Hypoxia stimulates human preproendothelin-1 promoter activity in transgenic mice

1997 ◽  
Vol 273 (4) ◽  
pp. L848-L855 ◽  
Author(s):  
Catherine R. Aversa ◽  
Suzanne Oparil ◽  
Jaime Caro ◽  
Huaibin Li ◽  
Shuang-Dan Sun ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgene Expression ◽  
Regulatory Mechanisms ◽  
Pulmonary Vasculature ◽  
Pulmonary Tissue ◽  
Hypoxic Induction ◽  
Flanking Sequences ◽  
Bronchiolar Epithelium

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (≈2.5 kb, 138 bp, or none of the 5′-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2tension.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Interactions of insulin-like growth factor (IGF)-II and growth hormone in vivo: circulating levels of IGF-I and IGF-binding proteins in transgenic mice

1997 ◽  
pp. 701-708 ◽  
Author(s):  
A Blackburn ◽  
RA Dressendorfer ◽  
WF Blum ◽  
M Erhard ◽  
G Brem ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgene Expression ◽  
High Protein Diet ◽  
High Protein ◽  
Protein Diet ◽  
Standard Diet ◽  
Igf I ◽  
Igf Binding ◽  
B Mice

To study interactions between insulin-like growth factor-II (IGF-II) and growth hormone (GH) in vivo, we crossed hemizygous transgenic mice carrying phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II fusion genes with hemizygous PEPCK-bovine GH (bGH) transgenic mice. Offspring harbouring both transgenes (IB), the IGF-II transgene (I) or the bGH transgene (B), and non-transgenic littermates (C) were obtained. Blood samples were taken before (end of week 12) and after (end of week 14) the mice had received a diet high in protein and low in carbohydrates to stimulate PEPCK promoter-controlled transgene expression. Mean serum GH concentrations of both B and IB mice corresponded to 900 ng/ml and increased more than twofold (P < 0.001) after 1 week of the high-protein diet. GH concentrations in controls and I mice were less than 20 ng/ml. Serum IGF-II concentrations in I and IB mice were three-to fourfold higher than those in C and B mice. Whereas IGF-II concentrations were not changed by the high-protein diet in the last two groups, serum IGF-II increased significantly in I (P < 0.001) and IB mice (P < 0.05). This increase was significantly (P < 0.05) less pronounced in IB than in C and I mice. Circulating IGF-I concentrations were about twofold (P < 0.001) higher in B and IB than in C and I mice, and showed a tendency to be lower in I than in C and in IB than in B mice when animals were maintained on the standard diet. The high-protein diet did not change circulating IGF-I concentrations in controls and B mice, but resulted in a significant reduction of serum IGF-I concentrations in I (P < 0.05) and IB mice (P < 0.001). Consequently, after PEPCK-IGF-II transgene expression was stimulated, serum IGF-I concentrations were significantly (P < 0.05) lower in I than in C and in IB than in B mice. Serum IGF-binding protein (IGFBP)-2 concentrations were significantly (P < 0.05) higher in I mice than in all other groups when mice were maintained on the standard diet, with a tendency to reduced IGFBP-2 concentrations in B mice. After the high-protein diet, serum IGFBP-2 concentrations did not change in C and I mice, but increased by two- to threefold in B and IB mice (P < 0.001). Serum IGFBP-3 concentrations tended to be greater in B and IB than in C and I mice, but these differences were mostly not significant. IGFBP-4 concentrations were significantly (P < 0.001) increased by GH overproduction in B and IB mice. Our data suggest that the reduction in circulating IGF-I concentrations by increased IGF-II is most probably due to the limited serum IGF binding capacity and the short half-life of free IGFs, rather than to a reduction in GH-dependent IGF-I production. Effects of GH overproduction on serum IGFBP-2 concentrations depend on dietary factors and may be both inhibitory and stimulatory.

Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

scholarly journals Amphotericin-B promotes leukocyte aggregation of nylon-wool-fiber- treated polymorphonuclear leukocytes

Blood ◽  
1981 ◽  
Vol 58 (3) ◽  
pp. 518-523 ◽  
Author(s):  
LA Boxer ◽  
LM Ingraham ◽  
J Allen ◽  
RS Oseas ◽  
RL Baehner
Keyword(s):  
Amphotericin B ◽  
Polymorphonuclear Leukocytes ◽  
Pulmonary Hemorrhage ◽  
Lactic Dehydrogenase ◽  
Wool Fiber ◽  
Pulmonary Vasculature ◽  
Pulmonary Tissue ◽  
Wool Fibers ◽  
Nylon Wool

Abstract Severe pulmonary reactions have been reported in patients receiving leukocyte transfusion and amphotericin-B. To study the interaction of amphotericin-B with polymorphonuclear leukocytes (PMN), purified human PMN were incubated with 200 mg of nylon wool fiber for 60 min either in the absence or presence of 2 mM EDTA. PMN were recovered in acid citrate dextrose solution and were suspended in balanced salt solution for determination of their aggregation properties. The cells exposed to nylon wool fibers without EDTA aggregated in response to concentration as low as 1.25 micrograms/ml of amphotericin-B. Cells initially treated with EDTA, however, failed to aggregate. Serum from a patient treated with amphotericin-B aggregated PMN exposed to nylon wool fiber but not control cells, whereas serum taken before amphotericin was given without effect on the PMN treated with nylon wool fiber. Amphotericin-B at 5 micrograms/ml failed to potentiate the release of beta- glucocuronidase or lactic dehydrogenase by PMN treated by nylon wool beyond that seen with exposure to the fibers alone. Rabbit peripheral blood was similarly incubated with nylon wool fibers and the recovered PMN were infused into recipient rabbits that had received 1 mg/kg of amphotericin-B intravenously 1 hr prior to the infusion of the leukocytes. Rabbits were sacrificed 30 min after transfusion of PMN, and their lungs were excised for histologic sectioning. Those rabbits receiving a combination of amphotericin-B and 4 x 10(7) nylon-wool- fiber-treated PMN had evidence of pulmonary hemorrhage and accumulation of leukocytes in the pulmonary vasculature whereas those animals who received such cells alone had normal appearing lung tissue. In summary, amphotericin-B at concentrations achievable in vivo enhanced the aggregation of PMN damaged by incubation with nylon fiber with subsequent accumulation of the phagocytes in pulmonary tissue.

scholarly journals Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).

1993 ◽  
Vol 120 (2) ◽  
pp. 493-502 ◽  
Author(s):  
N A Wall ◽  
M Blessing ◽  
C V Wright ◽  
B L Hogan
Keyword(s):  
Translational Regulation ◽  
Adult Mouse ◽  
Protein Localization ◽  
Cell Types ◽  
Morphogenetic Protein ◽  
Mouse Tissues ◽  
Bronchiolar Epithelium ◽  
Bone Morphogenetic Protein 6

DVR-6 (BMP-6 or Vgr-1) is a member of the TGF-beta superfamily of polypeptide signaling molecules. In situ hybridization studies have previously shown that DVR-6 RNA is expressed in a variety of cell types in the mouse embryo, but no information has been available on protein localization and biosynthesis. We have produced a polyclonal antibody to the proregion of DVR-6 and used it to localize the protein in whole mount and sectioned embryonic, newborn, and adult mouse tissues. DVR-6 protein is expressed in the mouse nervous system beginning at 9.5 days postcoitum (d.p.c.) and continues through adulthood. A variety of epithelial tissues also produce DVR-6 protein, including the suprabasal layer of the skin, bronchiolar epithelium, and the cornea. Additionally, a stably transfected cell line, BMGE+H/D6c4, is used to study the biosynthesis of DVR-6 protein and evidence is presented for translational regulation of DVR-6 expression.

