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The role of TGF-beta receptor and its ligand in leukocyte development, immune response and disease: Generation of a mouse model by conditional mutagenesis of TGF-betaRII

Cazac, Balthazar Bernard; (2000) The role of TGF-beta receptor and its ligand in leukocyte development, immune response and disease: Generation of a mouse model by conditional mutagenesis of TGF-betaRII. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The TGF-β family represents cytokines with profound regulatory effects on many biological processes. TGF-β1 deficient mice die of a multifocal inflammatory disease, involving most leukocyte subsets. To determine the role of TGF-β in leukocytes we have used the Cre/loxP system to create mouse models of cell type-specific TGF-β Receptor Type II (TGF-βRII) deficiency. The genomic locus of the mouse TGF-βRII was cloned and characterised. The information obtained was used to construct a targeting vector to flank, ("flox"), exon 3 of the TGF-βRII locus with loxP sites, through homologous recombination in mouse embryonic stem cells. Clones carrying a floxed copy of the TGF-βRII exon 3 (TGF-βRIIfl/+) were used to generate chimeric mice by morula aggregation. Chimeras transmitting the TGF-βRIIfl/+ mutation were crossed with mice expressing the Cre recombinase driven by the CD19 promoter to generate animals with a B cell-specific (TGF-βRIIfl/fl-CD19cre/+) TGF-βRII deficiency. B cell-specific Cre-mediated deletion of exon 3 was confirmed by PCR analysis of peripheral blood leukocytes and subsequently analysed by Southern blotting of genomic DNA from purified B and T lymphocytes. TGF-βRIIfl/fl-CD19cre/+ animals exhibited 97% deletion of exon 3 in a B cell specific manner. When analysing B cell development in conditional mutant and control littermates, no significant differences were found in the percentages of B cell populations from bone marrow, peripheral blood, spleen and lymph nodes. In contrast, FACS analysis of cells from the peritoneal cavity of TGF-βRIIfl/fl-CD19cre/+ mice, revealed a noticeable increase in total numbers of B-1 cells, in relation to the wild type littermates. To examine if TGF-β plays a role in B cell proliferation in vivo we next carried out a BrdU incorporation assay on TGF-βRIIfl/fl-CD19cre/+ and wild type control animals. This showed no difference in the turnover of bone marrow and peritoneal cavity B cells and a twofold increase in the turnover of TGF-βRII deficient splenic B lymphocytes. To assess if TGF-β is essential in directing switch recombination in the IgA immunoglobulin isotype, we carried out an intracellular staining of bone marrow and plasma cells from wild type and B cell specific TGF-βRIIfl deficient mice. Our results indicate that this immunoregulator is necessary for regular IgA class switch in vivo. This study proves the validity of our TGF-βRIIfl model in systematically addressing the roles of TGF-β in leukocyte commitment in vivo.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The role of TGF-beta receptor and its ligand in leukocyte development, immune response and disease: Generation of a mouse model by conditional mutagenesis of TGF-betaRII
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences; Cytokines
URI: https://discovery.ucl.ac.uk/id/eprint/10114206
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