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Sample records for netrin-1 transgene overexpression

  1. Down syndrome cell adhesion molecule (DSCAM) associates with uncoordinated-5C (UNC5C) in netrin-1-mediated growth cone collapse.

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    Purohit, Anish A; Li, Weiquan; Qu, Chao; Dwyer, Trisha; Shao, Qiangqiang; Guan, Kun-Liang; Liu, Guofa

    2012-08-03

    In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.

  2. Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein ResponseSummary

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    Thomas Lahlali

    2016-05-01

    Full Text Available Background & Aims: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. Methods: Isolation of translational complexes, determination of RNA secondary structures by selective 2’-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro–grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. Results: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA–like endoplasmic reticulum kinase (PERK, inositol requiring enzyme 1α (IRE1α, and activated transcription factor 6 (ATF6. The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region–driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb, netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C–mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin

  3. Netrin-1 Peptide Is a Chemorepellent in Tetrahymena thermophila

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    Heather Kuruvilla

    2016-01-01

    Full Text Available Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation is not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism.

  4. Mutations in the netrin-1 gene cause congenital mirror movements.

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    Méneret, Aurélie; Franz, Elizabeth A; Trouillard, Oriane; Oliver, Thomas C; Zagar, Yvrick; Robertson, Stephen P; Welniarz, Quentin; Gardner, R J MacKinlay; Gallea, Cécile; Srour, Myriam; Depienne, Christel; Jasoni, Christine L; Dubacq, Caroline; Riant, Florence; Lamy, Jean-Charles; Morel, Marie-Pierre; Guérois, Raphael; Andreani, Jessica; Fouquet, Coralie; Doulazmi, Mohamed; Vidailhet, Marie; Rouleau, Guy A; Brice, Alexis; Chédotal, Alain; Dusart, Isabelle; Roze, Emmanuel; Markie, David

    2017-09-25

    Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.

  5. Netrin-1 expression is an independent prognostic factor for poor patient survival in brain metastases.

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    Patrick N Harter

    Full Text Available The multifunctional molecule netrin-1 is upregulated in various malignancies and has recently been presented as a major general player in tumorigenesis leading to tumor progression and maintenance in various animal models. However, there is still a lack of clinico-epidemiological data related to netrin-1 expression. Therefore, the aim of our study was to elucidate the association of netrin-1 expression and patient survival in brain metastases since those constitute one of the most limiting factors for patient prognosis. We investigated 104 brain metastases cases for netrin-1 expression using in-situ hybridization and immunohistochemistry with regard to clinical parameters such as patient survival and MRI data. Our data show that netrin-1 is strongly upregulated in most cancer subtypes. Univariate analyses revealed netrin-1 expression as a significant factor associated with poor patient survival in the total cohort of brain metastasis patients and in sub-entities such as non-small cell lung carcinomas. Interestingly, many cancer samples showed a strong nuclear netrin-1 signal which was recently linked to a truncated netrin-1 variant that enhances tumor growth. Nuclear netrin-1 expression was associated with poor patient survival in univariate as well as in multivariate analyses. Our data indicate both total and nuclear netrin-1 expression as prognostic factors in brain metastases patients in contrast to other prognostic markers in oncology such as patient age, number of brain metastases or Ki67 proliferation index. Therefore, nuclear netrin-1 expression constitutes one of the first reported molecular biomarkers for patient survival in brain metastases. Furthermore, netrin-1 may constitute a promising target for future anti-cancer treatment approaches in brain metastases.

  6. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

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    Vinciane Régnier

    Full Text Available The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  7. Transgenic overexpression of a stable Plasminogen Activator Inhibitor-1 variant.

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    Fahim, Abigail T; Wang, He; Feng, Jining; Ginsburg, David

    2009-03-01

    Plasminogen Activator Inhibitor-1 (PAI-1) is a member of the Serine Protease Inhibitor (SERPIN) gene family and a key regulator of fibrinolysis. PAI-1 is unique among SERPINs in its spontaneous transition to a latent, inactive state, with a half-life of approximately 2 hours under physiologic conditions. The biologic importance of the PAI-1 transition to latency is unknown. This study aimed to engineer transgenic overexpression of a stable murine PAI-1 variant to examine the physiologic effects in vivo from delayed transition of PAI-1 to latency. Ten independent transgenic lines were generated with expression of a stable PAI-1 variant driven by the hybrid CMV/chicken beta-actin promoter. Plasma PAI-1 levels in the transgenic founders ranged from 3.1+/-0.1 ng/mL to 1268.8+/-717.0 ng/mL. Quantitative PCR analysis in 3 transgenic lines demonstrated elevated PAI-1 mRNA in multiple tissues, with the highest increases observed in liver, brain, heart, and kidney. The fold-increase in PAI-1 mRNA over wild-type ranged from 2-fold to >2000-fold. Immunohistochemistry showed increased PAI-1 in liver, kidney, heart, spleen, and lung. Histologic examination of transgenic mice showed no evidence of thrombosis. The two founders with the highest plasma PAI-1 levels failed to produce any transgenic offspring that survived to weaning, although genotyping of expired pups revealed successful transmission of the transgene. These results suggest that high expression of a stable variant of PAI-1 may be lethal in mice, while more moderate expression is generally well tolerated and produces no apparent thrombosis.

  8. Interfering with multimerization of netrin-1 receptors triggers tumor cell death

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    Mille, Frédéric; Llambi, Fabien; Guix, Catherine; Delloye-Bourgeois, Céline; Guenebeaud, Céline; Castro-Obregon, Susana; Bredesen, Dale E.; Thibert, Chantal; Mehlen, Patrick

    2009-01-01

    Netrin-1 was recently proposed to control tumorigenesis by inhibiting apoptosis induced by the dependence receptors DCC (Deleted in colorectal cancer) and UNC5H. While a loss of these dependence receptors’ expression has been described as a selective advantage for tumor growth and progression in numerous cancers, recent observations have shown that some tumors may utilize an alternative strategy to block dependence receptor-induced programmed cell death: the autocrine expression of netrin-1. This alternative strategy has been observed in a large fraction of aggressive breast cancers, neuroblastoma, pancreatic adenocarcinoma, and lung cancer. This observation is of potential interest regarding future targeted therapy, since in such cases interfering with the ability of netrin-1 to inhibit DCC or UNC5H-induced cell death is associated with apoptosis of netrin-1-expressing tumor cells in vitro, and with inhibition of tumor growth or metastasis in different mouse tumor models. The understanding of the mechanism by which netrin-1 inhibits cell death is therefore of interest. Here, we show that netrin-1 triggers the multimerization of both DCC and UNC5H2 receptors, and that multimerization of the intracellular domain of DCC and UNC5H2 is the critical step to inhibit the pro-apoptotic effects of both of these receptors. Taking advantage of this property, we utilized a recombinant specific domain of DCC that (i) interacts with netrin-1 and (ii) inhibits netrin-1- induced multimerization, to trigger apoptosis in netrin-dependent tumor cells. PMID:19543238

  9. Suppression of Retinal Neovascularization by Lentivirus-Mediated Netrin-1 Small Hairpin RNA

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    Xu, Huizhuo; Liu, Jiaolian; Xiong, Siqi; Le, Yun-zheng; Xia, Xiaobo

    2012-01-01

    Objectives The function of netrin-1 in pathological angiogenesis and its role in retinal neovascularization were investigated in the retinas of oxygen-induced retinopathy (OIR) mice by inhibition of netrin-1. Methods Expression of netrin-1 mRNA and protein in the retinas of OIR mice was analyzed by quantitative RT-PCR and immunoblotting. Inhibition of retinal neovascularization was achieved by lentivirus-mediated netrin-1 small hairpin RNA (shRNA) infection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections. Results Both mRNA and protein expression of netrin-1 were significantly upregulated in postnatal day 17 OIR mouse retinas. Treatment of OIR mice with specific lentivirus-mediated netrin-1 shRNA dramatically reduced neovascular outgrowth into the inner limiting membrane. Neovascular tufts and nonperfused areas were also reduced. Conclusions High expression of netrin-1 was detected in the retina under ischemic conditions and played a significant role in pathological retinal angiogenesis. Therefore, netrin-1 represents a potential therapeutic target for diabetic retinopathy, retinopathy of prematurity and other ocular neovascular diseases. PMID:22122983

  10. Serum netrin-1 in relation to gadolinium-enhanced magnetic resonance imaging in early multiple sclerosis.

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    Voortman, M M; Pekar, T; Bachmayer, D; Archelos, J-J; Stojakovic, T; Scharnagl, H; Ropele, S; Pichler, A; Enzinger, C; Fuchs, S; Fazekas, F; Seifert-Held, T; Khalil, M

    2017-01-01

    Netrin-1, a secreted laminin-related protein, is known to regulate not only axonal guidance and neuronal cell migration, but also blood-brain barrier integrity and inflammation. Two preliminary studies reported altered serum netrin-1 levels in multiple sclerosis; however, associations with longitudinal clinical and magnetic resonance imaging activity have not been investigated. We aimed to assess serum netrin-1 in multiple sclerosis and controls with respect to disease activity and its temporal dynamics. Serum netrin-1 was assessed by enzyme-linked immunosorbent assay in 79 patients with clinically isolated syndrome or multiple sclerosis, and 30 non-inflammatory neurological disease controls. In patients, serum samples were collected immediately prior to gadolinium-enhanced 3 T magnetic resonance imaging at two time points (initial contrast-enhancing gadolinium+ n = 47, non-enhancing gadolinium- n = 32; reference gadolinium- n = 70; median time-lag 1.4, interquartile range 1.0-2.3 years). Serum netrin-1 levels were similar in clinically isolated syndrome, multiple sclerosis and controls, and gadolinium+ and gadolinium- patients. Among gadolinium+ patients, serum netrin-1 was decreased in clinically active (n = 8) vs non-active patients (n = 39; p = 0.041). Serum netrin-1 showed no temporal dynamics in multiple sclerosis and was unrelated to clinical data. Serum netrin-1 levels show no multiple sclerosis specific changes and are not sensitive for detection of subclinical disease activity. Netrin-1 changes during relapses may deserve further examination.

  11. Netrin-1 protects against L-Arginine-induced acute pancreatitis in mice.

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    Ji Chen

    Full Text Available Acute pancreatitis (AP is a common inflammatory disease mediated by damage to acinar cells and subsequent pancreatic inflammation with infiltration of leukocytes. The neuronal guidance protein, netrin-1, has been shown to control leukocyte trafficking and modulate inflammatory responses in several inflammation-based diseases. The present study was aimed toward investigating the effects of netrin-1 in an in vivo model of AP in mice. AP was induced in C57BL/6 mice by administration of two intraperitoneal injections of L-Arginine (4 g/kg. Mice were treated with recombinant mouse netrin-1 at a dose of 1 µg/mouse or vehicle (0.1% BSA intravenously through the tail vein immediately after the second injection of L-Arginine, and every 24 h thereafter. Mice were sacrificed at several time intervals from 0 to 96 h after the induction of pancreatitis. Blood and tissue samples of pancreas and lung were collected and processed to determine the severity of pancreatitis biochemically and histologically. Immunohistochemical staining demonstrated that netrin-1 was mainly expressed in the islet cells of the normal pancreas and the AP model pancreas, and the pancreatic expression of netrin-1 was down-regulated at both the mRNA and protein levels during the course of AP. Exogenous netrin-1 administration significantly reduced plasma amylase levels, myeloperoxidase activity, pro-inflammatory cytokine production, and pancreas and lung tissue damages. Furthermore, netrin-1 administration did not cause significant inhibition of nuclear factor-kappa B activation in the pancreas of L-Arginine-induced AP. In conclusion, our novel data suggest that netrin-1 is capable of improving damage of pancreas and lung, and exerting anti-inflammatory effects in mice with severe acute pancreatitis. Thus, our results indicate that netrin-1 may constitute a novel target in the management of AP.

  12. Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1

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    Sympson, Carolyn J; Bissell, Mina J; Werb, Zena

    1995-06-01

    An intact basement membrane (BM) is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or loss of this BM occurs during normal development as well as in the disease state. To examine the importance of BM during mammary gland development in vivo, we generated transgenic mice that inappropriately express autoactivating isoforms of the matrix metalloproteinase stromelysin-1. The mammary glands from these mice are both functionally and morphologically altered throughout development. We have now documented a dramatic incidence of breast tumors in several independent lines of these mice. These data suggest that overexpression of stromelysin-1 and disruption of the BM may be a key step in the multi-step process of breast cancer.

  13. Complete Loss of Netrin-1 Results in Embryonic Lethality and Severe Axon Guidance Defects without Increased Neural Cell Death

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    Jenea M. Bin

    2015-08-01

    Full Text Available Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-β-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1−/− mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1−/− embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

  14. Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

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    Nishitani, Allison M; Ohta, Sho; Yung, Andrea R; Del Rio, Tony; Gordon, Michael I; Abraira, Victoria E; Avilés, Evelyn C; Schoenwolf, Gary C; Fekete, Donna M; Goodrich, Lisa V

    2017-09-15

    The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse. © 2017. Published by The Company of Biologists Ltd.

  15. A new transgenic mouse model for conditional overexpression of the Polycomb Group protein EZH2.

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    Koppens, Martijn A J; Tanger, Ellen; Nacerddine, Karim; Westerman, Bart; Song, Ji-Ying; van Lohuizen, Maarten

    2017-04-01

    The Polycomb Group protein EZH2 is upregulated in most prostate cancers, and its overexpression is associated with poor prognosis. Most insights into the functional role of EZH2 in prostate cancer have been gained using cell lines and EZH2 inactivation studies. However, the question remains whether overexpression of EZH2 can initiate prostate tumourigenesis or drive tumour progression. Appropriate transgenic mouse models that are required to answer such questions are lacking. We developed one such transgenic mouse model for conditional overexpression of Ezh2. In this transgene, Ezh2 and Luciferase are transcribed from a single open reading frame. The latter gene enables intravital bioluminescent imaging of tissues expressing this transgene, allowing the detection of tumour outgrowth and potential metastatic progression over time. Prostate-specific Ezh2 overexpression by crossbreeding with Probasin-Cre mice led to neoplastic prostate lesions at low incidence and with a long latency. Compounding a previously described Bmi1-transgene and Pten-deficiency prostate cancer mouse model with the Ezh2 transgene did not enhance tumour progression or drive metastasis formation. In conclusion, we here report the generation of a wildtype Ezh2 overexpression mouse model that allows for intravital surveillance of tissues with activated transgene. This model will be an invaluable tool for further unravelling the role of EZH2 in cancer.

  16. Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

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    Torres-Vega, Estefanía; Mancheño, Nuria; Cebrián-Silla, Arantxa; Herranz-Pérez, Vicente; Chumillas, María J; Moris, Germán; Joubert, Bastien; Honnorat, Jérôme; Sevilla, Teresa; Vílchez, Juan J; Dalmau, Josep; Graus, Francesc; García-Verdugo, José Manuel; Bataller, Luis

    2017-03-28

    To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma (p < 0.05). Coexisting Caspr2 and Netrin-1 receptor antibodies were associated with concurrent thymoma, myasthenia gravis, and neuromyotonia, often with Morvan syndrome (p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%). © 2017 American Academy of Neurology.

  17. Production of transgenic pigs over-expressing the antiviral gene Mx1

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    Quanmei Yan

    2014-01-01

    Full Text Available The myxovirus resistance gene (Mx1 has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15–25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV. Indirect immunofluorescence assay (IFA revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

  18. Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression.

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    Paradisi, Andrea; Maisse, Carine; Coissieux, Marie-May; Gadot, Nicolas; Lépinasse, Florian; Delloye-Bourgeois, Céline; Delcros, Jean-Guy; Svrcek, Magali; Neufert, Clemens; Fléjou, Jean-François; Scoazec, Jean-Yves; Mehlen, Patrick

    2009-10-06

    Chronic inflammation and cancer are intimately associated. This is particularly true for inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, which show a major increased risk for colorectal cancer. While the understanding of the molecular pathogenesis of IBD has recently improved, the mechanisms that link these chronic inflammatory states to colorectal cancer development are in large part unknown. One of these mechanisms is NF-kappaB pathway activation which in turn may contribute to tumor formation by providing anti-apoptotic survival signals to the epithelial cells. Based on the observation that netrin-1, the anti-apoptotic ligand for the dependence receptors DCC and UNC5H is up-regulated in colonic crypts in response to NF-kappaB, we show here that colorectal cancers from inflammatory bowel diseases patients have selected up-regulation of netrin-1. Moreover, we demonstrate that this inflammation-driven netrin-1 up-regulation is causal for colorectal cancer development as interference with netrin-1 autocrine loop in a mouse model for ulcerative colitis-associated colorectal cancer, while showing no effect on inflammation, inhibits colorectal cancer progression.

  19. Tau overexpression in transgenic mice induces glycogen synthase kinase 3beta and beta-catenin phosphorylation.

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    Shim, S B; Lim, H J; Chae, K R; Kim, C K; Hwang, D Y; Jee, S W; Lee, S H; Sin, J S; Leem, Y H; Lee, S H; Cho, J S; Lee, H H; Choi, S Y; Kim, Y K

    2007-05-11

    The abnormal phosphorylations of tau, GSK3beta, and beta-catenin have been shown to perform a crucial function in the neuropathology of Alzheimer's disease (AD). The primary objective of the current study was to determine the manner in which overexpressed htau23 interacts and regulates the behavior and phosphorylation characteristics of tau, GSK3beta, and beta-catenin. In order to accomplish this, transgenic mice expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE/htau23) were created. Transgenic mice evidenced the following: (i) tendency toward memory impairments at later stages, (ii) dramatic overexpression of the tau transgene, coupled with increased tau phosphorylation and paired helical filaments (PHFs), (iii) high levels of GSK3beta phosphorylation with advanced age, resulting in increases in the phosphorylations of tau and beta-catenin, (iv) an inhibitory effect of lithium on the phosphorylations of tau, GSK3beta, and beta-catenin, but not in the non-transgenic littermate group. Therefore, the overexpression of NSE/htau23 in the brains of transgenic mice induces abnormal phosphorylations of tau, GSK3beta, and beta-catenin, which are ultimately linked to neuronal degeneration in cases of AD. These transgenic mice are expected to prove useful for the development of new drugs for the treatment of AD.

  20. Hepatocellular alterations and dysregulation of oncogenic pathways in the liver of transgenic mice overexpressing growth hormone

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    Miquet, Johanna G.; Freund, Thomas; Martinez, Carolina S.; González, Lorena; Díaz, María E.; Micucci, Giannina P.; Zotta, Elsa; Boparai, Ravneet K.; Bartke, Andrzej; Turyn, Daniel; Sotelo, Ana I.

    2013-01-01

    Growth hormone (GH) overexpression throughout life in transgenic mice is associated with the development of liver tumors at old ages. The preneoplastic pathology observed in the liver of young adult GH-overexpressing mice is similar to that present in humans at high risk of hepatic cancer. To elucidate the molecular pathogenesis underlying the pro-oncogenic liver pathology induced by prolonged exposure to elevated GH levels, the activation and expression of several components of signal transduction pathways that have been implicated in hepatocellular carcinogenesis were evaluated in the liver of young adult GH-transgenic mice. In addition, males and females were analyzed in parallel in order to evaluate sexual dimorphism. Transgenic mice from both sexes exhibited hepatocyte hypertrophy with enlarged nuclear size and exacerbated hepatocellular proliferation, which were higher in males. Dysregulation of several oncogenic pathways was observed in the liver of GH-overexpressing transgenic mice. Many signaling mediators and effectors were upregulated in transgenic mice compared with normal controls, including Akt2, NFκB, GSK3β, β-catenin, cyclin D1, cyclin E, c-myc, c-jun and c-fos. The molecular alterations described did not exhibit sexual dimorphism in transgenic mice except for higher gene expression and nuclear localization of cyclin D1 in males. We conclude that prolonged exposure to GH induces in the liver alterations in signaling pathways involved in cell growth, proliferation and survival that resemble those found in many human tumors. PMID:23428905

  1. Hepatocellular alterations and dysregulation of oncogenic pathways in the liver of transgenic mice overexpressing growth hormone.

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    Miquet, Johanna G; Freund, Thomas; Martinez, Carolina S; González, Lorena; Díaz, María E; Micucci, Giannina P; Zotta, Elsa; Boparai, Ravneet K; Bartke, Andrzej; Turyn, Daniel; Sotelo, Ana I

    2013-04-01

    Growth hormone (GH) overexpression throughout life in transgenic mice is associated with the development of liver tumors at old ages. The preneoplastic pathology observed in the liver of young adult GH-overexpressing mice is similar to that present in humans at high risk of hepatic cancer. To elucidate the molecular pathogenesis underlying the pro-oncogenic liver pathology induced by prolonged exposure to elevated GH levels, the activation and expression of several components of signal transduction pathways that have been implicated in hepatocellular carcinogenesis were evaluated in the liver of young adult GH-transgenic mice. In addition, males and females were analyzed in parallel in order to evaluate sexual dimorphism. Transgenic mice from both sexes exhibited hepatocyte hypertrophy with enlarged nuclear size and exacerbated hepatocellular proliferation, which were higher in males. Dysregulation of several oncogenic pathways was observed in the liver of GH-overexpressing transgenic mice. Many signaling mediators and effectors were upregulated in transgenic mice compared with normal controls, including Akt2, NFκB, GSK3β, β-catenin, cyclin D1, cyclin E, c-myc, c-jun and c-fos. The molecular alterations described did not exhibit sexual dimorphism in transgenic mice except for higher gene expression and nuclear localization of cyclin D1 in males. We conclude that prolonged exposure to GH induces in the liver alterations in signaling pathways involved in cell growth, proliferation and survival that resemble those found in many human tumors.

  2. A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

    Science.gov (United States)

    Winkle, Cortney C.; McClain, Leslie M.; Valtschanoff, Juli G.; Park, Charles S.; Maglione, Christopher

    2014-01-01

    Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion. PMID:24778312

  3. Netrin-1 Mediates Neuronal Survival Through PIKE-L Interaction With the Dependence Receptor UNC5B

    Science.gov (United States)

    Tang, Xiaoling; Jang, Sung-Wuk; Okada, Masashi; Chan, Chi-Bun; Feng, Yue; Liu, Yu; Luo, Shi-Wen; Hong, Yan; Rama, Nicolas; Xiong, Wen-cheng; Mehlen, Patrick; Ye, Keqiang

    2010-01-01

    Netrins, a family of secreted molecules, play critical roles in axon guidance and cell migration during neuronal development 1,2. In addition to its role as a chemotropic molecule, netrin-1 also acts as a survival factor 3–7. Both UNC5 (i.e. UNC5A, B, C or D) and DCC are transmembrane receptors for netrin-18,9. In the absence of netrin-1, DCC and UNC5 act as dependence receptors and trigger apoptosis 3,6,10. However, how netrin-1 suppresses the apoptotic activity of the receptors remains elusive. Here, we show that netrin-1 induces interaction of UNC5B with the brain specific GTPase PIKE-L. This interaction triggers activation of PI 3-kinase signaling, prevents UNC5B’s pro-apoptotic activity and enhances neuronal survival. Moreover, this process tightly relies on Fyn as PIKE-L is tyrosine phosphorylated in response to netrin-1 and the netrin-1-mediated interaction of UNC5B with PIKE-L is inhibited in Fyn null mice. Thus, PIKE-L acts as a downstream survival effector for netrin-1 through UNC5B in the nervous system. PMID:18469807

  4. FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

    Directory of Open Access Journals (Sweden)

    Benjamin Rappaz

    Full Text Available Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

  5. FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones.

    Science.gov (United States)

    Rappaz, Benjamin; Lai Wing Sun, Karen; Correia, James P; Wiseman, Paul W; Kennedy, Timothy E

    2016-01-01

    Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

  6. Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas.

    Science.gov (United States)

    Iscache, Anne-Laure; Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Pedros, Christophe; Saoudi, Abdelhadi; Buelow, Roland; Anegon, Ignacio

    2011-10-01

    The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

  7. [Cotton laccase gene overexpression in transgenic Populus alba var. pyramidalis and its effects on the lignin biosynthesis in transgenic plants].

    Science.gov (United States)

    Wang, Ji; Zhu, Mu Lan; Wei, Zhi Ming

    2008-02-01

    Using petioles as explants, a cotton laccase cDNA (GaLA C1) was introduced into Populus alba var. pyramidalis by A. tumefaciens-mediated transformation. PCR and Southern blot analysis indicated that transgene was stably integrated into the genome of transformants. Enzyme assay showed that laccase activity was obviously increased in transformants. As compared with untransformed control, total lignin content in all tested transgenic lines was elevated in varying degrees (as highest as 21.5%). Histochemical staining of lignin further confirmed that overexpressing GaLA C1 could result in increased lignin content in transformants. Together, our data strongly suggested that GaLA C1 may participate in lignin synthesis and this is the first direct transgenic evidence for the involvement of plant laccases in lignification.

  8. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Directory of Open Access Journals (Sweden)

    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  9. Transgenic mice overexpressing renin exhibit glucose intolerance and diet-genotype interactions

    Directory of Open Access Journals (Sweden)

    Sarah J. Fletcher

    2013-01-01

    Full Text Available Numerous animal and clinical investigations have pointed to a potential role of the renin-angiotensin system (RAS in the development of insulin resistance and diabetes in conditions of expanded fat mass. However, the mechanisms underlying this association remain unclear. We used a transgenic mouse model overexpressing renin in the liver (RenTgMK to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity. Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II (4-6-fold increase vs. wild type. Surprisingly, RenTgMK mice developed glucose intolerance despite low levels of adiposity and insulinemia. The transgenics also had lower plasma triglyceride levels. Glucose intolerance in transgenic mice fed a low-fat diet was comparable to that observed in high fat-fed wild type mice. Glucose intolerance was exacerbated by high-fat feeding, only in female transgenic mice. These studies demonstrate that overexpression of renin and associated hyperangiotensinemia impair glucose tolerance in a diet-dependent manner and further support a consistent role of RAS in the pathogenesis of diabetes and insulin resistance, independent of changes in fat mass.

  10. Establishment of a transgenic mouse model of corneal dystrophy overexpressing human BIGH3.

    Science.gov (United States)

    Liao, Xin; Cui, Hongping; Wang, Fang

    2013-11-01

    This study aimed to establish a transgenic mouse model of corneal dystrophy (CD) overexpressing the human transforming growth factor, β-induced, 68 kDa (TGFBI, also known as BIGH3) gene. A purified and linearized recombinant plasmid carrying the expression cassette BIGH3‑IRES‑EGFP was microinjected into the pronuclei of C57BL/6J mouse fertilized eggs under the control of the phosphoglycerate kinase (PGK) promoter. The expression of human BIGH3 in the transgenic mice was confirmed by PCR using DNA extracted from tail tissue. Four founder transgenic mice were identified by PCR and the increased expression of BIGH3 was observed in the corneas of the transgenic mice by RT-PCR and western blot analysis. The abnormal corneas with central opacity were observed in the transgenic mice by corneal photography. We concluded that the exogenous gene, BIGH3, was integrated successfully into the mouse genome through microinjection. In addition, the phenotype observed in this BIGH3 transgenic mouse model was similar to CD. Therefore, this transgenic model may prove useful in the investigation of the pathogenesis of CD.

  11. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  12. Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase.

    Science.gov (United States)

    Zhang, Gui-Yun; Liu, Ru-Ru; Xu, Geng; Zhang, Peng; Li, Yin; Tang, Ke-Xuan; Liang, Guo-Hua; Liu, Qiao-Quan

    2013-02-01

    Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The α-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. γ-tocopherol methyltransferase (γ-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of γ- and δ-isomers to α- and β-isomers. In present study, the Arabidopsis γ-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the α-/γ-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the γ-isomers were converted to α-isomers, especially the γ- and δ-tocotrienol levels were dramatically decreased. As a result, the α-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the α-tocotrienol content. The results showed that the α-/γ-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on α-tocopherol level, which may reflect the fact that γ-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign γ-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world's most important food crops.

  13. Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

    Science.gov (United States)

    Kamkin, Andre; Kiseleva, Irina; Theres, Heinz; Eulert-Grehn, Jaime-Jürgen; Wagner, Kay-Dietrich; Scholz, Holger; Vetter, Roland

    2010-01-01

    BACKGROUND: Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca2+-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca2+ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart. OBJECTIVE: To explore whether enhanced Ca2+ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca2+ (ICa-L) currents. METHODS: The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals. RESULTS: The amplitudes of ICa-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of ICa-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced ICa-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca2+ with BAPTA. CONCLUSIONS: These findings indicate that enhanced Ca2+ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in ICa-L currents. Modified ICa-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats. PMID:21264068

  14. Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice

    OpenAIRE

    Imanishi, Yasuo; Hosokawa, Yoshitaka; Yoshimoto, Katsuhiko; Schipani, Ernestina; Mallya, Sanjay; Papanikolaou, Alexandros; Kifor, Olga; Tokura, Takehiko; Sablosky, Marilyn; Ledgard, Felicia; Gronowicz, Gloria; Wang, Timothy C.; Schmidt, Emmett V.; Hall, Charles; Brown, Edward M.

    2001-01-01

    The relationship between abnormal cell proliferation and aberrant control of hormonal secretion is a fundamental and poorly understood issue in endocrine cell neoplasia. Transgenic mice with parathyroid-targeted overexpression of the cyclin D1 oncogene, modeling a gene rearrangement found in human tumors, were created to determine whether a primary defect in this cell-cycle regulator can cause an abnormal relationship between serum calcium and parathyroid hormone response, as is typical of hu...

  15. Compensation of the AKT signaling by ERK signaling in transgenic mice hearts overexpressing TRIM72

    Energy Technology Data Exchange (ETDEWEB)

    Ham, Young-Mi, E-mail: youngmi_ham@hms.harvard.edu [College of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Department of Cell Biology, Harvard Medical School, Boston, MA 02115 (United States); Mahoney, Sarah Jane [Department of Cell Biology, Harvard Medical School, Boston, MA 02115 (United States)

    2013-06-10

    The AKT and ERK signaling pathways are known to be involved in cell hypertrophy, proliferation, survival and differentiation. Although there is evidence for crosstalk between these two signaling pathways in cellulo, there is less evidence for cross talk in vivo. Here, we show that crosstalk between AKT and ERK signaling in the hearts of TRIM72-overexpressing transgenic mice (TRIM72-Tg) with alpha-MHC promoter regulates and maintains their heart size. TRIM72, a heart- and skeletal muscle-specific protein, downregulates AKT-mTOR signaling via IRS-1 degradation and reduces the size of rat cardiomyocytes and the size of postnatal TRIM72-Tg hearts. TRIM72 expression was upregulated by hypertrophic inducers in cardiomyocytes, while IRS-1 was downregulated by IGF-1. TRIM72 specifically regulated IGF-1-dependent AKT-mTOR signaling, resulting in a reduction of the size of cardiomyocytes. Postnatal TRIM72-Tg hearts were smaller than control-treated hearts with inhibition of AKT-mTOR signaling. However, adult TRIM72-Tg hearts were larger than of control despite the suppression of AKT-mTOR signaling. Activation of ERK, PKC-α, and JNK were observed to be elevated in adult TRIM72-Tg, and these signals were mediated by ET-1 via the ET receptors A and B. Altogether, these results suggest that AKT signaling regulates cardiac hypertrophy in physiological conditions, and ERK signaling compensates for the absence of AKT signaling during TRIM72 overexpression, leading to pathological hypertrophy. -- Highlights: • TRIM72 inhibits AKT signaling through ubiquitination of IRS-1 in cardiac cells. • TRIM72 regulates the size of cardiac cells. • TRIM72 regulates size of postnatal TRIM72-overexpressing transgenic mice hearts. • Adult TRIM72-overexpressing transgenic mice hearts showed cardiac dysfunction. • Adult TRIM72 transgenic mice hearts showed higher expression of endothelin receptors.

  16. Overexpression AtNHX1 confers salt-tolerance of transgenic tall ...

    African Journals Online (AJOL)

    Saline soil is a serious problem worldwide, and it is necessary to improve the salt tolerance of plants so as to avoid the progressive deterioration of saline soil. Here we report that over-expression of AtNHX1 improves salt tolerance in transgenic tall fescue. The AtNHX1 gene driven with CaMV35S promoter was constructed ...

  17. Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1

    Science.gov (United States)

    Marsick, Bonnie M.; Flynn, Kevin C.; Santiago-Medina, Miguel; Bamburg, James R.; Letourneau, Paul C.

    2010-01-01

    Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity. PMID:20506164

  18. Netrin-1-dependent spinal interneuron subtypes are required for the formation of left-right alternating locomotor circuitry.

    Science.gov (United States)

    Rabe, Nadine; Gezelius, Henrik; Vallstedt, Anna; Memic, Fatima; Kullander, Klas

    2009-12-16

    Neuronal circuits in the spinal cord that produce the rhythmic and coordinated activities necessary for limb movements are referred to as locomotor central pattern generators (CPGs). The identities and preceding development of neurons essential for coordination between left and right limbs are not yet known. We show that the ventral floor plate chemoattractant Netrin-1 preferentially guides dorsally originating subtypes of commissural interneurons, the majority of which are inhibitory. In contrast, the excitatory and ventralmost V3 subtype of interneurons have a normal number of commissural fibers in Netrin-1 mutant mice, thus being entirely independent of Netrin-1-mediated attraction. This selective loss of commissural fibers in Netrin-1 mutant mice resulted in an abnormal circuitry manifested by a complete switch from alternating to synchronous fictive locomotor activity suggesting that the most ventral-originating excitatory commissural interneurons are an important component of a left-right synchrony circuit in the locomotor CPG. Thus, during development, Netrin-1 plays a critical role for the establishment of a functional balanced CPG.

  19. Over-expression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification.

    Science.gov (United States)

    Siritunga, Dimuth; Arias-Garzon, Diana; White, Wanda; Sayre, Richard T

    2004-01-01

    Cassava (Manihot esculenta, Crantz) roots are the primary source of calories for more than 500 million people, the majority of whom live in the developing countries of Africa. Cassava leaves and roots contain potentially toxic levels of cyanogenic glycosides. Consumption of residual cyanogens (linamarin or acetone cyanohydrin) in incompletely processed cassava roots can cause cyanide poisoning. Hydroxynitrile lyase (HNL), which catalyses the conversion of acetone cyanohydrin to cyanide, is expressed predominantly in the cell walls and laticifers of leaves. In contrast, roots have very low levels of HNL expression. We have over-expressed HNL in transgenic cassava plants under the control of a double 35S CaMV promoter. We show that HNL activity increased more than twofold in leaves and 13-fold in roots of transgenic plants relative to wild-type plants. Elevated HNL levels were correlated with substantially reduced acetone cyanohydrin levels and increased cyanide volatilization in processed or homogenized roots. Unlike acyanogenic cassava, transgenic plants over-expressing HNL in roots retain the herbivore deterrence of cyanogens while providing a safer food product.

  20. Digital gene expression analysis of mature seeds of transgenic maize overexpressing Aspergillus niger phyA2 and its non-transgenic counterpart.

    Science.gov (United States)

    Rao, Jun; Yang, Litao; Wang, Congmao; Zhang, Dabing; Shi, Jianxin

    2013-01-01

    The next generation sequencing technologies have been recently used for transcriptome analysis in many organisms because of the decreased sequencing cost and increased sequence output. In this study, we used digital gene expression (DGE) technique to compare the transcriptomic changes in mature seeds between transgenic maize overexpressing Aspergillus niger phyA2 and its non-transgenic counterpart. Deep sequencing of DGE libraries of the transgenic and its non-transgenic counterpart seeds generated 3,783,500 and 3,790,500 reads of 21-nucleotide, respectively, with frequencies spanning over four orders of magnitude. In transgenic maize, 53.97% of the unambiguous signature tags were mapped to the maize B73 reference genome, and 46.47% of genes were detected by at least two reads; in non-transgenic maize, the corresponding numbers were 51.38% and 47.39%. Compared with non-transgenic counterpart, about 12% of detected genes were differentially expressed in the transcriptome of transgenic maize seeds. Among these differentially expressed genes, there were 23 transcription factors in 14 families and no allergen genes. Pathway enrichment analysis revealed that 21 pathways were significantly affected by the transgenic event, in which the pathway involved in protein processing in endoplasmic reticulum was the most significantly affected. Results from this study indicated that both intended and unintended transcriptomic changes occurred in the transgenic maize, thus emphasizing the importance of transcriptome profiling in risk assessment of transgenic events.

