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T E Wagner

Publications and source records attributed to T E Wagner.

At least 73 records · Page 4Linked to original sources
PubMed1991

Glycine 119 of bovine growth hormone is critical for growth-promoting activity.

Bovine GH (bGH) analogs with single amino acid substitutions at positions 117 (bGH-E117L), 119 (bGH-G119R), and 122 (bGH-A122D) were generated. These analogs bind to mouse liver membrane preparations with affinities similar to native bGH. However, transgenic mice which express the analogs demonstrate different phenotypes ranging from dwarfism to gigantism. For example, expression of bGH or bGH-E117L result in large transgenic mice. In contrast, transgenic mice with a growth phenotype similar to nontransgenic animals result from expression of bGH-A122D. Surprisingly, transgenic mice with relatively high serum levels of bGH-G119R possessed a dwarf phenotype. Together these results suggest that Gly 119 and Ala 122 are involved in growth-promoting activity of GH.

Alanine↗
PubMed1990

Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.

Animals↗
PubMed1990

Studies on the thyroid in transgenic mice expressing the genes for human and bovine growth hormone.

The thyroid glands of transgenic mice (TM) expressing the genes for human (h) and bovine (b) growth hormone (GH) were studied. The percentages of larger follicles in hGH TM of either sex were significantly greater than in the corresponding normal littermates, and follicles ranging up to 350 microns in diameter were present in male hGH TM. In contrast, thyroid follicles were only slightly enlarged in male bGH TM, and were unchanged in female bGH TM. The serum concentrations of T4 were significantly decreased in male bGH TM and not altered in the other groups. Serum concentrations of T3 were slightly, but significantly increased in female hGH TM and female bGH TM, but were unaffected in male TM of either type. Since the principal difference between these foreign GHs in rodents is the additional lactogenic activity of human GH, these results may indicate that the effects of prolactin can influence the development of the thyroid.

Animals↗
PubMed1990

High level, regulated expression of the chimeric P-enolpyruvate carboxykinase (GTP)-bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene in transgenic mice.

Transgenic animals expressing genes capable of repairing DNA may be a valuable tool to study the effect of DNA-damaging agents on tissue-specific carcinogenesis. For this reason, we constructed a chimeric gene consisting of the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene linked to the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase and the polyadenylate region from the bovine growth hormone gene. The PEPCK promoter results in gene expression in liver and kidney and is induced by hormones, and its transcription is regulated by diet. The chimeric PEPCK ada gene was injected into the male pronucleus of fertilized eggs to produce transgenic mice. Six of 65 developing mice contained 5-10 copies of the intact trans gene per genome. Two founders transmitted the trans gene in a heterozygous manner, whereas 3 transmitted as germ line mosaics and 1 did not transmit to F1 offspring. All F1 offspring carrying the PEPCK ada trans gene expressed ada mRNA in liver and kidney and produced a functional alkyltransferase with a protein molecular weight of 39,000 originating from the bacterial gene. Total alkyltransferase activity was increased in the liver of F1 offspring from all founder mice, but offspring of only one founder had elevated renal alkyltransferase levels. A diet high in protein markedly increased ada mRNA and alkyltransferase activity within 1 week in both liver and kidney, whereas a high carbohydrate diet for 1 week markedly reduced expression of PEPCK ada and alkyltransferase levels. Nontransgenic animals were unaffected by these dietary manipulations. During induction with a high protein diet, hepatic alkyltransferase in transgenic mice was 16.6 +/- 1.5 units/micrograms DNA (mean +/- SE) compared to 5.3 +/- 0.6 units/micrograms DNA in control animals. This level of alkyltransferase is higher than that in any mammalian tissue noted previously except human liver. Transgenic animals expressing high levels of alkyltransferase should help define the role of DNA repair in protection from carcinogenesis induced by N-nitroso compounds.

