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Essential immunoregulatory role for BCAP in B cell development and function.

Yamazaki T, Takeda K, Gotoh K, Takeshima H, Akira S, Kurosaki T - J. Exp. Med. (2002)

Bottom Line: While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency.The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen.Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka, Japan.

ABSTRACT
BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

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Generation of BCAP−/− mice. (A) Schematic representation of the strategy used to target the BCAP locus. Exons are presented as shaded boxes and numbered, where the exon containing the initiation methionine corresponds to exon 1. Exon 8 (E8) harboring three YxxM motifs was replaced with the neomycin selection cassette (neo). Neo and thymidine kinase (tk) are shown as open arrows that indicate the direction of transcription. A probe used for Southern blot analysis is also shown as a bar. EV; EcoRV. (B) Representative Southern blot analysis of genomic DNA of progeny mice. Tail DNA was digested with EcoRV and probed with the external probe shown in panel A. +/+, +/−, and −/− indicate wild-type, heterozygous, and homozygous mice, respectively. (C) Northern blot analysis of total RNA from the spleen. A blot was hybridized with either BCAP full-length cDNA or β-actin. (D) Western blot analysis of BCAP protein from splenocytes. Immunoprecipitates from lysates with antiserum to BCAP or anti-BLNK antibody were immunoblotted with the same antibodies.
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fig1: Generation of BCAP−/− mice. (A) Schematic representation of the strategy used to target the BCAP locus. Exons are presented as shaded boxes and numbered, where the exon containing the initiation methionine corresponds to exon 1. Exon 8 (E8) harboring three YxxM motifs was replaced with the neomycin selection cassette (neo). Neo and thymidine kinase (tk) are shown as open arrows that indicate the direction of transcription. A probe used for Southern blot analysis is also shown as a bar. EV; EcoRV. (B) Representative Southern blot analysis of genomic DNA of progeny mice. Tail DNA was digested with EcoRV and probed with the external probe shown in panel A. +/+, +/−, and −/− indicate wild-type, heterozygous, and homozygous mice, respectively. (C) Northern blot analysis of total RNA from the spleen. A blot was hybridized with either BCAP full-length cDNA or β-actin. (D) Western blot analysis of BCAP protein from splenocytes. Immunoprecipitates from lysates with antiserum to BCAP or anti-BLNK antibody were immunoblotted with the same antibodies.

Mentions: We constructed a gene-targeting vector such that homologous recombination would replace an exon and its flanking introns with a neomycin resistance gene (neo), deleting three YxxM motifs that mediate binding to the p85 subunit of PI3K (Fig. 1 A). Nine ES clones with the appropriately targeted allele were obtained. Six clones were injected into C57BL/6 blastocysts and three of those yielded chimeric mice that transmitted the mutations to their offsprings. Southern blot analysis using an external probe confirmed the presence of the targeted locus (Fig. 1 B). Founder mice showing germline transmission were interbred to produce homozygous BCAP−/− mice, which were born at the expected Mendelian ratio. BCAP−/− mice appeared healthy and were fertile.


Essential immunoregulatory role for BCAP in B cell development and function.

Yamazaki T, Takeda K, Gotoh K, Takeshima H, Akira S, Kurosaki T - J. Exp. Med. (2002)

Generation of BCAP−/− mice. (A) Schematic representation of the strategy used to target the BCAP locus. Exons are presented as shaded boxes and numbered, where the exon containing the initiation methionine corresponds to exon 1. Exon 8 (E8) harboring three YxxM motifs was replaced with the neomycin selection cassette (neo). Neo and thymidine kinase (tk) are shown as open arrows that indicate the direction of transcription. A probe used for Southern blot analysis is also shown as a bar. EV; EcoRV. (B) Representative Southern blot analysis of genomic DNA of progeny mice. Tail DNA was digested with EcoRV and probed with the external probe shown in panel A. +/+, +/−, and −/− indicate wild-type, heterozygous, and homozygous mice, respectively. (C) Northern blot analysis of total RNA from the spleen. A blot was hybridized with either BCAP full-length cDNA or β-actin. (D) Western blot analysis of BCAP protein from splenocytes. Immunoprecipitates from lysates with antiserum to BCAP or anti-BLNK antibody were immunoblotted with the same antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193770&req=5

fig1: Generation of BCAP−/− mice. (A) Schematic representation of the strategy used to target the BCAP locus. Exons are presented as shaded boxes and numbered, where the exon containing the initiation methionine corresponds to exon 1. Exon 8 (E8) harboring three YxxM motifs was replaced with the neomycin selection cassette (neo). Neo and thymidine kinase (tk) are shown as open arrows that indicate the direction of transcription. A probe used for Southern blot analysis is also shown as a bar. EV; EcoRV. (B) Representative Southern blot analysis of genomic DNA of progeny mice. Tail DNA was digested with EcoRV and probed with the external probe shown in panel A. +/+, +/−, and −/− indicate wild-type, heterozygous, and homozygous mice, respectively. (C) Northern blot analysis of total RNA from the spleen. A blot was hybridized with either BCAP full-length cDNA or β-actin. (D) Western blot analysis of BCAP protein from splenocytes. Immunoprecipitates from lysates with antiserum to BCAP or anti-BLNK antibody were immunoblotted with the same antibodies.
Mentions: We constructed a gene-targeting vector such that homologous recombination would replace an exon and its flanking introns with a neomycin resistance gene (neo), deleting three YxxM motifs that mediate binding to the p85 subunit of PI3K (Fig. 1 A). Nine ES clones with the appropriately targeted allele were obtained. Six clones were injected into C57BL/6 blastocysts and three of those yielded chimeric mice that transmitted the mutations to their offsprings. Southern blot analysis using an external probe confirmed the presence of the targeted locus (Fig. 1 B). Founder mice showing germline transmission were interbred to produce homozygous BCAP−/− mice, which were born at the expected Mendelian ratio. BCAP−/− mice appeared healthy and were fertile.

Bottom Line: While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency.The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen.Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Osaka, Japan.

ABSTRACT
BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

Show MeSH
Related in: MedlinePlus