  1. Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration.

    Science.gov (United States)

    Passacquale, Gabriella; Phinikaridou, Alkystis; Warboys, Christina; Cooper, Margaret; Lavin, Begona; Alfieri, Alessio; Andia, Marcelo E; Botnar, Rene M; Ferro, Albert

    2015-07-01

    There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression. Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE(-/-) mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel. TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE(-/-) mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE(-/-) mice. Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE(-/-) mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes. © 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  2. Overexpressing MhNPR1 in transgenic Fuji apples enhances resistance to apple powdery mildew.

    Science.gov (United States)

    Chen, Xiu-Kong; Zhang, Ji-Yu; Zhang, Zhen; Du, Xiao-Li; Du, Bei-Bei; Qu, Shen-Chun

    2012-08-01

    Fuji is susceptible to fungal diseases like apple powdery mildew. Non-expressor of pathogenesis-related gene 1 (NPR1) plays a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). Previous studies show that overexpressing the Malus hupehensis-derived NPR1 (MhNPR1) gene in tobacco induces the transcript expression of pathogenesis-related genes (PRs) and resistance to the fungus Botrytis cinerea. In this study we introduced the MhNPR1 gene into the 'Fuji' apple via Agrobacterium-mediated transformation. Four transgenic apple lines were verified by PCR and RT-PCR. The semi-quantitative RT-PCR results showed that transcript overexpression of the MhNPR1 gene induced the expression of MdPRs and MdMLO genes known to interact with powdery mildew. Furthermore, the transgenic apple plants resisted infection by apple powdery mildew better than the wild-type plants. As a result, transcript overexpression of the MhNPR1 gene induced SAR and enhanced the Fuji apple's resistance to fungal disease.

  3. Effects of treadmill exercise on the expression of netrin-1 and its receptors in rat brain after cerebral ischemia.

    Science.gov (United States)

    Liu, N; Huang, H; Lin, F; Chen, A; Zhang, Y; Chen, R; Du, H

    2011-10-27

    Recent evidence suggests that exercise improves functional outcome in animal models of cerebral ischemia. Since netrin-1 and its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5B (Unc5B), act as important regulators in neural and vascular activities, we sought to determine whether netrin-1 and DCC and Unc5B are involved in the neuroprotective effects of exercise on rats with induced cerebral ischemia. A total of 108 rats were randomly distributed into three groups: sham-operated group (n = 12), middle cerebral artery occlusion (MCAO) group (n = 48), MCAO+treadmill exercise group (n = 48). Behavioral testing indicated that treadmill exercise could significantly improve neurologic deficits of rats with cerebral ischemia at day 14 and 28 after MCAO (n = 12, Pexercise enhanced netrin-1 and DCC expression, while it suppressed Unc5B expression in rat peri-ischemic brain area, especially at day 14 and 28 after MCAO (n = 4, Pexercise-induced neural circuit remodeling in the peri-ischemic area, and exercise may promote survival of neurons in this area by regulating netrin-1-Unc5B signaling. Additionally, netrin-1 may also play a role in brain-blood barrier via DCC-immunoreactive peri-vascular astrocytes. In conclusion, we demonstrate that treadmill exercise has beneficial effects that may be attributed, at least in part, to the involvement of netrin-1 and its receptors DCC and Unc5B in the neuronal and vascular activities in brain-ischemic rats. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats

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    Liangjie Bai

    2017-11-01

    Full Text Available Our previous findings indicated that treatment with Netrin-1 could improve functional recovery through the stimulation of autophagy, by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR signaling pathway in rats following spinal cord injury (SCI. However, the underlying mechanisms were not elucidated. The purpose of this study was to investigate the underlying mechanisms by which Netrin-1 promotes autophagy and improves functional recovery after SCI. Following controlled SCI in Sprague-Dawley rats, we observed that the autophagic flux in neurons was impaired, as reflected by the accumulation of light chain 3-II (LC3-II-positive and LC3-positive autophagosomes (APs, accompanied by the accumulation of the autophagic substrate, Sequestosome 1 (SQSTM1; also known as p62. Our results showed that treatment with Netrin-1 increases the levels of the lysosomal protease cathepsin D (CTSD and lysosomal-associated membrane protein 1 (LAMP1, through the regulation of the nuclear localization of Transcription factor EB (TFEB via the AMPK/mTOR signaling pathway. In addition, this enhancement of lysosomal biogenesis correlated strongly with the restoration of autophagic flux, inhibition of neural apoptosis and improved functional recovery. Suppression of lysosomal biogenesis via the inhibition of the nuclear translocation of TFEB by Compound C abolished this restoration of autophagic flux and the functional recovery effects of Netrin-1 following SCI. Taken together, these results indicate that Netrin-1 enhances lysosomal biogenesis by regulating the nuclear translocation of TFEB via the AMPK/mTOR signaling pathway. Furthermore, the enhancement of lysosomal biogenesis by Netrin-1 following SCI promotes autophagic flux and improves functional recovery in rats. Thus, the regulation of lysosomal biogenesis by modulating the nuclear localization of TFEB might be a novel approach for the treatment of SCI.

  5. Phenotypic Characterization of Transgenic Miscanthus sinensis Plants Overexpressing Arabidopsis Phytochrome B

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    Ok-Jin Hwang

    2014-01-01

    Full Text Available Phytochromes are dimeric pigment proteins with reversible photochromism between red and far-red light-absorbing forms. They are photoreceptors that regulate various aspects of plant growth and development and have been used for biotechnological applications to improve agricultural performance of crops. Miscanthus species have been suggested as one of the most promising energy crops. In this paper, Arabidopsis phytochrome B (PHYB gene was introduced into Miscanthus sinensis using Agrobacterium-mediated transformation method that we developed recently, with the herbicide resistance gene (BAR as a selection marker. After putative transgenic plants were selected using the herbicide resistance assay, genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and transgene expression was validated by Northern blot analysis. Compared to nontransformed control plants, transgenic plants overexpressing PHYB showed phenotypes with increased phytochrome B function, which includes increased chlorophyll content, decreased plant height, and delayed flowering. Therefore, these results suggest that Arabidopsis phytochrome B is functional in M. sinensis and provide a method to develop Miscanthus varieties with enhanced agricultural performance using phytochromes.

  6. Netrin-1 prevents the development of cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Wang, Nan; Cao, Yunshan; Zhu, Yan

    2016-03-01

    The aim of the present study was to examine whether netrin-1 is involved in the development of cardiac hypertrophy, induced by pressure overload. For this investigation, thoracic transverse aortic constriction (TAC) was performed in mice. A total of 18 mice were divided into three groups (n=6 per group): Sham, TAC and TAC + recombinant netrin-1. Neonatal rat cardiomyocytes were stimulated with endothelin-1 (ET-1), and samples were collected to examine the expression levels of netrin‑1 by western blot analysis and the mRNA expression of A‑type natriuretic peptide by reverse transcription‑quantitative polymerase chain reaction. It was found that the expression of netrin‑1 was decreased in the TAC mice and in the neonatal rat cardiomyocytes in response to ET‑1 stimulation. Netrin‑1 eliminated ventricular remodeling, cardiac dysfunction and DNA damage during pressure overload. Furthermore, analysis of the signaling events indicated that netrin‑1‑mediated protection against cardiac hypertrophy was attributed to interruption of the activation of mitogen‑activated protein kinase kinase (MEK) kinase‑1 (K1)‑dependent MEK‑extracellular signal‑regulated protein kinase 1/2 (ERK1/2) and c‑Jun N‑terminal kinase 1/2 (JNK1/2). Therefore, netrin‑1 prevented cardiac hypertrophy and heart failure through the negative regulation of the MEKK1-dependent MEK‑ERK1/2 and JNK1/2 signaling pathways.

  7. Cerebral glucose utilization in transgenic mice overexpressing heat shock protein 70 is altered by dizocilpine.

    Science.gov (United States)

    Kelly, Stephen; Bieneman, Alison; Uney, James B; McCulloch, James

    2002-03-01

    Heat shock protein (HSP70), a member of the 70 kDa HSP superfamily, has been widely implicated in the cellular stress response to numerous insults. HSP70 may be a significant factor in cell survival following stresses such as cerebral ischaemia. The precise mechanisms by which HSP70 facilitates cell survival remain unclear. The aim of this study was to ascertain whether any differences in local cerebral glucose utilization (LCGU) existed between transgenic mice overexpressing HSP70 (HSP70 Tg) and wild- type littermate (WT) mice. LCGU was assessed using (14)C-2-deoxyglucose in HSP70 Tg and WT mice under basal conditions (intraperitoneal injection of saline) and during metabolic activation produced by NMDA receptor blockade (intraperitoneal injection of dizocilpine, 1 mg/kg). No significant alterations in LCGU were observed between saline injected HSP70 Tg and WT mice in any of the 35 brain regions analyzed. Dizocilpine injection produced significant heterogeneous alterations in LCGU in HSP70 Tg mice (24 of 35 brain regions) and in WT mice (22 of 35 brain regions) compared with saline injected mice. The distribution of altered LCGU produced by dizocilpine was similar in HSP70 Tg and WT mice. However in five brain regions, dizocilpine injected HSP70 Tg mice displayed significantly altered LCGU compared to dizocilpine injected WT mice (anterior thalamic nucleus +27%, dorsal CA1 stratum lacunosum molecularae +22%, dorsal CA1 stratum oriens + 14%, superior olivary body -26%, and the nucleus of the lateral lemniscus -16%). These data highlight that while overexpression of HSP70 transgene does not significantly alter LCGU in the basal state, mice overexpressing the HSP70 transgene respond differently to metabolic stress produced by NMDA receptor blockade in some important brain regions.

  8. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1.

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    Sandra J Feeney

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

  9. Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

    Science.gov (United States)

    Schneider, Vanessa Karine; Soares-Costa, Andrea; Chakravarthi, Mohan; Ribeiro, Carolina; Chabregas, Sabrina Moutinho; Falco, Maria Cristina; Henrique-Silva, Flavio

    2017-01-01

    Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

  10. Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

    Science.gov (United States)

    Sun, Huanxing; Zhu, Yangyang; Pan, Hongyi; Chen, Xiaosong; Balestrini, Jenna L; Lam, TuKiet T; Kanyo, Jean E; Eichmann, Anne; Gulati, Mridu; Fares, Wassim H; Bai, Hanwen; Feghali-Bostwick, Carol A; Gan, Ye; Peng, Xueyan; Moore, Meagan W; White, Eric S; Sava, Parid; Gonzalez, Anjelica L; Cheng, Yuwei; Niklason, Laura E; Herzog, Erica L

    2016-05-01

    Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were

  11. Transgenic overexpression of LARGE induces α-dystroglycan hyperglycosylation in skeletal and cardiac muscle.

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    Martin Brockington

    2010-12-01

    Full Text Available LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation

  12. Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice

    DEFF Research Database (Denmark)

    Jørgensen, Louise Helskov; Jensen, Charlotte Harken; Wewer, Ulla M

    2007-01-01

    overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic......Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed...

  13. Trichostatin A suppresses lung adenocarcinoma development in Grg1 overexpressing transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ju, E-mail: ju.liu@sdu.edu.cn [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan (China); Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 (Canada); Li, Yan [Children' s Health Care Center, Shandong Provincial Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan, Shandong 250014 (China); Dong, Fengyun; Li, Liqun [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan (China); Masuda, Takahiro; Allen, Thaddeus D. [Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 (Canada); Lobe, Corrinne G. [Molecular and Cellular Biology Division, Sunnybrook Health Science Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5 (Canada); Miami Mice Research Corp., MaRS Centre, Heritage Bldg., 101 College Street, Toronto, Ontario M5G 1L7 (Canada)

    2015-08-07

    Trichostatin A (TSA) is a histone deacetylase inhibitor and a potential therapeutic for various malignancies. The in vivo effect of TSA, however, has not been investigated in a transgenic lung cancer model. Previously, we generated transgenic mice with overexpression of Groucho-related-gene 1 (Grg1) and these mice all developed mucinous lung adenocarcinoma. Grg1 is a transcriptional co-repressor protein, the function of which is thought to depend on HDAC activity. However, functions outside the nucleus have also been proposed. We tested the supposition that Grg1-induced tumorigenesis is HDAC-dependent by assaying the therapeutic effect of TSA in the Grg1 transgenic mouse model. We found that TSA significantly inhibited lung tumorigenesis in Grg1 transgenic mice (p < 0.01). TSA did not affect overall Grg1 protein levels, but instead reduced ErbB1 and ErbB2 expression, which are upregulated by Grg1 in the absence of TSA. We confirmed this effect in A549 cells. Furthermore, lapatinib, an inhibitor of both ErbB1 and ErbB2, effectively masked the effect of TSA on the inhibition of A549 cell proliferation and migration, suggesting TSA does work, at least in part, by downregulating ErbB receptors. We additionally found that TSA reduced the expression of VEGF and VEGFR2, but not basic FGF and FGFR1. Our findings indicate that TSA effectively inhibits Grg1-induced lung tumorigenesis through the down-regulation of ErbB1 and ErbB2, as well as reduced VEGF signaling. This suggests TSA and other HDAC inhibitors could have therapeutic value in the treatment of lung cancers with Grg1 overexpression. - Highlights: • TSA suppresses lung tumorigenesis in Grg1 overexpressing transgenic mice. • TSA does not affect overall Grg1 protein levels in the mice and in A549 cells. • TSA reduces ErbB1 and ErbB2 expression in the mice and in A549 cells. • Lapatinib masks TSA-induced inhibition of A549 cell proliferation and migration. • TSA inhibits VEGF signaling, but not basic FGF

  14. RNA-seq analysis of unintended effects in transgenic wheat overexpressing the transcription factor GmDREB1

    Directory of Open Access Journals (Sweden)

    Qiyan Jiang

    2017-06-01

    Full Text Available The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than the genetic modification for simple monogenic traits. For this reason, it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. We selected drought- and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, to investigate unintended pleiotropic effects using RNA-seq analysis. We compared the transcriptome alteration of transgenic plants with that of wild-type plants subjected to salt stress as a control. We found that GmDREB1 overexpression had a minimal impact on gene expression under normal conditions. GmDREB1 overexpression resulted in transcriptional reprogramming of the salt response, but many of the genes with differential expression are known to mitigate salt stress and contribute incrementally to the enhanced stress tolerance of transgenic wheat. GmDREB1 overexpression did not activate unintended gene networks with respect to gene expression in the roots of transgenic wheat. This work is important for establishing a method of detecting unintended effects of genetic engineering and the safety of such traits with the development of marketable transgenic crops in the near future.

  15. Overexpression of PSP1 enhances growth of transgenic Arabidopsis plants under ambient air conditions.

    Science.gov (United States)

    Han, Xiaofang; Peng, Keli; Wu, Haixia; Song, Shanshan; Zhu, Yerong; Bai, Yanling; Wang, Yong

    2017-07-01

    The importance of the phosphorylated pathway (PPSB) of L-serine (Ser) biosynthesis in plant growth and development has been demonstrated, but its specific role in leaves and interaction with photorespiration, the main leaf Ser biosynthetic pathway at daytime, are still unclear. To investigate whether changes in biosynthesis of Ser by the PPSB in leaves could have an impact on photorespiration and plant growth, we overexpressed PSP1, the last enzyme of this pathway, under control of the Cauliflower Mosaic Virus 35S promoter in Arabidopsis thaliana. Overexpressor plants grown in normal air displayed larger rosette diameter and leaf area as well as higher fresh and dry weight than the wild type. By contrast, no statistically significant differences to the wild type were observed when the overexpressor seedlings were transferred to elevated CO2, indicating a relationship between PSP1 overexpression and photorespiration. Additionally, the transgenic plants displayed higher photorespiration, an increase in CO2 net-uptake and stronger expression in the light of genes encoding enzymes involved in photorespiration. We further demonstrated that expression of many genes involved in nitrogen assimilation was also promoted in leaves of transgenic plants and that leaf nitrate reductase activity increased in the light, too, although not in the dark. Our results suggest a close correlation between the function of PPSB and photorespiration, and also nitrogen metabolism in leaves.

  16. Use of letrozole as a chemopreventive agent in aromatase overexpressing transgenic mice.

    Science.gov (United States)

    Luthra, Roopa; Kirma, Nameer; Jones, Jeremy; Tekmal, Rajeshwar Rao

    2003-09-01

    Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in mammary glands, and gynecomastia and testicular cancer in male aromatase transgenic mice. Our studies also have shown that aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of letrozole, an aromatase inhibitor without any effect on normal physiology. In the present study, we have examined the effect of prior low dose letrozole treatment on pregnancy and lactation. We have also investigated the effect of low dose letrozole treatment on subsequent mammary growth and biochemical changes in these animals. There was no change in the litter size, birth weight and no visible birth defects in letrozole-treated animals. Although, there was an insignificant increase in mammary growth in aged animals after 6 weeks of letrozole treatment, the levels of expression of estrogen receptor, progesterone receptor and genes involved in cell cycle and cell proliferation remained low compared to control untreated animals. These observations indicate that aromatase inhibitors such as letrozole can be used as chemopreventive agents without effecting normal physiology in aromatase transgenic mice.

  17. Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

    DEFF Research Database (Denmark)

    Ellegaard, Maria; Agca, Cansu; Petersen, Solveig

    2017-01-01

    It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R...... overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both...... mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased...

  18. Conditional tenomodulin overexpression favors tenogenic lineage differentiation of transgenic mouse derived cells.

    Science.gov (United States)

    Shi, Yuan; Xiong, Yao; Jiang, Yongkang; Zhang, Zhiyong; Zhou, Guangdong; Zhang, Wenjie; Cao, Yilin; He, Jing; Liu, Wei

    2017-01-20

    Tenomodulin (TNMD) is a type II transmembrane protein that is widely expressed in a variety of avascular connective tissues and fat tissue. Its function remains largely unknown except for a marker for mature tenocytes. This study reports the generation of tetracycline (Tet)-on driven conditional TNMD overexpressing mice and thus to provide a tool for systemic investigation of its role in regulating functions of various tissues. The current study focuses on in vitro comparison of tenogenic differentiation potentials induced by doxycycline (Dox) treatment among bone marrow derived stem cells (BMSCs), adipose derived stem cells (ASCs), dermal fibroblasts (DFs) and tenocytes (TCs) of the same transgenic mice. The results showed that BMSCs exhibited the best tenogenic potential than other three cell types (p0.05 for majority of markers). TCs were found the least capable of being induced for tenogenic gene expression. In addition, TNMD overexpression also significantly inhibited the differentiation towards osteogenic and chondrogenic lineages in both BMSCs and ASCs (p0.05), suggesting different gene regulation mechanisms may involve in different tissue types and thus leading to different functions, which is likely to be revealed with a transgenic mouse model. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Reduced grain chalkiness and its possible physiological mechanism in transgenic rice overexpressing l-GalLDH

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    Le Yu

    2015-04-01

    Full Text Available Chalkiness is one of the key factors determining rice quality and price. Ascorbic acid (Asc is a major plant antioxidant that performs many functions in plants. l-Galactono-1,4-lactone dehydrogenase (l-GalLDH, EC1.3.2.3 is an enzyme that catalyzes the final step of Asc biosynthesis in plants. Here we show that the l-GalLDH-overexpressing transgenic rice, GO-2, which has constitutively higher leaf Asc content than wild-type (WT plants, exhibits significantly reduced grain chalkiness. Higher foliar ascorbate/dehydroascorbate (Asc/DHA ratios at 40, 60, 80, and 100 days of plant age were observed in GO-2. Further investigation showed that the enhanced level of Asc resulted in a significantly higher ribulose-1,5-bisphosphate (RuBP carboxylase/oxygenase (Rubisco protein level in GO-2 at 80 days. In addition, levels of abscisic acid (ABA and jasmonic acid (JA were lower in GO-2 at 60, 80, and 100 days. The results we present here indicate that the enhanced level of Asc is likely responsible for changing redox homeostasis in key developmental stages associated with grain filling and alters grain chalkiness in the l-GalLDH-overexpressing transgenic by maintaining photosynthetic function and affecting phytohormones associated with grain filling.

  20. Transgenic overexpression of GLUT1 in mouse glomeruli produces renal disease resembling diabetic glomerulosclerosis

    Science.gov (United States)

    Wang, Youli; Heilig, Kathleen; Saunders, Thomas; Minto, Andrew; Deb, Dilip K.; Chang, Anthony; Brosius, Frank; Monteiro, Carmela

    2010-01-01

    Previous work identified an important role for hyperglycemia in diabetic nephropathy (The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977–986, 1993; UK Prospective Diabetes Study Group. Lancet 352: 837–853, 1998), and increased glomerular GLUT1 has been implicated. However, the roles of GLUT1 and intracellular glucose have not been determined. Here, we developed transgenic GLUT1-overexpressing mice (GT1S) to characterize the roles of GLUT1 and intracellular glucose in the development of glomerular disease without diabetes. GLUT1 was overexpressed in glomerular mesangial cells (MC) of C57BL6 mice, a line relatively resistant to diabetic nephropathy. Blood pressure, blood glucose, glomerular morphometry, matrix proteins, cell signaling, transcription factors, and selected growth factors were examined. Kidneys of GT1S mice overexpressed GLUT1 in glomerular MCs and small vessels, rather than renal tubules. GT1S mice were neither diabetic nor hypertensive. Glomerular GLUT1, glucose uptake, mean capillary diameter, and mean glomerular volume were all increased in the GT1S mice. Moderately severe glomerulosclerosis (GS) was established by 26 wk of age in GT1S mice, with increased glomerular type IV collagen and fibronectin. Modest increases in glomerular basement membrane thickness and albuminuria were detected with podocyte foot processes largely preserved, in the absence of podocyte GLUT1 overexpression. Activation of glomerular PKC, along with increased transforming growth factor-β1, VEGFR1, VEGFR2, and VEGF were all detected in glomeruli of GT1S mice, likely contributing to GS. The transcription factor NF-κB was also activated. Overexpression of glomerular GLUT1, mimicking the diabetic GLUT1 response, produced numerous features typical of diabetic glomerular disease, without diabetes or hypertension. This suggested GLUT1 may play an important role in the development of diabetic GS. PMID:20375117

  1. Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants.

    Science.gov (United States)

    Han, Junli; Wang, Hongzhen; Lundgren, Anneli; Brodelius, Peter E

    2014-06-01

    The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis.

    Science.gov (United States)

    Fu, Xiao-Yan; Zhao, Wei; Xiong, Ai-Sheng; Tian, Yong-Sheng; Zhu, Bo; Peng, Ri-He; Yao, Quan-Hong

    2013-02-01

    Triphenylmethane dyes are extensively utilized in textile industries, medicinal products, biological stains, and food processing industries, etc. They are generally considered as xenobiotic compounds, which are very recalcitrant to biodegradation. The widespread persistence of such compounds has generated concerns with regard to remediation of them because of their potential carcinogenicity, teratogenicity, and mutagenicity. In this study, we present a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of triphenylmethane reductase (TMR) from the Citrobacter sp. The morphology and growth of TMR transgenic Arabidopsis plants showed significantly enhanced tolerances to crystal violet (CV) and malachite green (MG). Further, HPLC and HPLC-MS analyses of samples before and after dye decolorization in culture media revealed that TMR transgenic plants exhibited strikingly higher capabilities of removing CV from their media and high efficiencies of converting CV to non-toxic leucocrystal violet (LCV). This work indicates that microbial degradative gene may be transgenically exploited in plants for bioremediation of triphenylmethane dyes in the environment.

  3. Salt tolerant SUV3 overexpressing transgenic rice plants conserve physicochemical properties and microbial communities of rhizosphere.

    Science.gov (United States)

    Sahoo, Ranjan K; Ansari, Mohammad W; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Key concerns in the ecological evaluation of GM crops are undesirably spread, gene flow, other environmental impacts, and consequences on soil microorganism's biodiversity. Numerous reports have highlighted the effects of transgenic plants on the physiology of non-targeted rhizospheric microbes and the food chain via causing adverse effects. Therefore, there is an urgent need to develop transgenics with insignificant toxic on environmental health. In the present study, SUV3 overexpressing salt tolerant transgenic rice evaluated in New Delhi and Cuttack soil conditions for their effects on physicochemical and biological properties of rhizosphere. Its cultivation does not affect soil properties viz., pH, Eh, organic C, P, K, N, Ca, Mg, S, Na and Fe(2+). Additionally, SUV3 rice plants do not cause any change in the phenotype, species characteristics and antibiotic sensitivity of rhizospheric bacteria. The population and/or number of soil organisms such as bacteria, fungi and nematodes were unchanged in the soil. Also, the activity of bacterial enzymes viz., dehydrogenase, invertase, phenol oxidases, acid phosphatases, ureases and proteases was not significantly affected. Further, plant growth promotion (PGP) functions of bacteria such as siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH3, phosphorus metabolism, ACC deaminase and iron tolerance were, considerably, not influenced. The present findings suggest ecologically pertinent of salt tolerant SUV3 rice to sustain the health and usual functions of the rhizospheric organisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum.

    Science.gov (United States)

    Tesfaye, M; Temple, S J; Allan, D L; Vance, C P; Samac, D A

    2001-12-01

    Al toxicity is a severe impediment to production of many crops in acid soil. Toxicity can be reduced through lime application to raise soil pH, however this amendment does not remedy subsoil acidity, and liming may not always be practical or cost-effective. Addition of organic acids to plant nutrient solutions alleviates phytotoxic Al effects, presumably by chelating Al and rendering it less toxic. In an effort to increase organic acid secretion and thereby enhance Al tolerance in alfalfa (Medicago sativa), we produced transgenic plants using nodule-enhanced forms of malate dehydrogenase and phosphoenolpyruvate carboxylase cDNAs under the control of the constitutive cauliflower mosaic virus 35S promoter. We report that a 1.6-fold increase in malate dehydrogenase enzyme specific activity in root tips of selected transgenic alfalfa led to a 4.2-fold increase in root concentration as well as a 7.1-fold increase in root exudation of citrate, oxalate, malate, succinate, and acetate compared with untransformed control alfalfa plants. Overexpression of phosphoenolpyruvate carboxylase enzyme specific activity in transgenic alfalfa did not result in increased root exudation of organic acids. The degree of Al tolerance by transformed plants in hydroponic solutions and in naturally acid soil corresponded with their patterns of organic acid exudation and supports the concept that enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and Al toxicity.

  5. Dspp-independent Effects of Transgenic Trps1 Overexpression on Dentin Formation

    Science.gov (United States)

    Mobley, C.G.; Kuzynski, M.; Zhang, H.; Jani, P.; Qin, C.

    2015-01-01

    The Trps1 transcription factor is highly expressed in dental mesenchyme and preodontoblasts, while in mature, secretory odontoblasts, it is expressed at low levels. Previously, we have shown that high Trps1 levels in mature odontoblasts impair their function in vitro and in vivo. Col1a1-Trps1 transgenic (Trps1-Tg) mice demonstrate defective dentin secretion and mineralization, which are associated with significantly decreased Dspp expression due to direct repression of the Dspp gene by Trps1. Here, by crossing Trps1-Tg and Col1a1-Dspp transgenic (Dspp-Tg) mice, we generated Col1a1-Trps1;Col1a1-Dspp double transgenic (double-Tg) mice in which Dspp was restored in odontoblasts overexpressing Trps1. Comparative micro–computed tomography analyses revealed partial correction of the dentin volume and no improvement of dentin mineralization in double transgenic mice in comparison with Trps1-Tg and wild-type (WT) mice. In addition, dentin of double-Tg mice has an irregular mineralization pattern characteristic for dentin in hypophosphatemic rickets. Consistent with this phenotype, decreased levels of Phex, Vdr, and Fam20c proteins are detected in both Trps1-Tg and double-Tg odontoblasts in comparison with WT and Dspp-Tg odontoblasts. This suggests that the Dspp-independent dentin mineralization defects in Trps1-Tg mice are a result of downregulation of a group of proteins critical for mineral deposition within the dentin matrix. In summary, by demonstrating that Trps1 functions as a repressor of later stages of dentinogenesis, we provide functional significance of the dynamic Trps1 expression pattern during dentinogenesis. PMID:25999324

  6. RNA-seq analysis of unintended effects in transgenic wheat overexpressing the transcription factor GmDREB1

    OpenAIRE

    Jiang, Qiyan; Niu, Fengjuan; Sun, Xianjun; Hu,Zheng; Li, Xinhai; Ma, Youzhi; Zhang, Hui

    2016-01-01

    The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than the genetic modification for simple monogenic traits. For this reason, it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. We selected drought- and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, to investigate uninten...

  7. Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

    Science.gov (United States)

    Elhiti, Mohamed; Yang, Cunchun; Chan, Ainsley; Durnin, Douglas C; Belmonte, Mark F; Ayele, Belay T; Tahir, Muhammad; Stasolla, Claudio

    2012-07-01

    SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

  8. Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene.

    Science.gov (United States)

    Wang, Guo-Kun; Zhang, Meng; Gong, Jiang-Feng; Guo, Qi-Fang; Feng, Ya-Nan; Wang, Wei

    2012-12-01

    Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

  9. Behavioural characterization of neuregulin 1 (NRG1) type I over-expressing transgenic mice

    Science.gov (United States)

    Deakin, Inga H.; Law, Amanda J.; Oliver, Peter L.; Schwab, Markus H.; Nave, Klaus Armin; Harrison, Paul J.; Bannerman, David M.

    2009-01-01

    Neuregulin 1 (NRG1) is a pleiotropic growth factor involved in diverse aspects of brain development and function. In schizophrenia, expression of the NRG1 type I isoform is selectively increased. However, virtually nothing is known about the roles of this isoform in brain. We have studied transgenic mice over-expressing type I NRG1 (NRG1type 1-tg) using a series of behavioural tests. NRG1type 1-tg mice have a tremor, are impaired on the accelerating rotarod, and have reduced prepulse inhibition in the context of an increased baseline startle response. There is no overall anxiety or activity phenotype, although female NRG1type 1-tg mice show mild increases in anxiety on some measures. The pattern of results shows both similarities and differences to those reported in hypomorphic NRG1 mice, and may be relevant for interpreting the increased NRG1 type I expression seen in schizophrenia. PMID:19829162

  10. Overexpression of a Chitinase Gene from Trichoderma asperellum Increases Disease Resistance in Transgenic Soybean.

    Science.gov (United States)

    Zhang, Fuli; Ruan, Xianle; Wang, Xian; Liu, Zhihua; Hu, Lizong; Li, Chengwei

    2016-12-01

    In the present study, a chi gene from Trichoderma asperellum, designated Tachi, was cloned and functionally characterized in soybean. Firstly, the effects of sodium thiosulfate on soybean Agrobacterium-mediated genetic transformation with embryonic tip regeneration system were investigated. The transformation frequency was improved by adding sodium thiosulfate in co-culture medium for three soybean genotypes. Transgenic soybean plants with constitutive expression of Tachi showed increased resistance to Sclerotinia sclerotiorum compared to WT plants. Meanwhile, overexpression of Tachi in soybean exhibited increased reactive oxygen species (ROS) level as well as peroxidase (POD) and catalase (SOD) activities, decreased malondialdehyde (MDA) content, along with diminished electrolytic leakage rate after S. sclerotiorum inoculation. These results suggest that Tachi can improve disease resistance in plants by enhancing ROS accumulation and activities of ROS scavenging enzymes and then diminishing cell death. Therefore, Tachi represents a candidate gene with potential application for increasing disease resistance in plants.

  11. Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

    2013-11-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

  12. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco.

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    Shiyi Zhou

    Full Text Available Aquaporin (AQP proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG, abscisic acid (ABA and H(2O(2. Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA and H(2O(2, and less ion leakage (IL, but higher relative water content (RWC and superoxide dismutase (SOD and catalase (CAT activities when compared with the wild type (WT under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants.

  13. Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Sandberg, Malin K; Al-Doujaily, Huda; Sigurdson, Christina J; Glatzel, Markus; O'Malley, Catherine; Powell, Caroline; Asante, Emmanuel A; Linehan, Jacqueline M; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John

    2010-10-01

    Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilized in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterized.

  14. Transgenic overexpression of miR-133a in skeletal muscle

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    Chen Jian-Fu

    2011-05-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a class of non-coding regulatory RNAs of ~22 nucleotides in length. miRNAs regulate gene expression post-transcriptionally, primarily by associating with the 3' untranslated region (UTR of their regulatory target mRNAs. Recent work has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Many miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related disorders. We have previously reported that the expression of muscle-specific miR-1 and miR-133 is induced during skeletal muscle differentiation and miR-1 and miR-133 play central regulatory roles in myoblast proliferation and differentiation in vitro. Methods In this study, we measured the expression of miRNAs in the skeletal muscle of mdx mice, an animal model for human muscular dystrophy. We also generated transgenic mice to overexpress miR-133a in skeletal muscle. Results We examined the expression of miRNAs in the skeletal muscle of mdx mice. We found that the expression of muscle miRNAs, including miR-1a, miR-133a and miR-206, was up-regulated in the skeletal muscle of mdx mice. In order to further investigate the function of miR-133a in skeletal muscle in vivo, we have created several independent transgenic founder lines. Surprisingly, skeletal muscle development and function appear to be unaffected in miR-133a transgenic mice. Conclusions Our results indicate that miR-133a is dispensable for the normal development and function of skeletal muscle.

  15. Overexpression of BID in thyroids of transgenic mice increases sensitivity to iodine-induced autoimmune thyroiditis.

    Science.gov (United States)

    Wang, Su He; Fan, Yongyi; Baker, James R

    2014-06-23

    BID functions as a bridge molecule between death-receptor and mitochondrial related apoptotic pathways to amplify apoptotic signaling. Our previous studies have demonstrated a substantial increase in BID expression in primary normal thyroid epithelia cells treated with inflammatory cytokines, including the combination of IFNγ and IL-1β or IFNγ and TNFα. The aim of this study was to determine whether an increase in BID expression in thyroid can induce autoimmune thyroiditis. A transgenic mouse line that expresses human BID in thyroid cells was established by fusing a mouse thyroglobulin (Tg) promoter upstream of human BID (Tg-BID). We tested whether the increased expression of pro-apoptotic BID in thyroid would induce autoimmune thyroiditis, both in the presence and absence of 0.3% iodine water. Our data show that Tg-BID mice in a CBA/J (H-2 k) background do not spontaneously develop autoimmune thyroiditis for over a year. However, upon ingestion of iodine in the drinking water, autoimmune thyroiditis does develop in Tg-BID transgenic mice, as shown by a significant increase in anti-Tg antibody and mononuclear cell infiltration in the thyroid glands in 30% of mice tested. Serum T4 levels, however, were similar between iodine-treated Tg-BID transgenic mice and the wild type mice. Our data demonstrate that increased thyroid expression of BID facilitates the development of autoimmune thyroiditis induced by iodine uptake. However, the overexpression of BID itself is not sufficient to initiate thyroiditis in CBA/J (H-2 k) mice.

  16. Overexpression of adiponectin targeted to adipose tissue in transgenic mice: impaired adipocyte differentiation.

    Science.gov (United States)

    Bauche, Isabelle B; El Mkadem, Samira Ait; Pottier, Anne-Marie; Senou, Maximin; Many, Marie-Christine; Rezsohazy, René; Penicaud, Luc; Maeda, Norikazu; Funahashi, Tohru; Brichard, Sonia M

    2007-04-01

    Adiponectin (ApN) is an adipokine whose expression and plasma levels are inversely related to obesity and insulin-resistant states. Chronic repercussions of ApN treatment or overexpression on adiposity and body weight are still controversial. Here, we generated a transgenic (Tg) mouse model allowing persistent and moderate overexpression of native full-length ApN targeted to white adipose tissue. Adipose mass and adipocyte size of Tg mice were reduced despite preserved calorie intake. This reduction resulted from increased energy expenditure and up-regulation of uncoupling proteins, and from abrogation of the adipocyte differentiation program, as shown by the loss of a key lipogenic enzyme and of adipocyte markers. Adipose mass remodeling favors enhanced insulin sensitivity and improved lipid profile of Tg mice. Alteration of the adipocyte phenotype was likely to result from increased expression of the preadipocyte factor-1 and from down-regulation of the transcription factor, CCAAT/enhancer binding protein-alpha, which orchestrates adipocyte differentiation. We further found that recombinant ApN directly stimulated pre- adipocyte factor-1 mRNA and attenuated CCAAT/enhancer binding protein-alpha expression in cultured 3T3-F442A cells. Conversely, opposite changes in the expression of these genes were observed in white fat of ApN-deficient mice. Thus, besides enhanced energy expenditure, our work shows that impairment of adipocyte differentiation contributes to the anti-adiposity effect of ApN.