Animals↗
PubMed1990

Effects of transgenes for human and bovine growth hormones on age-related changes in ovarian morphology in mice.

The expression of human growth hormone (GH) in female transgenic mice (TM) is accompanied by sterility, whereas females expressing the bovine GH gene are fertile. A light and electron microscopic study was conducted to examine whether expression of these foreign GH genes in mice is associated with structural changes in the ovaries of young adult (3-month-old) or middle-aged (7-month-old) mice. One ovary was serially sectioned for light microscopy, and the contralateral ovary was used for electron microscopy. The numbers of preantral (PAF) and antral (AF) follicles, with and without signs of atresia, as well as the number of corpora lutea (CL), were determined. As expected, body weights of both young and middle-aged TM of either kind were significantly increased over those of their normal littermates. However, the ovarian weights of TM and control mice did not differ. In the 3-month-old TM, the ovaries were grossly normal at the light microscopic level. However, significantly more CL were counted in the ovaries of human GH-TM than in those of the other two groups. The percentage of PAF with signs of atresia was significantly reduced in ovaries of bovine GH-TM compared with the other groups, while the percentages of AF undergoing atresia were significantly different in all groups, with the highest values in normal animals, intermediate ones in human GH-TM, and the lowest in bovine GH-TM. In the ovaries of 7-month-old human GH-TM, conspicuous clusters of large, foamy light cells were present in the cortex and the medulla. Ultrastructurally, these cells appeared as interstitial cells in various stages of degeneration, accumulating cholesterol crystal-like inclusions. Although degeneration of interstitial cells was observed also in the other types of animals, it involved usually only single cells and no cytoplasmic crystal inclusions. Moreover, in the ovaries of 7-month-old human GH-TM the percentages of PAF were significantly reduced and the percentages of AF significantly increased compared with those in the two other groups, which did not differ from each other with respect to these parameters. No significant differences in the numbers of CL were found between the groups. Percentages of atretic PAF were significantly reduced in bovine GH-TM and comparable in the other two groups, while percentages of atretic AF were not different between normal and bovine GH-TM, but were significantly increased in human GH-TM.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1990

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts.

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.

Journal Article↗
PubMed1990

Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.

To determine the importance of the third alpha-helix in bovine growth hormone (bGH) relative to growth-related biological activities, the following experimental approach was used: (i) mutagenesis of helix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies of wild-type and mutated bGH; and (iv) expression of the mutated gene in the transgenic mouse. An altered bGH gene (pBGH10 delta 6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGH10 delta 6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growth-suppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule.

Amino Acid Sequence↗
PubMed1990

Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulfur keratin promoter.

We have generated a transgenic mouse line by microinjection of a chimeric DNA fragment (KER-CAT) containing a hair-specific, murine ultra-high-sulfur keratin promoter (KER) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. A 671-base pair (bp) stretch of the 5' promoter region was used to direct the expression of the CAT gene in this construct. Of the tissues tested for CAT activity in these transgenic animals only skin with growing hair, isolated hair follicles, and microdissected vibrissae showed substantial levels of activity. These are the same tissues where the endogenous ultra-high-sulfur keratin gene is expressed as shown by in situ hybridization. Furthermore, analysis of the CAT activity during the developmental stages of the hair growth cycle shows that the chimeric gene is expressed during the anagen phase of the hair growth cycle; this is the expected time during development for its expression. From these results we conclude that 671 bp of the promoter sequence from the ultra-high-sulfur keratin gene is sufficient to direct the correct development-specific and tissue-specific expression of the reporter gene construct in transgenic mice. The appropriate expression of the KER-CAT construct in transgenic mice is an important step in understanding the regulation of this gene during hair organogenesis.

Animals↗
PubMed1990

Neuroendocrine function in transgenic male mice with human growth hormone expression.

