Christopher H. Clegg

Production of Transgenic Mice with Yeast Artificial Chromosomes

Abstract Techniques are now available that allow the transfer of intact yeast artificial chromosome (YAC) DNA into transgenic mice. Coupled with the ability to perform mutagenesis on YAC sequences by homologous recombination in yeast, they enable the analysis of large genes or multigenic loci in vivo. This system has been used to study the developmental regulation of the human β-globin locus.

Thymic Overexpression of CD40 Ligand Disrupts Normal Thymic Epithelial Organization

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.

Oncostatin M stimulates excessive extracellular matrix accumulation in a transgenic mouse model of connective tissue disease.

Abstract Oncostatin M (OM), a member of the IL-6 gene family, stimulates a variety of functions implicated in wound repair. Transgenic mice that express this cytokine in islet β-cells develop a connective tissue disorder that typifies excessive healing with severe fibrosis and lymphocytic infiltration. To compare this phenotype with the normal progression of connective tissue disease, we measured the expression patterns of genes encoding proinflammatory cytokines, fibrogenic cytokines, and ECM components by in situ hybridization. To test whether the OM effect was caused by its ability to regulate IL-6, we crossed the OM transgene into IL-6-deficient mice. Our data suggest that the fibrosis in these animals is not a secondary consequence of inflammation, or IL-6 expression, but is a direct effect by OM on extracellular matrix production. In a separate experiment, we observed that OM could regulate vasoactive intestinal peptide gene expression in the neurons that innervate the transgenic pancreas. This nerve healing response, in combination with its fibrogenic activity, suggests that OM functions downstream of inflammation in the wound repair cascade. These transgenic mice represent a useful model in which the fibroproliferative phase of connective tissue disease is uncoupled from inflammation.

Oncostatin M transforms lymphoid tissue function in transgenic mice by stimulating lymph node T-cell development and thymus autoantibody production.

Oncostatin M (OM) is a member of the IL-6 subfamily of cytokines that is expressed in primary lymphoid tissues such as bone marrow and thymus, as well as in secondary lymphoid tissues and activated leukocytes. We produced transgenic mice that overexpressed the human, bovine, or mouse OM genes and compared their relative ability to modulate lymphopoiesis. Each species of cytokine induced a similar extrathymic pathway of T-cell development involving the accumulation of immature T cells within lymph nodes. Reconstitution experiments utilizing lethally irradiated athymic mice indicated that OM had caused hematopoietic precursors within fetal liver and bone marrow to initiate lymph node T-cell development in the absence of a thymic environment. Breeding experiments with IL6-/- and IL-7r(alpha)-/- deficient mice, indicated that induction of this extrathymic pathway by the OM transgene occurred in the absence of IL-6, but was strictly dependent on IL-7 receptor signaling. Separately, OM stimulated the accumulation of immature B cells within the transgenic thymus and caused the subcapsular regions of the thymus to expand with mature B and T cells. This thymus conversion to secondary lymphoid tissue was responsible for a lethal autoimmune-like disease marked by high titers of circulating autoantibodies, proteinuria, and glomerulonephritis. The conserved phenotypes elicited by these three forms of OM indicate that this potent hematopoietic cytokine can regulate lymphoid tissue function and morphogenesis.

Promoter Sequences in the RI Subunit Gene of cAMP-dependent Protein Kinase Required for Transgene Expression in Mouse Brain

Neural-specific expression of the mouse regulatory type-Iβ (RIβ) subunit gene of cAMP-dependent protein kinase is controlled by a fragment of genomic DNA comprised of a TATA-less promoter flanked by 1.5 kilobases of 5′-upstream sequence and a 1.8-kilobase intron. This DNA contains a complex arrangement of transcription factor binding motifs, and previous experiments have shown that many of these are recognized by proteins found in brain nuclear extract. To identify sequences critical for RIβ expression in functional neurons, we performed a deletion analysis in transgenic mice. Evidence is presented that the GC-rich proximal promoter is responsible for cell type-specific expression in vivo because RIβ DNA containing as little as 17 base pairs (bp) of 5′-upstream sequence was functional in mouse brain. One likely regulatory element coincides with the start of transcription and includes an EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval. Maximal RIβ promoter activity required the adjacent 663 bp of 5′-upstream DNA where most, but not all, of the regulatory activity was localized between position −663 and −333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence. Intron I sequences were also tested, and deletion of a 388-bp region containing numerous Sp1-like sequences lowered transgene activity significantly. These results have identified specific regions of the RIβ promoter that are required for the expression of this signal transduction protein in mouse neurons.

Structural Features of the Murine Gene Encoding the RIβ Subunit of cAMP-Dependent Protein Kinase

The activation of cyclic AMP-dependent protein kinase is controlled by the regulatory (R) subunits of the holoenzyme. Here we present a characterization of the mouse RI beta subunit gene, which in contrast to other subunit genes of cyclic AMP-dependent protein kinase is expressed almost exclusively in neurons. It was determined that RI beta is relatively large with 11 exons spanning a minimum 75 kb. The mouse chromosomal locus (designated Prkar1b) was determined by interspecific backcross mapping and found to reside on the distal arm of chromosome 5. Previously, it was shown that 3.5 kb of DNA encompassing the RI beta promoter could direct neural-specific gene expression in transgenic mice. Analysis of this DNA suggests the presence of an unusually large number of binding sites for transcription factors ranging from tissue-specific regulators, immediate-early genes, and mediators of hormone action. In addition to 18 putative SP1 sites, we identified 27 consensus sequences for basic Helix-Loop-Helix, POU, and Pax family members, 5 AP1 sites, and over 40 half-sites for the superfamily of steroid hormone receptor. Gel mobility-shift assays employing brain nuclear extract and pure transcription factor protein established that many of these DNA sequences are functional in binding protein. The abundance and configuration of transcription factor binding sites within the promoter region of RI beta suggests that this gene is subject to complex modes of regulation in neurons.