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PubMed2005

Transgenic overexpression of prothymosin alpha induces development of polycystic kidney disease.

BACKGROUND: Polycystic kidney disease (PKD) is a genetic disorder characterized by development of renal cysts and progressive renal dysfunction. Renal tissues from both PKD patients and rodent models of PKD show elevated c-myc expression. Prothymosin alpha (ProT) is positively regulated by c-myc through binding to the E box of its promoter. Through creating transgenic mice and clinical studies, we sought to investigate whether ProT overexpression contributes to PKD development. METHODS: ProT heterozygous and homozygous transgenic mice were generated and characterized. Morphologic, histologic, immunohistochemical, and biochemical analyses of the transgenic mice were performed. RESULTS: Two transgenic lines that represented integration at two different loci of the chromosomes were generated. ProT overexpression in the kidneys of homozygous transgenic mice induced a PKD phenotype, which included polycystic kidneys, elevated blood urea nitrogen (BUN), and lethality at about 10 days of age. Similar overexpression pattern of ProT was noted in cystic kidneys of the transgenic mice as well as in human autosomal-recessive PKD (ARPKD) and autosomal-dominant PKD (ADPKD) kidneys. ProT protein levels in the kidneys and urine as well as renal mRNA level of epithelial growth factor receptor (EGFR) of homozygous ProT transgenic mice were significantly higher than heterozygous or nontransgenic littermates. Furthermore, the heterozygous transgenic mice at 17 months of age also developed mild cystic kidneys. CONCLUSION: Transgenic mice overexpressing ProT represent a novel model for PKD and may provide insights into PKD development. ProT, like c-myc and EGFR, may contribute to the development of renal cysts and may be a potential noninvasive diagnostic molecule of PKD.

Animals↗
PubMed1994

Transgenic glutathione peroxidase mouse models for neuroprotection studies.

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.

Animals↗
PubMed2006

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice.

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.

Alphapapillomavirus↗
PubMed1991

The polyomavirus early region gene in transgenic mice causes vascular and bone tumors.

Transgenic mice carrying the entire polyomavirus (Py) early region consistently develop both vascular and bone tumors. This tumor spectrum represents a subset of the tumors found in mice infected with Py and an expansion of the vascular tumor spectrum seen in Py middle T antigen (MT) transgenic mice (V. L. Bautch, S. Toda, J. A. Hassell, and D. Hanahan, Cell 51:529-538, 1987). Transgenic mice of three independent lineages develop these pathologies, and mice of individual lineages also develop lymphangiomas and fibrosarcomas. All tumors are of mesenchymal origin, and all tumor tissues express the Py transgene. Some unaffected tissues, including the testes of mice of all lineages, also express the Py transgene. The number of transgene expression sites in mice of a given lineage correlates with the severity and latency of the tumor phenotype in these animals. Analysis of transgene transcripts indicates that RNAs for Py large T antigen (LT), MT, and small T antigen (ST) are present in both tumors and testes of transgenic mice. The ratio of LT RNA to MT and ST RNAs, however, is higher in testes than in tumors and other unaffected tissues, indicating that tissue-specific differences in the relative amounts of the alternatively spliced Py RNAs exist. The finding that some sites of Py transgene expression are susceptible to tumor formation while other expressing tissues such as testes are refractory to tumorigenesis suggests that mechanisms of viral pathogenesis are influenced by a tissue specificity in the effects of the Py early region.

Animals↗
PubMed2001

Genetic immunization of wild-type and hepatitis C virus transgenic mice reveals a hierarchy of cellular immune response and tolerance induction against hepatitis C virus structural proteins.

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.

Adoptive Transfer↗
PubMed1993

Transcriptional insulation of the human keratin 18 gene in transgenic mice.

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes.

Animals↗
PubMed1995

Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice.