The neuroendocrine effects of human growth hormone (hGH) secretion were studied in adult male mice into which an hGH gene fused with mouse metallothionein 1 (mMT-1) promoter had been introduced. Intact transgenic mice had significantly greater plasma luteinizing hormone (LH) levels than did normal littermate controls. Castration increased LH levels in normal mice but was without effect on plasma LH levels in the transgenic mice. In vitro LH secretion and pituitary LH content were higher in the intact transgenic mice than in intact controls, while there was no significant difference in pituitary LH levels and in vitro LH secretion between the 2 groups of castrate animals. Intact transgenic mice exhibited a greater median eminence (ME) norepinephrine (NE) turnover than control animals, but ME NE turnover did not increase after castration in the transgenic animals as was the case in control mice. Castrate mice expressing the hGH gene had plasma levels of prolactin (PRL) similar to those seen in castrate controls, which was unexpected based on a previous study showing greatly attenuated PRL levels in intact hGH mice when compared to intact controls from the same line. Dopamine (DA) turnover in the ME was not significantly affected by the presence of the hGH gene, suggesting that the difference in plasma PRL levels between normal and transgenic mice is mediated through changes in PRL-regulating factors other than DA. In conclusion, the expression of the mMT-1/hGH hybrid gene in male mice leads to major alterations in LH secretion and lesser changes in PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

An immunocytochemical and ultrastructural study of adenohypophyses of mice transgenic for human growth hormone.

Adenohypophysial morphology in 12 mice transgenic for methallothionein-I-human (h) GH fusion gene was investigated by immunocytochemistry and electron microscopy. The sustained oversecretion of hGH stimulated body growth. The pituitary glands of 6-month-old transgenic mice were significantly decreased in weight and showed marked morphological changes in somatotrophs, lactotrophs, corticotrophs, and gonadotrophs. GH-immunoreactive cells were greatly reduced in size and midly decreased in number; by electron microscopy, the organelles implicated in hormone synthesis were inconspicuous in this cell type. Transgenic males were hypoprolactinemic, presumably due to lactogenic activity of hGH in rodents. Their pituitaries displayed few and slender PRL-immunoreactive cells; ultrastructurally, they belonged to immature (type II) lactotrophs. However, in females, PRL-containing cells showed no change in number, size, or distribution compared to controls. Prior biochemical studies demonstrated high blood levels of LH in males. Their pituitaries contained highly active gonadotrophs resembling gonadectomy cells, consistent with the view that these changes are related to PRL-like activity of hGH in mice. In both sexes, stimulated corticotrophs were present. The results indicate that some changes in adenohypophysial cells of mice transgenic for hGH can be attributed to protracted overproduction of the heterologous GH, whereas others can be explained by lactotrophic activity of hGH in mice. The divergent morphological responses of lactotrophs and gonadotrophs in the two sexes may reflect differences in the hormonal regulatory mechanisms between male and female mice.

Animals↗
PubMed1990

Stress-induced secretion of human growth hormone in transgenic mice.

The effect of stress on human growth hormone (hGH) secretion was studied in transgenic mice. Experiments were conducted on fourth, fifth, and sixth generation male mice carrying a fusion gene, consisting of the promoter sequence of the mouse metallothionein I gene ligated to the hGH structural gene (mMT-I/hGH). In animals adapted to a controlled photoperiod, basal (unstimulated) levels of plasma hGH exhibited a diurnal cycling, with peak values occurring during the later half of the light period (15.5 +/- 1.0 vs 10.7 +/- 0.9 ng/ml, mean +/- SE, light versus dark, respectively). Food deprivation (5 days) led to elevated levels of plasma hGH (11.0 +/- 0.7 vs 32.0 +/- 4.2 ng/ml, preversus post-fast, respectively) accompanied by weight loss (49.5 +/- 0.8 vs 34.3 +/- 0.7 g), and hypoglycemia (7.8 +/- 0.2 vs 5.0 +/- 0.3 mM); glucose administration (5% drinking solution ad libitum) blocked the changes in levels of plasma hGH (12.2 +/- 1.1 vs 13.8 +/- 0.8 ng/ml) and plasma glucose (7.4 +/- 0.3 vs 7.9 +/- 0.5 mM), although the animals still sustained significant weight loss (44.9 +/- 1.6 vs 35.2 +/- 1.1 g). Vigorous exercise (swimming, 4 hr) produced a small but significant increase in plasma hGH, 12.1 +/- 1.1 ng/ml (1 hr pre-swim) vs 16.7 +/- 0.6 ng/ml (immediately post-swim). These findings indicate that the mMT-I/hGH transgene is responsive to the physiologic status of the host animal. Taken together with information regarding the heterologous components of the fusion gene, these data are consistent with the view that the hGH (structural) sequence may play a role in the response to stress.

