The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.

A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.

Oncogene-induced liver neoplasia in transgenic mice.

Models of hepatocarcinogenesis were generated by directing the expression of SV40 T-antigens, an oncogenic mutant of c-H-ras, or c-myc to the liver of transgenic mice using the albumin enhancer/promoter. The majority of mice carrying the ras transgene (group A) were born with enlarged livers and atypical hepatic architecture, and these all died within several days of birth. The remaining ras transgenic mice (group B) had lower levels of hepatic ras expression, exhibited mild hepatic dysplasia but no liver enlargement, and all ultimately died from development of lung tumors. In contrast, the livers of mice expressing T-antigens were relatively normal at birth, by one month displayed marked dysplasia, and by three to seven months developed multiple nodular adenomas and carcinomas. Myc expression caused mild to severe hepatic dysplasia in young mice, and focal hepatic adenomas in some mice over fifteen months of age. Lines of mice expressing ras (group B), T-antigen, or myc were established and crossed with each other to generate dual transgenic mice expressing oncogene pairs. Each combination resulted in accelerated tumor development, suggesting that these oncoproteins can cooperate with one another during multistep hepatic transformation.

Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice.

The normal complement of neurotransmitters in noradrenergic neurons was altered by expressing the structural gene for the enzyme phenylethanolamine-N-methyltransferase (PNMT) under the control of the dopamine-beta-hydroxylase gene promoter in transgenic mice. This resulted in accumulation of large amounts of epinephrine in neurons of the sympathetic nervous system (SNS) and central nervous system (CNS) but did not reduce norepinephrine levels. Adrenalectomy reduced PNMT levels in the SNS and CNS, suggesting that the transgene is positively regulated by adrenal steroids. Epinephrine levels were unaffected by this treatment in the CNS, suggesting that PNMT is not rate limiting for epinephrine synthesis. However, catecholamines were elevated in a sympathetic ganglion and a target tissue of the SNS, perhaps due to up-regulation of tyrosine hydroxylase in response to adrenalectomy. These transgenic mice also reveal a marked difference in the ability of chromaffin cells and neurons to synthesize epinephrine.

Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice.

Serum levels of GH and insulin-like growth factor I (IGF-I) were genetically increased to investigate the physiological activities of these proteins. Lines of mice expressing chimeric genes composed of bovine GH, human GRF, or human IGF-I coding sequences fused to the mouse metallothionein I promoter were examined for consequences of chronic exposure to high levels of these peptides. Animals with elevated serum levels of GH (either bovine GH or mouse GH) have selective splanchnomegaly coupled with glomerular sclerosis and hepatocellularmegaly. Serum levels of insulin and cholesterol are increased. In contrast (with the exception of selective enlargement of organs), the chronic expression of IGF-I results in a different pattern of abnormalities. These findings suggest that the pathogenesis of GH-related disorders is not mediated solely by IGF-I.

Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice.

A line of transgenic mice expressing insulin-like growth factor-I (IGF-I) under the control of the mouse metallothionien-1 promoter was crossed to a line of dwarf transgenic mice lacking GH expressing cells that were genetically ablated by diphtheria toxin expression. Mice generated from this cross that carry both transgenes express IGF-I in the absence of GH. These mice grew larger than their GH-deficient transgenic littermates and exhibited weight and linear growth indistinguishable from that of their nontransgenic siblings. These results confirm the suspected role of IGF-I in mediating GH's stimulation of somatic growth, including that of long bones, and illustrates the essential role of GH and IGF-I in the modulation of postnatal growth. Analysis of differences in organ growth among these mice, however, suggests that GH and IGF-I also have growth promoting actions that are independent of one another; GH appears to be necessary for the attainment of normal liver size, while IGF-I can stimulate brain growth.

Transformation of liver by SV-40 T-antigen in transgenic mice is unaffected by metallothionein.

Transgenic mice expressing excess metallothionein-I and SV-40 T-antigen were generated to test the hypothesis that metallothionein may influence the rate of neoplastic transformation induced by T-antigen within the liver. The livers of the double transgenic mice grew at the same rate (to 32% body weight), had similar morphological and histological appearance, had similar chromosomal instability, and released identical amounts of serine and alanine aminotransferases into the blood as mice bearing SV-40 T-antigen alone, despite the fact that metallothionein levels were elevated five- to ten-fold. We conclude that elevated levels of metallothionein-I do not influence either the initial hyperplasia or the subsequent neoplastic transformation that is induced by T-antigen, which is thought to act by sequestering the P53 and retinoblastoma gene products.

Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development.