  17. Metabolic changes in transgenic maize mature seeds over-expressing the Aspergillus niger phyA2.

    Science.gov (United States)

    Rao, Jun; Yang, Litao; Guo, Jinchao; Quan, Sheng; Chen, Guihua; Zhao, Xiangxiang; Zhang, Dabing; Shi, Jianxin

    2016-02-01

    Non-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2. Genetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC-MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

  18. Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC).

    Science.gov (United States)

    Prieto, Catalina P; Ortiz, María Carolina; Villanueva, Andrea; Villarroel, Cynthia; Edwards, Sandra S; Elliott, Matías; Lattus, José; Aedo, Sócrates; Meza, Daniel; Lois, Pablo; Palma, Verónica

    2017-02-28

    Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6β1, expressed in

  19. Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands.

    Science.gov (United States)

    Gill, K; Kirma, N; Tekmal, R R

    2001-04-01

    Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.

  20. Deficiency and Also Transgenic Overexpression of Timp-3 Both Lead to Compromised Bone Mass and Architecture In Vivo.

    Directory of Open Access Journals (Sweden)

    Behzad Javaheri

    Full Text Available Tissue inhibitor of metalloproteinases-3 (TIMP-3 regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages.

  1. Netrin-1 - DCC Signaling Systems and Age-Related Macular Degeneration.

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    John Paul SanGiovanni

    Full Text Available We conducted a nested candidate gene study and pathway-based enrichment analysis on data from a multi-national 77,000-person project on the molecular genetics of age-related macular degeneration (AMD to identify AMD-associated DNA-sequence variants in genes encoding constituents of a netrin-1 (NTN1-based signaling pathway that converges on DNA-binding transcription complexes through a 3'-5'-cyclic adenosine monophosphate-calcineurin (cAMP-CN-dependent axis. AMD-associated single nucleotide polymorphisms (SNPs existed in 9 linkage disequilibrium-independent genomic regions; these included loci overlapping NTN1 (rs9899630, P ≤ 9.48 x 10(-5, DCC (Deleted in Colorectal Cancer--the gene encoding a primary NTN1 receptor (rs8097127, P ≤ 3.03 x 10(-5, and 6 other netrin-related genes. Analysis of the NTN1-DCC pathway with exact methods demonstrated robust enrichment with AMD-associated SNPs (corrected P-value = 0.038, supporting the idea that processes driven by NTN1-DCC signaling systems operate in advanced AMD. The NTN1-DCC pathway contains targets of FDA-approved drugs and may offer promise for guiding applied clinical research on preventive and therapeutic interventions for AMD.

  2. Overexpression of persimmon DkXTH1 enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants.

    Science.gov (United States)

    Han, Ye; Han, Shoukun; Ban, Qiuyan; He, Yiheng; Jin, Mijing; Rao, Jingping

    2017-04-01

    DkXTH1 promoted cell elongation and more strength to maintain structural integrity by involving in cell wall assembly, thus enhanced tolerance to abiotic stress with broader phenotype in transgenic plants. Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to play a key role in cell wall modifications by cleaving and re-joining xyloglucan, and participates in the diverse physiological processes. DkXTH1 was found to peak in immature expanding persimmon fruit, and its higher expression level exhibited along with firmer fruit during storage. In the present study, transgenic Arabidopsis and tomato plants were generated with DkXTH1 constitutively expressed. Overexpression of DkXTH1 enhanced tolerance to salt, ABA and drought stresses in transgenic Arabidopsis plants with respect to root and leaf growth, and survival. Transgenic tomatoes collected at the mature green stage, presented delayed fruit softening coupled with postponed color change, a later and lower ethylene peak, and higher firmness in comparison with the wild-type tomatoes during storage. Furthermore, broader leaves and tomato fruit with larger diameter were gained in transgenic Arabidopsis and tomato, respectively. Most importantly, transgenic plants exhibited more large and irregular cells with higher density of cell wall and intercellular spaces, resulting from the overactivity of XET enzymes involving in cell wall assembly. We suggest that DkXTH1 expression resulted in cells with more strength and thickness to maintain structural integrity, and thus enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants.

  3. Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis.

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    Suzhen Li

    Full Text Available Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP, is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.

  4. Analysis of the effects of overexpression of metallothionein-I in transgenic mice on the reproductive toxicology of cadmium.

    Science.gov (United States)

    Dalton, T; Fu, K; Enders, G C; Palmiter, R D; Andrews, G K

    1996-01-01

    Exposure to low levels of cadmium reduces fertility. In male mice spermatogenesis is highly sensitive to cadmium, whereas in females the peri-implantation period of pregnancy is sensitive. To examine the potential roles of the cadmium-binding protein, metallothionein (MT), in the reproductive toxicology of cadmium, we examined a transgenic mouse strain that overexpresses metallothionein-I (MT-I). These mice had dramatically increased steady-state levels of MT-I mRNA and MT in the testes and in the female reproductive tract during the peri-implantation period of pregnancy, and this overexpression occurred in a cell-specific and temporally regulated manner similar to that of the endogenous MT-I gene. Transgenic and control males were injected with cadmium, and the histology of the testes was examined. An injection of 7.5 mumol Cd/kg had no effect on histology of the testes in either transgenic or control mice. In contrast, an injection of 10 mumol Cd/kg caused rapid changes in the histology of the testes and resulted in pronounced testicular necrosis in both control and transgenic mice. Female transgenic and control mice were mated and then injected with cadmium (30-45 mumol Cd/kg) on the day of blastocyst implantation (day 4). In both of these groups, injection of cadmium reduced pregnancy rate, and no dramatic protection was afforded by maternal and/or embryonic overexpression of MT. Thus, overexpression of MT-I does not significantly protect against either of these cadmium-induced effects on fertility.

  5. Overexpression of IGF-I in skeletal muscle of transgenic mice does not prevent unloading-induced atrophy

    Science.gov (United States)

    Criswell, D. S.; Booth, F. W.; DeMayo, F.; Schwartz, R. J.; Gordon, S. E.; Fiorotto, M. L.

    1998-01-01

    This study examined the association between local insulin-like growth factor I (IGF-I) overexpression and atrophy in skeletal muscle. We hypothesized that endogenous skeletal muscle IGF-I mRNA expression would decrease with hindlimb unloading (HU) in mice, and that transgenic mice overexpressing human IGF-I (hIGF-I) specifically in skeletal muscle would exhibit less atrophy after HU. Male transgenic mice and nontransgenic mice from the parent strain (FVB) were divided into four groups (n = 10/group): 1) transgenic, weight-bearing (IGF-I/WB); 2) transgenic, hindlimb unloaded (IGF-I/HU); 3) nontransgenic, weight-bearing (FVB/WB); and 4) nontransgenic, hindlimb unloaded (FVB/HU). HU groups were hindlimb unloaded for 14 days. Body mass was reduced (P muscle of wild-type FVB mice. High-level expression of hIGF-I peptide and mRNA was confirmed in the GAST and tibialis anterior (TA) muscles of the transgenic mice. Nevertheless, masses of the GAST and TA muscles were reduced (P muscles did not differ between FVB and IGF-I mice. Therefore, skeletal muscle atrophy may not be associated with a reduction of endogenous rIGF-I mRNA level in 14-day HU mice. We conclude that high local expression of hIGF-I mRNA and peptide in skeletal muscle alone cannot attenuate unloading-induced atrophy of fast-twitch muscle in mice.

  6. Quantification of MRI signal of transgenic grafts overexpressing ferritin in murine myocardial infarcts.

    Science.gov (United States)

    Naumova, Anna V; Yarnykh, Vasily L; Balu, Niranjan; Reinecke, Hans; Murry, Charles E; Yuan, Chun

    2012-10-01

    The noninvasive detection of transplanted cells in damaged organs and the longitudinal follow-up of cell fate and graft size are important for the evaluation of cell therapy. We have shown previously that the overexpression of the natural iron storage protein, ferritin, permits the detection of engrafted cells in mouse heart by MRI, but further imaging optimization is required. Here, we report a systematic evaluation of ferritin-based stem cell imaging in infarcted mouse hearts in vivo using three cardiac-gated pulse sequences in a 3-T scanner: black-blood proton-density-weighted turbo spin echo (PD TSE BB), bright-blood T(2) -weighted gradient echo (GRE) and black-blood T(2) -weighted GRE with improved motion-sensitized-driven equilibrium (iMSDE) preparation. Transgenic C2C12 myoblast grafts overexpressing ferritin did not change MRI contrast in the PD TSE BB images, but showed a 20% reduction in signal intensity ratio in black-blood T(2) -weighted iMSDE (p < 0.05) and a 30% reduction in bright-blood T(2) -weighted GRE (p < 0.0001). Graft size measurements by T(2) iMSDE and T(2) GRE were highly correlated with histological assessments (r = 0.79 and r = 0.89, respectively). Unlabeled wild-type C2C12 cells transplanted to mouse heart did not change the MRI signal intensity, although endogenous hemosiderin was seen in some infarcts. These data support the use of ferritin to track the survival, growth and migration of stem cells transplanted into the injured heart. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Ectopic automaticity induced in ventricular myocytes by transgenic overexpression of HCN2.

    Science.gov (United States)

    Oshita, Kensuke; Itoh, Masayuki; Hirashima, Shingo; Kuwabara, Yoshihiro; Ishihara, Keiko; Kuwahara, Koichiro; Nakao, Kazuwa; Kimura, Takeshi; Nakamura, Kei-Ichiro; Ushijima, Kazuo; Takano, Makoto

    2015-03-01

    Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are expressed in the ventricles of fetal hearts but are normally down-regulated as development progresses. In the hypertrophied heart, however, these channels are re-expressed and generate a hyperpolarization-activated, nonselective cation current (Ih), which evidence suggests may increase susceptibility to arrhythmia. To test this hypothesis, we generated and analyzed transgenic mice overexpressing HCN2 specifically in their hearts (HCN2-Tg). Under physiological conditions, HCN2-Tg mice exhibited no discernible abnormalities. After the application of isoproterenol (ISO), however, ECG recordings from HCN2-Tg mice showed intermittent atrioventricular dissociation followed by idioventricular rhythm. Consistent with this observation, 0.3 μmol/L ISO-induced spontaneous action potentials (SAPs) in 76% of HCN2-Tg ventricular myocytes. In the remaining 24%, ISO significantly depolarized the resting membrane potential (RMP), and the late repolarization phase of evoked action potentials (APs) was significantly longer than in WT myocytes. Analysis of membrane currents revealed that these differences are attributable to the Ih tail current. These findings suggest HCN2 channel activity reduces the repolarization reserve of the ventricular action potential and increases ectopic automaticity under pathological conditions such as excessive β-adrenergic stimulation. Copyright © 2015. Published by Elsevier Ltd.

  8. Overexpression of the Galega orientalis gibberellin receptor improves biomass production in transgenic tobacco.

    Science.gov (United States)

    Li, Jun; Gao, Hongwen; Jiang, Jishan; Dzyubenko, Nikolay; Chapurin, Vladimir; Wang, Zan; Wang, Xuemin

    2013-12-01

    Gibberellins (GAs) are well-known phytohormones that contribute to a wide range of plant growth and development functions including stem elongation and leaf expansion. GA receptors perceive GA and transmit signals to activate GA-regulated reactions. In this study, a GA receptor gene with homology to other leguminous plants was isolated from Galega orientalis and termed GoGID. The 1732-bp full-length GoGID gene included an open reading frame of 1035 bp encoding a peptide of 344 amino acids. Sequence analysis indicated that GoGID shares conserved HGGS motif and active amino acid sites (Ser-Asp-Val/IIe) that are essential for maintaining it GA-binding activity. GoGID mRNA expression was more abundant in leaves than in roots or stems and could be up-regulated by the exogenous hormones. Overexpression of GoGID in transgenic tobacco plants promoted plant elongation and improved biomass production. These results suggested that GoGID functions as a GA receptor to alter GA-mediated signaling. GoGID may have a role in genetic engineering for the improvement of forage crops. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. Artesunate Tolerance in Transgenic Plasmodium falciparum Parasites Overexpressing a Tryptophan-Rich Protein▿†

    Science.gov (United States)

    Deplaine, Guillaume; Lavazec, Catherine; Bischoff, Emmanuel; Natalang, Onguma; Perrot, Sylvie; Guillotte-Blisnick, Micheline; Coppée, Jean-Yves; Pradines, Bruno; Mercereau-Puijalon, Odile; David, Peter H.

    2011-01-01

    Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins. PMID:21464256

  10. Overexpressing the Sedum alfredii Cu/Zn Superoxide Dismutase Increased Resistance to Oxidative Stress in Transgenic Arabidopsis

    Directory of Open Access Journals (Sweden)

    Zhen Li

    2017-06-01

    Full Text Available Superoxide dismutase (SOD is a very important reactive oxygen species (ROS-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD, from Sedum alfredii, a cadmium (Cd/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2 and superoxide radicals (O2•-. The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress.

  11. An immunological renal disease in transgenic mice that overexpress Fli-1, a member of the ets family of transcription factor genes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Liqun; Teng, Yen-Tung; Melet, F. [Univ. of Toronto, Ontario (Canada)] [and others

    1995-12-01

    This report describes how expression of the proto-oncogene Fli-1 is involved in the regulation of lymphopoiesis. Transgenic mice were generated which overexpressed the oncogene, leading to a high incidence of immunological renal diseases and death. The data suggests that overexpression of Fli-1 perturbs normal lymphoid cell function and programmed cell death, making these transgenic mice suitable as a biological model for autoimmune disease in humans. 35 refs., 6 figs., 4 tabs.

  12. Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tang, Lili; Cai, Hua; Ji, Wei; Luo, Xiao; Wang, Zhenyu; Wu, Jing; Wang, Xuedong; Cui, Lin; Wang, Yang; Zhu, Yanming; Bai, Xi

    2013-10-01

    GsZFP1 encodes a Cys2/His2-type zinc-finger protein. In our previous study, when GsZFP1 was heterologously expressed in Arabidopsis, the transgenic Arabidopsis plants exhibited enhanced drought and cold tolerance. However, it is still unknown whether GsZFP1 is also involved in salt stress. GsZFP1 is from the wild legume Glycine soja. Therefore, the aims of this study were to further elucidate the functions of the GsZFP1 gene under salt and drought stress in the forage legume alfalfa and to investigate its biochemical and physiological functions under these stress conditions. Our data showed that overexpression of GsZFP1 in alfalfa resulted in enhanced salt tolerance. Under high salinity stress, greater relative membrane permeability and malondialdehyde (MDA) content were observed and more free proline and soluble sugars accumulated in transgenic alfalfa than in the wild-type (WT) plants; in addition, the transgenic lines accumulated less Na(+) and more K(+) in both the shoots and roots. Overexpression of GsZFP1 also enhanced the drought tolerance of alfalfa. The fold-inductions of stress-responsive marker gene expression, including MtCOR47, MtRAB18, MtP5CS, and MtRD2, were greater in transgenic alfalfa than those of WT under drought stress conditions. In conclusion, the transgenic alfalfa plants generated in this study could be used for farming in salt-affected as well as arid and semi-arid areas. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  13. Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3.

    Science.gov (United States)

    Abdeen, Ashraf; Schnell, Jaimie; Miki, Brian

    2010-01-28

    Plants engineered for abiotic stress tolerance may soon be commercialized. The engineering of these plants typically involves the manipulation of complex multigene networks and may therefore have a greater potential to introduce pleiotropic effects than the simple monogenic traits that currently dominate the plant biotechnology market. While research on unintended effects in transgenic plant systems has been instrumental in demonstrating the substantial equivalence of many transgenic plant systems, it is essential that such analyses be extended to transgenic plants engineered for stress tolerance. Drought-tolerant Arabidopsis thaliana were engineered through overexpression of the transcription factor ABF3 in order to investigate unintended pleiotropic effects. In order to eliminate position effects, the Cre/lox recombination system was used to create control plant lines that contain identical T-DNA insertion sites but with the ABF3 transgene excised. This additionally allowed us to determine if Cre recombinase can cause unintended effects that impact the transcriptome. Microarray analysis of control plant lines that underwent Cre-mediated excision of the ABF3 transgene revealed only two genes that were differentially expressed in more than one plant line, suggesting that the impact of Cre recombinase on the transcriptome was minimal. In the absence of drought stress, overexpression of ABF3 had no effect on the transcriptome, but following drought stress, differences were observed in the gene expression patterns of plants overexpressing ABF3 relative to control plants. Examination of the functional distribution of the differentially expressed genes revealed strong similarity indicating that unintended pathways were not activated. The action of ABF3 is tightly controlled in Arabidopsis. In the absence of drought stress, ectopic activation of drought response pathways does not occur. In response to drought stress, overexpression of ABF3 results in a reprogramming of

  14. Transgenic overexpression of 14-3-3 zeta protects hippocampus against endoplasmic reticulum stress and status epilepticus in vivo.

    Directory of Open Access Journals (Sweden)

    Gary P Brennan

    Full Text Available 14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ isoform has been linked to endoplasmic reticulum (ER function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress.

  15. Hyperlipidemia and cutaneous abnormalities in transgenic mice overexpressing human apolipoprotein C1

    NARCIS (Netherlands)

    Jong, M. C.; Gijbels, M. J.; Dahlmans, V. E.; Gorp, P. J.; Koopman, S. J.; Ponec, M.; Hofker, M. H.; Havekes, L. M.

    1998-01-01

    Transgenic mice were generated with different levels of human apolipoprotein C1 (APOC1) expression in liver and skin. At 2 mo of age, serum levels of cholesterol, triglycerides (TG), and FFA were strongly elevated in APOC1 transgenic mice compared with wild-type mice. These elevated levels of serum

  16. Overexpression of a glycosyltransferase gene SrUGT74G1 from Stevia improved growth and yield of transgenic Arabidopsis by catechin accumulation.

    Science.gov (United States)

    Guleria, Praveen; Yadav, Sudesh Kumar

    2014-03-01

    Steviol glycoside and gibberellin biosynthetic routes are known as divergent branches of a common origin in Stevia. A UDP-glycosyltransferase encoded by SrUGT74G1 catalyses the conversion of steviolbioside into stevioside in Stevia rebaudiana leaves. In the present study, transgenic Arabidopsis thaliana overexpressing SrUGT74G1 cDNA from Stevia were developed to check the probability of stevioside biosynthesis in them. However, stevioside accumulation was not evident in transgenics. Also, the transgenic Arabidopsis showed no change in GA3 content on SrUGT74G1 overexpression. Surprisingly, significant accumulation of catechin was noticed in transgenics. The transgenics showed a considerable increase in shoot length, root length and rosette area. An increase in free radical scavenging activity of transgenics was noticed. Moreover, the seed yield of transgenics was also increased by 6-15% than control. Additionally, variation in trichome branching pattern on leaf surface of transgenics was observed. The trichome branching pattern was also validated by exogenous catechin exposure (10, 50, 100 ng ml(-1)) to control plants. Hence, present study reports the probable role of SrUGT74G1 from Stevia in catechin accumulation of transgenic Arabidopsis thaliana. Thus, detailed study in present perspective has revealed the role of Stevia SrUGT74G1 gene in trichome branching pattern, improved vegetative growth, scavenging potential and seed yield by catechin accumulation in transgenic Arabidopsis.

  17. Changes in amyloid-β and Tau in the cerebrospinal fluid of transgenic mice overexpressing amyloid precursor protein.

    Science.gov (United States)

    Maia, Luis F; Kaeser, Stephan A; Reichwald, Julia; Hruscha, Michael; Martus, Peter; Staufenbiel, Matthias; Jucker, Mathias

    2013-07-17

    Altered concentrations of amyloid-β (Aβ) peptide and Tau protein in the cerebrospinal fluid (CSF) are thought to be predictive markers for Alzheimer's disease (AD). Transgenic mice overexpressing human amyloid precursor protein (APP) have been used to model Aβ pathology, but concomitant changes in Aβ and Tau in CSF have been less well studied. We measured Aβ and Tau in the brains and CSF of two well-characterized transgenic mouse models of AD: one expressing human APP carrying the Swedish mutation (APP23) and the other expressing mutant human APP and mutant human presenilin-1 (APPPS1). Both mouse models exhibit Aβ deposition in the brain, but with different onset and progression trajectories. We found an age-related 50 to 80% decrease in Aβ42 peptide in mouse CSF and a smaller decrease in Aβ40, both inversely correlated with the brain Aβ load. Surprisingly, the same mice showed a threefold increase in total endogenous murine Tau in CSF at the stages when Aβ pathology became prominent. The results mirror the temporal sequence and magnitude of Aβ and Tau changes in the CSF of patients with sporadic and dominantly inherited AD. This observation indicates that APP transgenic mice may be useful as a translational tool for predicting changes in Aβ and Tau markers in the CSF of AD patients. These findings also suggest that APP transgenic mouse models may be useful in the search for new disease markers for AD.

  18. Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic ¤Nicotiana sylvestris¤

    DEFF Research Database (Denmark)

    Michalecka, A.M.; Agius, S.C.; Møller, I.M.

    2004-01-01

    The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD(P)H...... dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity...... for NADPH and dependent on calcium for activity. Transgenic lines overexpressing St-ndb1 had specifically increased protein levels for alternative oxidase and uncoupling protein, as compared to the WT and one co-suppressing line. This indicates cross-talk in the expressional control, or metabolic conditions...

  19. Analysis of the effects of overexpression of metallothionein-I in transgenic mice on the reproductive toxicology of cadmium.

    OpenAIRE

    Dalton, T; Fu, K; Enders, G C; Palmiter, R D; Andrews, G K

    1996-01-01

    Exposure to low levels of cadmium reduces fertility. In male mice spermatogenesis is highly sensitive to cadmium, whereas in females the peri-implantation period of pregnancy is sensitive. To examine the potential roles of the cadmium-binding protein, metallothionein (MT), in the reproductive toxicology of cadmium, we examined a transgenic mouse strain that overexpresses metallothionein-I (MT-I). These mice had dramatically increased steady-state levels of MT-I mRNA and MT in the testes and i...

  20. The Transgenic Overexpression of α-Synuclein and Not Its Related Pathology Associates with Complex I Inhibition*

    OpenAIRE

    Loeb, Virginie; Yakunin, Eugenia; Saada, Ann; Sharon, Ronit

    2010-01-01

    α-Synuclein (αS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance αS pathogenesis. Here we show that αS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T αS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I ac...

  1. In Vitro Preservation of Transgenic Tomato (Solanum lycopersicum L. Plants Overexpressing the Stress-Related SlAREB1 Transcription Factor

    Directory of Open Access Journals (Sweden)

    Ayed M. Al-Abdallat

    2017-07-01

    Full Text Available In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm and abscisic acid (0, 4, 8, 12 μm in Murashige and Skoog (MS media, while cryopreservation was conducted by using encapsulation dehydration, V-cryoplates and seeds. Significant differences were observed between tested lines grown on MS media supplemented with 200 mm sucrose where transgenic lines overexpressing SlAREB1 showed improved growth when compared with negative control. The addition of abscisic acid (ABA to the preservation media affected negatively transgenic lines growth and development when compared with ABA-free media. In encapsulation dehydration, non-cryopreserved transgenic lines overexpressing SlAREB1 pretreated in 0.8 M sucrose for 1 day and subjected to different dehydration periods showed significantly higher survival percentages when compared with negative control. For V-cryoplates technique, cryopreserved transgenic lines overexpressing SlAREB1 treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly higher survival percentages when compared with the negative control. Seed cryopreservation was performed successfully with a clear reduction in germination percentage in transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines overexpressing SlAREB1 were found to improve tolerance against different abiotic stresses associated with different in vitro preservation protocols.

  2. Overexpression of SbSI-1, A Nuclear Protein from Salicornia brachiata Confers Drought and Salt Stress Tolerance and Maintains Photosynthetic Efficiency in Transgenic Tobacco.

    Science.gov (United States)

    Kumari, Jyoti; Udawat, Pushpika; Dubey, Ashish K; Haque, Md Intesaful; Rathore, Mangal S; Jha, Bhavanath

    2017-01-01

    A novel Salicornia brachiata Salt Inducible (SbSI-1) gene was isolated and overexpressed in tobacco for in planta functional validation subjected to drought and salt stress. SbSI-1 is a nuclear protein. The transgenic tobacco overexpressing SbSI-1 gene exhibited better seed germination, growth performances, pigment contents, cell viability, starch accumulation, and tolerance index under drought and salt stress. Overexpression of SbSI-1 gene alleviated the build-up of reactive oxygen species (ROS) and curtailed the ROS-induced oxidative damages thus improved the physiological health of transgenic tobacco under stressed conditions. The higher activities of antioxidant enzymes, lower accumulation of ROS, higher membrane stability, relative water content, and polyphenol contents indicated the better survival of the transgenic tobacco than wild-type (WT) tobacco under stressed conditions. Transgenic tobacco had a higher net photosynthetic rate, PSII operating efficiency, and performance index under drought and salt stress. Higher accumulation of compatible solutes and K+/Na+ ratio in transgenic tobacco than WT showed the better osmotic and redox homeostasis under stressed conditions. The up-regulation of genes encoding antioxidant enzymes (NtSOD, NtAPX, and NtCAT) and transcription factors (NtDREB2 and NtAP2) in transgenic tobacco under stressed conditions showed the role of SbSI-1 in ROS alleviation and involvement of this gene in abiotic stress tolerance. Multivariate data analysis exhibited statistical distinction among growth responses, physiological health, osmotic adjustment, and photosynthetic responses of WT and transgenic tobacco under stressed conditions. The overexpression of SbSI-1 gene curtailed the ROS-induced oxidative damages and maintained the osmotic homeostasis under stress conditions thus improved physiological health and photosynthetic efficiencies of the transgenic tobacco overexpressing SbSI-1 gene.

  3. Overexpression of SbSI-1, A Nuclear Protein from Salicornia brachiata Confers Drought and Salt Stress Tolerance and Maintains Photosynthetic Efficiency in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    Jyoti Kumari

    2017-07-01

    Full Text Available A novel SalicorniabrachiataSalt Inducible (SbSI-1 gene was isolated and overexpressed in tobacco for in planta functional validation subjected to drought and salt stress. SbSI-1 is a nuclear protein. The transgenic tobacco overexpressing SbSI-1 gene exhibited better seed germination, growth performances, pigment contents, cell viability, starch accumulation, and tolerance index under drought and salt stress. Overexpression of SbSI-1 gene alleviated the build-up of reactive oxygen species (ROS and curtailed the ROS-induced oxidative damages thus improved the physiological health of transgenic tobacco under stressed conditions. The higher activities of antioxidant enzymes, lower accumulation of ROS, higher membrane stability, relative water content, and polyphenol contents indicated the better survival of the transgenic tobacco than wild-type (WT tobacco under stressed conditions. Transgenic tobacco had a higher net photosynthetic rate, PSII operating efficiency, and performance index under drought and salt stress. Higher accumulation of compatible solutes and K+/Na+ ratio in transgenic tobacco than WT showed the better osmotic and redox homeostasis under stressed conditions. The up-regulation of genes encoding antioxidant enzymes (NtSOD, NtAPX, and NtCAT and transcription factors (NtDREB2 and NtAP2 in transgenic tobacco under stressed conditions showed the role of SbSI-1 in ROS alleviation and involvement of this gene in abiotic stress tolerance. Multivariate data analysis exhibited statistical distinction among growth responses, physiological health, osmotic adjustment, and photosynthetic responses of WT and transgenic tobacco under stressed conditions. The overexpression of SbSI-1 gene curtailed the ROS-induced oxidative damages and maintained the osmotic homeostasis under stress conditions thus improved physiological health and photosynthetic efficiencies of the transgenic tobacco overexpressing SbSI-1 gene.

  4. Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

    Science.gov (United States)

    Ganapathi, Thumballi R.

    2015-01-01

    Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular. PMID:25962076

  5. Effects of transgenic overexpression of diapause hormone and diapause hormone receptor genes on non-diapause silkworm.

    Science.gov (United States)

    Gong, Chunying; Zeng, Wenhui; Zhang, Tianyang; Liu, Rongpeng; Ou, Yao; Ai, Junwen; Xiang, Zhonghuai; Xu, Hanfu

    2017-12-01

    Diapause is a state of developmental arrest that is most often observed in arthropods, especially insects. The domesticated silkworm, Bombyx mori, is a typical insect that enters diapause at an early embryonic stage. Previous studies have revealed that the diapause hormone (DH) signaling molecules, especially the core members DH and DH receptor 1 (DHR1), are crucial for the determination of embryonic diapause in diapause silkworm strains. However, whether they function in non-diapause silkworm strains remains largely unknown. Here, we generated two transgenic lines overexpressing DH or DHR1 genes in a non-diapause silkworm strain, Nistari. Our results showed that developmental expression patterns of DH and DHR1 are quite similar in transgenic silkworms: both genes are highly expressed in the mid to late stages of pupae and are most highly expressed in day-6 pupae but are expressed at very low levels in other developmental stages. Moreover, the overexpression of DH or DHR1 can affect the expression of diapause-related genes but is not sufficient to induce embryonic diapause in their offspring. This study provides new insights into the function of DH and DHR1 in a non-diapause silkworm strain.

  6. Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants.

    Directory of Open Access Journals (Sweden)

    Siddhesh B Ghag

    Full Text Available Micro RNAs (miRNAs are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.

  7. Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2015-01-01

    Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.

  8. A53T Human α-Synuclein Overexpression in Transgenic Mice Induces Pervasive Mitochondria Macroautophagy Defects Preceding Dopamine Neuron Degeneration

    Science.gov (United States)

    Xie, Zhiguo; Turkson, Susie

    2015-01-01

    In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies. PMID:25609609

  9. Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    2017-03-01

    Full Text Available Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF, a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

  10. Overexpression of the WOX gene STENOFOLIA improves biomass yield and sugar release in transgenic grasses and display altered cytokinin homeostasis.

    Science.gov (United States)

    Wang, Hui; Niu, Lifang; Fu, Chunxiang; Meng, Yingying; Sang, Dajun; Yin, Pengcheng; Wu, Jinxia; Tang, Yuhong; Lu, Tiegang; Wang, Zeng-Yu; Tadege, Million; Lin, Hao

    2017-03-01

    Lignocellulosic biomass can be a significant source of renewable clean energy with continued improvement in biomass yield and bioconversion strategies. In higher plants, the leaf blade is the central energy convertor where solar energy and CO2 are assimilated to make the building blocks for biomass production. Here we report that introducing the leaf blade development regulator STENOFOLIA (STF), a WOX family transcription factor, into the biofuel crop switchgrass, significantly improves both biomass yield and sugar release. We found that STF overexpressing switchgrass plants produced approximately 2-fold more dry biomass and release approximately 1.8-fold more solubilized sugars without pretreatment compared to controls. The biomass increase was attributed mainly to increased leaf width and stem thickness, which was also consistent in STF transgenic rice and Brachypodium, and appeared to be caused by enhanced cell proliferation. STF directly binds to multiple regions in the promoters of some cytokinin oxidase/dehydrogenase (CKX) genes and represses their expression in all three transgenic grasses. This repression was accompanied by a significant increase in active cytokinin content in transgenic rice leaves, suggesting that the increase in biomass productivity and sugar release could at least in part be associated with improved cytokinin levels caused by repression of cytokinin degrading enzymes. Our study provides a new tool for improving biomass feedstock yield in bioenergy crops, and uncovers a novel mechanistic insight in the function of STF, which may also apply to other repressive WOX genes that are master regulators of several key plant developmental programs.

  11. Netrin-1 Promotes Inflammation Resolution to Achieve Endothelialization of Small-Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ.

    Science.gov (United States)

    Li, Yanzhao; Wan, Simin; Liu, Ge; Cai, Wang; Huo, Da; Li, Gang; Yang, Mingcan; Wang, Yuxin; Guan, Ge; Ding, Ning; Liu, Feila; Zeng, Wen; Zhu, Chuhong

    2017-12-01

    The transplant of small-diameter tissue engineering blood vessels (small-diameter TEBVs) (vascular replacement therapy often fails because of early onset thrombosis and long-standing chronic inflammation. The specific inflammation state involved in small-diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small-diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin-1) is found present in endothelial cells of natural blood vessels and has anti-inflammatory effects. This work generates netrin-1-modified small-diameter TEBVs by using layer-by-layer self-assembly to resolve the inflammation. The results show that netrin-1 reprograms macrophages (MΦ) to assume an anti-inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small-diameter TEBVs modified by netrin-1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small-diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ-derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

  12. Populus euphratica XTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants

    Science.gov (United States)

    Chen, Shaoliang

    2013-01-01

    Populus euphratica is a salt-tolerant tree species that develops leaf succulence after a prolonged period of salinity stress. In the present study, a putative xyloglucan endotransglucosylase/hydrolase gene (PeXTH) from P. euphratica was isolated and transferred to tobacco plants. PeXTH localized exclusively to the endoplasmic reticulum and cell wall. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH overexpression. Compared with the wild type, PeXTH-transgenic plants contained 36% higher water content per unit area and 39% higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a 47–78% greater net photosynthesis under control and salt treatments (100–150mM NaCl). Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling. PMID:24085577

  13. Populus euphratica XTH overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants.

    Science.gov (United States)

    Han, Yansha; Wang, Wei; Sun, Jian; Ding, Mingquan; Zhao, Rui; Deng, Shurong; Wang, Feifei; Hu, Yue; Wang, Yang; Lu, Yanjun; Du, Liping; Hu, Zanmin; Diekmann, Heike; Shen, Xin; Polle, Andrea; Chen, Shaoliang

    2013-11-01

    Populus euphratica is a salt-tolerant tree species that develops leaf succulence after a prolonged period of salinity stress. In the present study, a putative xyloglucan endotransglucosylase/hydrolase gene (PeXTH) from P. euphratica was isolated and transferred to tobacco plants. PeXTH localized exclusively to the endoplasmic reticulum and cell wall. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH overexpression. Compared with the wild type, PeXTH-transgenic plants contained 36% higher water content per unit area and 39% higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a 47-78% greater net photosynthesis under control and salt treatments (100-150 mM NaCl). Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling.

  14. Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity.

    Directory of Open Access Journals (Sweden)

    Justin Wai-Chung Leung

    Full Text Available Endothelin-1 (ET-1 is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1 with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A receptor (BQ123 but not by ET(B receptor (BQ788 antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.

  15. Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS.

    Science.gov (United States)

    Siu, Kin Lung; Lotz, Christopher; Ping, Peipei; Cai, Hua

    2015-01-01

    Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this "pharmacological" treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may

  16. Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits

    Directory of Open Access Journals (Sweden)

    Shoucui Gao

    2017-01-01

    Full Text Available High levels of plasma high-density lipoprotein-cholesterol (HDL-C are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.

  17. Transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

    Science.gov (United States)

    Liu, Bigang; Gong, Shuai; Li, Qiuhui; Chen, Xin; Moore, John; Suraneni, Mahipal V; Badeaux, Mark D; Jeter, Collene R; Shen, Jianjun; Mehmood, Rashid; Fan, Qingxia; Tang, Dean G

    2017-08-08

    This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo , we generated a NanogP8 transgenic mouse model, in which the ARR 2 PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR 2 PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR 2 PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR 2 PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR 2 PB-NanogP8 transgenic mice with ARR 2 PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR 2 PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

  18. Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

    Science.gov (United States)

    Kim, Won-Seok; Chronis, Demosthenis; Juergens, Matthew; Schroeder, Amy C; Hyun, Seung Won; Jez, Joseph M; Krishnan, Hari B

    2012-01-01

    Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

  19. Transgenic Overexpression of the Proprotein Convertase Furin Enhances Skin Tumor Growth

    Directory of Open Access Journals (Sweden)

    Jian Fu

    2012-04-01

    Full Text Available Furin, one of the members of the family of proprotein convertases (PCs, ubiquitously expressed as a type I membrane-bound proteinase, activates several proteins that contribute to tumor progression. In vitro studies using cancer cell lines and clinical specimens demonstrated that furin processes important substrates such as insulin-like growth factor 1 receptor (IGF-1R and transforming growth factor β, leading to increased tumor growth and progression. Despite the numerous studies associating furin with tumor development, its effects in preclinical models has not been comprehensively studied. In this study, we sought to determine the protumorigenic role of furin in vivo after a two-stage chemical carcinogenesis protocol in transgenic mice in which furin expression was targeted to the epidermal basal layer. We found that processing of the PC substrate IGF-1R and the proliferation rate of mouse epidermis was enhanced in transgenic mice when compared with their WT counterparts. Histopathologic diagnoses of the tumors demonstrated that furin transgenic mice (line F47 developed twice as many squamous carcinomas as the control, WT mice (P < .002. Similarly, tumors cells from transgenic mice were able to process PC substrates more efficiently than tumor cells from WT mice. Furthermore, furin expression resulted in a higher SCC volume in transgenic mice as well as an increase in the percentage of high-grade SCC, including poorly differentiated and spindle cell carcinomas. In conclusion, expression of furin in the basal layer of the epidermis increased tumor development and enhanced tumor growth, supporting the consideration of furin as a potential target for cancer treatment.