Animals↗
PubMed1990

The human growth hormone transgene: expression in hemizygous and homozygous mice.

Female transgenic mice carrying the mouse metallothionein-I/human growth hormone (hGH) fusion gene are sterile. Transmission of the transgene has been limited to the male germ line, resulting in the production of hemizygous (He) progeny containing only a single (paternal) copy of the gene. Using ovary transfer, we have developed procedures for producing homozygous (Ho) TG mice, viz., male TG mice were mated with control (non-TG) females carrying ovaries donated by female TG mice. In both He and Ho TG animals, serum levels of hGH were higher (1.5-fold) in males than in females, tended to decrease with age of the animal, and were increased (about 5-fold) by zinc induction. However, in comparison to He animals of the same sex, the Ho TG mice attained a greater body weight and had more than 2-fold higher levels of liver hGH-mRNA and serum hGH, both under basal conditions and in response to zinc induction. That is, the expression of the transgene was qualitatively similar in He and Ho TG mice, but the level of transgene activity was greater in the Ho animals. We interpret this to indicate that both copies (maternal and paternal) of the transgene were active and expressed additively (or cooperatively) in the Ho TG animal.

Analysis of Variance↗
PubMed1990

Developmental regulation and tissue-specific expression of a chimaeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic animals.

Expression of the bovine growth hormone (bGH) gene, directed by the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter, in transgenic animals was investigated. Different lengths of the 5' PEPCK promoter-regulatory domain were utilized to control bGH expression; these included -2000/+73, -460/+73, -355/+73, and -174/+73. The chimaeric PEPCK/bGH gene containing -460/+73 of PEPCK 5' flanking sequence (PEPCK/bGH(460] is regulated by cAMP, insulin, and dexamethasone in the same manner as the endogenous PEPCK gene. This PEPCK promoter-regulatory domain also controls the tissue-specific expression of the bGH gene to liver, kidney, adipose tissue, jejunum and mammary gland. Furthermore, the correct developmental pattern of expression is observed in the mouse lines which contain PEPCK/bGH(460). The transgene mRNA is not detected during fetal development until Day 19. At Day 1 after birth, due to alterations in the insulin:glucagon ratio, the amounts of transgene mRNA are greatly increased, similar to the endogenous PEPCK gene.

Animals↗
PubMed1990

In-vitro mutagenesis of the bovine growth hormone gene.

The biological activities of bovine growth hormone (bGH) were studied in a transgenic mouse model system. The following experimental design was used: (1) in-vitro mutagenesis of the bGH gene; (2) expression of the mutated gene in cultured mouse cells under transcriptional regulation of the mouse metallothionein I promoter; (3) binding studies of the mutated and wild-type protein to mouse liver membrane preparations; (4) generation of transgenic mice which express the mutant hormone; and (5) growth rate analysis of transgenic mice. Removal of the alanine +1 codon from the bGH gene or a substitution of serine for cysteine 189 does not affect the ability of the mutant protein to influence transgenic mouse growth. Also, mutations which increase the hydrophobicity within the bGH alpha helix 3 region (amino acid residues 109-126) do not alter the enhanced growth rate in transgenic mice which express these mutated bGH proteins.