Transgenic mice expressing polyomavirus large T antigen (PVLT) in cardiomyocytes develop a cardiac hypertrophy in adulthood. Morphometric analysis identified cardiomyocytes enlarged up to ninefold in cross-sectional area in the adult transgenic hearts compared with normal age-matched nontransgenic hearts. Most enlarged cardiomyocytes were found in the subendocardium, whereas normal-sized cardiomyocytes were localized to the midmyocardium. Transgenic hearts did not express detectable skeletal muscle actin mRNA or protein, or skeletal troponin I isoform mRNA. Some, but not all, transgenic hearts expressed an increase in the beta-myosin heavy chain mRNA. All five transgenic mice tested had increased expression of atrial natriuretic factor (ANF) mRNA. Whereas normal hearts expressed three myosin light chain proteins of 19, 16, and 15 kDa, we found that the 19-kDa myosin light chain was not observed in the transgenic hearts. We conclude that adult, PVLT-expressing, transgenic mice developed enlarged cardiomyocytes with an increase in beta-myosin heavy chain and ANF mRNA expression, but a widespread skeletal isoform usage was not present in these transgenic mice. The adult transgenic hearts thus display histological and molecular changes similar to those found in hypertrophy induced by a pressure overload in vivo.

Animals↗
PubMed2000

Chronologic changes of cerebral ventricular size in a transgenic model of hydrocephalus.

We have maintained a transgenic mouse model of hydrocephalus created to overproduce the cytokine, transforming growth factor-beta1 (TGF-beta1) in the central nervous system (CNS). The aim of the present study was to estimate the embryonic period when the transgenic mice would develop hydrocephalus, by investigating the chronological developmental changes of the cerebral ventricles. Qualitative analysis of ventricular size was performed on sections cut in the coronal plane of embryos at the 15th (E15) and 18th (E18) embryonic days, and postnatal mice aged 4 days (P4). The presence of the TGF-beta1 transgene was determined by performing polymerase chain reaction (PCR) analysis. We have examined 24 embryos and 14 postnatal mice. By performing PCR analysis, the TGF-beta1 transgene was determined to be present in 16 (42.1%). Five of 16 embryos at E15 carried the transgene, and showed a slight enlargement of the lateral ventricles. Three of 8 embryos at E18 carried the transgene, and had remarkable enlargement of the lateral ventricles. Eight of 14 pups at P4 carried the transgene, and 7 of 8 pups with the transgene developed hydrocephalus. In pups that were positive for the transgene, massive enlargement of the lateral ventricles was observed and there was an associated thinning of the overlying cerebral cortex. These results suggest that congenital hydrocephalus may develop at an important embryonic time period, which coincides with the stage of neural stem cell proliferation and differentiation in the CNS.

Animals↗
PubMed1993

Transgenic mouse models of vasopressin expression.

Arginine vasopressin is a nine-amino acid neuropeptide hormone important in the regulation of water metabolism. It also may have a role in other physiological functions, such as blood pressure regulation and the response to stress. Whole animal studies have provided a good understanding of vasopressin physiology and regulation of the normal vasopressin gene, and in vitro cell culture studies have demonstrated important features of the intracellular regulation of vasopressin gene expression. Transgenic mice provide useful models for the study of the in vivo regulation of gene expression. Previously reported mouse lines transgenic with vasopressin gene constructs have not expressed the transgene in a tissue distribution similar to that detected for the endogenous mouse vasopressin gene. An 8.2-kb genomic construct of the rat vasopressin gene, including 3 kb each of 5' and 3' flanking sequences, has been used to develop a line of transgenic mice. These animals express the transgene in a tissue-specific manner, demonstrate appropriate osmotic regulation of transgenic vasopressin mRNA, and have normal water metabolism. Animals homozygous for the 8.2-kb transgene have increased basal plasma levels of vasopressin peptide but have no apparent change in basal water metabolism. The findings with this and other previously reported mouse lines transgenic for vasopressin constructs provide a basis for developing future transgenic lines to study the in vivo regulation of the vasopressin gene.

Animals↗
PubMed1995

In vivo demonstration of red cell-endothelial interaction, sickling and altered microvascular response to oxygen in the sickle transgenic mouse.