  20. Overexpression of a weed (Solanum americanum) proteinase inhibitor in transgenic tobacco results in increased glandular trichome density and enhanced resistance to Helicoverpa armigera and Spodoptera litura.

    Science.gov (United States)

    Luo, Ming; Wang, Zhaoyu; Li, Huapeng; Xia, Kuai-Fei; Cai, Yinpeng; Xu, Zeng-Fu

    2009-04-23

    In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

  1. Overexpression of a Weed (Solanum americanum Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu

    2009-04-01

    Full Text Available In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm(Helicoverpa armigera and tobacco cutworm(Spodoptera litura larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

  2. Double overexpression of DREB and PIF transcription factors improves drought stress tolerance and cell elongation in transgenic plants.

    Science.gov (United States)

    Kudo, Madoka; Kidokoro, Satoshi; Yoshida, Takuya; Mizoi, Junya; Todaka, Daisuke; Fernie, Alisdair R; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2017-04-01

    Although a variety of transgenic plants that are tolerant to drought stress have been generated, many of these plants show growth retardation. To improve drought tolerance and plant growth, we applied a gene-stacking approach using two transcription factor genes: DEHYDRATION-RESPONSIVE ELEMENT-BINDING 1A (DREB1A) and rice PHYTOCHROME-INTERACTING FACTOR-LIKE 1 (OsPIL1). The overexpression of DREB1A has been reported to improve drought stress tolerance in various crops, although it also causes a severe dwarf phenotype. OsPIL1 is a rice homologue of Arabidopsis PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), and it enhances cell elongation by activating cell wall-related gene expression. We found that the OsPIL1 protein was more stable than PIF4 under light conditions in Arabidopsis protoplasts. Transactivation analyses revealed that DREB1A and OsPIL1 did not negatively affect each other's transcriptional activities. The transgenic plants overexpressing both OsPIL1 and DREB1A showed the improved drought stress tolerance similar to that of DREB1A overexpressors. Furthermore, double overexpressors showed the enhanced hypocotyl elongation and floral induction compared with the DREB1A overexpressors. Metabolome analyses indicated that compatible solutes, such as sugars and amino acids, accumulated in the double overexpressors, which was similar to the observations of the DREB1A overexpressors. Transcriptome analyses showed an increased expression of abiotic stress-inducible DREB1A downstream genes and cell elongation-related OsPIL1 downstream genes in the double overexpressors, which suggests that these two transcription factors function independently in the transgenic plants despite the trade-offs required to balance plant growth and stress tolerance. Our study provides a basis for plant genetic engineering designed to overcome growth retardation in drought-tolerant transgenic plants. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology

  3. Transgenic overexpression of expansin influences particle size distribution and improves viscosity of tomato juice and paste

    NARCIS (Netherlands)

    Kalamaki, M.S.; Powell, A.L.T.; Struijs, K.; Labavitch, J.M.; Reid, D.S.; Bennett, A.B.

    2003-01-01

    Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products,

  4. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants

    Science.gov (United States)

    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-01-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na+/H+) antiporter that transports Na+ into the vacuole and exports H+ into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na+/H+ antiporter that exports Na+ to the extracellular space and imports H+ into the plant cell. Plants rely on these enzymes either to keep Na+ out of the cell or to sequester Na+ into vacuoles to avoid the toxic level of Na+ in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. PMID:26985021

  5. Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1

    DEFF Research Database (Denmark)

    Zhao, Liang; Svingen, Terje; Ting Ng, Ee

    2015-01-01

    Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable...... for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal...... testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans....

  6. Parvalbumin overexpression alters immune-mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis

    Science.gov (United States)

    Beers, D. R.; Ho, B. K.; Siklos, L.; Alexianu, M. E.; Mosier, D. R.; Mohamed, A. H.; Otsuka, Y.; Kozovska, M. E.; McAlhany, R. E.; Smith, R. G.; hide

    2001-01-01

    Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium-binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated 'toxic-gain-of-function' in transgenic mice.

  7. Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk.

    Science.gov (United States)

    Cui, Dan; Li, Jia; Zhang, Linlin; Liu, Shen; Wen, Xiao; Li, Qiuyan; Zhao, Yaofeng; Hu, Xiaoxiang; Zhang, Ran; Li, Ning

    2015-04-01

    Intensive swine production industry uses antibiotics to treat diseases and improve pig growth. This can not only cause antibiotic resistance, but can also pollute the environment or eventually affect human public health. To date, human lactoferrin (hLF) and human lysozyme (hLZ) have been known as non-adaptive but interactive antimicrobial members and could act in concert against bacteria, which contribute to host defense. Therefore, their expression in pigs might be an alternative strategy for replacing antibiotics in the pig production industry. In our study, we produced hLF and hLZ bi-transgenic pigs and assessed the milk's antibacterial ability. Integration of both transgenes was confirmed by PCR and southern blot. Both the hLF and hLZ were expressed in the mammary gland of bi-transgenic pigs, as detected by western blotting. The expression amounts were 6.5 g/L for hLF and 1.1 mg/L for hLZ using ELISA. Interestingly, pig milk containing hLF and hLZ had synergistic antimicrobial activity. Our results suggest an alternative approach for avoiding the use of antibiotics in the pig industry, which would be of great benefit to the commercial swine production.

  8. Increased thermoregulation in cold-exposed transgenic mice overexpressing lipoprotein lipase in skeletal muscle: an avian phenotype?

    Science.gov (United States)

    Jensen, Dalan R; Knaub, Leslie A; Konhilas, John P; Leinwand, Leslie A; MacLean, Paul S; Eckel, Robert H

    2008-04-01

    LPL is an enzyme involved in the breakdown and uptake of lipoprotein triglycerides. In the present study, we examined how the transgenic (Tg) overexpression of human LPL in mouse skeletal muscle affected tolerance to cold temperatures, cold-induced thermogenesis, and fuel utilization during this response. Tg mice and their nontransgenic controls were placed in an environmental chamber and housed in metabolic chambers that monitored oxygen consumption and carbon dioxide production with calorimetry. When exposed to 4 degrees C, an attenuation in the decline in body temperature in Tg mice was accompanied by an increased metabolic rate (15%; P cold tolerance and thermogenesis in Tg mice. The more oxidative type IIa fibers were favored over the more glycolytic type IIb fibers (P cold tolerance by enhancing the capacity for fat oxidation, producing an avian-like phenotype in which skeletal muscle contributes significantly to the thermogenic response to cold temperatures.

  9. Overexpression of Poplar Pyrabactin Resistance-Like Abscisic Acid Receptors Promotes Abscisic Acid Sensitivity and Drought Resistance in Transgenic Arabidopsis.

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    Jingling Yu

    Full Text Available Drought stress is an important environmental factor limiting productivity of plants, especially fast growing species with high water consumption like poplar. Abscisic acid (ABA is a phytohormone that positively regulates seed dormancy and drought resistance. The PYR1 (Pyrabactin Resistance 1/ PYRL (PYR-Like/ RCAR (Regulatory Component of ABA Receptor (PYR/PYL/RCAR ABA receptor family has been identified and widely characterized in Arabidopsis thaliana. However, their functions in poplars remain unknown. Here, we report that 2 of 14 PYR/PYL/RCAR orthologues in poplar (Populus trichocarpa (PtPYRLs function as a positive regulator of the ABA signal transduction pathway. The Arabidopsis transient expression and yeast two-hybrid assays showed the interaction among PtPYRL1 and PtPYRL5, a clade A protein phosphatase 2C, and a SnRK2, suggesting that a core signalling complex for ABA signaling pathway exists in poplars. Phenotypic analysis of PtPYRL1 and PtPYRL5 transgenic Arabidopsis showed that these two genes positively regulated the ABA responses during the seed germination. More importantly, the overexpression of PtPYRL1 and PtPYRL5 substantially improved ABA sensitivity and drought stress tolerance in transgenic plants. In summary, we comprehensively uncovered the properties of PtPYRL1 and PtPYRL5, which might be good target genes to genetically engineer drought-Resistant plants.

  10. Establishment and evaluation of a transgenic mouse model of arthritis induced by overexpressing human tumor necrosis factor alpha

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    Ge Li

    2016-04-01

    Full Text Available Tumor necrosis factor alpha (TNFα plays a key role in the pathogenesis of rheumatoid arthritis (RA. Blockade of TNFα by monoclonal antibody has been widely used for the therapy of RA since the 1990s; however, its mechanism of efficacy, and potential safety concerns of the treatment are still not fully understood. This study sought to establish a transgenic arthritic mouse model by overexpressing human TNFα (hTNFα and to apply this model as a means to evaluate therapeutic consequences of TNFα inhibitors. The transgenic mouse line (TgTC with FVB background was generated by incorporating 3′-modified hTNFα gene sequences. A progressively erosive polyarthritis developed in the TgTC mice, with many characteristics observed in human rheumatoid arthritis, including polyarticular swelling, impairment of movement, synovial hyperplasia, and cartilage and bone erosion. Gene expression analysis demonstrated that hTNFα is not only expressed in hyperplastic synovial membrane, but also in tissues without lesions, including brain, lung and kidney. Treatment of the TgTC mice with anti-hTNFα monoclonal antibodies (mAb significantly decreased the level of hTNFα in the diseased joint and effectively prevented development of arthritis in a dose-dependent response fashion. Our results indicated that the TgTC mice represent a genetic model which can be used to comprehensively investigate the pathogenesis and therapeutics of TNFα-related diseases.

  11. An overexpression of chalcone reductase of Pueraria montana var. lobata alters biosynthesis of anthocyanin and 5'-deoxyflavonoids in transgenic tobacco.

    Science.gov (United States)

    Joung, Jae-youl; Kasthuri, G Mangai; Park, Ji-young; Kang, Won-jin; Kim, Hyun-soon; Yoon, Bong-sik; Joung, Hyouk; Jeon, Jae-heung

    2003-03-28

    We isolated the chalcone reductase (pl-chr) gene of Pueraria montana var. lobata by using a PCR strategy from cDNA pools of storage roots. A high level of expression of RNA was found in both stems and roots. The genomic Southern blot result suggests that pl-chr exists as a member of a small gene family. By introducing a pl-chr gene under the control of the 35S CaMV promoter into the pink-flowering Xanthi line of Nicotiana tabacum, the flower color was changed from pink to white-to-pink. The contents of anthocyanin in the flowers of the transgenic lines were dramatically decreased by 40%, but the total UV absorption compounds remained unchanged. The production of liquiritigenin in pl-chr overexpressed transgenic tobacco lines was confirmed by HPLC and MS analysis. The introduction of pl-chr gene provides a method to redirect the flavonoid pathway into 5'-deoxyflavonoid production in non-legume crops, in order to manipulate the phenylpropanoid pathway for isoflavonoid production.

  12. Involvement of autophagy in cardiac remodeling in transgenic mice with cardiac specific over-expression of human programmed cell death 5.

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    Lin An

    Full Text Available Programmed cell death 5 (PDCD5 is a cytosolic protein suppressing growth of multiple types of cancer cells through activating p53. We hypothesized that PDCD5 plays an essential role in cardiac remodeling and function. PDCD5 was significantly up-regulated in the hearts from mice subjected to angiotensin II treatment or transverse aortic constriction. Thus, we generated transgenic mice over-expressing human PDCD5 under the control of alpha myosin heavy chain promoter to examine the role of PDCD5 in cardiac remodeling. Transgenic founder died spontaneously displayed enlarged heart. The high PDCD5 over-expressing line (10-fold showed reduced survival rate, increase in heart weight normalized to body weight. Real-Time RT-PCR analysis revealed fetal gene program was up-regulated. Echocardiography and histopathological examination showed characteristics of dilated cardiomyopathy and heart failure in transgenic mice. Western blot and immunohistochemistry analysis showed autophagy was dramatically increased in transgenic mice as compared to WT littermates control mice, while apoptosis remained unchanged. The enhanced autophagy in high over-expressing line was associated with significant increase in p53 activity and its downstream target damage-regulated autophagy modulator expression. The low over-expressing line (3.5-fold appeared normal, but was more susceptible to angiotensin II-induced cardiac hypertrophy. This study is the first providing evidence that PDCD5 plays an important role in cardiac remodeling.

  13. Comparative analysis of chemical compositions between non-transgenic soybean seeds and those from plants over-expressing AtJMT, the gene for jasmonic acid carboxyl methyltransferase.

    Science.gov (United States)

    Nam, Kyong-Hee; Kim, Do Young; Pack, In-Soon; Park, Jung-Ho; Seo, Jun Sung; Choi, Yang Do; Cheong, Jong-Joo; Kim, Chung Ho; Kim, Chang-Gi

    2016-04-01

    Transgenic overexpression of the Arabidopsis gene for jasmonic acid carboxyl methyltransferase (AtJMT) is involved in regulating jasmonate-related plant responses. To examine its role in the compositional profile of soybean (Glycine max), we compared the seeds from field-grown plants that over-express AtJMT with those of the non-transgenic, wild-type (WT) counterpart. Our analysis of chemical compositions included proximates, amino acids, fatty acids, isoflavones, and antinutrients. Overexpression of AtJMT in the seeds resulted in decreased amounts of tryptophan, palmitic acid, linolenic acid, and stachyose, but increased levels of gadoleic acid and genistein. In particular, seeds from the transgenic soybeans contained 120.0-130.5% more genistein and 60.5-82.1% less stachyose than the WT. A separate evaluation of ingredient values showed that all were within the reference ranges reported for commercially available soybeans, thereby demonstrating the substantial equivalence of these transgenic and non-transgenic seeds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Transgenic Mice Overexpressing the Divalent Metal Transporter 1 Exhibit Iron Accumulation and Enhanced Parkin Expression in the Brain.

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    Zhang, Cheng-Wu; Tai, Yee Kit; Chai, Bing-Han; Chew, Katherine C M; Ang, Eng-Tat; Tsang, Fai; Tan, Bryce W Q; Hong, Eugenia T E; Asad, Abu Bakar Ali; Chuang, Kai-Hsiang; Lim, Kah-Leong; Soong, Tuck Wah

    2017-07-10

    Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson's disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD.

  15. Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice

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    Gong Ping

    2011-12-01

    Full Text Available Abstract Background p23 belongs to the highly conserved p24 family of type I transmembrane proteins, which participate in the bidirectional protein transport between the endoplasmic reticulum and Golgi apparatus. Mammalian p23 has been shown to interact with γ-secretase complex, and modulate secretory trafficking as well as intramembranous processing of amyloid precursor protein in cultured cells. Negative modulation of β-amyloid production by p23 in cultured cell lines suggested that elevation of p23 expression in neurons might mitigate cerebral amyloid burden. Results We generated several lines of transgenic mice expressing human p23 in neurons under the control of Thy-1.2 promoter. We found that even a 50% increase in p23 levels in the central nervous system of mice causes post-natal growth retardation, severe neurological problems characterized by tremors, seizure, ataxia, and uncoordinated movements, and premature death. The severity of the phenotype closely correlated with the level of p23 overexpression in multiple transgenic lines. While the number and general morphology of neurons in Hup23 mice appeared to be normal throughout the brain, abnormal non-Golgi p23 localization was observed in a subset of neurons with high transgene expression in brainstem. Moreover, detailed immunofluorescence analysis revealed marked proliferation of astrocytes, activation of microglia, and thinning of myelinated bundles in brainstem of Hup23 mice. Conclusions These results demonstrate that proper level of p23 expression is critical for neuronal function, and perturbing p23 function by overexpression initiates a cascade of cellular reactions in brainstem that leads to severe motor deficits and other neurological problems, which culminate in premature death. The neurological phenotype observed in Hup23 mice highlights significant adverse effects associated with manipulating neuronal expression of p23, a previously described negative modulator of

  16. Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus) Over-Expressing MicroRNA394

    Science.gov (United States)

    Song, Jian Bo; Shu, Xia Xia; Shen, Qi; Li, Bo Wen; Song, Jun; Yang, Zhi Min

    2015-01-01

    Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus) miR394 with its target gene Brassica napus LEAF CURLING RESPONSIVENESS (BnLCR) to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS) and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development. PMID:25978066

  17. Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7

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    Hennig Lars

    2010-10-01

    Full Text Available Abstract Background The clock-controlled RNA-binding protein AtGRP7 influences circadian oscillations of its own transcript at the post-transcriptional level. To identify additional targets that are regulated by AtGRP7, transcript profiles of transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox and wild type plants were compared. Results Approximately 1.4% of the transcripts represented on the Affymetrix ATH1 microarray showed changes in steady-state abundance upon AtGRP7 overexpression. One third of the differentially expressed genes are controlled by the circadian clock, and they show a distinct bias of their phase: The up-regulated genes preferentially peak around dawn, roughly opposite to the AtGRP7 peak abundance whereas the down-regulated genes preferentially peak at the end of the day. Further, transcripts responsive to abiotic and biotic stimuli were enriched among AtGRP7 targets. Transcripts encoding the pathogenesis-related PR1 and PR2 proteins were elevated in AtGRP7-ox plants but not in plants overexpressing AtGRP7 with a point mutation in the RNA-binding domain, indicating that the regulation involves RNA binding activity of AtGRP7. Gene set enrichment analysis uncovered components involved in ribosome function and RNA metabolism among groups of genes upregulated in AtGRP7-ox plants, consistent with its role in post-transcriptional regulation. Conclusion Apart from regulating a suite of circadian transcripts in a time-of-day dependent manner AtGRP7, both directly and indirectly, affects other transcripts including transcripts responsive to abiotic and biotic stimuli. This suggests a regulatory role of AtGRP7 in the output of the endogenous clock and a complex network of transcripts responsive to external stimuli downstream of the AtGRP7 autoregulatory circuit.

  18. Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus Over-Expressing MicroRNA394.

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    Jian Bo Song

    Full Text Available Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus miR394 with its target gene Brassica napus leaf curling responsiveness (BnLCR to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including leafy cotyledon1 (BnLEC1, BnLEC2, and FUSCA3 (FUS3. Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development.

  19. Overexpression of native ferritin gene MusaFer1 enhances iron content and oxidative stress tolerance in transgenic banana plants.

    Science.gov (United States)

    Yadav, Karuna; Patel, Prashanti; Srivastava, Ashish Kumar; Ganapathi, T R

    2017-01-01

    Iron is an indispensable element for plant growth and defense and hence it is essential to improve the plant's ability to accumulate iron. Besides, it is also an important aspect for human health. In view of this, we attempted to increase the iron content in banana cultivar Rasthali using MusaFer1 as a candidate gene. Initially, the expression of all five genes of the MusaFer family (MusaFer1-5) was quantified under iron-excess and -deficient conditions. The supplementation of 250 and 350 μM iron enhanced expression of all MusaFer genes; however, MusaFer1 was increased maximally by 2- and 4- fold in leaves and roots respectively. Under iron deficient condition, all five MusaFer genes were downregulated, indicating their iron dependent regulation. In MusaFer1 overexpressing lines, iron content was increased by 2- and 3-fold in leaves and roots respectively, as compared with that of untransformed lines. The increased iron was mainly localized in the epidermal regions of petiole. The analysis of MusaFer1 promoter indicated that it might control the expression of iron metabolism related genes and also other genes of MusaFer family. MusaFer1 overexpression led to downregulated expression of MusaFer3, MusaFer4 and MusaFer5 in transgenic leaves which might be associated with the plant's compensatory mechanism in response to iron flux. Other iron metabolism genes like Ferric reductase (FRO), transporters (IRT, VIT and YSL) and chelators (NAS, DMAS and NAAT) were also differentially expressed in transgenic leaf and root, suggesting the multifaceted impact of MusaFer1 towards iron uptake and organ distribution. Additionally, MusaFer1 overexpression increased plant tolerance against methyl viologen and excess iron which was quantified in terms of photosynthetic efficiency and malondialdehyde content. Thus, the study not only broadens our understanding about iron metabolism but also highlights MusaFer1 as a suitable candidate gene for iron fortification in banana.

  20. Overexpression of native ferritin gene MusaFer1 enhances iron content and oxidative stress tolerance in transgenic banana plants.

    Directory of Open Access Journals (Sweden)

    Karuna Yadav

    Full Text Available Iron is an indispensable element for plant growth and defense and hence it is essential to improve the plant's ability to accumulate iron. Besides, it is also an important aspect for human health. In view of this, we attempted to increase the iron content in banana cultivar Rasthali using MusaFer1 as a candidate gene. Initially, the expression of all five genes of the MusaFer family (MusaFer1-5 was quantified under iron-excess and -deficient conditions. The supplementation of 250 and 350 μM iron enhanced expression of all MusaFer genes; however, MusaFer1 was increased maximally by 2- and 4- fold in leaves and roots respectively. Under iron deficient condition, all five MusaFer genes were downregulated, indicating their iron dependent regulation. In MusaFer1 overexpressing lines, iron content was increased by 2- and 3-fold in leaves and roots respectively, as compared with that of untransformed lines. The increased iron was mainly localized in the epidermal regions of petiole. The analysis of MusaFer1 promoter indicated that it might control the expression of iron metabolism related genes and also other genes of MusaFer family. MusaFer1 overexpression led to downregulated expression of MusaFer3, MusaFer4 and MusaFer5 in transgenic leaves which might be associated with the plant's compensatory mechanism in response to iron flux. Other iron metabolism genes like Ferric reductase (FRO, transporters (IRT, VIT and YSL and chelators (NAS, DMAS and NAAT were also differentially expressed in transgenic leaf and root, suggesting the multifaceted impact of MusaFer1 towards iron uptake and organ distribution. Additionally, MusaFer1 overexpression increased plant tolerance against methyl viologen and excess iron which was quantified in terms of photosynthetic efficiency and malondialdehyde content. Thus, the study not only broadens our understanding about iron metabolism but also highlights MusaFer1 as a suitable candidate gene for iron fortification in

  1. Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis

    Science.gov (United States)

    Won, Hee-Young; Jang, Ki-Seok; Min, Kyueng-Whan; Jang, Si-Hyong; Woo, Jong-Kyu; Oh, Seung Hyun; Kong, Gu

    2015-01-01

    Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development. PMID:25938540

  2. Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.

    Science.gov (United States)

    Zhao, Liang; Svingen, Terje; Ng, Ee Ting; Koopman, Peter

    2015-03-15

    Genes related to Dmrt1, which encodes a DNA-binding DM domain transcription factor, act as triggers for primary sex determination in a broad range of metazoan species. However, this role is fulfilled in mammals by Sry, a newly evolved gene on the Y chromosome, such that Dmrt1 has become dispensable for primary sex determination and instead maintains Sertoli cell phenotype in postnatal testes. Here, we report that enforced expression of Dmrt1 in XX mouse fetal gonads using a Wt1-BAC transgene system is sufficient to drive testicular differentiation and male secondary sex development. XX transgenic fetal gonads showed typical testicular size and vasculature. Key ovarian markers, including Wnt4 and Foxl2, were repressed. Sertoli cells expressing the hallmark testis-determining gene Sox9 were formed, although they did not assemble into normal testis cords. Other bipotential lineages differentiated into testicular cell types, including steroidogenic fetal Leydig cells and non-meiotic germ cells. As a consequence, male internal and external reproductive organs developed postnatally, with an absence of female reproductive tissues. These results reveal that Dmrt1 has retained its ability to act as the primary testis-determining trigger in mammals, even though this function is no longer normally required. Thus, Dmrt1 provides a common thread in the evolution of sex determination mechanisms in metazoans. © 2015. Published by The Company of Biologists Ltd.

  3. Overexpression of rice NAC gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton.

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    Guanze Liu

    Full Text Available The SNAC1 gene belongs to the stress-related NAC superfamily of transcription factors. It was identified from rice and overexpressed in cotton cultivar YZ1 by Agrobacterium tumefaciens-mediated transformation. SNAC1-overexpressing cotton plants showed more vigorous growth, especially in terms of root development, than the wild-type plants in the presence of 250 mM NaCl under hydroponic growth conditions. The content of proline was enhanced but the MDA content was decreased in the transgenic cotton seedlings under drought and salt treatments compared to the wild-type. Furthermore, SNAC1-overexpressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in the greenhouse. The performances of the SNAC1-overexpressing lines under drought and salt stress were significantly better than those of the wild-type in terms of the boll number. During the drought and salt treatments, the transpiration rate of transgenic plants significantly decreased in comparison to the wild-type, but the photosynthesis rate maintained the same at the flowering stage in the transgenic plants. These results suggested that overexpression of SNAC1 improve more tolerance to drought and salt in cotton through enhanced root development and reduced transpiration rates.

  4. Promotion of nicotine biosynthesis in transgenic tobacco by overexpressing allene oxide cyclase from Hyoscyamus niger.

    Science.gov (United States)

    Jiang, Keji; Pi, Yan; Hou, Rong; Jiang, Lili; Sun, Xiaofen; Tang, Kexuan

    2009-04-01

    Plant secondary metabolites are a wide variety of low-molecular weight compounds whose productions are often enhanced in response to both biotic and abiotic stresses. Many of the responses are mediated by a class of hormones, named as jasmonates. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most crucial step. Here a heterologous AOC gene from Hyoscyamus niger L. (black henbane), named HnAOC (GenBank accession No. AY708383), was overexpressed in Nicotiana tabacum cv. Petit Havana to investigate the consequence on nicotine content. This study revealed that the transcription of HnAOC in tobacco resulted in overexpression of nicotine biosynthetic pathway genes and higher yield of nicotine, with the maximum of 4.8-fold over control. Therefore, it indicated that without the cost of extrinsic hormones, genetic manipulation of jasmonate biosynthetic pathway genes could be an alternative approach in metabolic engineering for the production of valuable secondary metabolites, which were induced by jasmonates.

  5. Transgenic mice overexpressing the ALS-linked protein Matrin 3 develop a profound muscle phenotype.

    Science.gov (United States)

    Moloney, Christina; Rayaprolu, Sruti; Howard, John; Fromholt, Susan; Brown, Hilda; Collins, Matt; Cabrera, Mariela; Duffy, Colin; Siemienski, Zoe; Miller, Dave; Swanson, Maurice S; Notterpek, Lucia; Borchelt, David R; Lewis, Jada

    2016-11-18

    Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder of upper and lower motor neurons. Mutations in the gene encoding the nuclear matrix protein Matrin 3 have been found in familial cases of ALS, as well as autosomal dominant distal myopathy with vocal cord and pharyngeal weakness. We previously found that spinal cord and muscle, organs involved in either ALS or distal myopathy, have relatively lower levels of Matrin 3 compared to the brain and other peripheral organs in the murine system. This suggests that these organs may be vulnerable to any changes in Matrin 3. In order to determine the role of Matrin 3 in these diseases, we created a transgenic mouse model for human wild-type Matrin 3 using the mouse prion promoter (MoPrP) on a FVB background.We identified three founder transgenic lines that produced offspring in which mice developed either hindlimb paresis or paralysis with hindlimb and forelimb muscle atrophy. Muscles of affected mice showed a striking increase in nuclear Matrin 3, as well as the presence of rounded fibers, vacuoles, nuclear chains, and subsarcolemmal nuclei. Immunoblot analysis of the gastrocnemius muscle from phenotypic mice showed increased levels of Matrin 3 products migrating at approximately 120 (doublet), 90, 70, and 55 kDa. While there was no significant change in the levels of Matrin 3 in the spinal cord in the phenotypic mice, the ventral horn contained individual cells with cytoplasmic redistribution of Matrin 3, as well as gliosis. The phenotypes of these mice indicate that dysregulation of Matrin 3 levels is deleterious to neuromuscular function.

  6. Downregulation of the ACE2/Ang-(1-7)/Mas axis in transgenic mice overexpressing GH.

    Science.gov (United States)

    Muñoz, Marina C; Burghi, Valeria; Miquet, Johanna G; Giani, Jorge F; Banegas, Ricardo D; Toblli, Jorge E; Fang, Yimin; Wang, Feiya; Bartke, Andrzej; Dominici, Fernando P

    2014-05-01

    The renin-angiotensin system (RAS) plays a crucial role in the regulation of physiological homeostasis and diseases such as hypertension, coronary artery disease, and chronic renal failure. In this cascade, the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/AT1 receptor axis induces pathological effects, such as vasoconstriction, cell proliferation, and fibrosis, while the ACE2/Ang-(1-7)/Mas receptor axis is protective for end-organ damage. The altered function of the RAS could be a contributing factor to the cardiac and renal alterations induced by GH excess. To further explore this issue, we evaluated the consequences of chronic GH exposure on the in vivo levels of Ang II, Ang-(1-7), ACE, ACE2, and Mas receptor in the heart and the kidney of GH-transgenic mice (bovine GH (bGH) mice). At the age of 7-8 months, female bGH mice displayed increased systolic blood pressure (SBP), a high degree of both cardiac and renal fibrosis, as well as increased levels of markers of tubular and glomerular damage. Angiotensinogen abundance was increased in the liver and the heart of bGH mice, along with a concomitant increase in cardiac Ang II levels. Importantly, the levels of ACE2, Ang-(1-7), and Mas receptor were markedly decreased in both tissues. In addition, Ang-(1-7) administration reduced SBP to control values in GH-transgenic mice, indicating that the ACE2/Ang-(1-7)/Mas axis is involved in GH-mediated hypertension. The data indicate that the altered expression profile of the ACE2/Ang-(1-7)/Mas axis in the heart and the kidney of bGH mice could contribute to the increased incidence of hypertension, cardiovascular, and renal alterations observed in these animals.

  7. Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-06-01

    Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by

  8. Global Overexpression of ET-1 Decreases Blood Pressure - A Systematic Review and Meta-Analysis of ET-1 Transgenic Mice

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    Yong Ping Lu

    2016-11-01

    Full Text Available Background/Aims: ET-1 has independent effects on blood pressure regulation in vivo, it is involved in tubular water and salt excretion, promotes constriction of smooth muscle cells, modulates sympathetic nerve activity, and activates the liberation of nitric oxide. To determine the net effect of these partially counteracting mechanisms on blood pressure, a systematic meta-analysis was performed. Methods: Based on the principles of Cochrane systematic reviews, we searched in major literature databases - MEDLINE (PubMed, Embase, Google Scholar, and the China Biological Medicine Database (CBM-disc - for articles relevant to the topic of the blood pressure phenotype of endothelin-1 transgenic (ET-1+/+ mice from January 1, 1988 to March 31, 2016. Review Manager Version 5.0 (Rev-Man 5.0 software was applied for statistical analysis. In total thirteen studies reported blood pressure data. Results: The meta-analysis of blood pressure data showed that homozygous ET-1 transgenic mice (ET-1+/+ mice had a significantly lower blood pressure as compared to WT mice (mean difference: -2.57 mmHg, 95% CI: -4.98∼ -0.16, P = 0.04, with minimal heterogeneity (P = 0.86. A subgroup analysis of mice older than 6 months revealed that the blood pressure difference between ET-1+/+ mice and WT mice was even more pronounced (mean difference: -6.19 mmHg, 95% CI: -10.76∼ -1.62, P = 0.008, with minimal heterogeneity (P = 0.91. Conclusion: This meta-analysis provides robust evidence that global ET-1 overexpression in mice lowers blood pressure in an age-dependent manner. Older ET-1+/+ mice have a somewhat more pronounced reduction of blood pressure.

  9. Resistance to BmNPV via Overexpression of an Exogenous Gene Controlled by an Inducible Promoter and Enhancer in Transgenic Silkworm, Bombyx mori

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    Jiang, Liang; Cheng, Tingcai; Zhao, Ping; Yang, Qiong; Wang, Genhong; Jin, Shengkai; Lin, Ping; Xiao, Yang; Xia, Qingyou

    2012-01-01

    The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 105 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity. PMID:22870254

  10. Resistance to BmNPV via overexpression of an exogenous gene controlled by an inducible promoter and enhancer in transgenic silkworm, Bombyx mori.

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    Liang Jiang

    Full Text Available The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP, Bombyx mori A4 promoter (A4P, hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG. After oral inoculation of BmNPV with 3 × 10(5 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

  11. Enhanced water and salt intake in transgenic mice with brain-restricted overexpression of angiotensin (AT1) receptors.

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    Lazartigues, Eric; Sinnayah, Puspha; Augoyard, Ginette; Gharib, Claude; Johnson, Alan Kim; Davisson, Robin L

    2008-11-01

    To address the relative contribution of central and peripheral angiotensin II (ANG II) type 1A receptors (AT(1A)) to blood pressure and volume homeostasis, we generated a transgenic mouse model [neuron-specific enolase (NSE)-AT(1A)] with brain-restricted overexpression of AT(1A) receptors. These mice are normotensive at baseline but have dramatically enhanced pressor and bradycardic responses to intracerebroventricular ANG II or activation of endogenous ANG II production. Here our goal was to examine the water and sodium intake in this model under basal conditions and in response to increased ANG II levels. Baseline water and NaCl (0.3 M) intakes were significantly elevated in NSE-AT(1A) compared with nontransgenic littermates, and bolus intracerebroventricular injections of ANG II (200 ng in 200 nl) caused further enhanced water intake in NSE-AT(1A). Activation of endogenous ANG II production by sodium depletion (10 days low-sodium diet followed by furosemide, 1 mg sc) enhanced NaCl intake in NSE-AT(1A) mice compared with wild types. Fos immunohistochemistry, used to assess neuronal activation, demonstrated sodium depletion-enhanced activity in the anteroventral third ventricle region of the brain in NSE-AT(1A) mice compared with control animals. The results show that brain-selective overexpression of AT(1A) receptors results in enhanced salt appetite and altered water intake. This model provides a new tool for studying the mechanisms of brain AT(1A)-dependent water and salt consumption.

  12. Overexpression of a Weed (Solanum americanum) Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

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    Luo, Ming; Wang, Zhaoyu; Li, Huapeng; Xia, Kuai-Fei; Cai, Yinpeng; Xu, Zeng-Fu

    2009-01-01

    In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for i...

  13. [Transgenic mice overexpressing PACAP in pancreatic beta-cells: acute and chronic effects on insulin and glucose homeostasis].

    Science.gov (United States)

    Tomimoto, Shuhei; Hashimoto, Hitoshi; Shintani, Norihito; Baba, Akemichi

    2004-04-01

    PACAP belongs to the vasoactive intestinal polypeptide (VIP)/secretin/glucagon superfamily, which also includes glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). PACAP shares an insulinotropic property with the latter two peptides; for instance, it stimulates insulin secretion from islets in a glucose-dependent manner at femtomolar concentrations. However, the pathophysiological significance of PACAP in diabetes remains largely unknown, for several reasons, including a lack of low-molecular weight PACAP ligands and a lack of suitable animal models. As an approach to understanding PACAP's pancreatic function in vivo, we have recently generated transgenic mice overexpressing PACAP in islet beta cells under the control of human insulin promoter (Tg mice). As a consequence, it has been demonstrated that in addition to stimulating insulin secretion, PACAP has long-term effects on pancreatic endocrine cells, including proliferation of beta cells during streptozotocin-induced diabetes development as well as aging. These observations provide additional information to support the possibility that drugs associated with PACAP-signaling pathways might be of therapeutic value for the treatment of diabetes. In this review, we briefly summarize these previous studies using Tg mice and also focus on the physiological and pathophysiological roles mediated by PACAP during diabetes development.

  14. Overexpression of Ran gene from Lepidium latifolium L. (LlaRan) renders transgenic tobacco plants hypersensitive to cold stress.