Animals↗
PubMed1990

Production of transgenic pigs harbouring a rat phosphoenolpyruvate carboxykinase-bovine growth hormone fusion gene.

Over the past 5 years, reports detailing the production of transgenic pigs have focussed on enhanced growth performance. Phenotypic side-effects observed in pigs harbouring chimaeric constructs containing metallothionein or Moloney murine leukaemia virus transcriptional activators fused to growth hormone (GH) structural genes have been attributed to chronic overexpression of GH. In an effort to regulate a transgene product more effectively, a liver specific 460 bp 5' flanking sequence of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene was ligated to a BamHI site of the first exon of the genomic bovine GH (bGH) structural gene. Following micro-injection of the PEPCK/bGH construct into 1- and 2-cell pig zygotes. 124 offspring were produced of which 7 pigs were determined to be transgenic by dot-blot and Southern analysis. The PEPCK gene expression, in terms of tissue and developmental specificity, appears similar to that observed in PEPCK/bGH transgenic mice. Germ-line transmission was identified in 1 of 3 mated founders. Dramatic influences on backfat thickness were observed including a 41% reduction in backfat depth when compared to non-transgenic sex-matched littermate control pigs. Both the regulation and characterization of gene expression in PEPCK/bGH transgenic pigs are under investigation.

Animals↗
PubMed1989

Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells.

A mouse metallothionein-I/human growth hormone fusion gene was microinjected into fertilized mouse eggs, the embryos were implanted into pseudopregnant foster mothers, and the offspring analysed. Five of twenty-six mice born after one series of injections contained from one to eight copies of the fusion gene stably integrated into their genomes and had human growth hormone in their serum. When several of these transgenic mice and transgenic offspring were treated with glucocorticoids, serum growth hormone levels were elevated from 1.5- to 6.3-fold. A fourfold induction in fusion gene mRNA in the liver of one of the five mice was also observed after treatment with glucocorticoids. When the fusion gene was transiently transfected into mouse L cells, dexamethasone caused a three- to fourfold induction of fusion gene mRNA and secreted human growth hormone. A deletion analysis of regulatory elements required for inducibility in L cells shows that DNA sequences responsible for the observed inductions are located within the transcribed region of the human growth hormone gene. However, a previously described glucocorticoid receptor binding site in the first intron of the gene is not required for response to the hormone.

Animals↗
PubMed1988

Infertility in transgenic female mice with human growth hormone expression: evidence for luteal failure.

Introduction of the human growth hormone (hGH) gene fused with mouse metallothionein I promoter into domestic mice leads to ectopic synthesis of hGH, marked stimulation of somatic growth, and female sterility. Transgenic females (produced by mating transgenic males to normal females) mated but failed to become pregnant or pseudopregnant as evidenced by the recurrence of vaginal plugs every 5-7 days. Daily injections of 1 mg progesterone, starting on day 1 postcoitum (p.c.), maintained pregnancy, suggesting that the sterility of these animals is due to inadequate luteal function. In ovariectomized female transgenic mice, median eminence (ME) turnover of dopamine (DA) was increased, and plasma prolactin (PRL) levels were reduced, presumably because of the known lactogenic activity of hGH in rodents. From these observations we suspected that either 1) the corpora lutea of these animals are unresponsive to lactogenic hormones, or 2) hGH by stimulating tuberoinfundibular dopaminergic (TIDA) neurons interferes with the increase in PRL release that normally follows mating and this, in turn, leads to luteal failure. To distinguish between these possibilities, transgenic females were treated with PRL-secreting ectopic pituitary transplants from normal females of the same strain on day 1 p.c. Eight of ten treated females became pregnant and delivered litters. We conclude that infertility of transgenic female mice with hGH expression is due to activation of the TIDA system, suppression of endogenous PRL release, and luteal deficiency.

Animals↗