Intravascular sickling, red cell-endothelium interaction, and altered microvascular responses have been suggested to contribute to the pathophysiology of human sickle cell disease, but have never been demonstrated under in vivo flow. To address this issue, we have examined a transgenic mouse line, alphaHbetaSbetaS-Antilles [betaMDD] which has a combined high (78%) expression of beta S and beta S-Antilles globins. In vivo microcirculatory studies using the cremaster muscle preparation showed adhesion of red cells, restricted to postcapillary venules, in transgenic mice but not in control mice. Electron microscopy revealed distinct contacts between the red cell membrane and the endothelium surface. Some red cells exhibiting sickling were regularly observed in the venular flow. Infusion of transgenic mouse red cells into the ex vivo mesocecum vasculature also showed adhesion of mouse red cells exclusively in venules. Under resting conditions (pO2, 15-20 mmHg), there were no differences in the cremaster microvascular diameters of control and transgenic mice; however, transgenic mice showed a drastic reduction in microvascular red cell velocities (Vrbc) with maximal Vrbc decrease (> 60%) occurring in venules, the sites of red cell adhesion and sickling. Local, transient hyperoxia (pO2, 150 mmHg) resulted in striking differences between control and transgenic mice. In controls, oxygen caused a 69% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic mice, hyperoxia resulted in only 8% decrease in the arteriolar diameter and in 68% increase in VrBC; the latter is probably due to an improved flow behavior of red cells as a consequence of unsickling. In summary, the high expression of human sickle hemoglobin in the mouse results not only in intravascular sickling but also red cell-endothelium interaction. The altered microvascular response to oxygen could be secondary to blood rheological changes, although possible intrinsic differences in the endothelial cell/vascular smooth muscle function in the transgenic mouse may also contribute. These sickle transgenic mice could serve as a useful model to investigate vasoocclusive mechanisms, as well as to test potential therapies.

Anemia, Sickle Cell↗
PubMed2006

Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.

BACKGROUND: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage lambda red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step. RESULTS: We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion. CONCLUSION: This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct.

Bacteriophage lambda↗
PubMed2006

Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice.

BACKGROUND: The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located approximately 10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model. RESULTS: We constructed a approximately 20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions. CONCLUSION: We have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe that these transgenic mice will provide a useful model for further characterizing the E11 and E1 promoter in vivo under different physiological and pathological circumstances such as chronic opioid treatment and chronic pain models.

Animals↗
PubMed1992

Elevated beta-cell calmodulin produces a unique insulin secretory defect in transgenic mice.

Transgenic mice with elevated levels of beta-cell calmodulin develop severe diabetes even though pancreatic beta-cells contain reserve levels of insulin. Electron microscopic examination of transgenic pancreas confirmed the presence of abundant insulin secretory granules and failed to reveal obvious morphological abnormalities. These observations suggested that excess calmodulin may specifically impair the secretory process. To directly assess the effect of excess calmodulin on beta-cell function we have isolated pancreatic islets from transgenic animals. Transgenic islets from 6- to 8-day-old mice used 40% less glucose than normal islets and contained 58% of the normal insulin content, 90% of the normal glucagon content, and 5-fold higher levels of calmodulin than islets from control mice of the same age. Parallel perifusions of normal and transgenic islets confirmed that excess calmodulin inhibited glucose-stimulated insulin secretion; first phase secretion was reduced by 60%, and second phase secretion was essentially absent. Static assays were performed to assess the response to other secretagogues. All fuel secretagogues tested were ineffective in stimulating insulin secretion from transgenic islets. Secretion in response to depolarizing levels of potassium was also severely impaired. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine increased transgenic secretion, but not to the level obtained in normal islets. Of the compounds examined, only phorbol 12-myristate 13-acetate and carbachol, two substances thought to act in beta-cells by stimulation of protein kinase-C, produced equivalent secretion in normal and transgenic islets. Phorbol 12-myristate 13-acetate also appeared to restore second phase secretion in transgenic islets. These results indicate that the initial period of calmodulin-induced diabetes is due to a secretory defect. This defect appears to be distal to membrane depolarization and is selective for the second phase of insulin secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Overexpression of insulin-like growth factor-binding protein-4 (IGFBP-4) in smooth muscle cells of transgenic mice through a smooth muscle alpha-actin-IGFBP-4 fusion gene induces smooth muscle hypoplasia.

Insulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a -724 bp fragment of the mouse smooth muscle alpha-actin 5'-flanking region (SMP2-BP-4) or -1074 bp, 63 bp of 5'-untranslated region, and 2.5 kb of intron 1 of smooth muscle alpha-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were characterized. Mating of hemizygous SMP8-BP-4 mice with controls produced about 50% transgenic offspring, with equal sex distribution. Expression of IGFBP-4 mRNA in nontransgenic littermates was maximal in liver and kidney. By contrast, transgenic IGFBP-4 mRNA expression, distinguished because of a smaller transcript size, was confined to SMC-containing tissues, with the following hierarchy: bladder > aorta > stomach = uterus. There was no transgene expression in skeletal muscle, brain, or cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand blotting or by immunoblotting, was 8- to 10-fold higher in aorta and bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with no change in plasma IGFBP-4 levels. Transgenic mice exhibited a significant reduction in wet weight of SMC-rich tissues, including bladder, intestine, aorta, uterus, and stomach, with no change in total body or carcass weight. In situ hybridization showed that transgene expression was targeted exclusively to the muscular layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a reciprocal phenotype to that of transgenic mice overexpressing IGF-I under control of the same promoter (SMP8-IGF-I). Double transgenic mice derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest decrease in wet weight at selected SMC tissues. Although we cannot exclude that the effects of IGFBP-4 may be IGF independent, these data suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC in vivo.

Actins↗
PubMed1999

Reduced blood pressure and increased sensitivity of the vasculature to parathyroid hormone-related protein (PTHrP) in transgenic mice overexpressing the PTH/PTHrP receptor in vascular smooth muscle.

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is postulated to exert vasorelaxant properties by activation of the PTH/PTHrP type 1 receptor. As a model for studying the actions of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress the PTH/PTHrP receptor (PTHrP-R) in smooth muscle. Oocyte injection with a SMP8-PTHrP-R fusion construct yielded six founder mice. F1 offspring were viable and demonstrated selective overexpression of the SMP8-PTHP-R messenger RNA in smooth muscle-rich tissues. Baseline blood pressure measured in conscious mice by tail sphygmomanometry was significantly lower in the receptor-overexpressing mice than that in controls (117 +/- 4 vs. 133 +/- 3 mm Hg; P < 0.05). In anesthetized animals, iv infusion of PTHrP-(1-34)NH2 caused a significantly greater reduction in blood pressure and total peripheral resistance in transgenic mice than in control animals. Vascular contractility was studied in paired, isometrically mounted aortas from 9-week-old transgenic and wild-type mice. The force of contraction in response to phenlyephrine was not significantly different between transgenic and wild-type mice. However, PTHrP-(1-34) NH2 relaxed aortic vessel preparations from transgenic mice to a greater extent than in controls (77.1 +/- 3% vs. 38.4 +/- 4%; P < 0.001). To determine the impact of overexpression of PTH/PTHrP type 1 receptor and its ligand on the development of the cardiovascular system, double transgenic mice were created by crossing SMP8-PTHrP-R transgenic mice with mice overexpressing PTHrP (SMP8-PTHrP). Double transgenic mice died around day E9 with abnormalities in the developing heart. In conclusion, overexpression of PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure, probably through sustained activation of the receptor by endogenous ligand. The cardiovascular defects observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP may play a role in the normal development of the cardiovascular system.

Actins↗
PubMed1999

The human growth hormone-releasing hormone transgenic mouse as a model of modest obesity: differential changes in leptin receptor (OBR) gene expression in the anterior pituitary and hypothalamus after fasting and OBR localization in somatotrophs.