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    Sinha, Vimlendu Bhushan; Grover, Atul; Singh, Sadhana; Pande, Veena; Ahmed, Zakwan

    2014-09-01

    Ran is a multifunctional small GTPase involved in important cellular activities like nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope formation, etc., but is also known to be differentially expressed in response to abiotic stress, particularly low temperature. We have over-expressed Lepidium latifolium (Fam. Brassicaceae) Ran gene in tobacco to study the response of the plants to cold stress (24 h; 4 °C). Transformation of the tobacco plants was verified using PCR targeting Ran gene and co-transformed selectable marker gene nptII. Segregation in Mendelian ratios was validated in five transgenic lines by germination of T1 and T2 seeds on moist filter papers containing 150 mg/l kanamycin. Higher levels of electrolyte leakage and lipid peroxidation pointed towards hypersensitivity of plants. Similarly, lesser proline accumulation compared to wild types also indicated susceptibility of plants to death under chilling conditions. Specific activity of antioxidant enzymes superoxide dismutase and glutathione reductase was also measured under stressed and control conditions. A variation was observed across the different lines, and four out of five lines showed lesser specific activity compared to wild type plants, thus indicating reduced capability of scavenging free radicals. In totality, a strong evidence on induced hypersensitivity to cold stress has been collected which may further be helpful in designing appropriate strategies for engineering crop plants for survival under cold stress conditions.

  15. Overexpression of miR-155 in the liver of transgenic mice alters the expression profiling of hepatic genes associated with lipid metabolism.

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    Xiaolin Lin

    Full Text Available Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG and high-density lipoprotein (HDL, as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into

  16. Overexpression of a Plasma Membrane-Localized SbSRP-Like Protein Enhances Salinity and Osmotic Stress Tolerance in Transgenic Tobacco

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    Avinash Mishra

    2017-04-01

    Full Text Available An obligate halophyte, Salicornia brachiata grows in salt marshes and is considered to be a potential resource of salt- and drought-responsive genes. It is important to develop an understanding of the mechanisms behind enhanced salt tolerance. To increase this understanding, a novel SbSRP gene was cloned, characterized, over-expressed, and functionally validated in the model plant Nicotiana tabacum. The genome of the halophyte S. brachiata contains two homologs of an intronless SbSRP gene of 1,262 bp in length that encodes for a stress-related protein. An in vivo localization study confirmed that SbSRP is localized on the plasma membrane. Transgenic tobacco plants (T1 that constitutively over-express the SbSRP gene showed improved salinity and osmotic stress tolerance. In comparison to Wild Type (WT and Vector Control (VC plants, transgenic lines showed elevated relative water and chlorophyll content, lower malondialdehyde content, lower electrolyte leakage and higher accumulation of proline, free amino acids, sugars, polyphenols, and starch under abiotic stress treatments. Furthermore, a lower build-up of H2O2 content and superoxide-radicals was found in transgenic lines compared to WT and VC plants under stress conditions. Transcript expression of Nt-APX (ascorbate peroxidase, Nt-CAT (catalase, Nt-SOD (superoxide dismutase, Nt-DREB (dehydration responsive element binding factor, and Nt-AP2 (apetala2 genes was higher in transgenic lines under stress compared to WT and VC plants. The results suggested that overexpression of membrane-localized SbSRP mitigates salt and osmotic stress in the transgenic tobacco plant. It was hypothesized that SbSRP can be a transporter protein to transmit the environmental stimuli downward through the plasma membrane. However, a detailed study is required to ascertain its exact role in the abiotic stress tolerance mechanism. Overall, SbSRP is a potential candidate to be used for engineering salt and osmotic

  17. Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses.

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    Sreedharan, Shareena; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2013-10-01

    Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause-effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse-maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress-inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons

  18. Netrin-1 hyperexpression in mouse brain promotes angiogenesis and long-term neurological recovery after transient focal ischemia.

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    Lu, Haiyan; Wang, Yongting; He, Xiaosong; Yuan, Falei; Lin, Xiaojie; Xie, Bohua; Tang, Guanghui; Huang, Jun; Tang, Yaohui; Jin, Kunlin; Chen, Shengdi; Yang, Guo-Yuan

    2012-03-01

    Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focal neovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression on focal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO). Adeno-associated viral vectors carrying either the NT-1 gene (AAV-NT-1) or GFP (AAV-GFP) were generated and injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7-28 days of reperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7-28 days after tMCAO. Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiation microangiography and immunohistochemistry. Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achieved in both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily in neurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison to saline or AAV-GFP-treated groups (PAAV-NT-1-treated mice compared with mice in saline or AAV-GFP-treated groups (PAAV-NT-1-treated mice compared with the other 2 groups (PAAV-NT-1-treated mice than in other groups. AAV-NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.

  19. Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications.

    Science.gov (United States)

    Shafi, Amrina; Dogra, Vivek; Gill, Tejpal; Ahuja, Paramvir Singh; Sreenivasulu, Yelam

    2014-01-01

    Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.

  20. Overexpression of angiotensinogen downregulates aquaporin 1 expression via modulation of Nrf2–HO-1 pathway in renal proximal tubular cells of transgenic mice

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    Shiao-Ying Chang

    2016-09-01

    Full Text Available Introduction: We aimed to examine the regulation of aquaporin 1 expression in an angiotensinogen transgenic mouse model, focusing on underlying mechanisms. Methods: Male transgenic mice overexpressing rat angiotensinogen in their renal proximal tubular cells (RPTCs and rat immortalised RPTCs stably transfected with rat angiotensinogen cDNA were used. Results: Angiotensinogen-transgenic mice developed hypertension and nephropathy, changes that were either partially or completely attenuated by treatment with losartan or dual renin–angiotensin system blockade (losartan and perindopril, respectively, while hydralazine prevented hypertension but not nephropathy. Decreased expression of aquaporin 1 and heme oxygenase-1 and increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2 and sodium–hydrogen exchanger 3 were observed in RPTCs of angiotensinogen-transgenic mice and in angiotensinogen-transfected immortalised RPTCs. These parameters were normalised by dual renin–angiotensin system blockade. Both in vivo and in vitro studies identified a novel mechanism in which angiotensinogen overexpression in RPTCs enhances the cytosolic accumulation of Nrf2 via the phosphorylation of pGSK3β Y216. Consequently, lower intranuclear Nrf2 levels are less efficient to trigger heme oxygenase-1 expression as a defence mechanism, which subsequently diminishes aquaporin 1 expression in RPTCs. Conclusions: Angiotensinogen-mediated downregulation of aquaporin 1 and Nrf2 signalling may play an important role in intrarenal renin–angiotensin system-induced hypertension and kidney injury.

  1. The overexpression of insect endogenous small RNAs in transgenic rice inhibits growth and delays pupation of striped stem borer (Chilo suppressalis).

    Science.gov (United States)

    Jiang, Shan; Wu, Hao; Liu, Haoju; Zheng, Jie; Lin, Yongjun; Chen, Hao

    2017-07-01

    The striped stem borer (SSB), Chilo suppressalis Walker, is a major rice insect pest worldwide. RNA interference (RNAi) has become a promising strategy for developing insect-resistant crops. In a previous study, five double-stranded RNAs (dsRNAs) targeting important SSB housekeeping genes were overexpressed in rice, but none of the acquired dsRNA-transgenic rice plants showed significant effects on SSB. Thirteen selected SSB endogenous small RNAs, predicted as SSB novel microRNAs (miRNAs), were overexpressed in rice using artificial miRNA (amiRNA) expression technology. Feeding tests showed that two out of 13 selected SSB novel miRNAs caused significant growth inhibition for feeding SSB larvae based on transgenic rice expression. Pupation was delayed 4 days when SSB larvae consecutively fed on transgenic rice expressing the SSB novel miRNA candidate csu-novel-miR15 (csu-15 rice). Gene expression analysis confirmed that the expression levels of at least six SSB unigenes significantly changed (i.e., were up- or down-regulated) after feeding on csu-15 rice. Our research demonstrated a novel RNAi strategy using SSB endogenous small RNAs to develop RNAi crops for pest management; this strategy is different from the common RNAi resulting from transgenic dsRNAs or amiRNAs targeting certain insect endogenous genes. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  2. RanBP9 overexpression accelerates loss of pre and postsynaptic proteins in the APΔE9 transgenic mouse brain.

    Science.gov (United States)

    Wang, Hongjie; Wang, Ruizhi; Xu, Shaohua; Lakshmana, Madepalli K

    2014-01-01

    There is now compelling evidence that the neurodegenerative process in Alzheimer's disease (AD) begins in synapses. Loss of synaptic proteins and functional synapses in the amyloid precursor protein (APP) transgenic mouse models of AD is well established. However, what is the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to robustly increased amyloid β peptide (Aβ) generation leading to enhanced amyloid plaque burden in a mouse model of AD. In this study we compared synaptic protein levels among four genotypes of mice, i.e., RanBP9 single transgenic (Ran), APΔE9 double transgenic (Dbl), APΔE9/RanBP9 triple transgenic (Tpl) and wild-type (WT) controls. We found significant reductions in the levels of synaptic proteins in both cortex and hippocampus of 5- and 6-months-old but not 3- or 4-months-old mice. Specifically, at 5-months of age, rab3A was reduced in the triple transgenic mice only in the cortex by 25% (pproteins in the mouse brain.

  3. Increased abscisic acid levels in transgenic tobacco over-expressing 9 cis-epoxycarotenoid dioxygenase influence H2O2 and NO production and antioxidant defences.

    Science.gov (United States)

    Zhang, Yiming; Tan, Jiali; Guo, Zhenfei; Lu, Shaoyun; He, Sijian; Shu, Wei; Zhou, Biyan

    2009-05-01

    Abscisic acid (ABA) regulates the plant's adaptive responses to abiotic stresses. Over-expression of the 9-cis-epoxycarotenoid dioxygenase gene (SgNCED1) in the transgenic tobaccos increased ABA content and tolerance to drought and salt stresses. H2O2 and nitric oxide (NO) contents were enhanced in guard cells and mesophyll cells of the transgenic plants, accompanied with increased transcripts and activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). The enhancements of H2O2 and NO and transcripts and activities of antioxidant enzymes in the transgenic plants were blocked by pre-treatments with inhibitor of ABA biosynthesis, scavengers of H2O2 and NO, and inhibitors of NADPH oxidase and NO synthase-like (NOS-like). The elevated production of NO in the transgenic plants was blocked by scavenger of H2O2 and inhibitors of NADPH oxidase, whereas H2O2 level was not affected by scavenger of NO and inhibitor of NOS-like, indicating that H2O2 is essential for the elevated production of NO. The results demonstrate that the increased drought and salt tolerance in the transgenic plants is associated with ABA-induced production of H2O2 via NADPH oxidase and NO via NOS-like, which sequentially induce transcripts and activities of SOD, CAT, APX and GR.

  4. Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice.

    Science.gov (United States)

    Ma, Dezun; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Jiang, Shengwang; Xiao, Gaojun; Cui, Wentao

    2015-08-24

    Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

  5. Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidial MEP pathway.

    Science.gov (United States)

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2014-04-15

    The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Assessment of the phenolic profile, antimicrobial activity and oxidative stability of transgenic Perilla frutescens L.overexpressing tocopherol methyltransferase (γ-tmt) gene.

    Science.gov (United States)

    Ghimire, Bimal Kumar; Yu, Chang Yeon; Chung, Ill-Min

    2017-09-01

    This study evaluated the effects of enhanced concentrations of α-tocopherol and phenolic compounds on the resistance and stability of Perilla oil in transgenic Perilla frutescens plants against various tested pathogenic bacteria by over-expressing the γ-tmt gene. The concentration of phenolic compounds in the non-transgenic samples was 9313.198 ± 18.887 μg g(-1) dry weight (DW), whereas the total concentration of the transgenic samples ranged from 9118.015 ± 18.822 to 10527.612 ± 20.411 μg g(-1) DW. The largest increases in phenolic compounds in the transgenic plants in comparison with the control plants were observed in gallic acid, pyrogallol, 5-sulfosalicylic acid, catechin, chlorogenic acid, vanillin, syringic acid, naringenin, salicylic acid, quercetin, o-coumaric acid, kaempferol, and hesperetin. o-coumaric and benzoic acid acid were the most abundant phenolic acids found in the transgenic plants. Gram-negative bacteria (Salmonella typhimurium) were the most susceptible microorganism against transgenic ethyl acetate extracts with lower measurement of minimum inhibitory concentration (MICs) (0.25 ± 0.03 mg/ml) at an extract concentration of 2 mg/ml in dried plant material. The same extracts were more effective against gram-positive bacteria (Bacillus subtilis) when compared to control plants with MICs values of 0.52 ± 0.02 mg/ml. The suplementation of 20 μg of α-tocopherol (1000 ppm) in combination with ethyl acetate extracts enhanced the antimicrobial activity against S. typhimurium and B. subtilis, compared to the non-transgenic plants. The acid value of transgenic Perilla oil improved by 91.2% and 35.54% relative to the non-transgenic control oil and commercial Perilla oil, respectively. The low acid value suggests that the oil will be less susceptible to lipase action, and more economically viable and thus, may also improve the oil quality for industrial purposes. In addition, extracts obtained from transgenic plants could

  7. Constitutive and stress-inducible overexpression of a native aquaporin gene (MusaPIP2;6) in transgenic banana plants signals its pivotal role in salt tolerance.

    Science.gov (United States)

    Sreedharan, Shareena; Shekhawat, Upendra K Singh; Ganapathi, Thumballi R

    2015-05-01

    High soil salinity constitutes a major abiotic stress and an important limiting factor in cultivation of crop plants worldwide. Here, we report the identification and characterization of a aquaporin gene, MusaPIP2;6 which is involved in salt stress signaling in banana. MusaPIP2;6 was firstly identified based on comparative analysis of stressed and non-stressed banana tissue derived EST data sets and later overexpression in transgenic banana plants was performed to study its tangible functions in banana plants. The overexpression of MusaPIP2;6 in transgenic banana plants using constitutive or inducible promoter led to higher salt tolerance as compared to equivalent untransformed control plants. Cellular localization assay performed using transiently transformed onion peel cells indicated that MusaPIP2;6 protein tagged with green fluorescent protein was translocated to the plasma membrane. MusaPIP2;6-overexpressing banana plants displayed better photosynthetic efficiency and lower membrane damage under salt stress conditions. Our results suggest that MusaPIP2;6 is involved in salt stress signaling and tolerance in banana.

  8. Transgenic Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) Overexpressing S-Adenosylmethionine Decarboxylase (SAMDC) Gene for Improved Cold Tolerance Through Involvement of H2O2 and NO Signaling.

    Science.gov (United States)

    Luo, Jianhao; Liu, Mingxi; Zhang, Chendong; Zhang, Peipei; Chen, Jingjing; Guo, Zhenfei; Lu, Shaoyun

    2017-01-01

    Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass species. Transgenic centipedgrass plants overexpressing S-adenosylmethionine decarboxylase from bermudagrass (CdSAMDC1) that was induced in response to cold were generated in this study. Higher levels of CdSAMDC1 transcript and sperimidine (Spd) and spermin (Spm) concentrations and enhanced freezing and chilling tolerance were observed in transgenic plants as compared with the wild type (WT). Transgenic plants had higher levels of polyamine oxidase (PAO) activity and H2O2 than WT, which were blocked by pretreatment with methylglyoxal bis (guanylhydrazone) or MGBG, inhibitor of SAMDC, indicating that the increased PAO and H2O2 were a result of expression of CdSAMDC1. In addition, transgenic plants had higher levels of nitrate reductase (NR) activity and nitric oxide (NO) concentration. The increased NR activity were blocked by pretreatment with MGBG and ascorbic acid (AsA), scavenger of H2O2, while the increased NO level was blocked by MGBG, AsA, and inhibitors of NR, indicating that the enhanced NR-derived NO was dependent upon H2O2, as a result of expression CdSAMDC1. Elevated superoxide dismutase (SOD) and catalase (CAT) activities were observed in transgenic plants than in WT, which were blocked by pretreatment with MGBG, AsA, inhibitors of NR and scavenger of NO, indicating that the increased activities of SOD and CAT depends on expression of CdSAMDC1, H2O2, and NR-derived NO. Our results suggest that the elevated cold tolerance was associated with PAO catalyzed production of H2O2, which in turn led to NR-derived NO production and induced antioxidant enzyme activities in transgenic plants.

  9. Urine neutrophil gelatinase-associated lipocalin (uNGAL) and netrin-1: are they effectively improving the clinical management of sepsis-induced acute kidney injury (AKI)?

    Science.gov (United States)

    Mussap, Michele; Noto, Antonio; Fravega, Marco; Fanos, Vassilios

    2011-10-01

    Neutrophil gelatinase-associated lipocalin (NGAL) and Netrin-1 have been proposed over the past years as emergent biomarkers for the early and accurate diagnosis and monitoring of acute kidney injury (AKI). During the early phases of AKI, a rapid and massive up-regulation of NGAL mRNA takes place in the thick ascending limb of Henle's loop and in the collecting ducts, and therefore, changes in urinary NGAL (uNGAL) excretion seem to be more specific than plasma NGAL in assessing early kidney injury. The availability of a new automated immunoassay for measuring uNGAL facilitates its introduction in the clinical routine, especially in an emergency setting. However, in critically ill newborns AKI often develops during sepsis, which in turn induces an up-regulation of NGAL mRNA in neutrophils. To improve the effectiveness of therapeutic treatment in septic newborns with AKI, there is the need to accurately distinguish NGAL molecular forms originating within the distal nephron from those originating from neutrophils. This concise review summarizes properties and perspectives of uNGAL and Netrin-1 for their appropriate clinical utilization.

  10. Overexpression of a maize E3 ubiquitin ligase gene enhances drought tolerance through regulating stomatal aperture and antioxidant system in transgenic tobacco.

    Science.gov (United States)

    Liu, Jianjun; Xia, Zongliang; Wang, Meiping; Zhang, Xiaoquan; Yang, Tiezhao; Wu, Jianyu

    2013-12-01

    Drought is one of the most important limiting factors in crop production. In our previous study, a putative Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (AtSDIR1) homolog encoding a RING-finger protein from Zea mays (ZmRFP1) was cloned and its expression pattern and Ub E3 ligase activity were characterized. However, it is uncertain that ZmRFP1 acts as a positive regulator during drought stress. In this study, we further characterized ZmRFP1 in transgenic tobacco to investigate drought tolerance and possible function mechanisms. Overexpression of ZmRFP1 enhanced drought tolerance in tobacco. The transgenic tobacco lines had more closed stomatal pores, higher proline accumulation, but lower levels of malondialdehyde (MDA) when compared with the wild type (WT) under drought stress. Further investigation showed that ZmRFP1 transgenic plants displayed higher SOD and CAT activities, increased NtSOD and NtCAT transcript levels, and decreased reactive oxygen species (ROS) accumulation under drought stress. Taken together, our results demonstrate that ZmRFP1 confers drought stress tolerance in transgenic tobacco not only by increasing the ability to retain water, but also by reducing ROS accumulation and membrane damage through enhancing the antioxidant system. ZmRFP1 might serve as a candidate gene in genetic improvement for drought tolerance engineering in cereal crop plants. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  11. Recent advances using FcRn overexpression in transgenic animals to overcome impediments of standard antibody technologies to improve the generation of specific antibodies

    Science.gov (United States)

    Cervenak, Judit; Erdei, Anna; Goldsby, Richard A; Butler, John E

    2011-01-01

    This review illustrates the salutary effects of neonatal Fc receptor (FcRn) overexpression in significantly improving humoral immune responses in the generation of antibodies for immunotherapy and diagnostics. These include: (1) improved IgG protection; (2) augmented antigen-specific humoral immune response with larger numbers of antigen specific B cells, thus offering a wider spectrum of clones; (3) generation of antibodies against weakly immunogenic antigens; (4) significant improvements in the number and substantial developments in the diversity of hybridomas. FcRn transgenesis thus confers a number of practical benefits, including faster antibody production, higher antibody yields and improved generation of hybridomas for monoclonal antibody production. Notably, these efficiencies in polyclonal antibody production were also demonstrated in FcRn transgenic rabbits. Overall, FcRn transgenic animals yield more antibodies and provide a route to the generation of antibodies against antigens of low immunogenicity that are difficult to obtain using currently available methods. PMID:22048692

  12. Increasing anthraquinone production by overexpression of 1-deoxy-D: -xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia.

    Science.gov (United States)

    Quevedo, Carla; Perassolo, María; Alechine, Eugenia; Corach, Daniel; Giulietti, Ana María; Talou, Julián Rodriguez

    2010-07-01

    A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

  13. Over-expression of a glutathione S-transferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Ji, Wei; Zhu, Yanming; Li, Yong; Yang, Liang; Zhao, Xiaowen; Cai, Hua; Bai, Xi

    2010-08-01

    Glycine soja is a species of soybean that survives in adverse environments including high salt and drought conditions. We constructed a cDNA library from G. soja seedlings treated with NaCl and isolated a glutathione S-transferase gene (GsGST: GQ265911) from the library. The cDNA encoding GsGST contains an open reading frame of 660 bp and the predicted protein belongs to the tau class of GST family proteins. Tobacco plants over-expressing the GsGST gene showed sixfold higher GST activity than wild-type plants. Transgenic tobacco plants exhibited enhanced dehydration tolerance. T(2) transgenic tobacco plants showed higher tolerance at the seedling stage than wild-type plants to salt and mannitol as demonstrated by longer root length and less growth retardation.

  14. Inhibition of Lipolysis in the Novel Transgenic Quail Model Overexpressing G0/G1 Switch Gene 2 in the Adipose Tissue during Feed Restriction

    Science.gov (United States)

    Shin, Sangsu; Choi, Young Min; Han, Jae Yong; Lee, Kichoon

    2014-01-01

    In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G0/G1 switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) in vitro, its role in vivo has not been fully clarified. This study was conducted to investigate the role of G0S2 gene in vivo by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under ad libitum feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis in vivo during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity. PMID:24964090

  15. Transcriptomic changes reveal gene networks responding to the overexpression of a blueberry DWARF AND DELAYED FLOWERING 1 gene in transgenic blueberry plants.

    Science.gov (United States)

    Song, Guo-Qing; Gao, Xuan

    2017-06-19

    Constitutive expression of the CBF/DREB1 for increasing freezing tolerance in woody plants is often associated with other phenotypic changes including dwarf plant and delayed flowering. These phenotypic changes have been observed when Arabidopsis DWARF AND DELAYED FLOWERING 1 (DDF1) was overexpressed in A. thaliana plants. To date, the DDF1 orthologues have not been studied in woody plants. The aim of this study is to investigate transcriptomic responses to the overexpression of blueberry (Vaccinium corymbosum) DDF1 (herein, VcDDF1-OX). The VcDDF1-OX resulted in enhanced freezing tolerance in tetraploid blueberry plants and did not result in significant changes in plant size, chilling requirement, and flowering time. Comparative transcriptome analysis of transgenic 'Legacy-VcDDF1-OX' plants containing an overexpressed VcDDF1 with non-transgenic highbush blueberry 'Legacy' plants revealed the VcDDF1-OX derived differentially expressed (DE) genes and transcripts in the pathways of cold-response, plant flowering, DELLA proteins, and plant phytohormones. The increase in freezing tolerance was associated to the expression of cold-regulated genes (CORs) and the ethylene pathway genes. The unchanged plant size, dormancy and flowering were due to the minimal effect of the VcDDF1-OX on the expression of DELLA proteins, flowering pathway genes, and the other phytohormone genes related to plant growth and development. The DE genes in auxin and cytokinin pathways suggest that the VcDDF1-OX has also altered plant tolerance to drought and high salinity. A DDF1 orthologue in blueberry functioned differently from the DDF1 reported in Arabidopsis. The overexpression of VcDDF1 or its orthologues is a new approach to increase freezing tolerance of deciduous woody plant species with no obvious effect on plant size and plant flowering time.

  16. Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants.

    Science.gov (United States)

    Zhou, Li; Wang, Na-Na; Gong, Si-Ying; Lu, Rui; Li, Yang; Li, Xue-Bao

    2015-11-01

    Soil salinity is one of the most serious threats in world agriculture, and often influences cotton growth and development, resulting in a significant loss in cotton crop yield. WRKY transcription factors are involved in plant response to high salinity stress, but little is known about the role of WRKY transcription factors in cotton so far. In this study, a member (GhWRKY34) of cotton WRKY family was functionally characterized. This protein containing a WRKY domain and a zinc-finger motif belongs to group III of cotton WRKY family. Subcellular localization assay indicated that GhWRKY34 is localized to the cell nucleus. Overexpression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to salt stress. Several parameters (such as seed germination, green cotyledons, root length and chlorophyll content) in the GhWRKY34 transgenic lines were significantly higher than those in wild type under NaCl treatment. On the contrary, the GhWRKY34 transgenic plants exhibited a substantially lower ratio of Na(+)/K(+) in leaves and roots dealing with salt stress, compared with wild type. Growth status of the GhWRKY34 transgenic plants was much better than that of wild type under salt stress. Expressions of the stress-related genes were remarkably up-regulated in the transgenic plants under salt stress, compared with those in wild type. Based on the data presented in this study, we hypothesize that GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Na(+)/K(+) homeostasis as well as activating the salt stress-related genes in cells. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  17. Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants.

    Directory of Open Access Journals (Sweden)

    Yanhui Chen

    Full Text Available Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA content, relative electrical conductivity, and reactive oxygen species (ROS accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.

  18. Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

    Science.gov (United States)

    Chen, Yanhui; Han, Yangyang; Zhang, Meng; Zhou, Shan; Kong, Xiangzhu; Wang, Wei

    2016-01-01

    Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants. PMID:27073898

  19. Overexpression of Phenylalanine Ammonia-Lyase in Transgenic Roots of Coleus blumei Alters Growth and Rosmarinic Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Hrvoje Fulgosi

    2011-01-01

    Full Text Available Most natural phenolic compounds in plants are derived from trans-cinnamic acid, formed by the nonoxidative deamination of L-phenylalanine by phenylalanine ammonia-lyase (EC 4.3.1.24. Although a strict line between primary and secondary metabolism cannot be drawn, phenylalanine ammonia-lyase is considered to play a pivotal role in channelling carbon flux from primary metabolism to phenolic synthesis. The objective of this work is to evaluate the impact of phenylalanine ammonia-lyase activity on phenolic production in Coleus blumei. Transgenic roots of C. blumei, harbouring the Arabidopsis thaliana PAL1 gene, under the control of the CaMV 35S promoter, along with empty vector and wild-type roots, were regenerated. Transgenic root lines had disparate phenylalanine ammonia-lyase activities ranging from 67 to 350 %, compared to wild-type roots. Growth rates significantly differed, with the lowest in transgenic roots exerting augmented phenylalanine ammonia-lyase activity. Transgenic roots with high phenylalanine ammonia-lyase activity had lower growth rates, lower amounts of total phenolics, rosmarinic acid (the major phenolic compound in C. blumei and chlorogenic acid, but increased amounts of caffeic acid. There was no increase in total phenolics and rosmarinic acid content after feeding transgenic roots with casein enzymatic hydrolysate and L-tyrosine. This shows that augmented phenylalanine ammonia-lyase activity inhibits growth and phenolic metabolism, and the probable regulator of these processes is trans-cinnamic acid.

  20. Overexpression of a cotton gene that encodes a putative transcription factor of AP2/EREBP family in Arabidopsis affects growth and development of transgenic plants.

    Science.gov (United States)

    Zhou, Ying; Xia, Hui; Li, Xiao-Jie; Hu, Rong; Chen, Yun; Li, Xue-Bao

    2013-01-01

    In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling.

  1. Transgenic mice overexpressing the complement inhibitor crry as a soluble protein are protected from antibody-induced glomerular injury.

    Science.gov (United States)

    Quigg, R J; He, C; Lim, A; Berthiaume, D; Alexander, J J; Kraus, D; Holers, V M

    1998-10-05

    Complement receptor 1-related gene/protein y (Crry) is a potent murine membrane complement regulator that inhibits classical and alternative pathway C3 convertases. In nephrotoxic serum (NTS) nephritis, injected antibodies (Abs) bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. To study the phenotypic effects of continuous complement pathway blockade, transgenic mice were created that express recombinant soluble (rs) Crry directed by the broadly active and heavy metal-inducible metallothionein-I promoter. One transgenic line expressing high levels of rsCrry was propagated. Serum rsCrry levels were 18.7 +/- 2.7 microg/ml (n = 5) at basal level and increased to 118.1 +/- 20.6 microg/ml 4 d after addition of zinc to the drinking water. By reverse transcription polymerase chain reaction (RT-PCR), transgene messenger (m)RNA was present in liver, kidney, brain, lung, and spleen, but not in heart. By in situ RT-PCR analysis of kidneys, transgene mRNA was widely expressed both in renal glomeruli and tubules. Urinary excretion of rsCrry was 113.4 +/- 22.4 microg/ml with a fractional excretion relative to creatinine of 13.2 +/- 2.7%, consistent with local renal production of rsCrry and secretion into urine. The founder and all transgene positive adult animals have remained healthy with no mortality or apparent phenotypic abnormalities, including infection or immune complex disease. To determine whether rsCrry blocked complement-mediated injury, NTS nephritis was induced by injection of NTS immunoglobulin (Ig)G, followed by an 18-h urine collection to quantitate the excretion of albumin as a measure of glomerular injury. In transgene-negative littermates (n = 15), transgene-positive animals (n = 10), and transgene-positive animals fed zinc (n = 10), albuminuria was 4,393 +/- 948, 1,783 +/- 454, and 1,057 +/- 277 microg/mg creatinine, respectively (P Ab-induced disease model in vivo. These results support the hypothesis that

  2. A semi-automated motion-tracking analysis of locomotion speed in the C. elegans transgenics overexpressing beta-amyloid in neurons

    Science.gov (United States)

    Machino, Kevin; Link, Christopher D.; Wang, Susan; Murakami, Hana; Murakami, Shin

    2014-01-01

    Multi-Worm Tracker (MWT) is a real-time computer vision system that can simultaneously quantify motional patterns of multiple worms. MWT provides several behavioral parameters, including analysis of accurate real-time locomotion speed in the nematode, Caenorhabditis elegans. Here, we determined locomotion speed of the Alzheimer's disease (AD) transgenic strain that over-expresses human beta-amyloid1-42 (Aβ) in the neurons. The MWT analysis showed that the AD strain logged a slower average speed than the wild type (WT) worms. The results may be consistent with the observation that the AD patients with dementia tend to show deficits in physical activities, including frequent falls. The AD strain showed reduced ability of the eggs to hatch and slowed hatching of the eggs. Thus, over-expression of Aβ in neurons causes negative effects on locomotion and hatchability. This study sheds light on new examples of detrimental effects that Aβ deposits can exhibit using C. elegans as a model system. The information gathered from this study indicates that the motion tracking analysis is a cost-effective, efficient way to assess the deficits of Aβ over-expression in the C. elegans system. PMID:25071831

  3. Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes.

    Science.gov (United States)

    Kim, Myoung Duck; Kim, Yun-Hee; Kwon, Suk-Yoon; Yun, Dae-Jin; Kwak, Sang-Soo; Lee, Haeng-Soon

    2010-10-01

    Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)-induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non-transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV-treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses. Copyright © Physiologia Plantarum 2010.

  4. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation.

    Science.gov (United States)

    Das, Natasha; Bhattacharya, Surajit; Maiti, Mrinal K

    2016-08-01

    One of the most grievous heavy metal pollutants in the environment is cadmium (Cd), which is not only responsible for the crop yield loss owing to its phytotoxicity, but also for the human health hazards as the toxic elements usually accumulate in the consumable parts of crop plants. In the present study, we aimed to isolate and functionally characterize the OsMTP1 gene from indica rice (Oryza sativa L. cv. IR64) to study its potential application for efficient phytoremediation of Cd. The 1257 bp coding DNA sequence (CDS) of OsMTP1 encodes a ∼46 kDa protein belonging to the cation diffusion facilitator (CDF) or metal tolerance/transport protein (MTP) family. The OsMTP1 transcript in rice plant was found to respond during external Cd stress. Heterologous expression of OsMTP1 in tobacco resulted in the reduction of Cd stress-induced phytotoxic effects, including growth inhibition, lipid peroxidation, and cell death. Compared to untransformed control, the transgenic tobacco plants showed enhanced vacuolar thiol content, indicating vacuolar localization of the sequestered Cd. The transgenic tobacco plants exhibited significantly higher biomass growth (2.2-2.8-folds) and hyperaccumulation of Cd (1.96-2.22-folds) compared to untransformed control under Cd exposure. The transgenic plants also showed moderate tolerance and accumulation of arsenic (As) upon exogenous As stress, signifying broad substrate specificity of OsMTP1. Together, findings of our research suggest that the transgenic tobacco plants overexpressing OsMTP1 with its hyperaccumulating activity and increased growth rate could be useful for future phytoremediation applications to clean up the Cd-contaminated soil. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Pushpika eUdawat

    2016-04-01

    Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

  6. Metallothionein-I overexpression decreases brain pathology in transgenic mice with astrocyte-targeted expression of interleukin-6

    DEFF Research Database (Denmark)

    Molinero, Amalia; Penkowa, Milena; Hernández, Joaquín

    2003-01-01

    Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) causes significant damage and alters the expression of many genes, including a dramatic upregulation of metallothionein-I (MT-I). The findings...

  7. Starch characteristics of transgenic wheat (Triticum aestivum L.) overexpressing the Dx5 high molecular weight glutenin subunit are substantially equivalent to those in nonmodified wheat.

    Science.gov (United States)

    Beckles, Diane M; Tananuwong, Kanitha; Shoemaker, Charles F

    2012-04-01

    The effects of engineering higher levels of the High Molecular Weight Glutenin Dx5 subunit on starch characteristics in transgenic wheat (Triticum aestivum L.) grain were evaluated. This is important because of the interrelationship between starch and protein accumulation in grain, the strong biotechnological interest in modulating Dx5 levels and the increasing likelihood that transgenic wheat will be commercialized in the U.S. Unintended effects of Dx5 overexpression on starch could affect wheat marketability and therefore should be examined. Two controls with native levels of Dx5 were used: (i) the nontransformed Bobwhite cultivar, and (ii) a transgenic line (Bar-D) expressing a herbicide resistant (bar) gene, and they were compared with 2 transgenic lines (Dx5G and Dx5J) containing bar and additional copies of Dx5. There were few changes between Bar-D and Dx5G compared to Bobwhite. However, Dx5J, the line with the highest Dx5 protein (×3.5) accumulated 140% more hexose, 25% less starch and the starch had a higher frequency of longer amylopectin chains. These differences were not of sufficient magnitude to influence starch functionality, because granule morphology, crystallinity, amylose-to-amylopectin ratio, and the enthalpy of starch gelatinization and the amylose-lipid complex melting were similar to the control (P > 0.05). This overall similarity was borne out by Partial Least Squares-Discriminant Function Analysis, which could not distinguish among genotypes. Collectively our data imply that higher Dx5 can affect starch accumulation and some aspects of starch molecular structure but that the starches of the Dx5 transgenic wheat lines are substantially equivalent to the controls. Transgenic manipulation of biochemical pathways is an effective way to enhance food sensory quality, but it can also lead to unintended effects. These spurious changes are a concern to Government Regulatory Agencies and to those Industries that market the product. In this study we

  8. Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 (-) deficiency in transgenic tobacco plants.

    Science.gov (United States)

    Zhuo, Chunliu; Wang, Ting; Guo, Zhenfei; Lu, Shaoyun

    2016-06-14

    Plasma membrane intrinsic proteins (PIPs), which belong to aquaporins (AQPs) superfamily, are subdivided into two groups, PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but have a role in water permeability through interacting with PIP2. A cold responsive PIP2 named as MfPIP2-7 was isolated from Medicago falcata (hereafter falcata), a forage legume with great cold tolerance, and transgenic tobacco plants overexpressing MfPIP2-7 were analyzed in tolerance to multiple stresses including freezing, chilling, and nitrate reduction in this study. MfPIP2-7 transcript was induced by 4 to 12 h of cold treatment and 2 h of abscisic acid (ABA) treatment. Pretreatment with inhibitor of ABA synthesis blocked the cold induced MfPIP2-7 transcript, indicating that ABA was involved in cold induced transcription of MfPIP2-7 in falcata. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing, chilling and NO3 (-) deficiency in transgenic tobacco (Nicotiana tabacum L.) plants as compared with the wild type. Moreover, MfPIP2-7 was demonstrated to facilitate H2O2 diffusion in yeast. Higher transcript levels of several stress responsive genes, such as NtERD10B, NtERD10C, NtDREB1, and 2, and nitrate reductase (NR) encoding genes (NtNIA1, and NtNIA2) were observed in transgenic plants as compared with the wild type with dependence upon H2O2. In addition, NR activity was increased in transgenic plants, which led to alterations in free amino acid components and concentrations. The results suggest that MfPIP2-7 plays an important role in plant tolerance to freezing, chilling, and NO3 (-) deficiency by promoted H2O2 diffusion that in turn up-regulates expression of NIAs and multiple stress responsive genes.

