteric lymph nodes (C) of wild-type (left) and Deltex-1^Δ/Δ (right) mice were stained with hematoxylin, eosin, and saffron and then photographed under a light microscope at a magnification of ×40. (B) Higher magnification of the images shown in panel A, showing primary B-cell follicles (Fo) and adjacent marginal zones (MZ). GC, germinal center.

Deltex-1^Δ/Δ mice have normal basal immunoglobulin levels in serum and mount normal humoral responses. The plotted values represent concentrations in serum for each mouse relative to the average concentration of all wild-type mice. (A) Concentrations of indicated serum immunoglobulin isotypes of 10 Deltex-1^Δ/Δ (filled circles) and 10 littermate (open circles) control mice as measured by ELISA. (B) T-independent type 2 response. Seven wild-type and seven Deltex-1^Δ/Δ mice were immunized with TNP-Ficoll, and their sera were assayed for the presence of TNP-specific antibodies of the IgM and IgG3 isotypes by ELISA. (C) T-dependent response. Eight wild-type and eight Deltex-1^Δ/Δ mice were immunized with TNP-KLH and boosted at day 21, as marked by arrows. Sera were quantified for the presence of TNP-specific antibodies of the IgM, IgG1, IgG2a, IgG2b, and IgG3 isotypes by ELISA.

Expression profiles of Deltex-1, Deltex-2, and Deltex-4 genes. (A) Northern blot analysis of mouse Deltex-1, Deltex-2, and Deltex-4 expression. Spleens, brains, testes, mouse embryonic fibroblasts (MEF), and LPS-stimulated B cells (LPS-B) were prepared from Deltex-1^+/Δ mice. The exposure times were 7 days (Deltex-1), 5 days (Deltex-2), and 6 days (Deltex-4). Normalization was performed by use of an actin probe. (B) Semiquantitative RT-PCR analysis of Deltex-1, Deltex-2, and Deltex-4 expression in follicular B cells (FoB; CD19⁺ CD21^int CD23^hi) and marginal-zone B cells (MZ-B; C19⁺ CD21^hi CD23^−/lo). Threefold serial dilutions of reverse transcription products were analyzed. The number of cycles used for each reaction is indicated. (C) Semiquantitative RT-PCR analysis of Deltex-2 and Deltex-4 expression in splenic cells from Deltex-1^+/+ and Deltex-1^Δ/Δ mice. Tenfold serial dilutions of reverse transcription products were analyzed. The number of cycles used for each reaction is indicated.