Tyrosine hydroxylase catalyses the initial, rate-limiting step in the catecholamine biosynthetic pathway. Catecholamines, which include dopamine, noradrenaline, and adrenaline, are important neurotransmitters and hormones that regulate visceral functions, motor coordination and arousal in adults. The gene encoding tyrosine hydroxylase becomes transcriptionally active in developing neuroblasts during mid-gestation of rodent embryos, before the onset of neurotransmission. Here we show that inactivation of both tyrosine hydroxylase alleles results in mid-gestational lethality: about 90% of mutant embryos die between embryonic days 11.5 and 15.5, apparently of cardiovascular failure. Administration of L-DOPA (dihydroxyphenylalanine), the product of the tyrosine hydroxylase reaction, to pregnant females results in complete rescue of mutant mice in utero. Without further treatment, however, they die before weaning. We conclude that catecholamines are essential for mouse fetal development and postnatal survival.

Metallothionein I and II protect against zinc deficiency and zinc toxicity in mice.

Metallothionein (MT)-bound zinc accumulates when animals are exposed to excess zinc and is depleted under conditions of zinc deficiency, suggesting that MT serves as a means of sequestering excess zinc as well as a zinc reservoir that can be utilized when zinc is deficient. To examine the importance of MT for these processes, mice with null alleles of both MT I and MT II genes were created and the zinc concentration and histological appearance of multiple organs assessed. At birth, the hepatic zinc concentration of these MT-null mice was lower than that of wild-type controls (0.27 +/- 0.02 vs. 0.65 +/- 0.11 micromol zinc/g tissue, P < 0.05). During the next 3 wk of suckling zinc-replete (95 micrograms zinc/g diet) dams, the hepatic zinc concentration of controls fell to 0.42 +/- 0.04 micromol/g but was unchanged in the MT-null mice (0.28 +/- 0.04 micromol/g). The most prominent histological anomaly observed at 3 wk of age was the presence of swollen Bowman's capsules in the kidneys of MT-null mice. When nursing MT-null dams were fed a severely zinc-deficient (1.5 microg/g) diet, kidney development in the MT-null pups was retarded as indicated by the retention of the nephrogenic zone and incomplete tubule development. We suggest that the lack of a hepatic reservoir of zinc jeopardizes the developing kidney in the MT-null mice. In addition to being more sensitive to dietary zinc restriction, MT-null mice are more sensitive to zinc toxicity. When adult mice were challenged with a ramping dose of zinc up to a total of 3700 micromol zinc/kg body weight, MT-null mice had a greater incidence of pancreatic acinar cell degeneration compared with control mice despite accumulating less zinc (2.72 +/- 0.46 vs. 1.23 +/- 0.52 micromol zinc/g pancreas, control and MT-null, respectively, P < 0.05). The results of these experiments suggest that MT I and MT II can protect against both zinc deficiency and zinc toxicity.

ZnT-3, a putative transporter of zinc into synaptic vesicles.

The murine ZnT3 gene was cloned by virtue of its homology to the ZnT2 gene, which encodes a membrane protein that facilitates sequestration of zinc in endosomal vesicles. ZnT-3 protein is predicted to have six transmembrane domains and shares 52% amino acid identity with ZnT-2, with the homology extending throughout the two sequences. Human ZnT-3 cDNAs were also cloned; the amino acid sequence is 86% identical to murine ZnT-3. The mouse ZnT3 gene has 8 exons and maps to chromosome 5. Northern blot and reverse transcriptase-PCR analyses demonstrate that murine ZnT-3 expression is restricted to the brain and testis. In situ hybridization reveals that within the brain, ZnT-3 mRNA is most abundant in the hippocampus and cerebral cortex. Antibodies raised against the C-terminal tail of mouse ZnT-3 react with the projections from these neurons and produce a pattern similar to that obtained with Timm's reaction, which reveals histochemically reactive zinc within synaptic vesicles. We propose that ZnT-3 facilitates the accumulation of zinc in synaptic vesicles.

Pancreatic tumor pathogenesis reflects the causative genetic lesion.

Transgenic mice in which c-myc expression is targeted to pancreatic acinar cells develop mixed acinar/ductal pancreatic adenocarcinomas between 2 and 7 months of age. This contrasts with the effect on pancreas of the simian virus 40 tumor antigen or activated ras, which in adult mice causes lesions composed exclusively of acinar-like cells. Furthermore, during an early stage of myc-induced pathology, transformed acinar-derived cells appear within islets, suggesting that islet hormones may influence the progression of these exocrine pancreatic tumors. These findings demonstrate that the initial oncogenic alteration can influence the pattern of subsequent tumor pathogenesis and, given that human exocrine pancreatic tumors are predominantly ductal adenocarcinomas, support the suggestion that transformed acinar cells may contribute to the genesis of this serious disease in man.

Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.

A method of deleting specific cell lineages has been developed that entails microinjection into fertilized eggs of a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. We show that microinjection of a construct in which the elastase I promoter/enhancer is fused to a gene for diphtheria toxin A polypeptide results in birth of mice lacking a normal pancreas because of expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, containing islet and duct-like cells, was observed in some of the transgenic mice. This method provides a new approach for studying cell-lineage relationships and for analyzing cellular interactions during development.

Pancreatic neoplasia induced by ras expression in acinar cells of transgenic mice.