  9. Transgenic overexpression of human DMPK accumulates into hypertrophic cardiomyopathy, myotonic myopathy and hypotension traits of myotonic dystrophy.

    NARCIS (Netherlands)

    O'Cochlain, D.F.; Perez-Terzic, C.; Reyes, S.; Kane, G.C.; Behfar, A.; Hodgson, D.M.; Strommen, J.A.; Liu, X.K.; Broek, W.J.A.A. van den; Wansink, D.G.; Wieringa, B.; Terzic, A.

    2004-01-01

    Abnormal expression of human myotonic dystrophy protein kinase (hDMPK) gene products has been implicated in myotonic dystrophy type 1 (DM1), yet the impact of distress accumulation produced by persistent overexpression of this poorly understood member of the Rho kinase-related protein kinase

  10. Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1.

    Science.gov (United States)

    Samuilov, Sladjana; Lang, Friedericke; Djukic, Matilda; Djunisijevic-Bojovic, Danijela; Rennenberg, Heinz

    2016-09-01

    Growth and development of plants largely depends on their adaptation ability in a changing climate. This is particularly true on heavy metal contaminated soils, but the interaction of heavy metal stress and climate on plant performance has not been intensively investigated. The aim of the present study was to elucidate if transgenic poplars (Populus tremula x P. alba) with enhanced glutathione content possess an enhanced tolerance to drought and lead (Pb) exposure (single and in combination) and if they are good candidates for phytoremediation of Pb contaminated soil. Lead exposure reduced growth and biomass accumulation only in above-ground tissue of wild type poplar, although most of lead accumulated in the roots. Drought caused a decline of the water content rather than reduced biomass production, while Pb counteracted this decline in the combined exposure. Apparently, metals such as Pb possess a protective function against drought, because they interact with abscisic acid dependent stomatal closure. Lead exposure decreased while drought increased glutathione content in leaves of both plant types. Lead accumulation was higher in the roots of transgenic plants, presumably as a result of chelation by glutathione. Water deprivation enhanced Pb accumulation in the roots, but Pb was subject to leakage out of the roots after re-watering. Transgenic plants showed better adaptation under mild drought plus Pb exposure partially due to improved glutathione synthesis. However, the transgenic plants cannot be considered as a good candidate for phytoremediation of Pb, due to its small translocation to the shoots and its leakage out of the roots upon re-watering. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development

    DEFF Research Database (Denmark)

    Conover, Cheryl A; Mason, Megan A; Bale, Laurie K

    2010-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development...... of atherosclerotic lesions, we generated transgenic mice that express human PAPP-A in arterial smooth muscle. Four founder lines were characterized for transgenic human PAPP-A mRNA and protein expression, IGFBP-4 protease activity, and tissue specificity. In study I, apolipoprotein E knockout (ApoE KO) mice, a well......-characterized mouse model of atherosclerosis, and ApoE KO mice expressing the human PAPP-A transgene at relatively high levels (ApoE KO/Tg) were fed a high-fat diet. At harvest, aortas were dissected and opened longitudinally for en face staining of lipid-rich lesions. Lesion area was increased 3.5-fold in aortas...

  12. Assessment of heat shock protein 70 induction by heat in alfalfa varieties and constitutive overexpression in transgenic plants.

    Directory of Open Access Journals (Sweden)

    Nicoletta Ferradini

    Full Text Available Heat shock proteins (HSPs are molecular chaperones involved in many cellular functions. It has been shown that mammalian cytosolic HSP70 binds antigenic peptides mediating the activation of the immune system, and that it plays a determining role in tumour immunogenicity. This suggests that HSP70 may be used for the production of conjugated vaccines. Human and plant HSPs share high sequence similarity and some important biological functions in vitro. In addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from plants for use as vaccine adjuvant requires enhancing its concentration in plant tissues. In this work, we explored the possibility to produce HSP70 in both transgenic and non-transgenic plants, using alfalfa as a model species. First, a transcriptional analysis of a constitutive and an inducible HSP70 genes was conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form was cloned and introduced into alfalfa by Agrobacterium-mediated transformation, and the accumulation of the protein in leaf tissue of transgenic plants was demonstrated. We also tested diverse alfalfa varieties for heat-inducible expression of endogenous HSP70, revealing variety-specific responses to heat shock.

  13. Overexpression of the transgene for manganese superoxide dismutase (MnSOD) in 32D cl 3 cells prevents apoptosis induction by TNF-alpha, IL-3 withdrawal, and ionizing radiation.

    Science.gov (United States)

    Epperly, Michael W; Bernarding, Michael; Gretton, Joan; Jefferson, Mia; Nie, Suhua; Greenberger, Joel S

    2003-06-01

    Stabilization of the mitochondria in IL-3-dependent hematopoietic progenitor cell line 32D cl 3 by overexpression of the transgene for manganese superoxide dismutase (MnSOD) prior to ionizing radiation prevents apoptosis. We now demonstrate that overexpression of the MnSOD transgene also protects 32D cl 3 cells from apoptosis caused by exposure to tumor necrosis factor-alpha (TNF-alpha) or withdrawal of interleukin (IL)-3. The hematopoietic progenitor cell line, 32D cl 3, and subclones overexpressing the human MnSOD transgene, 1F2 or 2C6, were radiated to 1000 cGy or were exposed to TNF-alpha (0 to 100 etag/mL) or were subjected to IL-3 withdrawal. The cells were then examined at several time points for DNA strand breaks using a comet assay, depolarization of the mitochondrial membrane, activation of caspase-3, PARP cleavage, and apoptosis, and also for changes in cell cycle distribution. Overexpression of the transgene for MnSOD resulted in increased survival following exposure to radiation, exposure to TNF-alpha, or IL-3 withdrawal. The cell lines overexpressing MnSOD (1F2 or 2C6) displayed decreased radiation-induced, TNF-alpha-induced, or IL-3 withdrawal-induced mitochondrial membrane permeability, caspase-3 and PARP activation, and apoptosis. Overexpression of the human MnSOD transgene in 32D cl 3 cells results in stabilization of the mitochondria and reduction in radiation-, TNF-alpha-, or IL-3 withdrawal-induced damage. Thus, MnSOD stabilization of the mitochondrial membrane is relevant to reduction of apoptosis by several classes of oxidative stress inducers.

  14. Diversion of carbon flux from gibberellin to steviol biosynthesis by over-expressing SrKA13H induced dwarfism and abnormality in pollen germination and seed set behaviour of transgenic Arabidopsis.

    Science.gov (United States)

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2015-07-01

    This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 μg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 μg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Simultaneous Overexpression of the HhERF2 and PeDREB2a Genes Enhanced Tolerances to Salt and Drought in Transgenic Cotton.

    Science.gov (United States)

    Li, Jin Bo; Ni Dong, Xue; Lei, Zhi; Liang Li, Yong; Yang Yang, Pei; Tao, Fei; Zhao, Liang; Li, Shi-Gang; Du, Lin Feng; Shao, Ji Rong; Wu, Yan Min

    2016-01-01

    A desert-grown Halimodendron halodendron ethylene-responsive element binding factor gene (HhERF2), which encodes a 245 amino acids protein containing a conserved AP2/EREBP domain, was isolated through the rapid amplification cDNA end (RACE) method. Sequence and phylogenetic analysis indicated that HhERF2 was classified into the B-2 group of ERF subfamily. Semiquantitative RT-PCR showed that HhERF2 was greatly induced by treatments with high-salt, drought and low temperature in H. halodendron seedlings. The expression vector containing HhERF2 and Populus euphratica dehydration- responsive element binding protein (PeDREB2a) genes driven by rd29A promoter was constructed and transferred into cotton (Gossypium hirsutum L.) by non-tissue culture Agrobacterium-mediated genetic transformation system. The transformation and expression of HhERF2 and PeDREB2a were identified by PCR and RT-PCR. Analyses of physiological function indicated that transgenic cottons had improved seeds germination, tolerance to drought and highsalt stresses. Agronomic evaluation in the field exhibited that transgenic lines presented a dwarf phenotype and improved further in the yield and characters. These results demonstrated that overexpressed both HhERF2 and PeDREB2a transcription factor genes in cotton induced elevated tolerance to drought and high-salt stresses.

  16. Over-expression of the peroxisomal ascorbate peroxidase (SbpAPX) gene cloned from halophyte Salicornia brachiata confers salt and drought stress tolerance in transgenic tobacco.

    Science.gov (United States)

    Singh, Natwar; Mishra, Avinash; Jha, Bhavanath

    2014-06-01

    Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.

  17. Psc-AFP from Psoralea corylifolia L. overexpressed in Pichia pastoris increases antimicrobial activity and enhances disease resistance of transgenic tobacco.

    Science.gov (United States)

    Luo, Xiu-Mei; Xie, Cheng-Jian; Wang, De; Wei, Yun-Min; Cai, Jie; Cheng, Shan-Shan; Yang, Xing -Yong; Sui, An -Ping

    2017-02-01

    Psc-AFP, isolated from the seeds of Psoralea corylifolia L., is an antimicrobial protein with trypsin inhibitor activity. Its encoding gene was cloned by 3'- rapid amplification of cDNA ends (RACE) combined with Y-shaped adaptor-dependent extension (YADE) method. The gene Psc-AFP encodes a protein of 203 amino acids with a deduced signal peptide of 24 residues. The growth inhibition effect exerted by the heterologously expressed Psc-AFP in Pichia pastoris revealed that the recombinant Psc-AFP inhibited mycelium growth of Aspergillus niger, Rhizoctonia solani, and Alternaria brassicae and conidial germination of Alternaria alternata. The recombinant Psc-AFP also showed protease inhibitor activity manifested by the inhibition of trypsin. The transgenic tobacco bioassays confirmed that overexpressing Psc-AFP significantly enhanced the disease resistance of tobacco and that some of the transgenic lines were almost fully tolerant to Ralstonia solanacearum and A. alternata, whereas no apparent alteration in plant growth and development was observed. Collectively, these results indicate that the recombinant Psc-AFP is an active antimicrobial protein, with protease inhibitor activity that can be successfully produced in the yeast and tobacco and, therefore, maybe a potential antimicrobial candidate for practical use.

  18. Ectopic overexpression of a novel Glycine soja stress-induced plasma membrane intrinsic protein increases sensitivity to salt and dehydration in transgenic Arabidopsis thaliana plants.

    Science.gov (United States)

    Wang, Xi; Cai, Hua; Li, Yong; Zhu, Yanming; Ji, Wei; Bai, Xi; Zhu, Dan; Sun, Xiaoli

    2015-01-01

    Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5'-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants.

  19. Ectopic overexpression of SsCBF1, a CRT/DRE-binding factor from the nightshade plant Solanum lycopersicoides, confers freezing and salt tolerance in transgenic Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lili Zhang

    Full Text Available The C-repeat (CRT/dehydration-responsive element (DRE binding factor (CBF/DREB1 transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1 was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193-228 (SsCBF1(193-228. The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

  20. Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight.

    Science.gov (United States)

    Hutabarat, Olly Sanny; Flachowsky, Henryk; Regos, Ionela; Miosic, Silvija; Kaufmann, Christine; Faramarzi, Shadab; Alam, Mohammed Zobayer; Gosch, Christian; Peil, Andreas; Richter, Klaus; Hanke, Magda-Viola; Treutter, Dieter; Stich, Karl; Halbwirth, Heidi

    2016-05-01

    Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab. The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3'-hydroxylase (F3'H). Using recombinant F3'H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3'H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.

  1. Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

    Science.gov (United States)

    Matsushita, Misako; Nakatake, Yuhki; Arai, Itaru; Ibata, Keiji; Kohda, Kazuhisa; Goparaju, Sravan K; Murakami, Miyako; Sakota, Miki; Chikazawa-Nohtomi, Nana; Ko, Shigeru B H; Kanai, Takanori; Yuzaki, Michisuke; Ko, Minoru S H

    2017-08-19

    Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori.

    Science.gov (United States)

    Xu, Hanfu; Liu, Yaowen; Wang, Feng; Yuan, Lin; Wang, Yuancheng; Ma, Sanyuan; Beneš, Helen; Xia, QingYou

    2014-08-01

    In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5-2.0 and 5.5-6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84% of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.

  3. Generation and Characterization of Transgenic Mice Expressing Mouse Ins1 Promoter for Pancreatic β-Cell-Specific Gene Overexpression and Knockout.

    Science.gov (United States)

    Cheng, Yulong; Su, Yutong; Shan, Aijing; Jiang, Xiuli; Ma, Qinyun; Wang, Weiqing; Ning, Guang; Cao, Yanan

    2015-07-01

    The technologies for pancreatic β-cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at embryonic day 13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be used to knock out or overexpress target genes in embryos and adults to facilitate β-cell researches.

  4. Transgenic mice overexpressing glia maturation factor-β, an oxidative stress inducible gene, show premature aging due to Zmpste24 down-regulation.

    Science.gov (United States)

    Imai, Rika; Asai, Kanae; Hanai, Jun-ichi; Takenaka, Masaru

    2015-07-01

    Glia Maturation Factor-β (GMF), a brain specific protein, is induced by proteinuria in renal tubules. Ectopic GMF overexpression causes apoptosisin vitro via cellular vulnerability to oxidative stress. In order to examine the roles of GMF in non-brain tissue, we constructed transgenic mice overexpressing GMF (GMF-TG). The GMF-TG mice exhibited appearance phenotypes associated with premature aging. The GMF-TG mice also demonstrated short lifespans and reduced hair regrowth, suggesting an accelerated aging process. The production of an abnormal lamin A, a nuclear envelope protein, plays a causal role in both normal aging and accelerated aging diseases, known as laminopathies. Importantly, we identified the abnormal lamin A (prelamin A), accompanied by a down-regulation of a lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The GMF-TG mice showed accelerated aging in the kidney, compared with wild-type mice, showing increased TGF-β1, CTGF gene and serum creatinine. The gene expression of p21/waf1 was increased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at a later stage, at 60 weeks. In conclusion, we propose that GMF-TG mice might be a novel mouse model of accelerated aging, due to the abnormal lamin A.

  5. Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Yan eYan

    2015-09-01

    Full Text Available WRKY proteins constitute transcriptional regulators involved in various biological processes, especially in coping with diverse biotic and abiotic stresses. However, in contrast to other well-characterized WRKY groups, the functions of group III WRKY transcription factors are poorly understood in the economically important crop cotton (Gossypium hirsutum. In this study, a group III WRKY gene from cotton, GhWRKY27a, was isolated and characterized. Our data indicated that GhWRKY27a localized to the nucleus and that GhWRKY27a expression could be strongly induced by abiotic stresses, pathogen infection, and multiple defense-related signaling molecules. Virus-induced gene silencing (VIGS of GhWRKY27a enhanced tolerance to drought stress in cotton. In contrast, GhWRKY27a overexpression in Nicotiana benthamiana markedly reduced plant tolerance to drought stress, as determined through physiological analyses of leaf water loss, survival rates and the stomatal aperture. This susceptibility was coupled with reduced stomatal closure in response to abscisic acid and decreased expression of stress-related genes. In addition, GhWRKY27a-overexpressing plants exhibited reduced resistance to Rhizoctonia solani infection, mainly demonstrated by the transgenic lines exhibiting more severe disease symptoms, accompanied by attenuated expression of defense-related genes in Nicotiana benthamiana. Taken together, these findings indicated that GhWRKY27a functions in negative responses to drought tolerance and in resistance to Rhizoctonia solani infection.

  6. TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model

    Directory of Open Access Journals (Sweden)

    Gulsen Gultekin

    2011-10-01

    Full Text Available Abstract Background The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14ARF-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer. Results In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses NFκBIB, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells. Conclusions Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary

  7. Ectopic overexpression of SlHsfA3, a heat stress transcription factor from tomato, confers increased thermotolerance and salt hypersensitivity in germination in transgenic Arabidopsis.

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    Zhenjun Li

    Full Text Available Plant heat stress transcription factors (Hsfs are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3 was isolated from the cultivated tomato (Solanum lycopersicum, Sl and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA. The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions.

  8. Retraction Note: Transgenic mice overexpressing the ALS-linked protein Matrin 3 develop a profound muscle phenotype.

    Science.gov (United States)

    Moloney, Christina; Rayaprolu, Sruti; Howard, John; Fromholt, Susan; Brown, Hilda; Collins, Matt; Cabrera, Mariela; Duffy, Colin; Siemienski, Zoe; Miller, Dave; Swanson, Maurice S; Notterpek, Lucia; Borchelt, David R; Lewis, Jada

    2017-12-13

    The authors are retracting this article. The article describes mice expressing wild-type human MATR3. However, since publication the authors have become aware that all of the lines of mice described express human MATR3 containing the F115C mutation. Transgenic mice expressing wild-type and mutant Matrin were created simultaneously in their laboratory and, at a crucial stage of generating the DNA for embryo injection, as confirmed by an investigation by the University of Florida, the DNA preparations were accidentally mislabelled. All of the founders created were mosaic, requiring extensive breeding to isolate stable lines. Mice mislabelled as expressing wild-type MATR3 were the first to produce lines that stably transmitted the transgene and thus were the first to be characterized. However, as lines of mice that were mislabelled as expressing the mutant (F115C) MATR3 were ultimately established, the data began to suggest that an error had been made. Sequence analysis of amplified tail DNA from mice descended from the lines reported in the article have revealed that they express the F115C variant. The data described are therefore an accurate description of the pathology of mice that express the F115C variant of MATR3, but not of mice expressing wild-type MATR3. The authors are preparing a new manuscript reporting data from both mice expressing the F115C variant of MATR3 and mice expressing wild-type MATR3.

  9. Transgenic Overexpression of Tcfap2c/AP-2gamma Results in Liver Failure and Intestinal Dysplasia

    Science.gov (United States)

    Woynecki, Tatiana; Egert, Angela; Becker, Astrid; Huss, Sebastian; Stabenow, Dirk; Zimmer, Andreas; Knolle, Percy; Tolba, René; Fischer, Hans-Peter; Schorle, Hubert

    2011-01-01

    Background The transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown. Methodology/Principal Findings Therefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis) and lead to rapid mortality within 3–7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa. Conclusions The transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9 known to be expressed

  10. Transgenic overexpression of Tcfap2c/AP-2gamma results in liver failure and intestinal dysplasia.

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    Daniel Holl

    Full Text Available BACKGROUND: The transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Therefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis and lead to rapid mortality within 3-7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa. CONCLUSIONS: The transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9

  11. Transgenic tobacco overexpressing Brassica juncea HMG-CoA synthase 1 shows increased plant growth, pod size and seed yield.

    Science.gov (United States)

    Liao, Pan; Wang, Hui; Wang, Mingfu; Hsiao, An-Shan; Bach, Thomas J; Chye, Mee-Len

    2014-01-01

    Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the mevalonate (MVA) pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt) and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A) BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi) of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i) phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1) in comparison to vector-transformed tobacco, (ii) higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii) induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant reproduction

  12. Transgenic tobacco overexpressing Brassica juncea HMG-CoA synthase 1 shows increased plant growth, pod size and seed yield.

    Directory of Open Access Journals (Sweden)

    Pan Liao

    Full Text Available Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS, the second enzyme in the mevalonate (MVA pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1 in comparison to vector-transformed tobacco, (ii higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant

  13. Severe drug-induced repetitive behaviors and striatal overexpression of VAChT in ChAT-ChR2-EYFP BAC transgenic mice

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    Jill R Crittenden

    2014-05-01

    Full Text Available In drug users, drug-related cues alone can induce dopamine release in the dorsal striatum. Instructive cues activate inputs to the striatum from both dopaminergic and cholinergic neurons, which are thought to work together to support motor learning and motivated behaviors. Imbalances in these neuromodulatory influences can impair normal action selection and might thus contribute to pathologically repetitive and compulsive behaviors such as drug addiction. Dopamine and acetylcholine can have either antagonistic or synergistic effects on behavior, depending on the state of the animal and the receptor signaling systems at play. Semi-synchronized activation of cholinergic interneurons in the dorsal striatum drives dopamine release via presynaptic nicotinic acetylcholine receptors located on dopamine terminals. Nicotinic receptor blockade is known to diminish abnormal repetitive behaviors (stereotypies induced by psychomotor stimulants. By contrast, blockade of postsynaptic acetylcholine muscarinic receptors in the dorsomedial striatum exacerbates drug-induced stereotypy, exemplifying how different acetylcholine receptors can also have opposing effects. Although acetylcholine release is known to be altered in animal models of drug addiction, predicting whether these changes will augment or diminish drug-induced behaviors thus remains a challenge. Here, we measured amphetamine-induced stereotypy in BAC transgenic mice that have been shown to overexpress the vesicular acetylcholine transporter (VAChT with consequent increased acetylcholine release. We found that drug-induced stereotypies, consisting of confined sniffing and licking behaviors, were greatly increased in the transgenic mice relative to sibling controls, as was striatal VAChT protein. These findings suggest that VAChT-mediated increases in acetylcholine could be critical in exacerbating drug-induced stereotypic behaviors and promoting exaggerated behavioral fixity.

  14. Over-expression in the nucleotide-binding site-leucine rich repeat gene DEPG1 increases susceptibility to bacterial leaf streak disease in transgenic rice plants.

    Science.gov (United States)

    Guo, Lijia; Li, Min; Wang, Wujing; Wang, Lijuan; Hao, Guojing; Guo, Chiming; Chen, Liang

    2012-04-01

    Bacterial leaf streak of rice (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a widely-spread disease in the main rice-producing areas of the world. Investigating the genes that play roles in rice-Xoc interactions helps us to understand the defense signaling pathway in rice. Here we report a differentially expressed protein gene (DEPG1), which regulates susceptibility to BLS. DEPG1 is a nucleotide-binding site (NBS)-leucine rich repeat (LRR) gene, and the deduced protein sequence of DEPG1 has approximately 64% identity with that of the disease resistance gene Pi37. Phylogenetic analysis of DEPG1 and the 18 characterized NBS-LRR genes revealed that DEPG1 is more closely related to Pi37. DEPG1 protein is located to the cytoplasm, which was confirmed by transient expression of DEPG1-GFP (green fluorescent protein) fusion construct in onion epidermal cells. Semi-quantitative PCR assays showed that DEPG1 is widely expressed in rice, and is preferentially expressed in internodes, leaf blades, leaf sheaths and flag leaves. Observation of cross sections of leaves from the transgenic plants with a DEPG1-promoter::glucuronidase (GUS) fusion gene revealed that DEPG1 is also highly expressed in mesophyll tissues where Xoc mainly colonizes. Additionally, Xoc negatively regulates expression of DEPG1 at the early stage of the pathogen infection, and so do the three defense-signal compounds including salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic-acid (ACC). Transgenic rice plants overexpressing DEPG1 exhibit enhanced susceptibility to Xoc compared to the wild-type controls. Moreover, enhanced susceptibility to Xoc may be mediated by inhibition of the expression of some SA biosynthesis-related genes and pathogenesis-related genes that may contribute to the disease resistance. Taken together, DEPG1 plays roles in the interactions between rice and BLS pathogen Xoc.

  15. Severe drug-induced repetitive behaviors and striatal overexpression of VAChT in ChAT-ChR2-EYFP BAC transgenic mice.

    Science.gov (United States)

    Crittenden, Jill R; Lacey, Carolyn J; Lee, Tyrone; Bowden, Hilary A; Graybiel, Ann M

    2014-01-01

    In drug users, drug-related cues alone can induce dopamine release in the dorsal striatum. Instructive cues activate inputs to the striatum from both dopaminergic and cholinergic neurons, which are thought to work together to support motor learning and motivated behaviors. Imbalances in these neuromodulatory influences can impair normal action selection and might thus contribute to pathologically repetitive and compulsive behaviors such as drug addiction. Dopamine and acetylcholine can have either antagonistic or synergistic effects on behavior, depending on the state of the animal and the receptor signaling systems at play. Semi-synchronized activation of cholinergic interneurons in the dorsal striatum drives dopamine release via presynaptic nicotinic acetylcholine receptors located on dopamine terminals. Nicotinic receptor blockade is known to diminish abnormal repetitive behaviors (stereotypies) induced by psychomotor stimulants. By contrast, blockade of postsynaptic acetylcholine muscarinic receptors in the dorsomedial striatum exacerbates drug-induced stereotypy, exemplifying how different acetylcholine receptors can also have opposing effects. Although acetylcholine release is known to be altered in animal models of drug addiction, predicting whether these changes will augment or diminish drug-induced behaviors thus remains a challenge. Here, we measured amphetamine-induced stereotypy in BAC transgenic mice that have been shown to overexpress the vesicular acetylcholine transporter (VAChT) with consequent increased acetylcholine release. We found that drug-induced stereotypies, consisting of confined sniffing and licking behaviors, were greatly increased in the transgenic mice relative to sibling controls, as was striatal VAChT protein. These findings suggest that VAChT-mediated increases in acetylcholine could be critical in exacerbating drug-induced stereotypic behaviors and promoting exaggerated behavioral fixity.

  16. Creation and characterization of BAC-transgenic mice with physiological overexpression of epitope-tagged RCAN1 (DSCR1).

    Science.gov (United States)

    Xing, Luzhou; Salas, Martha; Zhang, Hong; Gittler, Julia; Ludwig, Thomas; Lin, Chyuan-Sheng; Murty, Vundavalli V; Silverman, Wayne; Arancio, Ottavio; Tycko, Benjamin

    2013-02-01

    The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg was expressed at levels only moderately higher than the native Rcan1 gene: approximately 1.5-fold in RCAN1 (BAC-Tg1) and twofold in RCAN1 (BAC-Tg2). Affinity purification of the RCAN1 protein complex from brains of these mice revealed a core complex of RCAN1 with CaN, glycogen synthase kinase 3-beta (Gsk3b), and calmodulin, with substoichiometric components, including LOC73419. The BAC-Tg mice are fully viable, but long-term synaptic potentiation is impaired in proportion to BAC-Tg dosage in hippocampal brain slices from these mice. RCAN1 can act as a tumor suppressor in some systems, but we found that the RCAN1 BAC-Tg did not reduce mammary cancer growth when present at a low copy number in Tp53;WAP-Cre mice. This work establishes a useful mouse model for investigating the biochemistry and dose-dependent functions of the RCAN1 protein in vivo.

  17. Mammary specific transgenic over-expression of insulin-like growth factor-I (IGF-I) increases pig milk IGF-I and IGF binding proteins, with no effect on milk composition or yield.

    Science.gov (United States)

    Monaco, Marcia H; Gronlund, Derek E; Bleck, Gregory T; Hurley, Walter L; Wheeler, Matthew B; Donovan, Sharon M

    2005-10-01

    IGF-I regulates lactation by stimulating mammary mitogenesis, inhibiting apoptosis, and partially mediating the effects of growth hormone on lactogenesis. Herein, lactation performance during first and second parity was assessed in transgenic swine (TG) that over-expressed human IGF-I in milk under the control of the bovine alpha-lactalbumin promoter, regulatory regions and signal peptide coding sequence. Milk samples were collected throughout lactation (farrowing to d24) from TG sows and non-transgenic littermates (CON) and IGF-I, IGF-II, and IGFBP determined. Colostral (mature milk (d2-24 postpartum). There was no effect of parity on milk IGF-I content. Milk IGF-II concentration was unaffected by IGF-I over-expression. Low molecular weight IGFBP (IGFBP-2 and -5) in the milk of TG sows were higher (p=0.02) than CON in the early postpartum period, but did not differ in mature milk. Milk yield, determined by weigh-suckle-weigh, was similar in TG and CON as was litter weight gain. Milk nutrient composition was not significantly affected by IGF over-expression. Thus, mammary specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, but did not impact lactation performance in swine.

  18. Aging-associated changes in oxidative stress, cell proliferation, and apoptosis are prevented in the prostate of transgenic rats overexpressing regucalcin.

    Science.gov (United States)

    Vaz, Cátia V; Marques, Ricardo; Maia, Cláudio J; Socorro, Sílvia

    2015-12-01

    Regucalcin (RGN) is a calcium (Ca(2+))-binding protein that displays a characteristic downregulated expression with aging in several tissues. Besides its role in regulating intracellular Ca(2+) homeostasis, RGN has been associated with the control of oxidative stress, cell proliferation, and apoptosis. Thus, the diminished expression of RGN with aging may contribute to the age-associated deterioration of cell function. In the present study, we hypothesized that the maintenance of high expression levels of RGN may prevent age-related alterations in the processes mentioned previously. First, we confirmed that RGN expression is significantly diminished in the prostate of 8-, 9-, 12-, and 24-months wild-type rats. Then, the effect of aging on lipid peroxidation, antioxidant defenses, cell proliferation, and apoptosis in the prostate of wild-type controls and transgenic rats overexpressing RGN (Tg-RGN) was investigated. The activity of glutathione and the antioxidant capacity were increased in Tg-RGN rats in response to the age-associated increase in thiobarbituric acid reactive substances levels, an effect not seen in wild type. Overexpression of RGN also counteracted the effect of aging increasing prostate cell proliferation. In contrast to wild-type animals, the prostate weight of Tg-RGN did not change with aging and was underpinned by the diminished expression of stem cell factor and c-kit, and increased expression of p53. In addition, aged Tg-RGN animals displayed increased expression (activity) of apoptosis regulators, therefore not showing the age-induced resistance to apoptosis observed in wild type. Altogether, these findings indicate the protective role of RGN against the development of age-related pathologies, such as, for example, prostate cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor.

    Science.gov (United States)

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Shanker, Karuna; Mathur, Ajay K

    2015-01-01

    Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus have been successfully over-expressed in the rol gene integrated cell suspensions of V. minor. Thirty seconds SAAT (sonication-assisted Agrobacterium transformation) treatment of plant cell suspension with LBA1119 having construct () generated three stable TDC + STR over-expressing cell lines--PVG1, PVG2, and PVG3. The transgenes were confirmed by β-glucuronidase GUS histochemical assay and PCR amplification of rol genes/GUS gene. All the three cell suspension lines were found to be slow growing. In comparison to the control cell suspensions (GI = 241.0 ± 5.8), PVG3 cell line registered a growth index (GI) of 208.0 ± 10.0 followed by PVG1 (GI = 140.0 ± 14.2) and PVG2 (GI = 85.0 ± 9.6). The PVG3 cell line was also up-scaled in the 5-l stirred tank bioreactor with GI of 745.6 ± 35.3 under optimized parameters. Only PVG3 line registered a twofold increase in total alkaloid content (2.1 ± 0.1% dry wt.) and showed vincamine presence (0.003 ± 0.001% dry wt.) which was further enhanced at the bioreactor level (2.7 ± 0.3 and 0.005 ± 0.001% dry wt., respectively). Real-time (RT) qPCR analysis of PVG3 showed more than sevenfold to eightfold increase in TDC and STR expression [relative quantity value (RQ) = 7.6 ± 0.8 (TDC); RQ = 8.5 ± 0.9 (STR)].

  20. Transgenic Overexpression of the Type I Isoform of Neuregulin 1 Affects Working Memory and Hippocampal Oscillations but not Long-term Potentiation

    Science.gov (United States)

    Deakin, Inga H.; Nissen, Wiebke; Law, Amanda J.; Lane, Tracy; Kanso, Riam; Schwab, Markus H.; Nave, Klaus-Armin; Lamsa, Karri P.; Paulsen, Ole; Bannerman, David M.

    2012-01-01

    Neuregulin 1 (NRG1) is a growth factor involved in neurodevelopment and plasticity. It is a schizophrenia candidate gene, and hippocampal expression of the NRG1 type I isoform is increased in the disorder. We have studied transgenic mice overexpressing NRG1 type I (NRG1tg-type I) and their wild-type littermates and measured hippocampal electrophysiological and behavioral phenotypes. Young NRG1tg-type I mice showed normal memory performance, but in older NRG1tg-type I mice, hippocampus-dependent spatial working memory was selectively impaired. Hippocampal slice preparations from NRG1tg-type I mice exhibited a reduced frequency of carbachol-induced gamma oscillations and an increased tendency to epileptiform activity. Long-term potentiation in NRG1tg-type I mice was normal. The results provide evidence that NRG1 type I impacts on hippocampal function and circuitry. The effects are likely mediated via inhibitory interneurons and may be relevant to the involvement of NRG1 in schizophrenia. However, the findings, in concert with those from other genetic and pharmacological manipulations of NRG1, emphasize the complex and pleiotropic nature of the gene, even with regard to a single isoform. PMID:21878485

  1. Overexpression of a tobacco small G protein gene NtRop1 causes salt sensitivity and hydrogen peroxide production in transgenic plants.

    Science.gov (United States)

    Cao, YangRong; Li, ZhiGang; Chen, Tao; Zhang, ZhiGang; Zhang, JinSong; Chen, ShouYi

    2008-05-01

    The small GTPases of Rop/Rho family is central regulators of important cellular processes in plants. Tobacco small G protein gene NtRop1 has been isolated; however, its roles in stress responses were unknown. In the present study, the genomic sequence of NtRop1 was cloned, which has seven exons and six introns, similar to the Rop gene structure from Arabidopsis. The NtRop1 gene was constitutively expressed in the different organs whereas the other six Rop genes from tobacco had differential expression patterns. The expression of the NtRop1 gene was moderately induced by methyl viologen, NaCl, and ACC treatments, but slightly inhibited by ABA treatment, with no significant induction by NAA treatment. The transgenic Arabidopsis plants overexpressing the NtRop1 showed increased salt sensitivity as can be seen from the reduced root growth and elevated relative electrolyte leakage. The hydrogen peroxide production was also promoted in the NtRop1-trangenic plants in comparison with wild type plants. These results imply that the NtRop1 may confer salt sensitivity through activation of H2O2 production during plant response to salt stress.

  2. Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Michael Hart, C; Chandel, Navdeep; Scott Budinger, G R; Kamp, David W

    2016-12-01

    Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Crocidolite asbestos (100µg/50µL), TiO2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role for

  3. Primary over-expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK-AβPP transgenic mouse.

    Science.gov (United States)

    Luo, Yue-Bei; Johnsen, Russell D; Griffiths, Lisa; Needham, Merrilee; Fabian, Victoria A; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-12-01

    The aim of this study is to determine whether primary over-expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK-AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild-type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over-expression of full length AβPP in transgenic mouse muscles, deposition of amyloid-β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase-deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild-type animals was the presence of tubular aggregates which was age-related and largely restricted to male animals. Expression of full length AβPP in this MCK-AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid-β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re-deriving transgenic mouse strains in different laboratories. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

  4. Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

    Science.gov (United States)

    Gao, Qiong; Li, Xiaoxia; Jia, Junting; Zhao, Pincang; Liu, Panpan; Liu, Zhujiang; Ge, Liangfa; Chen, Shuangyan; Qi, Dongmei; Deng, Bo; Lee, Byung-Hyun; Liu, Gongshe; Cheng, Liqin

    2016-03-01

    As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Overexpression of CYP710A1 and CYP710A4 in transgenic Arabidopsis plants increases the level of stigmasterol at the expense of sitosterol.

    Science.gov (United States)

    Arnqvist, Lisa; Persson, Mattias; Jonsson, Lisbeth; Dutta, Paresh C; Sitbon, Folke

    2008-01-01

    Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008-1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008-1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.

  6. Transgenic Rabbits That Overexpress the Neonatal Fc Receptor (FcRn) Generate Higher Quantities and Improved Qualities of Anti-Thymocyte Globulin (ATG)

    Science.gov (United States)

    Baranyi, Mária; Cervenak, Judit; Bender, Balázs; Kacskovics, Imre

    2013-01-01

    Immune suppression with rabbit anti-thymocyte globulin (rATG) is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn) and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α1-antitrypsin was performed, resulting in significantly greater numbers of antigen-specific B-cells in the FcRn Tg rabbits as compared with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies. PMID:24194847

  7. Sperm parameters and epididymis function in transgenic rats overexpressing the Ca2+-binding protein regucalcin: a hidden role for Ca2+ in sperm maturation?

