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Biomedical subjects

R E Hammer

Publications and source records attributed to R E Hammer.

At least 163 records · Page 9Linked to original sources
PubMed1986

Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene.

The pituitaries of transgenic mice that express a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin-14 exclusively in the gonadotrophic cells. The purpose of this study was to determine whether somatostatin expressed from the foreign fusion gene enters the normal secretory pathway within these cells. Immuno-gold labeling of serial thin sections localized somatostatin to the secretory granules of gonadotropin-producing cells. The gonadotroph-specific hypophysiotropic factor, luteinizing hormone-releasing hormone caused a dose-dependent secretion of somatostatin when applied to primary pituitary cultures from these mice. Growth hormone-releasing hormone, thyrotropin-releasing hormone, corticotropin releasing factor, and dopamine did not affect somatostatin secretion. These experiments demonstrate that a neurosecretory peptide encoded by a foreign gene can enter the regulated secretory pathway of pituitary cells from transgenic mice.

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PubMed1986

Stimulation of growth hormone synthesis by glucose in islets of Langerhans isolated from transgenic mice.

To examine further the mechanism by which the synthesis of proteins translated on the rough endoplasmic reticulum is regulated in pancreatic beta-cells, the synthesis of growth hormone in islets from transgenic mice carrying the metallothionein-rat growth hormone gene fusion was studied. High glucose (17 mM) stimulated the synthesis and secretion of an apparently normally processed growth hormone. The stimulation of synthesis of growth hormone was less efficient than the stimulation of insulin synthesis in these islets, whereas the stimulation of release of labeled growth hormone paralleled that of insulin. These results are consistent with the hypothesis that signal recognition particle-mediated mechanism(s) may be involved in regulating the translational efficiency of secreted proteins in isolated islets (Welsh, M., Scherberg, N., Gilmore, R., and Steiner, D. F. (1986) Biochem. J. 235, 459-467). Furthermore, in the beta-cells, growth hormone follows the normal regulated pathway of secretory granule transport and exocytosis.

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PubMed1986

Gonadotroph-specific expression of metallothionein fusion genes in pituitaries of transgenic mice.

Transgenic mice expressing a metallothionein-somatostatin fusion gene contain high concentrations of somatostatin in the anterior pituitary gland, a tissue that does not normally produce somatostatin. Immunoreactive somatostatin within the anterior pituitaries was found exclusively within gonadotrophs. Similarly, a metallothionein-human growth-hormone fusion gene was also expressed selectively in gonadotrophs. It is proposed that sequences common to the two fusion genes are responsible for the gonadotroph-specific expression.

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PubMed1986

Glucocorticoid regulation of metallothionein during murine development.

During the second half of gestation in the mouse there is a rise in both fetal (4-fold) and maternal (10-fold) metallothionein-I (MT-I) mRNA in the liver (but not in the kidney). There is a large increase in plasma corticosterone (the predominant murine glucocorticoid hormone), as well as an increase in hepatic zinc, which is coincident with the induction of MT-I mRNA. Considering that both of these compounds are known to be effective inducers of MT-I mRNA, we set out to determine whether either one or both were involved in the developmental regulation of MT-I genes. Several lines of evidence suggest that corticosterone is the principal inducer of fetal MT-I mRNA: The induction of MT-I mRNA in the liver, but not in the kidney, mimics glucocorticoid regulation but not metal regulation. Reduction of maternal corticosterone levels by treating mice with metyrapone lowered MT-I mRNA levels but had no effect on zinc levels. A line of transgenic mice carrying a metallothionein-growth hormone fusion gene that is responsive to metals but unresponsive to glucocorticoids was not developmentally regulated. Based on these observations, we propose that corticosterone is responsible for the induction of MT-I mRNA and that the resulting MT sequesters zinc and copper which may be used later in development.

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PubMed1986

Transgenic progeny inherit tissue-specific expression of rat elastase I genes.

Six different lines of transgenic mice bearing rat elastase I genes stably inherit both the high-level pancreatic expression and low-level nonpancreatic expression characteristic of each original founding mouse. The high pancreatic expression of the introduced rat genes is transcriptionally determined. In response to high rat elastase I mRNA content in the pancreas, rat elastase I protein is synthesized and secreted at high levels. The stable inheritance of expression of the foreign rat elastase I genes in transgenic mice allows the development of animal lines that synthesize and secrete high levels of foreign protein by the pancreas.

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PubMed1986

Estrogen regulation of the avian transferrin gene in transgenic mice.

The intact chicken transferrin gene was microinjected into fertilized mouse eggs, and the resulting transgenic animals were used to produce lines of mice containing integrated copies of the chicken gene. The levels of expression of the chicken gene were quantitated in various tissues, and the response of the gene to estrogen stimulation was measured after chronic or acute estrogen exposure. Two of the three mouse lines studied maintained stable levels of expression in successive generations of offspring, and the third line had two- to threefold-higher levels in offspring than in the original parent. In the third line, the original transgenic parent was found to be a mosaic. The chicken transferrin gene was expressed at 10- to 20-fold-higher levels in liver than in any other tissue; however, the levels of chicken transferrin mRNA in kidney were higher than expected, indicating that the tissue specificity was only partial. In all three lines, the foreign gene was induced by estrogen administration. After 10 days of estrogen administration, there was a twofold increase in both transferrin mRNA and transcription of the chicken transferrin gene. A single injection of estradiol led to a fourfold increase in transferrin mRNA synthesis at 4h. As a control the levels of mouse albumin were measured, and both the level of albumin mRNA and its rate of transcription declined about twofold after estrogen administration. Our results indicate that the intact chicken gene with 2.2 kilobases of 5' flanking sequence contains signals for both tissue specificity and steroid regulation that can be recognized in mice.