We reported previously an increase in leptin receptor (OBR) gene expression in the anterior pituitary of human GH-releasing hormone (hGHRH) transgenic mice. The primary goal of this study was to investigate the possible mechanisms regulating OBR expression in these mice. Compared with normal sibling controls, hGHRH transgenic mice had significantly greater amounts of abdominal fat, higher levels of leptin messenger RNA (mRNA), and a 2-fold increase in plasma leptin concentrations. Despite normal plasma glucose levels, hGHRH transgenic mice had 4.5-fold elevated levels of plasma insulin. Using a ribonuclease protection assay, we measured the mRNA levels of the OBR long form (OBR(L)) in the anterior pituitary and hypothalamus after 48 h of fasting. In the anterior pituitary, food deprivation induced dramatic increases in OBR(L) mRNA levels in both normal and transgenic mice. In contrast, in the hypothalamus, fasting resulted in a significant decrease in OBR(L) gene expression in normal mice, and no changes were detected in hGHRH transgenic mice. Using dual in situ hybridization, OBR(L) mRNA was detected in somatotrophs. Moreover, the number of OBR(L)-positive pituitary cells as well as the percentage of OBR(L)-positive cells that express GH mRNA were increased in transgenic mice. In conclusion, 1) the modest obesity in hGHRH transgenic mice is associated with increases in leptin synthesis and secretion as well as insulin secretion; 2) GH and/or GHRH as well as leptin and insulin may differentially contribute to the changes in OBR(L) gene expression in the anterior pituitary and the hypothalamus; 3) the response of OBR(L) gene expression in the hypothalamus to fasting is absent in the modestly obese hGHRH transgenic mice; and 4) somatotrophs are target cells for leptin, and the increase in OBR(L) gene expression in the pituitary of hGHRH transgenic mice is due at least in part to the increase in the number of cells expressing OBR(L).

Adipose Tissue↗
PubMed2001

Enhanced spontaneous locomotor activity in bovine GH transgenic mice involves peripheral mechanisms.

Clinical and experimental studies indicate a role for GH in mechanisms related to anhedonia/hedonia, psychic energy, and reward. Recently we showed that transgenic mice with general overexpression of bovine GH display increased spontaneous locomotor activity. In the present study, we investigated whether this behavioral change is owing to a direct action of GH in the central nervous system or to peripheral GH actions. A transgenic construct, containing the glial fibrillary acidic protein promoter directing specific expression of bovine GH to the central nervous system, was designed. The central nervous system-specific expression of bovine GH in the glial fibrillary acidic protein-bovine GH transgenic mice was confirmed, but no effect on spontaneous locomotor activity was observed. Serum bovine GH levels were increased in glial fibrillary acidic protein-bovine GH transgenic mice but clearly lower than in transgenic mice with general overexpression of bovine GH. In contrast to the transgenic mice with general overexpression of bovine GH, glial fibrillary acidic protein-bovine GH mice did not display any difference in serum IGF-I levels. The levels of free T(3) and the conversion of the free T(4) to free T(3) were only increased in transgenic mice with general overexpression of bovine GH, but serum corticosterone levels were similarly increased in both transgenic models. These results suggest that free T(3) and/or IGF-I, affecting dopamine and serotonin systems in the central nervous system, may mediate the enhanced locomotor activity observed in transgenic mice with general overexpression of bovine GH.

Animals↗
PubMed1990

Position-specific activity of the Hox1.1 promoter in transgenic mice.

During development, positional values have to be assigned to groups of cells. The murine Hox genes are a class of genes that are predicted to be involved at some stage in this process. During embryogenesis they are expressed in distinct overlapping region- and stage-specific patterns and therefore must be regulated in response to positional information. In this study, we have analysed the activity of Hox1.1 promoter sequences in transgenic mice. The use of lacZ as a marker allows a detailed analysis of expression at the single cell level during early embryonic development. We show that 3.6 kbp of promoter and 1.7 kbp of 3' sequences provide sufficient regulatory information to express a transgene in a spatial and temporal manner indistinguishable from the endogenous Hox1.1 gene during the period of development when Hox1.1 expression is established. The activation occurs in a strict order in specific ectodermal and mesodermal domains. Within each of these domains the transgene is activated over a period of four hours apparently randomly in single cells. In a following second period, Hox1.1 and transgene expression patterns diverge. In this period, transgene expression persists in many mesodermally derived cells that do not express Hox1.1 indicating the absence of a negative regulatory element in the transgene. The anterior boundary of transgene expression is identical to that of Hox1.1. However, no posterior boundary of transgene expression is set, suggesting that a separate element absent from the transgene specifies this boundary.

Animals↗