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The regeneration of islet cells in a transgenic mouse strain harboring the interferon-gamma gene (IFN-gamma) linked to the insulin promoter DNA fragment (ins-IFN-gamma) is described. The regeneration follows the loss of islets by an immune response provoked by IFN-gamma and manifests in the proliferation of duct cells, the presence of progenitor cells, and the formation of buds and isletlike structure. All three types (A, B, and D) of four endocrine cells identified by immunolabeling are present. The progenitor cells express neuronal enzymes, tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD), as revealed by specific antibodies. The results indicate that the islet regeneration closely resembles the embryonic islet differentiation and serves as a model for studying islet development. The expression of neuronal enzymes by islet progenitor cells signifies yet unknown relationships to the nervous tissue. GAD, recognized as an autoantigen in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome, may provide a clue to the mechanism of autoimmune disease.

Investigation of intracellular pathways of stimulus-secretion signaling in vivo is possible by transgenic expression of agents known to influence specific biochemical interactions in the cells. The objective of the present study was to establish an experimental model for analyzing signal transduction mechanisms in pancreatic beta-cells in vivo, by expressing the cholera toxin A1 subunit under control of the insulin promoter, intending a constant activation of the Gs-protein, and thereby constant generation of cAMP. Surprisingly, the transgenic mice demonstrated mild hyperglycemia and hypoinsulinemia in vivo, and diminished glucose-induced insulin release from the in vitro perfused pancreas, whereas the pancreatic insulin content was normal. These observations suggest a deficiency in either the insulin release mechanisms or glucose recognition. Although the translated cholera toxin A1 subunit was biologically active, there was no increase in the islet content of cAMP. We conclude that the observed phenotype in the cholera toxin transgenic mice may be caused by a deleterious effect of the transgene itself on beta-cell function, or that counter regulatory mechanisms may compensate for the transgene-induced changes in intracellular enzymatic pathways.

The ability of transgenic mice to express a specific protein in a specific tissue has enabled the mechanisms of self-tolerance and autoimmunity to be elucidated. Further studies, combined with more sophisticated methods of gene targeting, will provide insights into the pathway leading from loss of self-tolerance to autoimmunity.

We have shown that the pancreatic duct cells of IFN-gamma mouse are actively multiplying and that many duct cells differentiate to become endocrine cells. This islet regenerating process closely parallels the islet development during normal organogenesis in the fetus and offers a model for studying the cell lineage relationships of islet cells. The subject has received wide interest and intensive research in recent years. One of the noteworthy results of this study is the finding that duct cells retain the ability to proliferate and to differentiate into islet cells. Under normal conditions, duct cells do not continue to multiply or to differentiate. The results suggest that in the transgenic mice, the progenitor cells of embryonic multipotential duct cells transform into adult cells, but in the presence of appropriate signals or stimuli can resume their multipotential property. The appearance of hepatocytes indicates that while the cell proliferation observed largely results in endocrine cells, other differentiation pathways are occasionally possible. We also detect a few large cells containing albumin and alpha-fetoprotein in the periductal area. Pancreatic hepatocytes have also been observed in the rat after recovery from copper deficiency diet. Thus, the regeneration of islet cells in transgenic mice provides a model system for the study of factors modulating the growth pattern as well as the differentiation pathway.

Several recent studies have utilized transgenic technology to explore basic questions in the pathophysiology of diabetes mellitus. The ultimate expression of altered glucose homeostasis is a theme common to them. The experimental models have been diverse, however, and, in some instances, resulted in unexpected biologic effects. Many of the studies have examined the autoimmune etiology of insulin-dependent diabetes mellitus by expressing regulatory molecules of the immune system as transgenes in islet beta cells. The molecules have included products of the major histocompatibility complex (MHC), cytokines, and other cell surface antigens. Ectopic expression of these transgenes resulted in altered immune responses directed against islets, and these transgenic mice now serve as important models to study mechanisms of immunologic tolerance. Transgenic technology is also being used to explore basic aspects of islet beta-cell physiology and insulin metabolism. beta-cell function is disrupted by transgenic beta-cell expression of molecules such as calmodulin and H-ras. Hyperexpression of insulin as a transgene can result in a syndrome resembling features of non-insulin-dependent diabetes.

The sexual difference in the incidence of autoimmune diseases has remained an enigma for many years. In the examination of the induction of autoimmunity in transgenic mice, evidence has been obtained further implicating the lymphokine interferon-gamma in the etiology of autoimmunity. Sex steroid regulation of the production of this molecule, as well as other cytokines, may help explain the gender-specific differences in the immune system, including autoimmunity.

We have investigated the structure and properties of a chromosomal product recovered from a rare recombination event between a t haplotype and a wild-type form of mouse chromosome 17. Our embryological and molecular studies indicate that this chromosome (twLub2) is characterized by both a deletion and duplication of adjacent genetic material. The deletion appears to be responsible for a dominant lethal maternal effect and a recessive embryonic lethality. The duplication provides an explanation for the twLub2 suppression of the dominant T locus phenotype. A reanalysis of previously described results with another chromosome 17 variant called TtOrl indicates a structure for this chromosome that is reciprocal to that observed for twLub2. We have postulated the existence of an inversion over the proximal portion of all complete t haplotypes in order to explain the generation of the partial t haplotypes twLub2 and TtOrl. This proximal inversion and the previously described distal inversion are sufficient to account for all of the recombination properties that are characteristic of complete t haplotypes. The structures determined for twLub2 and TtOrl indicate that rare recombination can occur between nonequivalent genomic sequences within the inverted proximal t region when wild-type and t chromosomes are paired in a linear, nonhomologous configuration.

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.

We have characterized a novel mouse gene (D17Si11) on chromosome 17 that expresses a major transcript observed uniquely in the testes. The D17Si11 locus has been mapped to the central region of chromosome 17 between H-2 and C3. Sequence analysis demonstrates several unusual features of this locus and its transcript: first is the presence of complementary sets of alternating purine and pyrimidine residues within the 3' region of the transcript that could form double-stranded, hairpin-like secondary structures with properties similar to that of Z-DNA; second is the existence of a hypothetical, long open reading frame in the nucleotide strand that is complementary to the testes transcripts. This complementary strand open reading frame is three times the size of the longest potential open reading frame present in the transcript itself. Although a function for D17Si11 has yet to be determined, the gene is relatively nonpolymorphic in mice and appears conserved in mammals.