Expression of an activated human c-H-ras oncogene under control of rat elastase I regulating elements leads to neoplasia of the fetal exocrine pancreas. In most transgenic mice bearing this gene construct, massive tumors involving all the pancreatic acinar cells develop within a few days of pancreatic differentiation. Expression of the normal c-H-ras proto-oncogene in acinar cells leads to more subtle anomalies, but no tumors develop. Thus modest amounts of the mutant ras proteins are sufficient, in an otherwise normal genetic background, to lead to neoplastic transformation of differentiating pancreatic acinar cells. In contrast, a comparable elastase-myc construct produces no pancreatic tumors in transgenic mice.

Ectopic expression of metallothionein-III causes pancreatic acinar cell necrosis in transgenic mice.

Mice express four distinct metallothioneins (MTs) that have similar metal-binding properties. MT-I and MT-II are expressed coordinately in most organs, whereas MT-III is expressed predominantly in a subset of neurons and MT-IV is expressed in certain stratified epithelia. The restricted expression of MT-III suggests that it may severe a specialized function. To test this hypothesis, transgenic mice were generated that express MT-III in the wider expression domain of MT-I. Similar transgenic lines expressing extra MT-I under the same regulation were generated as controls for the effect of over-expression of MT. Transgenic mice that express MT-III ectopically frequently die at 2-3 months of age. The pancreata of moribund mice were abnormally small and histological examination, at various ages, revealed a progressive degeneration of the acinar cells. At early stages multifocal acinar cell eosinophilia and swollen nuclei were seen and this pathology progressed to multifocal acinar cell necrosis and fibrosis. The terminal stages were characterized by a loss of the acinar compartment, leaving the islets embedded in a fibrotic remnant. Other organs of these mice were grossly and histologically normal. All organs examined from mice expressing excess MT-I were unremarkable even though expression of either MT-I or MT-III transgenes resulted in similar accumulations of zinc and copper in the pancreata. This study indicates that pancreatic acinar cells are unusually sensitive to chronic expression of MT-III. The mechanism by which MT-III disrupts pancreatic function is unclear, but the results provide further evidence that MT isoforms exhibit distinct properties and probably serve distinct biological functions.

Metallothionein overexpression suppresses hepatic hyperplasia induced by hepatitis B surface antigen.

Transgenic mice that express the viral coat proteins of hepatitis B virus (HBV) in the liver display hepatocellular damage, inflammation, regeneration, hyperplasia, and, eventually, neoplasia that is similar to that of people with chronic, active hepatitis caused by HBV infection. Hepatocellular regeneration, in the context of chronic injury and inflammation, is thought to expose dividing cells to excessive oxygen radicals, which are believed to lead to DNA damage and, ultimately, neoplasia. Because metallothioneins scavenge free radicals in vitro, we generated mice that express excess (>10-fold) metallothionein I (MT-I* mice) and the HBV surface antigens (HBsAg) to ascertain whether MT-I* would ameliorate aspects of the pathology induced by HBsAg. Markers of hepatocyte injury and tumorigenesis in HBsAg mice were compared to those in double transgenic (HBsAg and MT-I*) mice. Hepatic hyperplasia, histology, aneuploidy, and accumulation of an oxidative DNA adduct, 8-oxo-2'-deoxyguanosine, were examined. Although hepatitis and neoplasia were not prevented by MT-I* expression in the HBsAg mice, there was less hyperplasia and less aneuploidy. We conclude that MT-I produces a beneficial effect in this in vivo model of HBV-induced hepatitis.

Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.

We inactivated the mouse metallothionein (MT)-I and MT-II genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal laboratory conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo.

Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia.

A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III gene in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia associated with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakeratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. We suggest that MT-IV plays a special role in regulating zinc metabolism during the differentiation of stratified epithelia.

Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles.

MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons. The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA. MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum. Transgenic mice generated using 11.5 kb of the mouse MT-III 5' flanking region fused to the E. coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization. MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies. There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles. MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc. These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.

Induction of mouse metallothionein-I mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones.

++Induction of metallothionein-I (MT-I) mRNA by bacterial endotoxin (LPS) was examined. A single injection of LPS induced MT-I mRNA accumulation in both liver and kidney comparable to that induced by heavy metals. Maximal message levels were achieved 6 hr after LPS administration, prior to any increase in either hepatic or renal Zn or Cu. Experiments in which LPS was administered to transgenic mice harboring recombinant genes made by fusing the MT-I gene promoter to the herpes simplex virus thymidine kinase structural gene revealed that the response to LPS is independent of glucocorticoid hormones. These experiments begin to define the region of the MT-I gene promoter required for the LPS response.

Visualization and ablation of phenylethanolamine N-methyltransferase producing cells in transgenic mice.

We cloned and sequenced the mouse phenylethanolamine N-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5' flanking sequences from the cloned mouse PNMT gene can direct expression of the Escherichia coli beta-galactosidase (lacZ) gene to predicted regions of the adrenal, eye and brain in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT8 kb-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.