Impaired Lentiviral Transgene Expression In Vivo Caused by Massive Methylation of SFFV Promoter Sequences.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3760-3760 ◽  
Author(s):  
Friederike Herbst ◽  
Claudia R Ball ◽  
Francesca Tuorto ◽  
Wei Wang ◽  
Ulrich Kloz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Es Cells ◽  
Methylation Status ◽  
Lentiviral Vectors ◽  
Control Vector ◽  
Lentiviral Integration

Abstract Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Real-time measurement of non-lethal platelet thromboembolic responses in the anaesthetized mouse

2008 ◽  
Vol 99 (02) ◽  
pp. 435-440 ◽  
Author(s):  
Charalambos Tymvios ◽  
Sarah Jones ◽  
Christopher Moore ◽  
Simon C. Pitchford ◽  
Clive P. Page ◽  
...  
Keyword(s):  
Real Time ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Inhibitory Effect ◽  
Pulmonary Vasculature ◽  
Real Time Measurement ◽  
Platelet Aggregates ◽  
Dose Responses

SummaryIdentifying and evaluating new therapeutic targets in platelets requires advanced animal models in which platelet responses can be measured directly and in situ.This is important because platelet function is strongly influenced by external factors such as those originating from the vascular endothelium. Our objectives were to record graded, non-lethal thromboembolic platelet responses to platelet agonists in situ in the mouse and to demonstrate an inhibitory effect of aspirin in our model. Radiolabelled platelets were infused into anaesthetized mice and responses to ADP, collagen and thrombin measured as changes in platelet associated counts in miniaturized detection probes placed over the thoracic region. All agonists induced dose-dependent changes in platelet counts due to accumulation of thrombi in the pulmonary vasculature. We confirmed a specific platelet effect by comparing platelet and erythrocyte responses and showing platelet aggregates in the lung vasculature histologically. Simultaneous injection of collagen and adrenaline induced increased and protracted synergistic platelet responses compared with individual injection of these agents and aspirin inhibited collagen-induced responses. We confirmed the clinical relevance of our model by showing that platelet thromboembolism in the mouse, like pulmonary embolism in humans, impaired cardiovascular performance. We present a refined method for measuring platelet agonist dose-responses and thromboembolism in real-time without inducing mortality in the mouse. Our technique will be useful in investigating the molecular determinants of physiological and pathophysiological platelet function in an in-vivo context and will enable investigations of both platelet and non-platelets mediators of thrombus formation.

scholarly journals Leukocyte integrin CD11b promoter directs expression in lymphocytes and granulocytes in transgenic mice

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 1017-1024 ◽  
Author(s):  
A Back ◽  
K East ◽  
D Hickstein
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Human Leukocyte ◽  
Surface Expression ◽  
Regulatory Elements ◽  
Specific Pattern ◽  
Integrin Subunit ◽  
Cd11b Expression

The human leukocyte integrin subunit CD11b is expressed predominantly on myelomonocytic cells. To identify CD11b promoter sequences important for myelomonocytic gene expression and to assess the utility of the CD11b promoter for expressing heterologous genes in vivo, we generated transgenic mice with a human CD4 reporter gene driven by CD11b promoter constructs composed of 1.5, 0.3, or 0.1 kb of DNA sequence 5′ to the transcription start site. Using flow cytometry to detect the human CD4 reporter on murine leukocytes, two of three 1.5-kb CD11b promoter founder lines showed surface expression of the human CD4 transgene in granulocytes and lymphocytes. The transgene expression observed in lymphocytes was inappropriate relative to the normal pattern of CD11b expression. Of the eight 0.3-kb or 0.1-kb founder lines, only one 0.1- kb founder line showed transgene expression. The overall pattern of transgene expression among the 11 founder lines does not parallel expression of the endogenous CD11b gene. These studies indicate that additional CD11b regulatory elements will be required to express a reporter gene in vivo in a lineage-specific pattern that mimics the endogenous CD11b gene.

scholarly journals Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo

2009 ◽  
Vol 181 (2) ◽  
pp. 212-226 ◽  
Author(s):  
Stan D. Atkin ◽  
Sundip Patel ◽  
Ara Kocharyan ◽  
Lynne A. Holtzclaw ◽  
Susanna H. Weerth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Glial Cell ◽  
Schwann Cells

scholarly journals Identification of DNA elements cooperatively activating proopiomelanocortin gene expression in the pituitary glands of transgenic mice.

1992 ◽  
Vol 12 (9) ◽  
pp. 3978-3990 ◽  
Author(s):  
B Liu ◽  
G D Hammer ◽  
M Rubinstein ◽  
M Mortrud ◽  
M J Low
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Intermediate Lobe ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dna Elements ◽  
Pomc Gene

The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.

Sign in / Sign up

Export Citation Format

Share Document