    Science.gov (United States)

    Correia, S; Oliveira, P F; Guerreiro, P M; Lopes, G; Alves, M G; Canário, A V M; Cavaco, J E; Socorro, Sílvia

    2013-09-01

    Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca(2+)) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca(2+)-binding protein, regulating the activity of Ca(2+)-channels and Ca(2+)-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca(2+) fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with (45)Ca(2+) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca(2+)-influx, indicating unbalanced Ca(2+) concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca(2+)-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.

  8. Overexpression of Nictaba-Like Lectin Genes from Glycine max Confers Tolerance towards Pseudomonas syringae Infection, Aphid Infestation and Salt Stress in Transgenic Arabidopsis Plants

    Directory of Open Access Journals (Sweden)

    Sofie Van Holle

    2016-10-01

    Full Text Available Plants have evolved a sophisticated immune system that allows them to recognize invading pathogens by specialized receptors. Carbohydrate-binding proteins or lectins are part of this immune system and especially the lectins that reside in the nucleocytoplasmic compartment are known to be implicated in biotic and abiotic stress responses. The class of Nictaba-like lectins (NLL groups all proteins with homology to the tobacco (Nicotiana tabacum lectin, known as a stress-inducible lectin. Here we focus on two Nictaba homologs from soybean (Glycine max, referred to as GmNLL1 and GmNLL2. Confocal laser scanning microscopy of fusion constructs with the green fluorescent protein either transiently expressed in Nicotiana benthamiana leaves or stably transformed in tobacco BY-2 suspension cells revealed a nucleocytoplasmic localization for the GmNLLs under study. RT-qPCR analysis of the transcript levels for the Nictaba-like lectins in soybean demonstrated that the genes are expressed in several tissues throughout the development of the plant. Furthermore, it was shown that salt treatment, Phytophthora sojae infection and Aphis glycines infestation trigger the expression of particular NLL genes. Stress experiments with Arabidopsis lines overexpressing the NLLs from soybean yielded an enhanced tolerance of the plant towards bacterial infection (Pseudomonas syringae, insect infestation (Myzus persicae and salinity. Our data showed a better performance of the transgenic lines compared to wild type plants, indicating that the NLLs from soybean are implicated in the stress response. These data can help to further elucidate the physiological importance of the Nictaba-like lectins from soybean, which can ultimately lead to the design of crop plants with a better tolerance to changing environmental conditions.

  9. Overexpression of TaNAC2D Displays Opposite Responses to Abiotic Stresses between Seedling and Mature Stage of Transgenic Arabidopsis

    Science.gov (United States)

    Huang, Quanjun; Wang, Yan

    2016-01-01

    Environmental stresses frequently affect plant growth and development, and many genes have been found to be induced by unfavorable environmental conditions. Here, we reported the biological functions of TaNAC2D, a stress-related NAC (NAM, ATAF, and CUC) gene from wheat. TaNAC2D showed transcriptional activator activity in yeast. TaNAC2D-GFP fusion protein was localized in the nucleus of wheat mesophyll protoplasts. TaNAC2D transcript abundance was significantly induced by NaCl, PEG6000, and abscisic acid (ABA) at seedling stage, and repressed by NaCl and PEG6000 at mature plant stage. When TaNAC2D was introduced into Arabidopsis, the 35-day-old soil-grown TaNAC2D-overexpression (TaNAC2D-OX) plants displayed slower stomatal closure, higher water loss rate, and more sensitivity to salt and drought stresses compared with WT plants. In contrast, TaNAC2D-OX seedlings, grown on 1/2 MS medium supplemented with different concentrations of NaCl, Mannitol, and MV, had enhanced tolerances to salt, osmotic and oxidative stresses during seed germination and post-germination periods. The opposite stress-responsive phenotypes of transgenic Arabidopsis were consistent with the expression patterns of TaNAC2D in wheat. Moreover, under high salinity and dehydration conditions, three marker genes, including NCED3, RD29A, and RD29B, were down-regulated in 35-day-old TaNAC2D-OX plants grown in soil and up-regulated in 14-day-old TaNAC2D-OX seedlings grown on 1/2 MS medium. Our results suggest that the change in growth stages and environmental conditions may regulate TaNAC2D’s function. PMID:27933076

  10. Evaluating water deficit and glyphosate treatment on the accumulation of phenolic compounds and photosynthesis rate in transgenic Codonopsis lanceolata (Siebold & Zucc.) Trautv. over-expressing γ-tocopherol methyltransferase (γ-tmt) gene.

    Science.gov (United States)

    Ghimire, Bimal Kumar; Son, Na-Young; Kim, Seung-Hyun; Yu, Chang Yeon; Chung, Ill-Min

    2017-07-01

    The effect of water stress and herbicide treatment on the phenolic compound concentration and photosynthesis rate in transgenic Codonopsis lanceolata plants over-expressing the γ-tmt gene was investigated and compared to that in control non-transgenic C. lanceolata plants. The total phenolic compound content was investigated using high-performance liquid chromatography combined with diode array detection in C. lanceolata seedlings 3 weeks after water stress and treatment with glyphosate. Changes in the composition of phenolic compounds were observed in leaf and root extracts from transformed C. lanceolata plants following water stress and treatment with glyphosate. The total concentration of phenolic compounds in the leaf extracts of transgenic samples after water stress ranged from 3455.13 ± 40.48 to 8695.00 ± 45.44 µg g-1 dry weight (DW), whereas the total concentration phenolic compound in the leaf extracts of non-transgenic control samples was 5630.83 ± 45.91 µg g-1 DW. The predominant phenolic compounds that increased after the water stress in the transgenic leaf were (+) catechin, benzoic acid, chlorogenic acid, ferulic acid, gallic acid, rutin, vanillic acid, and veratric acid. The total concentration of phenolic compounds in the leaf extracts of transgenic samples after glyphosate treatment ranged from 4744.37 ± 81.81 to 12,051.02 ± 75.00 µg g-1 DW, whereas the total concentration of the leaf extracts of non-transgenic control samples after glyphosate treatment was 3778.28 ± 59.73 µg g-1 DW. Major phenolic compounds that increased in the transgenic C. lanceolata plants after glyphosate treatment included kaempherol, gallic acid, myricetin, p-hydroxybenzjoic acid, quercetin, salicylic acid, t-cinnamic acid, catechin, benzoicacid, ferulic acid, protocatechuic acid, veratric acid, and vanillic acid. Among these, vanillic acid showed the greatest increase in both leaf and root extracts from transgenic plants relative to those

  11. Assessment of stress tolerance, productivity, and forage quality in T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum

    Directory of Open Access Journals (Sweden)

    Peng Kang

    2016-10-01

    Full Text Available Salinization, desertification, and soil nutrient deprivation are threatening the production of alfalfa (Medicago sativa L. in northern China. We have previously generated T0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop this excellent breeding material into the new forage cultivar, stress tolerance, productivity, and forage quality of T1 transgenic alfalfa (GM were assessed in this study. The GM inherited the traits of salt and drought tolerance from T0 generation. Most importantly, co-overexpression of ZxNHX and ZxVP1-1 enhanced the tolerance to Pi deficiency in GM, which was associated with more Pi accumulation in plants. Meanwhile, T1 transgenic alfalfa developed a larger root system with increased root size, root dry weight and root/shoot ratio, which may be one important reason for the improvement of phosphorus nutrition and high biomass accumulation in GM under various conditions. GM also accumulated more crude protein, crude fibre, crude fat, and crude ash than wild-type (WT plants, especially under stress conditions and in the field. More interestingly, the crude fat contents sharply dropped in WT (by 66%-74%, whereas showed no change or decreased less in GM, when subjected to salinity, drought or low-Pi. Our results indicate that T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 shows stronger stress tolerance, higher productivity and better forage quality. This study provides a solid foundation for creating the alfalfa cultivars with high yield, good quality and wide adaptability on saline, dry and nutrient-deprived marginal lands of northern China.

  12. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

    DEFF Research Database (Denmark)

    Sanmartin, Maite; Drogoudi, Pavlina D.; Lyons, Tom

    2003-01-01

    Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O3). Three homozygous transgenic lines, chosen on the basis o...

  13. Nonsymbiotic Hemoglobin-2 Leads to an Elevated Energy State and to a Combined Increase in Polyunsaturated Fatty Acids and Total Oil Content When Overexpressed in Developing Seeds of Transgenic Arabidopsis Plants1[OA

    Science.gov (United States)

    Vigeolas, Helene; Hühn, Daniela; Geigenberger, Peter

    2011-01-01

    Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds. PMID:21205621

  14. Nonsymbiotic hemoglobin-2 leads to an elevated energy state and to a combined increase in polyunsaturated fatty acids and total oil content when overexpressed in developing seeds of transgenic Arabidopsis plants.

    Science.gov (United States)

    Vigeolas, Helene; Hühn, Daniela; Geigenberger, Peter

    2011-03-01

    Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds.

  15. Stress Tolerance Profiling of a Collection of Extant Salt-Tolerant Rice Varieties and Transgenic Plants Overexpressing Abiotic Stress Tolerance Genes

    National Research Council Canada - National Science Library

    Kurotani, Ken-ichi; Yamanaka, Kazumasa; Toda, Yosuke; Ogawa, Daisuke; Tanaka, Maiko; Kozawa, Hirotsugu; Nakamura, Hidemitsu; Hakata, Makoto; Ichikawa, Hiroaki; Hattori, Tsukaho; Takeda, Shin

    2015-01-01

    .... In this study, we investigated the profiles of stress tolerance of nine salt-tolerant rice varieties and transgenic rice lines carrying constitutively expressed genes that are potentially involved...

  16. Overexpression of TaLEA gene from Tamarix androssowii improves salt and drought tolerance in transgenic poplar (Populus simonii × P. nigra.

    Directory of Open Access Journals (Sweden)

    Weidong Gao

    Full Text Available Late embryogenesis abundant (LEA genes were confirmed to confer resistance to drought and water deficiency. An LEA gene from Tamarixandrossowii (named TaLEA was transformed into Xiaohei poplar (Populussimonii × P. nigra via Agrobacterium. Twenty-five independent transgenic lines were obtained that were resistant to kanamycin, and 11 transgenic lines were randomly selected for further analysis. The polymerase chain reaction (PCR and ribonucleic acid (RNA gel blot indicated that the TaLEA gene had been integrated into the poplar genome. The height growth rate, malondialdehyde (MDA content, relative electrolyte leakage and damages due to salt or drought to transgenic and non-transgenic plants were compared under salt and drought stress conditions. The results showed that the constitutive expression of the TaLEA gene in transgenic poplars could induce an increase in height growth rate and a decrease in number and severity of wilted leaves under the salt and drought stresses. The MDA content and relative electrolyte leakage in transgenic lines under salt and drought stresses were significantly lower compared to those in non-transgenic plants, indicating that the TaLEA gene may enhance salt and drought tolerance by protecting cell membranes from damage. Moreover, amongst the lines analyzed for stress tolerance, the transgenic line 11 (T11 showed the highest tolerance levels under both salinity and drought stress conditions. These results indicated that the TaLEA gene could be a salt and drought tolerance candidate gene and could confer a broad spectrum of tolerance under abiotic stresses in poplars.

  17. Overexpression of an acidic endo-β-1,3-1,4-glucanase in transgenic maize seed for direct utilization in animal feed.

    Directory of Open Access Journals (Sweden)

    Yuhong Zhang

    Full Text Available BACKGROUND: Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed. METHODOLOGY/PRINCIPAL FINDINGS: Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0-8.0, which is in agreement with the normal environment of digestive tract. CONCLUSION/SIGNIFICANCE: Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

  18. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    Science.gov (United States)

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  19. Overexpression of an acidic endo-β-1,3-1,4-glucanase in transgenic maize seed for direct utilization in animal feed.

    Science.gov (United States)

    Zhang, Yuhong; Xu, Xiaolu; Zhou, Xiaojin; Chen, Rumei; Yang, Peilong; Meng, Qingchang; Meng, Kun; Luo, Huiying; Yuan, Jianhua; Yao, Bin; Zhang, Wei

    2013-01-01

    Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed. Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0-8.0, which is in agreement with the normal environment of digestive tract. Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.

  20. Gender-specific effects on food intake but no inhibition of age-related fat accretion in transgenic mice overexpressing human IGFBP-2 lacking the Cardin-Weintraub sequence motif.

    Science.gov (United States)

    Wiedmer, Petra; Schwarz, Franziska; Große, Birgit; Schindler, Nancy; Tuchscherer, Armin; Russo, Vincenzo C; Tschöp, Matthias H; Hoeflich, Andreas

    2015-06-01

    IGFBP-2 affects growth and metabolism and is thought to impact on energy homeostasis and the accretion of body fat via its heparin binding domains (HBD). In order to assess the function of the HBD present in the linker domain (HBD1) we have generated transgenic mice overexpressing mutant human IGFBP-2 lacking the PKKLRP sequence and carrying a PNNLAP sequence instead. Transgenic mice expressed high amounts of human IGFBP-2, while endogenous IGFBP-2 or IGF-I serum concentrations were not affected. In both genders we performed a longitudinal analysis of growth and metabolism including at least 4 separate time points between the age of 10 and 52 weeks. Body composition was assessed by nuclear magnetic resonance (NMR) analysis. Food intake was recorded by an automated online-monitoring. We describe negative effects of mutant human IGFBP-2 on body weight, longitudinal growth and lean body mass (p transgenic mice of both genders (p transgene expression significantly increased absolute mass of total body fat over all age groups (p Food intake was increased in female but decreased in male transgenic mice at an age of 11 weeks. Thus our study clearly provides gender- and time-specific effects of HBD1-deficient hIGFBP-2 (H1d-BP-2) on fat mass accretion and food intake. While our data are in principal agreement with current knowledge on the role of HB-domains for fat accretion we now may also speculate on a role of HBD1 for the control of eating behavior.

  1. Overexpression of Poplar PtrWRKY89 in Transgenic Arabidopsis Leads to a Reduction of Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling.

    Directory of Open Access Journals (Sweden)

    Yuanzhong Jiang

    Full Text Available The plant hormones jasmonic acid (JA and salicylic acid (SA play key roles in plant defenses against pathogens and several WRKY transcription factors have been shown to have a role in SA/JA crosstalk. In a previous study, overexpression of the poplar WRKY gene PtrWRKY89 enhanced resistance to pathogens in transgenic poplars. In this study, the promoter of PtrWRKY89 (ProPtrWRKY89 was isolated and used to drive GUS reporter gene. High GUS activity was observed in old leaves of transgenic Arabidopsis containing ProPtrWRKY89-GUS construct and GUS expression was extremely induced by SA solution and SA+MeJA mixture but not by MeJA treatment. Subcellular localization and transactivation assays showed that PtrWRKY89 acted as a transcription activator in the nucleus. Constitutive expression of PtrWRKY89 in Arabidopsis resulted in more susceptible to Pseudomonas syringae and Botrytis cinerea compared to wild-type plants. Quantitative real-time PCR (qRT-PCR analysis confirmed that marker genes of SA and JA pathways were down-regulated in transgenic Arabidopsis after pathogen inoculations. Overall, our results indicated that PtrWRKY89 modulates a cross talk in resistance to P. syringe and B. cinerea by negatively regulating both SA and JA pathways in Arabidopsis.

  2. Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

    Science.gov (United States)

    Singh, H Ranjit; Deka, Manab; Das, Sudripta

    2015-07-01

    Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

  3. Metallothionein-I overexpression alters brain inflammation and stimulates brain repair in transgenic mice with astrocyte-targeted interleukin-6 expression

    DEFF Research Database (Denmark)

    Penkowa, Milena; Camats, Jordi; Giralt, Mercedes

    2003-01-01

    Transgenic expression of IL-6 in the CNS under the control of the GFAP gene promoter, glial fibrillary acidic protein-interleukin-6 (GFAP-IL-6) mice, raises an inflammatory response and causes significant brain damage. However, the results obtained in the GFAP-IL-6 mice after a traumatic brain...

  4. Cloning of Gossypium hirsutum Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (GhSnRK2) and Its Overexpression in Transgenic Arabidopsis Escalates Drought and Low Temperature Tolerance

    Science.gov (United States)

    Bello, Babatunde; Zhang, Xueyan; Liu, Chuanliang; Yang, Zhaoen; Yang, Zuoren; Wang, Qianhua; Zhao, Ge; Li, Fuguang

    2014-01-01

    The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance. PMID:25393623

  5. Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots.

    Science.gov (United States)

    Matthews, Benjamin F; Beard, Hunter; Brewer, Eric; Kabir, Sara; MacDonald, Margaret H; Youssef, Reham M

    2014-04-16

    Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.

  6. Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells

    NARCIS (Netherlands)

    Fielitz, K. (Kathrin); Althoff, K. (Kristina); De Preter, K. (Katleen); J. Nonnekens (Julie); Ohli, J. (Jasmin); Elges, S. (Sandra); Hartmann, W. (Wolfgang); G. Kloppel (Günter); Knösel, T. (Thomas); Schulte, M. (Marc); L. Klein-Hitpass (Ludger); Beisser, D. (Daniela); Reis, H. (Henning); Eyking, A. (Annette); Cario, E. (Elke); J.H. Schulte (Johannes); A. Schramm (Alexander); U. Schüller (Ulrich)

    2016-01-01

    textabstractAmplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the

  7. Diffusion Kurtosis Imaging Detects Microstructural Alterations in Brain of alfa-Synuclein Overexpressing Transgenic Mouse Model of Parkinson’s Disease: A Pilot Study

    Czech Academy of Sciences Publication Activity Database

    Khainar, A.; Latta, P.; Dražanová, Eva; Rudá-Kučerová, J.; Szabó, N.; Arab, A.; Hutter-Paier, B.; Havas, D.; Windisch, M.; Šulcová, A.; Starčuk jr., Zenon; Rektorová, I.

    2015-01-01

    Roč. 28, č. 4 (2015), 281-289 ISSN 1029-8428 R&D Projects: GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : diffusion kurtosis imaging * alfa-Synuclein * TNWT-61 * Parkinson’s disease * Transgenic mice * TBSS Subject RIV: BH - Optics, Masers, Lasers Impact factor: 3.140, year: 2015

  8. Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

    Directory of Open Access Journals (Sweden)

    Yingying Li

    Full Text Available This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon-Wiener (H and the Simpson (1-D indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.

  9. Overexpression of SoCYP85A1, a Spinach Cytochrome p450 Gene in Transgenic Tobacco Enhances Root Development and Drought Stress Tolerance

    Directory of Open Access Journals (Sweden)

    Fangmeng Duan

    2017-11-01

    Full Text Available Brassinosteroids (BRs play an essential role in plant growth, development, and responses to diverse abiotic stresses. However, previous studies mainly analyzed how exogenous BRs influenced plant physiological reactions to drought stress, therefore, genetic evidences for the endogenous BRs-mediated regulation of plant responses still remain elusive. In this study, a key BRs biosynthetic gene, SoCYP85A1 was cloned from Spinacia oleracea, which has a complete open reading frame of 1,392 bp encoding a 464 amino acid peptide and shares high sequence similarities with CYP85A1 from other plants. The expression of SoCYP85A1 which was higher in leaf compared with root and stem, was induced by treatments of PEG6000, abscisic acid (ABA, low temperature and high salt. Increases in both SoCYP85A1 transcripts and endogenous BRs in transgenic tobacco which resulted in longer primary root and more lateral roots enhanced drought tolerance compared with wild types. The transgenic tobacco accumulated much lower levels of reactive oxygen species and malondialdehyde (MDA than wild types did, accompanied by significantly higher content of proline and notably enhanced activities of antioxidant enzymes. Besides, transcriptional expressions of six stress-responsive genes were regulated to higher levels in transgenic lines under drought stress. Taken together, our results demonstrated that SoCYP85A1 involves in response to drought stress by promoting root development, scavenging ROS, and regulating expressions of stress-responsive genes.

  10. Elevated H(2)O (2) production via overexpression of a chloroplastic Cu/ZnSOD gene of lily (Lilium oriental hybrid 'Marco Polo') triggers ethylene synthesis in transgenic potato.

    Science.gov (United States)

    Kim, Yoon-Sik; Kim, Hyun-Soon; Lee, Yong-Hwa; Kim, Mi-Sun; Oh, Hyun-Woo; Hahn, Kyu-Woong; Joung, Hyouk; Jeon, Jae-Heung

    2008-06-01

    Transgenic potato plants (SS2 and SS4) that overexpressed a chloroplastic copper/zinc superoxide dismutase lily gene were utilized as an H(2)O(2)-inducible system in order to study the role of H(2)O(2) as a signaling molecule in the biosynthesis of ethylene. SS2 and SS4 plants grown in vitro under sealed microenvironment (SME) conditions displayed anomalous phenotypes including reduction of stem elongation, radial stem growth, and promotion of root hair formation in the generated root, which were similar to ethylene-induced responses. In addition, SS4 plants showed severe vitrification in developing leaves and elevated ethylene production under SME conditions. After the ethylene action inhibitor AgNO(3), 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) inhibitor CoCl(2), and ACC synthase inhibitor L -aminoethoxyvinylglycine were added to the growth media, the anomalous phenotypes in SS4 plants reverted to their normal phenotype with a concurrent decrease in ethylene production. Northern blot analysis showed that ACO transcripts in SS4 plants were constantly at high levels under normal and SME conditions, indicating that a high level of H(2)O(2) in SS4 plants up-regulates ACO transcripts. Moreover, the direct treatment of H(2)O(2) in potato plants confirmed the elevated expression of the ACO gene. Taken together, these data suggest that the high concentration of H(2)O(2) in transgenic potato plants stimulates ethylene biosynthesis by activating ACO gene expression.

  11. Distinct transmissibility features of TSE sources derived from ruminant prion diseases by the oral route in a transgenic mouse model (TgOvPrP4 overexpressing the ovine prion protein.

    Directory of Open Access Journals (Sweden)

    Jean-Noël Arsac

    Full Text Available Transmissible spongiform encephalopathies (TSEs are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP. Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE, transmissible mink encephalopathy (TME, kuru and variant Creutzfeldt-Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4 overexpressing the ovine prion protein (A136R154Q171 under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.

  12. Impact of concurrent overexpression of cytosolic glutamine synthetase (GS1) and sucrose phosphate synthase (SPS) on growth and development in transgenic tobacco.

    Science.gov (United States)

    Seger, Mark; Gebril, Sayed; Tabilona, Jules; Peel, Amanda; Sengupta-Gopalan, Champa

    2015-01-01

    The outcome of simultaneously increasing SPS and GS activities in transgenic tobacco, suggests that sucrose is the major determinant of growth and development, and is not affected by changes in N assimilation. Carbon (C) and nitrogen (N) are the major components required for plant growth and the metabolic pathways for C and N assimilation are very closely interlinked. Maintaining an appropriate balance or ratio of sugar to nitrogen metabolites in the cell, is important for the regulation of plant growth and development. To understand how C and N metabolism interact, we manipulated the expression of key genes in C and N metabolism individually and concurrently and checked for the repercussions. Transgenic tobacco plants with a cytosolic soybean glutamine synthetase (GS1) gene and a sucrose phosphate synthase (SPS) gene from maize, both driven by the CaMV 35S promoter were produced. Co-transformants, with both the transgenes were produced by sexual crosses. While GS is the key enzyme in N assimilation, involved in the synthesis of glutamine, SPS plays a key role in C metabolism by catalyzing the synthesis of sucrose. Moreover, to check if nitrate has any role in this interaction, the plants were grown under both low and high nitrogen. The SPS enzyme activity in the SPS and SPS/GS1 co-transformants were the same under both nitrogen regimens. However, the GS activity was lower in the co-transformants compared to the GS1 transformants, specifically under low nitrogen conditions. The GS1/SPS transformants showed a phenotype similar to the SPS transformants, suggesting that sucrose is the major determinant of growth and development in tobacco, and its effect is only marginally affected by increased N assimilation. Sucrose may be functioning in a metabolic capacity or as a signaling molecule.

  13. Creation and characterization of BAC-transgenic mice with physiological over-expression of epitope-tagged RCAN1 (DSCR1)

    OpenAIRE

    Xing, Luzhou; Salas , Martha; Zhang, Hong; Gittler, Julia; Ludwig, Thomas; Lin, Chyuan-Sheng; Murty, Vundavalli V.; Silverman, Wayne; Arancio, Ottavio; Tycko, Benjamin

    2012-01-01

    The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg...

  14. Overexpression of WsSGTL1 gene of Withania somnifera enhances salt tolerance, heat tolerance and cold acclimation ability in transgenic Arabidopsis plants.

    Directory of Open Access Journals (Sweden)

    Manoj K Mishra

    Full Text Available BACKGROUND: Sterol glycosyltrnasferases (SGT are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant's adaptation to abiotic stress. METHODOLOGY: The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses--salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA and the 3D structures were predicted by using Discovery Studio Ver. 2.5. RESULTS: The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana. CONCLUSIONS: Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found

  15. Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Misra, Aparna; Khan, Kasim; Niranjan, Abhishek; Nath, Pravendra; Sane, Vidhu A

    2013-12-01

    The increasing consumption of fossil fuels and petroleum products is leading to their rapid depletion and is a matter of concern around the globe. Substitutes of fossil fuels are required to sustain the pace of economic development. In this context, oil from the non food crops (biofuel) has shown potential to substitute fossil fuels. Jatropha curcas is an excellent shrub spread and naturalized across the globe. Its oil contains a high percentage of unsaturated fatty acids (about 78-84% of total fatty acid content) making the oil suitable for biodiesel production. Despite its high oil content, it has been poorly studied in terms of important enzymes/genes responsible for oil biosynthesis. Here, we describe the isolation of the full length cDNA clone of JcDGAT1, a key enzyme involved in oil biosynthesis, from J. curcas seeds and manipulation of oil content and composition in transgenic Arabidopsis plants by its expression. Transcript analysis of JcDGAT1 reveals a gradual increase from early seed development to its maturation. Homozygous transgenic Arabidopsis lines expressing JcDGAT1 both under CaMV35S promoter and a seed specific promoter show an enhanced level of total oil content (up by 30-41%) in seeds but do not show any phenotypic differences. In addition, our studies also show alterations in the oil composition through JcDGAT1 expression. While the levels of saturated FAs such as palmitate and stearate in the oil do not change, there is significant reproducible decrease in the levels of oleic acid and a concomitant increase in levels of linolenic acid both under the CaMV35S promoter as well as the seed specific promoter. Our studies thus confirm that DGAT is involved in flux control in oil biosynthesis and show that JcDGAT1 could be used specifically to manipulate and improve oil content and composition in plants. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots

    Science.gov (United States)

    2014-01-01

    Background Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. Results Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). Conclusions Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes. PMID:24739302

  17. Overexpression of PvPin1, a Bamboo Homolog of PIN1-Type Parvulin 1, Delays Flowering Time in Transgenic Arabidopsis and Rice

    Directory of Open Access Journals (Sweden)

    Zhigang Zheng

    2017-09-01

    Full Text Available Because of the long and unpredictable flowering period in bamboo, the molecular mechanism of bamboo flowering is unclear. Recent study showed that Arabidopsis PIN1-type parvulin 1 (Pin1At is an important floral activator and regulates floral transition by facilitating the cis/trans isomerization of the phosphorylated Ser/Thr residues preceding proline motifs in suppressor of overexpression of CO 1 (SOC1 and agamous-like 24 (AGL24. Whether bamboo has a Pin1 homolog and whether it works in bamboo flowering are still unknown. In this study, we cloned PvPin1, a homolog of Pin1At, from Phyllostachys violascens (Bambusoideae. Bioinformatics analysis showed that PvPin1 is closely related to Pin1-like proteins in monocots. PvPin1 was widely expressed in all tested bamboo tissues, with the highest expression in young leaf and lowest in floral bud. Moreover, PvPin1 expression was high in leaves before bamboo flowering then declined during flower development. Overexpression of PvPin1 significantly delayed flowering time by downregulating SOC1 and AGL24 expression in Arabidopsis under greenhouse conditions and conferred a significantly late flowering phenotype by upregulating OsMADS56 in rice under field conditions. PvPin1 showed subcellular localization in both the nucleus and cytolemma. The 1500-bp sequence of the PvPin1 promoter was cloned, and cis-acting element prediction showed that ABRE and TGACG-motif elements, which responded to abscisic acid (ABA and methyl jasmonate (MeJA, respectively, were characteristic of P. violascens in comparison with Arabidopsis. On promoter activity analysis, exogenous ABA and MeJA could significantly inhibit PvPin1 expression. These findings suggested that PvPin1 may be a repressor in flowering, and its delay of flowering time could be regulated by ABA and MeJA in bamboo.

  18. Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats.

    Science.gov (United States)

    Vetter, Roland; Rehfeld, Uwe; Reissfelder, Christoph; Fechner, Henry; Seppet, Enn; Kreutz, Reinhold

    2011-03-01

    The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P hypothyroid TG (P hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

  19. Paracrine effects of IGF-1 overexpression on the functional decline due to skeletal muscle disuse: molecular and functional evaluation in hindlimb unloaded MLC/mIgf-1 transgenic mice.

    Science.gov (United States)

    Pierno, Sabata; Camerino, Giulia M; Cannone, Maria; Liantonio, Antonella; De Bellis, Michela; Digennaro, Claudio; Gramegna, Gianluca; De Luca, Annamaria; Germinario, Elena; Danieli-Betto, Daniela; Betto, Romeo; Dobrowolny, Gabriella; Rizzuto, Emanuele; Musarò, Antonio; Desaphy, Jean-François; Camerino, Diana Conte

    2014-01-01

    Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5

  20. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

    Science.gov (United States)

    Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2014-08-01

    The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  1. Overexpression of miR-483-5p/3p cooperate to inhibit mouse liver fibrosis by suppressing the TGF-β stimulated HSCs in transgenic mice.

    Science.gov (United States)

    Li, Fuyuan; Ma, Ning; Zhao, Ruiqi; Wu, Guodong; Zhang, Yanfen; Qiao, Yu; Han, Dong; Xu, Ya; Xiang, Ying; Yan, Bingzhu; Jin, Jianfeng; Lv, Guixiang; Wang, Lei; Xu, Changqing; Gao, Xu; Luo, Shanshun

    2014-06-01

    The transition from liver fibrosis to hepatocellular carcinoma (HCC) has been suggested to be a continuous and developmental pathological process. MicroRNAs (miRNAs) are recently discovered molecules that regulate the expression of genes involved in liver disease. Many reports demonstrate that miR-483-5p and miR-483-3p, which originate from miR-483, are up-regulated in HCC, and their oncogenic targets have been identified. However, recent studies have suggested that miR-483-5p/3p is partially down-regulated in HCC samples and is down-regulated in rat liver fibrosis. Therefore, the aberrant expression and function of miR-483 in liver fibrosis remains elusive. In this study, we demonstrate that overexpression of miR-483 in vivo inhibits mouse liver fibrosis induced by CCl4 . We demonstrate that miR-483-5p/3p acts together to target two pro-fibrosis factors, platelet-derived growth factor-β and tissue inhibitor of metalloproteinase 2, which suppress the activation of hepatic stellate cells (HSC) LX-2. Our work identifies the pathway that regulates liver fibrosis by inhibiting the activation of HSCs. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Overexpression of gibberellin 20-oxidase1 from Pinus densiflora results in enhanced wood formation with gelatinous fiber development in a transgenic hybrid poplar.

    Science.gov (United States)

    Park, Eung-Jun; Kim, Hyun-Tae; Choi, Young-Im; Lee, Chanhui; Nguyen, Van Phap; Jeon, Hyung-Woo; Cho, Jin-Seong; Funada, Ryo; Pharis, Richard P; Kurepin, Leonid V; Ko, Jae-Heung

    2015-11-01

    Gibberellins (GAs) are important regulators of plant shoot biomass growth, and GA 20-oxidase (GA20ox) is one of the major regulatory enzymes in the GA biosynthetic pathway. Previously, we showed that the expression levels of a putative GA20ox1 (i.e., PdGA20ox1) in stem tissue of 3-month-old seedlings of 12 families of Pinus densiflora were positively correlated with stem diameter growth across those same families growing in an even-aged 32-year-old pine forest (Park EJ, Lee WY, Kurepin LV, Zhang R, Janzen L, Pharis RP (2015) Plant hormone-assisted early family selection in Pinus densiflora via a retrospective approach. Tree Physiol 35:86-94). To further investigate the molecular function of this gene in the stem wood growth of forest trees, we produced transgenic poplar lines expressing PdGA20ox1 under the control of the 35S promoter (designated as 35S::PdGA20ox1). By age 3 months, most of the 35S::PdGA20ox1 poplar trees were showing an exceptional enhancement of stem wood growth, i.e., up to fourfold increases in stem dry weight, compared with the nontransformed control poplar plants. Significant increases in endogenous GA1, its immediate precursor (GA20) and its catabolite (GA8) in elongating internode tissue accompanied the increased stem growth in the transgenic lines. Additionally, the development of gelatinous fibers occurred in vertically grown stems of the 35S::PdGA20ox1 poplars. An analysis of the cell wall monosaccharide composition of the 35S::PdGA20ox1 poplars showed significant increases in xylose and glucose contents, indicating a qualitative increase in secondary wall depositions. Microarray analyses led us to find a total of 276 probe sets that were upregulated (using threefold as a threshold) in the stem tissues of 35S::PdGA20ox1 poplars relative to the controls. 'Cell organization or biogenesis'- and 'cell wall'-related genes were overrepresented, including many of genes that are involved in cell wall modification. Several transcriptional

  3. Creation and characterization of BAC-transgenic mice with physiological over-expression of epitope-tagged RCAN1 (DSCR1)

    Science.gov (United States)

    Xing, Luzhou; Salas, Martha; Zhang, Hong; Gittler, Julia; Ludwig, Thomas; Lin, Chyuan-Sheng; Murty, Vundavalli V.; Silverman, Wayne; Arancio, Ottavio; Tycko, Benjamin

    2012-01-01

    The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg was expressed at levels only moderately higher than the native Rcan1 gene; approximately 1.5-fold in RCAN1BAC-Tg1 and 2-fold in RCAN1BAC-Tg2. Affinity purification of the RCAN1 protein complex from brains of these mice revealed a core complex of RCAN1 with calcineurin (CaN), glycogen synthase kinase 3-beta (Gsk3b), and calmodulin, with sub-stoichiometric components including LOC73419. The BAC-Tg mice are fully viable, but long-term synaptic potentiation (LTP) is impaired in proportion to BAC-Tg dosage in hippocampal brain slices from these mice. RCAN1 can act as a tumor suppressor in some systems, but we found that the RCAN1 BAC-Tg did not reduce mammary cancer growth when present at a low copy number in Tp53;WAP-Cre mice. This work establishes a useful mouse model for investigating the biochemistry and dose-dependent functions of the RCAN1 protein in vivo. PMID:23096997

  4. Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dy(W) mouse model of merosin-deficient congenital muscular dystrophy type 1A.

    Science.gov (United States)

    Doe, Jinger A; Wuebbles, Ryan D; Allred, Erika T; Rooney, Jachinta E; Elorza, Margaret; Burkin, Dean J

    2011-07-01

    Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7β1 integrin; however, it is unclear how the secondary loss of α7β1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy(W⁻/⁻) mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7β1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dy(W⁻/⁻) mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A.

  5. RANK Overexpression in Transgenic Mice with Mouse Mammary Tumor Virus Promoter-Controlled RANK Increases Proliferation and Impairs Alveolar Differentiation in the Mammary Epithelia and Disrupts Lumen Formation in Cultured Epithelial Acini▿ †

    Science.gov (United States)

    Gonzalez-Suarez, Eva; Branstetter, Daniel; Armstrong, Allison; Dinh, Huyen; Blumberg, Hal; Dougall, William C.

    2007-01-01

    RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins β-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis. PMID:17145767

  6. RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini.

    Science.gov (United States)

    Gonzalez-Suarez, Eva; Branstetter, Daniel; Armstrong, Allison; Dinh, Huyen; Blumberg, Hal; Dougall, William C

    2007-02-01

    RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins beta-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.

  7. Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dyW mouse model of merosin-deficient congenital muscular dystrophy type 1A

    Science.gov (United States)

    Doe, Jinger A.; Wuebbles, Ryan D.; Allred, Erika T.; Rooney, Jachinta E.; Elorza, Margaret; Burkin, Dean J.

