Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing ␤ cells and glucagon-positive ␣ cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcriptioncoupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of ␦ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.Insulin is synthesized, stored, and secreted by the pancreatic islet ␤ cells in a highly regulated manner and plays a vital role in glucose homeostasis. Insulin action also results in several other pleiotropic effects that are less well documented. Embryonic insulin synthesis begins early in gestation, but fetal glycemia closely follows maternal blood glucose levels. The question, therefore, arises as to what function embryonic insulin might fulfill during development. For instance, one might ask whether insulin plays an autocrine or paracrine role in pancreatic islet cell growth and differentiation, since insulin is synthesized with other hormones in developing islet cell types (1-3). Recently, this question has been addressed in a few transgenic studies. For instance, the gene encoding PDX-1 (4, 5), a homeodomain transcription factor synthesized in adult ␤ cells and capable of transactivating insulin gene expression, has been inactivated by targeted disruption (6, 7). Agenesis of pancreas resulting from PDX-1 deficiency precluded from addressing the question of the possible role of insulin in islet cell growth and differentiation. Similarly, mice lacking the LIM homeodomain transcription factor ISL1, synthesized in all classes of islet cells in the adult, were arrested in development soon after embryonic day 9.5 (8). The requirement of ISL1 in pancreatic epithelium for the differentiation of all islet cell types was, however, demonstrated by in vitro culture of explants from ISL1-deficient embryonic day 9.5 embryos that gave rise to cells that were negative for glucagon, insulin, and somatostatin. In another study, transgenic mouse embryos expressing the gene encoding the diphteria toxin A chain under control of the rat Ins2 promoter were generated (9). The resulting genetic ablation of the insulin-producing cells did not appear to alter the development of the nontarget...

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, ␤2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and ␤2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

Ten transgenic mouse lines harboring the -346/-103 fragment of the rat insulin I enhancer linked to a heterologous promoter and a reporter gene (Eins-Ptk-CAT construct) were produced. Expression of the hybrid transgene was essentially observed in pancreas and to a lesser extent in brain. These results indicate that the rat insulin I promoter is dispensable for pancreatic expression. This insulin gene sequence is the shortest fragment described as conferring tissue-specific expression in transgenic mice. Two short homologous sequences in the rat insulin I enhancer fragment used, IEB2 and IEB1, have been described as playing a dominant role in the regulation of HIT hamster insulinoma cell-specific transcription of the insulin gene (1). We investigated whether the combination of IEB2 and IEB1 sequences is sufficient to confer specific expression in transgenic mice to a IEB2-IEB1-Ptk-CAT gene construct. No CAT activity was observed neither in pancreas nor in any other organ examined in 19 different transgenic mice. Moreover in transient expression experiments in RIN2A rat insulinoma cells, the IEB sequences had a very weak or no enhancer activity. These observations contribute to the conclusion that DNA regulatory elements other than the IEB sequences are necessary for gene expression in vivo.