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PubMed1986

Genetic engineering of mammalian embryos.

A gene consisting of the mouse metallothionein I promoter/regulator (MT) fused to the human growth hormone (hGH) structural gene (MThGH) was microinjected into rabbit, sheep and pig eggs. Visualization of nuclear structures was accomplished by interference-contrast (I-C) microscopy for rabbit and sheep eggs and by centrifugation and I-C microscopy for pig eggs. The gene integrated into the chromosomes of each species with an efficiency of 13% in rabbits, 1% in sheep and 10% in pigs. Human GH mRNA was detected in the liver of transgenic rabbits as well as tail and ear samples of pigs. Immunoreactive hGH was present in the serum of a transgenic rabbit and plasma of most transgenic pigs. In several pigs hGH levels increased between birth and 90 d of age. The presence of substantial quantities of hGH in plasma of pigs did not increase postnatal somatic growth rates. Founder animals will be bred and their transgenic and control progeny used to assess the effects of hGH on feed efficiency and carcass composition. These experiments demonstrate the feasibility of introducing foreign genes into the genome of several animal species by microinjection of eggs.

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PubMed1985

A simple method for counting nuclei in the preimplantation mouse embryo.

An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.

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PubMed1985

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice.

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.

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PubMed1985

Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei.

The obscured pronuclei or nuclei in living one- and two-celled pig ova were revealed after centrifugation for 3 min at 15,000 X g. To determine viability of centrifuged ova, one- and two-celled pig ova were collected from superovulated gilts; half of the ova were centrifuged and all ova were transferred into recipient gilts. Prior to transfer all embryos were stained with tetramethylrhodamine isothiocyanate (TRITC) to distinguish the experimental embryos from the recipients's own ova. Centrifuged ova were transferred into one oviduct of recipient gilts and uncentrifuged ova were deposited into the opposite oviduct. Embryos were recovered 4 days after transfer and were stained with lacmoid or Hoechst 33342 to assess their stage of development. Centrifugation had no detectable influence on survival of the recovered embryos to 4 days. Centrifugation is a simple, reliable method for revealing pronuclei and nuclei of one- and two-celled pig ova and apparently does not alter subsequent preimplantation development.

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PubMed1985

Developmental regulation of alpha-fetoprotein genes in transgenic mice.

The mouse alpha-fetoprotein gene is activated in embryonic development in the visceral endoderm of the extraembryonic yolk sac and the fetal liver and gut. Transcription of the gene is subsequently repressed in the neonatal liver. To ask whether the DNA sequence elements required for tissue-specific activation are the same or different from those required for postnatal developmental regulation of the gene, modified copies of the alpha-fetoprotein gene were microinjected into fertilized mouse eggs. Those animals which developed to term and carried integrated copies of the modified gene were analyzed for expression. In approximately 50% of such animals, the introduced gene was active only in the three cell lineages which expressed the authentic alpha-fetoprotein gene. Furthermore, its expression was repressed in the neonatal liver. Thus, we conclude that the modified genes, which included either 7 or 14 kilobase pairs of 5'-flanking DNA, contained the DNA sequence information to direct both tissue-specific expression and developmental regulation. The observation that 50% of the mice which carried the modified gene did not express it in any tissue, combined with the fact that the level of expression was highly variable between expressing transgenic animals, suggested that the gene was susceptible to its site of integration in the mouse genome.

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PubMed1985

Effects of nicotine on oviducal blood flow and embryo development in the rat.

Nicotine (5.0 mg/kg) was injected (s.c.) twice daily on Day 1 or Days 1-4 or 1-5 of pregnancy. Cumulative doses of nicotine retarded embryo cell cleavage and substantially reduced embryo cell number (saline vs nicotine: 42.5 +/- 1.7 vs 22.1 +/- 1.9 nuclei/embryo, at 12:00 h on Day 5; P less than 0.05). However, treatment for even 1 day (Day 1) significantly reduced cell number (saline vs nicotine: 42.5 +/- 1.7 vs 30.5 +/- 0.9, at 12:00 h day on Day 5; P less than 0.01). Nicotine injection also resulted in a marked and prolonged reduction in oviduct blood flow (pretreatment vs 90 min after nicotine: 0.61 +/- 0.06 vs 0.37 +/- 0.10 ml/min . g-1; P less than 0.005). The results indicate that, in the rat, even a brief exposure to nicotine, the chief alkaloid of tobacco, reduces oviducal blood flow and the rate of embryo cell proliferation. The embryo is therefore susceptible to the effects of nicotine before implantation.

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PubMed1984

Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice.

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10(3)-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.

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