    2011-01-01

    Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7β1 integrin; however, it is unclear how the secondary loss of α7β1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dyW−/− mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7β1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dyW−/− mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A. PMID:21652631

  8. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

    Science.gov (United States)

    2011-01-01

    Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of M. persicae as a result

  9. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

    Directory of Open Access Journals (Sweden)

    Sun Weiwei

    2011-01-01

    Full Text Available Abstract Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2, one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of

  10. Expression Profiling of Ribosomal Protein Gene Family in Dehydration Stress Responses and Characterization of Transgenic Rice Plants Overexpressing RPL23A for Water-Use Efficiency and Tolerance to Drought and Salt Stresses

    Directory of Open Access Journals (Sweden)

    Mazahar Moin

    2017-11-01

    Full Text Available Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a. In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP gene family including both small (RPS and large (RPL subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24, and 31 and RPS4, 10 and 18a. To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ13C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt

  11. Phytoremediation with transgenic trees

    Energy Technology Data Exchange (ETDEWEB)

    Peuke, A.D.; Rennenberg, H. [Inst. fuer Forstbotanik und Baumphysiologie, Professur fuer Baumphysiologie, Freiburg im Breisgau (Germany)

    2005-04-01

    In the present paper actual trends in the use of transgenic trees for phytoremediation of contaminated soils are reviewed. In this context a current field trial in which transgenic poplars with enhanced GSH synthesis and hence elevated capacity for phytochelatin production are compared with wildtype plants for the removal of heavy metals at different levels of contamination and under different climatic conditions. The studies are carried out with grey poplar (Populus tremula x P. alba), wildtype plants and plants overexpressing the gene for {gamma}-glutamylcysteine synthetase (gshI) from E. coli in the cytosol. The expression of this gene in poplar leads to two- to four-fold enhanced GSH concentrations in the leaves. In greenhouse experiments under controlled conditions these transgenic poplars showed a high potential for uptake and detoxification of heavy metals and pesticides. This capacity is evaluated in field experiments. Further aims of the project are to elucidate (a) the stability of the transgene under field conditions and (b) the possibility of horizontal gene transfer to microorganisms in the rhizosphere. The results will help to assess the biosafety risk of the use of transgenic poplar for phytoremediation of soils. (orig.)

  12. Improved nutritive quality and salt resistance in transgenic maize by simultaneously overexpression of a natural lysine-rich protein gene, SBgLR, and an ERF transcription factor gene, TSRF1.

    Science.gov (United States)

    Wang, Meizhen; Liu, Chen; Li, Shixue; Zhu, Dengyun; Zhao, Qian; Yu, Jingjuan

    2013-04-29

    Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9-10 and 19-11, ZmMYC1 in line 19-11 and ZmSDR in line 19-11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment

  13. Improved Nutritive Quality and Salt Resistance in Transgenic Maize by Simultaneously Overexpression of a Natural Lysine-Rich Protein Gene, SBgLR, and an ERF Transcription Factor Gene, TSRF1

    Directory of Open Access Journals (Sweden)

    Meizhen Wang

    2013-04-01

    Full Text Available Maize (Zea mays L., as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9–10 and 19–11, ZmMYC1 in line 19–11 and ZmSDR in line 19–11, the expression of other stress-related genes were inhibited in transgenic lines under normal

  14. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    Directory of Open Access Journals (Sweden)

    Rodrigo S Lacruz

    Full Text Available We have previously identified amelotin (AMTN as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL and ameloblastin (AMBN was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  15. Targeted overexpression of amelotin disrupts the microstructure of dental enamel.

    Science.gov (United States)

    Lacruz, Rodrigo S; Nakayama, Yohei; Holcroft, James; Nguyen, Van; Somogyi-Ganss, Eszter; Snead, Malcolm L; White, Shane N; Paine, Michael L; Ganss, Bernhard

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

  16. YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2.

    Science.gov (United States)

    Li, Zheng-Yi; Chung, Yoon Hee; Shin, Eun-Joo; Dang, Duy-Khanh; Jeong, Ji Hoon; Ko, Sung Kwon; Nah, Seung-Yeol; Baik, Tae Gon; Jhoo, Jin Hyeong; Ong, Wei-Yi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2017-04-27

    Ginkgo biloba has been reported to possess free radical-scavenging antioxidant activity and anti-inflammatory properties. In our pilot study, YY-1224, a terpene trilactone-strengthened extract of G. biloba, showed anti-inflammatory, neurotrophic, and antioxidant effects. We investigated the pharmacological potential of YY-1224 in β-amyloid (Aβ) (1-42)-induced memory impairment using cyclooxygenase-2 (COX-2) knockout (-/-) and APPswe/PS1dE9 transgenic (APP/PS1 Tg) mice. Repeated treatment with YY-1224 significantly attenuated Aβ (1-42)-induced memory impairment in COX-2 (+/+) mice, but not in COX-2 (-/-) mice. YY-1224 significantly attenuated Aβ (1-42)-induced upregulation of platelet-activating factor (PAF) receptor gene expression, reactive oxygen species, and pro-inflammatory factors. In addition, YY-1224 significantly inhibited Aβ (1-42)-induced downregulation of PAF-acetylhydrolase-1 (PAF-AH-1) and peroxisome proliferator-activated receptor γ (PPARγ) gene expression. These changes were more pronounced in COX-2 (+/+) mice than in COX-2 (-/-) mice. YY-1224 significantly attenuated learning impairment, Aβ deposition, and pro-inflammatory microglial activation in APP/PS1 Tg mice, whereas it significantly enhanced PAF-AH and PPARγ expression. A preferential COX-2 inhibitor, meloxicam, did not affect the pharmacological activity by YY-1224, suggesting that the COX-2 gene is a critical mediator of the neuroprotective effects of YY-1224. The protective activity of YY-1224 appeared to be more efficacious than a standard G. biloba extract (Gb) against Aβ insult. Our results suggest that the protective effects of YY-1224 against Aβ toxicity may be associated with its PAF antagonistic- and PPARγ agonistic-potential as well as inhibition of the Aβ-mediated pro-inflammatory switch of microglia phenotypes through suppression of COX-2 expression.

  17. Transgenic plants with enhanced growth characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2016-09-06

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  18. Transgenic plants with enhanced growth characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J.; Anderson, Penelope S.; Knight, Thomas J.

    2018-01-09

    The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. In one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) are provided. The GPT+GS double-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with T0 generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

  19. Targeted Overexpression of Amelotin Disrupts the Microstructure of Dental Enamel

    OpenAIRE

    Lacruz, Rodrigo S.; Yohei Nakayama; James Holcroft; Van, Nguyen.; Eszter Somogyi-Ganss; Snead, Malcolm L.; WHITE, Shane N.; Paine, Michael L.; Bernhard Ganss

    2012-01-01

    We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. ...

  20. Generation of transgenic Hydra by embryo microinjection.

    Science.gov (United States)

    Juliano, Celina E; Lin, Haifan; Steele, Robert E

    2014-09-11

    As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

  1. MusaWRKY71 Overexpression in Banana Plants Leads to Altered Abiotic and Biotic Stress Responses

    OpenAIRE

    Upendra K S Shekhawat; Ganapathi, Thumballi R.

    2013-01-01

    WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by S...

  2. GH/STAT5 signaling during the growth period in livers of mice overexpressing GH

    OpenAIRE

    Martinez, Carolina Soledad; Piazza, Verónica Gabriela; Díaz, María Eugenia; Boparal, Raavnet K.; Arum, Oge; Ramirez, Maria Cecilia; Gonzalez, Lorena; Becu, Damasia; Bartke, Andrzej; Turyn, Daniel; Miquet, Johanna Gabriela; Sotelo, Ana Isabel

    2016-01-01

    GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing m...

  3. Overexpression of GbERF confers alteration of ethylene-responsive ...

    Indian Academy of Sciences (India)

    To study the function of GbERF in the process of biotic stress, transgenic tobacco plants expressing GbERF were generated. Overexpression of GbERF did not change transgenic plant's phenotype and endogenous ethylene level. However, the expression profile of some ethylene-inducible GCC-box and non-GCC-box ...

  4. Overexpression of mineralocorticoid receptors does not affect memory and anxiety-like behavior in female mice

    NARCIS (Netherlands)

    Kanatsou, S.; Kuil, L.E.; Arp, M.; Oitzl, M.S.; Harris, A.P.; Seckl, J.R.; Krugers, H.J.; Joels, M.

    2015-01-01

    Mineralocorticoid receptors (MRs) have been implicated in behavioral adaptation and learning and memory. Since-at least in humans-MR function seems to be sex-dependent, we examined the behavioral relevance of MR in female mice exhibiting transgenic MR overexpression in the forebrain. Transgenic MR

  5. Transgenic plants for phytoremediation.

    Science.gov (United States)

    Maestri, Elena; Marmiroli, Nelson

    2011-01-01

    Phytoremediation is a green, sustainable and promising solution to problems of environmental contamination. It entails the use of plants for uptake, sequestration, detoxification or volatilization of inorganic and organic pollutants from soils, water, sediments and possibly air. Phytoremediation was born from the observation that plants possessed physiological properties useful for environmental remediation. This was shortly followed by the application of breeding techniques and artificial selection to genetically improve some of the more promising and interesting species. Now, after nearly 20 years of research, transgenic plants for phytoremediation have been produced, but none have reached commercial existence. Three main approaches have been developed: (1) transformation with genes from other organisms (mammals, bacteria, etc.); (2) transformation with genes from other plant species; and (3) overexpression of genes from the same plant species. Many encouraging results have been reported, even though in some instances results have been contrary to expectations. This review will illustrate the main examples with a critical discussion of what we have learnt from them.

  6. Transgene silencing and transgene-derived siRNA production in tobacco plants homozygous for an introduced AtMYB90 construct

    Science.gov (United States)

    Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines Myb27 and Myb237 accumulated large quantities of anthoc...

  7. Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice

    NARCIS (Netherlands)

    M.J. van Haperen (Rien); A. van Tol (Arie); P. Vermeulen; M. Jauhiainen; T. van Gent (Teus); P.M. van den Berg (Paul); S. Ehnholm (Sonja); A.W.M. van der Kamp (Arthur); M.P.G. de Crom (Rini); F.G. Grosveld (Frank)

    2000-01-01

    textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress

  8. Transgenic Rescue of the LARGEmyd Mouse: A LARGE Therapeutic Window?

    Directory of Open Access Journals (Sweden)

    J C W Hildyard

    Full Text Available LARGE is a glycosyltransferase involved in glycosylation of α-dystroglycan (α-DG. Absence of this protein in the LARGEmyd mouse results in α-DG hypoglycosylation, and is associated with central nervous system abnormalities and progressive muscular dystrophy. Up-regulation of LARGE has previously been proposed as a therapy for the secondary dystroglycanopathies: overexpression in cells compensates for defects in multiple dystroglycanopathy genes. Counterintuitively, LARGE overexpression in an FKRP-deficient mouse exacerbates pathology, suggesting that modulation of α-DG glycosylation requires further investigation. Here we demonstrate that transgenic expression of human LARGE (LARGE-LV5 in the LARGEmyd mouse restores α-DG glycosylation (with marked hyperglycosylation in muscle and that this corrects both the muscle pathology and brain architecture. By quantitative analyses of LARGE transcripts we also here show that levels of transgenic and endogenous LARGE in the brains of transgenic animals are comparable, but that the transgene is markedly overexpressed in heart and particularly skeletal muscle (20-100 fold over endogenous. Our data suggest LARGE overexpression may only be deleterious under a forced regenerative context, such as that resulting from a reduction in FKRP: in the absence of such a defect we show that systemic expression of LARGE can indeed act therapeutically, and that even dramatic LARGE overexpression is well-tolerated in heart and skeletal muscle. Moreover, correction of LARGEmyd brain pathology with only moderate, near-physiological LARGE expression suggests a generous therapeutic window.

  9. Transgenic plants with increased calcium stores

    Science.gov (United States)

    Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Robertson, Dominique (Inventor); Boss, Wendy (Inventor)

    2004-01-01

    The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

  10. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    LI ZHANG

    Kawakami, Division of Molecular and Developmental. Biology, National Institute of Genetics, Mishima, Shizuoka,. Japan. The pT2AL200R150G (Tol2) vector contains a green fluorescent protein (GFP) expression cassette com- posed of the Xenopus EF1 enhancer/promoter for ubiquitous expression, the rabbit globin intron, ...

  11. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    Fertilized eggs were obtained by natural mating, incubated at the same temperature and light condi- tions as adults. The animal ..... showed traits of prematurity with a distinct tail and a yolk extension elongating away from the yolk (figure 2A, d). We believe this prematurity was attributed to BAFF overexpres- sion because a ...

  12. Transgenic overexpression of BAFF regulates the expression of ...

    Indian Academy of Sciences (India)

    Hebei Key Lab of Laboratory Animal Science, Hebei Medical University, Shijiazhuang 050017, People's Republic of China; Department of Medical Genetics, Tongji Medical College; Huazhong University of Science and Technology, Hubei 430030, People's Republic of China; Second Hospital of Hebei Medical University, ...

  13. Overexpression of Arabidopsis MYB96 confers drought resistance in Camelina sativa via cuticular wax accumulation.

    Science.gov (United States)

    Lee, Saet Buyl; Kim, Hyojin; Kim, Ryeo Jin; Suh, Mi Chung

    2014-09-01

    Camelina has been highlighted as an emerging oilseed crop. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited drought resistance by activating expression of Camelina wax biosynthetic genes and accumulating wax load. Camelina (Camelina sativa L.) is an oilseed crop in the Brassicaeae family with potential to expand biofuel production to marginal land. The aerial portion of all land plants is covered with cuticular wax to protect them from desiccation. In this study, the Arabidopsis MYB96 gene was overexpressed in Camelina under the control of the CaMV35S promoter. Transgenic Camelina plants overexpressing Arabidopsis MYB96 exhibited normal growth and development and enhanced tolerance to drought. Deposition of epicuticular wax crystals and total wax loads increased significantly on the surfaces of transgenic leaves compared with that of non-transgenic plants. The levels of alkanes and primary alcohols prominently increased in transgenic Camelina plants relative to non-transgenic plants. Cuticular transpiration occurred more slowly in transgenic leaves than that in non-transgenic plants. Genome-wide identification of Camelina wax biosynthetic genes enabled us to determine that the expression levels of CsKCS2, CsKCS6, CsKCR1-1, CsKCR1-2, CsECR, and CsMAH1 were approximately two to sevenfold higher in transgenic Camelina leaves than those in non-transgenic leaves. These results indicate that MYB96-mediated transcriptional regulation of wax biosynthetic genes is an approach applicable to generating drought-resistant transgenic crops. Transgenic Camelina plants with enhanced drought tolerance could be cultivated on marginal land to produce renewable biofuels and biomaterials.

  14. ADAM 12 protease induces adipogenesis in transgenic mice

    DEFF Research Database (Denmark)

    Kawaguchi, Nobuko; Xu, Xiufeng; Tajima, Rie

    2002-01-01

    -anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent...... in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did...... not exhibit increased serum insulin, cholesterol, or triglycerides. Male transgenics were slightly overweight and also developed herniation but did not become obese. Transgenic mice expressing a truncated form of ADAM 12-S lacking the prodomain and the metalloprotease domain did not develop this adipogenic...

  15. Overexpression of SIRT1 in Mouse Forebrain Impairs Lipid/Glucose Metabolism and Motor Function: e21759

    National Research Council Canada - National Science Library

    Dongmei Wu; Yifu Qiu; Xiang Gao; Xiao-Bing Yuan; Qiwei Zhai

    2011-01-01

    .... And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter...

  16. Overexpression of mineralocorticoid receptors does not affect memory and anxiety-like behavior in female mice

    Directory of Open Access Journals (Sweden)

    Sofia eKanatsou

    2015-07-01

    Full Text Available Mineralocorticoid receptors (MRs have been implicated in behavioral adaptation and learning and memory. Since – at least in humans - MR function seems to be sex-dependent, we examined the behavioral relevance of MR in female mice exhibiting transgenic MR overexpression in the forebrain. Transgenic MR overexpression did not affect contextual fear memory or cued fear learning and memory. Moreover, MR overexpressing and control mice discriminated equally well between fear responses in a combined cue and context fear conditioning paradigm. Also context-memory in an object recognition task was unaffected in MR overexpressing mice. We conclude that MR overexpression in female animals does not affect fear conditioned responses and object recognition memory.

  17. Phosphate uptake and growth characteristics of transgenic rice with ...

    African Journals Online (AJOL)

    Yomi

    2012-04-03

    Apr 3, 2012 ... Farmers have used phosphate fertilizer to provide sufficient yields. However, overuse of phosphorus accumulate in soil and causes soil and water pollution. We evaluated the phosphate acquisition and growth characteristics of OsPT1 transgenic rice (OsPT1-OX, over-expressing the high affinity phosphate ...

  18. Phosphate uptake and growth characteristics of transgenic rice with ...

    African Journals Online (AJOL)

    Farmers have used phosphate fertilizer to provide sufficient yields. However, overuse of phosphorus accumulate in soil and causes soil and water pollution. We evaluated the phosphate acquisition and growth characteristics of OsPT1 transgenic rice (OsPT1-OX, over-expressing the high affinity phosphate transporter 1) in ...

  19. Molecular Analyses of Transgenic Plants.

    Science.gov (United States)

    Trijatmiko, Kurniawan Rudi; Arines, Felichi Mae; Oliva, Norman; Slamet-Loedin, Inez Hortense; Kohli, Ajay

    2016-01-01

    One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue.

  20. E2F-1-Induced p53-independent apoptosis in transgenic mice

    DEFF Research Database (Denmark)

    Holmberg, Christian Henrik; Helin, K.; Sehested, M.

    1998-01-01

    The E2F transcription factors are key targets for the retinoblastoma protein, pRB. By inactivation of E2Fs, pRB prevents progression to the S phase. To test proliferative functions of E2F, we generated transgenic mice expressing human E2F-1 and/or human DP-1. When the hydroxymethyl glutaryl...... involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered...

  1. Behavioral characterization of a mouse model overexpressing DSCR1/ RCAN1

    OpenAIRE

    Mara Dierssen; Gloria Arqué; Jerome McDonald; Nuria Andreu; Carmen Martínez-Cué; Jesús Flórez; Cristina Fillat

    2011-01-01

    DSCR1/ RCAN1 is a chromosome 21 gene found to be overexpressed in the brains of Down syndrome (DS) and postulated as a good candidate to contribute to mental disability. However, even though Rcan1 knockout mice have pronounced spatial learning and memory deficits, the possible deleterious effects of its overexpression in DS are not well understood. We have generated a transgenic mouse model overexpressing DSCR1/RCAN1 in the brain and analyzed the effect of RCAN1 overexpression on cognitive fu...

  2. Neuroglobin Overexpression Inhibits AMPK Signaling and Promotes Cell Anabolism.

    Science.gov (United States)

    Cai, Bin; Li, Wenjun; Mao, XiaoOu; Winters, Ali; Ryou, Myoung-Gwi; Liu, Ran; Greenberg, David A; Wang, Ning; Jin, Kunlin; Yang, Shao-Hua

    2016-03-01

    Neuroglobin (Ngb) is a recently discovered globin with preferential localization to neurons. Growing evidence indicates that Ngb has distinct physiological functions separate from the oxygen storage and transport roles of other globins, such as hemoglobin and myoglobin. We found increased ATP production and decreased glycolysis in Ngb-overexpressing immortalized murine hippocampal cell line (HT-22), in parallel with inhibition of AMP-activated protein kinase (AMPK) signaling and activation of acetyl-CoA carboxylase (ACC). In addition, lipid and glycogen content was increased in Ngb-overexpressing HT-22 cells. AMPK signaling was also inhibited in the brain and heart from Ngb-overexpressing transgenic mice. Although Ngb overexpression did not change glycogen content in whole brain, glycogen synthase was activated in cortical neurons of Ngb-overexpressing mouse brain and Ngb overexpression primary neurons. Moreover, lipid and glycogen content was increased in hearts derived from Ngb-overexpressing mice. These findings suggest that Ngb functions as a metabolic regulator and enhances cellular anabolism through the inhibition of AMPK signaling.

  3. Transgenic soya bean seeds accumulating β‐carotene exhibit the collateral enhancements of oleate and protein content traits

    National Research Council Canada - National Science Library

    Schmidt, Monica A; Parrott, Wayne A; Hildebrand, David F; Berg, R. Howard; Cooksey, Amanda; Pendarvis, Ken; He, Yonghua; McCarthy, Fiona; Herman, Eliot M

    2015-01-01

    Transgenic soya bean ( Glycine max ) plants overexpressing a seed‐specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 μg β carotene g...

  4. Over-expressing Salicornia europaea (SeNHX1) gene in tobacco ...

    African Journals Online (AJOL)

    When exposed to either 138 mM or 172 mM NaCl in vitro, the transgenic apical meristem and stem segments with buds exhibited stronger salt-tolerance than their wild-type counterparts. In pot soil with 10.2 mg g-1 DW Na+ stress, transgenic plants over-expressing SeNHX1 accumulated 1.2 mg g-1 FW Na+ greater than ...

  5. Odontoblast-targeted Bcl-2 Overexpression Promotes Dentin Damage Repair

    Science.gov (United States)

    Zhang, Wenjian; Ju, Jun

    2011-01-01

    Objective Bcl-2 is widely expressed in a developing tooth organ and regulates tooth morphogenesis. However, whether Bcl-2 is related to tooth damage repair is unknown yet. Using an odontoblast-targeted Bcl-2 overexpression transgenic mouse (Col2.3Bcl-2) and artificial cavity preparation as a model system, the relationship between Bcl-2 and reparative dentinogenesis is investigated in this study. Methods The odontoblastic-like cell cultures derived from mouse molar pulps were established. The expression of transgenic human Bcl-2 (hBcl-2) and endogenous mouse Bcl-2 (mBcl-2) and mouse Bax (mBax, a Bcl-2 antagonist) was detected in vivo and in vitro by Western blot and immunocytochemistry, respectively. Basal level and artificial cavity-induced odontoblast apoptosis was detected by the Deoxynucleotidyl Transferase (TdT) dUTP Nick End labeling (TUNEL) technique. Reparative dentin formation induced by artificial cavity drilled to a half dentin thickness on mesial cervical region of mandibular first molars 2, 4, and 6 weeks post-op was evaluated histologically and via micro-CT. Results The transgenic hBcl-2 was stably expressed in odontoblasts of the transgenic animals without interference with the expression of mBcl-2 and mBax. Basal level as well as artificial cavity- induced odontoblast apoptosis was prevented by the transgene. Compared to the wild type, the transgenic animals produced reparative dentin with significantly higher mineral density 6 weeks after the operation. Conclusions Bcl-2 overexpression prevents odontoblast apoptosis and promotes dentin damage repair, indicating that genetic manipulation of Bcl-2 may be a novel strategy to maintain the vitality and function of dentine-pulp complex under detrimental mechanical stimuli. PMID:21930259

  6. Effects of Mineralocorticoid Receptor Overexpression on Anxiety and Memory after Early Life Stress in Female Mice.

    Science.gov (United States)

    Kanatsou, Sofia; Ter Horst, Judith P; Harris, Anjanette P; Seckl, Jonathan R; Krugers, Harmen J; Joëls, Marian

    2015-01-01

    Early-life stress (ELS) is a risk factor for the development of psychopathology, particularly in women. Human studies have shown that certain haplotypes of NR3C2, encoding the mineralocorticoid receptor (MR), that result in gain of function, may protect against the consequences of stress exposure, including childhood trauma. Here, we tested the hypothesis that forebrain-specific overexpression of MR in female mice would ameliorate the effects of ELS on anxiety and memory in adulthood. We found that ELS increased anxiety, did not alter spatial discrimination and reduced contextual fear memory in adult female mice. Transgenic overexpression of MR did not alter anxiety but affected spatial memory performance and enhanced contextual fear memory formation. The effects of ELS on anxiety and contextual fear were not affected by transgenic overexpression of MR. Thus, MR overexpression in the forebrain does not represent a major resilience factor to early life adversity in female mice.

  7. Autotaxin overexpression causes embryonic lethality and vascular defects.

    Directory of Open Access Journals (Sweden)

    Hiroshi Yukiura

    Full Text Available Autotaxin (ATX is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA, and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation.

  8. Autotaxin Overexpression Causes Embryonic Lethality and Vascular Defects

    Science.gov (United States)

    Yukiura, Hiroshi; Kano, Kuniyuki; Kise, Ryoji; Inoue, Asuka; Aoki, Junken

    2015-01-01

    Autotaxin (ATX) is a secretory protein, which converts lysophospholipids to lysophosphatidic acid (LPA), and is essential for embryonic vascular formation. ATX is abundantly detected in various biological fluids and its level is elevated in some pathophysiological conditions. However, the roles of elevated ATX levels remain to be elucidated. In this study, we generated conditional transgenic (Tg) mice overexpressing ATX and examined the effects of excess LPA signalling. We found that ATX overexpression in the embryonic period caused severe vascular defects and was lethal around E9.5. ATX was conditionally overexpressed in the neonatal period using the Cre/loxP system, which resulted in a marked increase in the plasma LPA level. This resulted in retinal vascular defects including abnormal vascular plexus and increased vascular regression. Our findings indicate that the ATX level must be carefully regulated to ensure coordinated vascular formation PMID:25992708

  9. Podocyte Injury and Albuminuria in Mice with Podocyte-Specific Overexpression of the Ste20-Like Kinase, SLK

    Science.gov (United States)

    Cybulsky, Andrey V.; Takano, Tomoko; Papillon, Joan; Guillemette, Julie; Herzenberg, Andrew M.; Kennedy, Chris R. J.

    2010-01-01

    SLK expression and activity are increased during kidney development and recovery from renal ischemia-reperfusion injury. In cultured cells, SLK promotes F-actin destabilization as well as apoptosis, partially via the p38 kinase pathway. To better understand the effects of SLK in vivo, a transgenic mouse model was developed where SLK was expressed in a podocyte-specific manner using the mouse nephrin promoter. Offspring of four founder mice carried the SLK transgene. Among male transgenic mice, 66% developed albuminuria at approximately 3 months of age, and the albuminuric mice originated from three of four founders. Overall, the male transgenic mice demonstrated about fivefold greater urinary albumin/creatinine compared with male non-transgenic mice. Transgenic and non-transgenic female mice did not develop albuminuria, suggesting that females were less susceptible to glomerular filtration barrier damage than their male counterparts. In transgenic mice, electron microscopy revealed striking podocyte injury, including poorly formed or effaced foot processes, and edematous and vacuolated cell bodies. By immunoblotting, nephrin expression was decreased in glomeruli of the albuminuric transgenic mice. Activation-specific phosphorylation of p38 was increased in transgenic mice compared with non-transgenic animals. Glomeruli of SLK transgenic mice showed around 30% fewer podocytes, and a reduction in F-actin compared with control glomeruli. Thus, podocyte SLK overexpression in vivo results in injury and podocyte loss, consistent with the effects of SLK in cultured cells. PMID:20889563

  10. PP005. Vitamin D depletion aggravates hypertension in transgenic rats

    DEFF Research Database (Denmark)

    Bjørkholt Andersen, Louise; Herse, Florian; Christesen, Henrik Thybo

    2013-01-01

    INTRODUCTION: Vitamin D may ameliorate hypertension and kidney disease through genomic and extra-genomic pathways. OBJECTIVE: To investigate the impact of vitamin D in a transgenic rat model of angiotensin II-mediated hypertensive organ failure. METHODS: In 4-week-old age-matched rats overexpress......INTRODUCTION: Vitamin D may ameliorate hypertension and kidney disease through genomic and extra-genomic pathways. OBJECTIVE: To investigate the impact of vitamin D in a transgenic rat model of angiotensin II-mediated hypertensive organ failure. METHODS: In 4-week-old age-matched rats...

  11. Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

    Science.gov (United States)

    Coruzzi, Gloria [New York, NY; Gutierrez, Rodrigo A [Santiago, CL; Nero, Damion C [Woodside, NY

    2012-04-10

    Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

  12. THE TRIAL OF TRANSGENICS

    Directory of Open Access Journals (Sweden)

    Antonio f. Díaz García

    2015-04-01

    Full Text Available This paper discloses the uncertainty with which transgenic uses are authorized.  It provides a list of reasons showing that there is no absolute proof of the benefits of transgenic use.  Moreover it discusses the need to provide more credibility to safety studies and reports on results of various tests of GMOs.  Finally it proposes the establishment of higher penalties for specialists that omit relevant information in their studies and reports on this matter.

  13. Tubular overexpression of gremlin induces renal damage susceptibility in mice.

    Directory of Open Access Journals (Sweden)

    Alejandra Droguett

    Full Text Available A growing number of patients are recognized worldwide to have chronic kidney disease. Glomerular and interstitial fibrosis are hallmarks of renal progression. However, fibrosis of the kidney remains an unresolved challenge, and its molecular mechanisms are still not fully understood. Gremlin is an embryogenic gene that has been shown to play a key role in nephrogenesis, and its expression is generally low in the normal adult kidney. However, gremlin expression is elevated in many human renal diseases, including diabetic nephropathy, pauci-immune glomerulonephritis and chronic allograft nephropathy. Several studies have proposed that gremlin may be involved in renal damage by acting as a downstream mediator of TGF-β. To examine the in vivo role of gremlin in kidney pathophysiology, we generated seven viable transgenic mouse lines expressing human gremlin (GREM1 specifically in renal proximal tubular epithelial cells under the control of an androgen-regulated promoter. These lines demonstrated 1.2- to 200-fold increased GREM1 expression. GREM1 transgenic mice presented a normal phenotype and were without proteinuria and renal function involvement. In response to the acute renal damage cause by folic acid nephrotoxicity, tubule-specific GREM1 transgenic mice developed increased proteinuria after 7 and 14 days compared with wild-type treated mice. At 14 days tubular lesions, such as dilatation, epithelium flattening and hyaline casts, with interstitial cell infiltration and mild fibrosis were significantly more prominent in transgenic mice than wild-type mice. Tubular GREM1 overexpression was correlated with the renal upregulation of profibrotic factors, such as TGF-β and αSMA, and with increased numbers of monocytes/macrophages and lymphocytes compared to wild-type mice. Taken together, our results suggest that GREM1-overexpressing mice have an increased susceptibility to renal damage, supporting the involvement of gremlin in renal damage

  14. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    Science.gov (United States)

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  15. Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress

    NARCIS (Netherlands)

    Leborgne-Castel, N.; Jelitto- Dooren, E.P.W.M. van; Crofts, A.J.; Denecke, J.

    1999-01-01

    To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of

  16. Over-expression of ZmPti1, a homologue to Pti1, increases salt ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-02-01

    Feb 1, 2010 ... terized. One week after salt treatment on MS agar plates, the. WT plants wilted to death, while S1-1 and S2-1 kept alive and their leaves were still green (Figure 2). This result primarily showed that ZmPti1-transgenic Arabidopsis in- creased salt tolerance. To obtain further evidence that over-expression of.

  17. ß-Cell Specific Overexpression of GPR39 Protects against Streptozotocin-Induced Hyperglycemia

    DEFF Research Database (Denmark)

    Egerod, Kristoffer Lihme; Jin, Chunyu; Petersen, Pia Steen

    2011-01-01

    promoter to selectively overexpress GPR39 in the ß cells in a double transgenic mouse strain and challenge them with multiple low doses of streptozotocin, which in the wild-type littermates leads to a gradual increase in nonfasting glucose levels and glucose intolerance observed during both food intake...

  18. Improvement in Fruit Quality by Overexpressing miR399a in Woodland Strawberry.

    Science.gov (United States)

    Wang, Yan; Zhang, Junxiang; Cui, Weixu; Guan, Chunyue; Mao, Wenjuan; Zhang, Zhihong

    2017-08-30

    Fruit quality is an important trait in strawberry and is determined by many factors. The soluble solid content in strawberry fruits is positively related to the phosphorus content. MicroRNA399 (miR399) is involved in the regulation of phosphate (Pi) homeostasis. However, the effect of miR399 on strawberry quality remains unknown. In this study, miR399a-overexpressing transgenic woodland strawberries (Fragaria vesca) were obtained via an Agrobacterium-mediated transformation. The phosphorus (P) content was 1.1-fold to 2.1-fold higher in the leaves and fruits of the miR399a-overexpressing plants than in the wild type (WT). However, the P content in the miR399a-overexpressing plants was decreased by 25% to 45% in the roots. The primary root length of the transgenic lines in both the high-Pi and low-Pi media was shorter than that of the WT. Interestingly, the transgenic lines in pots under Pi-sufficient conditions grew better than the WT, and the fruit quality, including the contents of fructose and glucose and soluble solid, was significantly higher in the transgenic lines than in the WT. The overexpression of miR399a in strawberry can be used to improve the parameters involved in fruit quality and provides information regarding breeding nutrient-improved strawberry.

  19. Over-expression of thymosin beta 4 promotes abnormal tooth development and stimulation of hair growth.

    Science.gov (United States)

    Cha, Hee-Jae; Philp, Deborah; Lee, Soo-Hyun; Moon, Hye-Sung; Kleinman, Hynda K; Nakamura, Takashi

    2010-01-01

    Thymosin beta 4 has multi-functional roles in cell physiology. It accelerates wound healing, hair growth and angiogenesis, and increases laminin-5 expression in corneal epithelium. Furthermore, thymosin beta 4 stimulates tumor growth and metastasis by induction of cell migration and vascular endothelial growth factor-mediated angiogenesis. Using a construct on the skin-specific keratin-5 promoter, we have developed thymosin beta 4 over-expressing transgenic mice to further study its functional roles. Thymosin beta 4 in adult skin and in embryonic stages of the transgenic mouse was analyzed by both Western blot and immunohistochemistry. The over-expression of thymosin beta 4 was observed especially around hair follicles and in the teeth in the transgenic mice. We examined the phenotype of the thymosin beta 4 over-expressing mice. Hair growth was accelerated. In addition, the transgenic mice had abnormally-shaped white teeth and dull incisors. We found that the expression of laminin-5 was up-regulated in the skin of the transgenic mice. We conclude that thymosin beta 4 has an important physiological role in hair growth and in tooth development.

  20. GH/STAT5 signaling during the growth period in livers of mice overexpressing GH.

    Science.gov (United States)

    Martinez, Carolina S; Piazza, Verónica G; Díaz, María E; Boparai, Ravneet K; Arum, Oge; Ramírez, María C; González, Lorena; Becú-Villalobos, Damasia; Bartke, Andrzej; Turyn, Daniel; Miquet, Johanna G; Sotelo, Ana I

    2015-04-01

    GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing mice. GH receptor and the positive modulators glucocorticoid receptor and HNF1 display greater abundance in transgenic animals, supporting the activity of STAT5. The negative modulators cytokine-induced suppressor and PTP1B are increased in GH-overexpressing mice. The suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, indicating that they are being actively degraded. Therefore, STAT5 signaling is increased in the liver in GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in accelerated but controlled growth. © 2015 Society for Endocrinology.

  1. Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase.

    Science.gov (United States)

    Byeon, Yeong; Park, Sangkyu; Lee, Hyoung Yool; Kim, Young-Soon; Back, Kyoungwhan

    2014-04-01

    A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well-known bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Transgene mus som sygdomsmodeller

    DEFF Research Database (Denmark)

    Schuster, Mikkel Bruhn; Porse, Bo Torben

    2003-01-01

    Transgenic animal models have proven to be useful tools in understanding both basic biology and the events associated with disease. Recent technical advances in the area of genomic manipulation in combination with the availability of the human and murine genomic sequences now allow the precise ta...... tailoring of the mouse genome. In this review we describe a few systems in which transgenic animal models have been employed for the purpose of studying the etiology of human diseases. Udgivelsesdato: 2003-Feb-17......Transgenic animal models have proven to be useful tools in understanding both basic biology and the events associated with disease. Recent technical advances in the area of genomic manipulation in combination with the availability of the human and murine genomic sequences now allow the precise...

  3. Transgenics in Agriculture

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 6; Issue 2. Transgenics in Agriculture. D Rex Arunraj B Gajendra Babu. Classroom Volume 6 Issue 2 February 2001 pp 83-92. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/006/02/0083-0092 ...

  4. iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes.

    Science.gov (United States)

    Martinez-Fernandez, Almudena; Nelson, Timothy J; Reyes, Santiago; Alekseev, Alexey E; Secreto, Frank; Perez-Terzic, Carmen; Beraldi, Rosanna; Sung, Hoon-Ki; Nagy, Andras; Terzic, Andre

    2014-10-01

    Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/β-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation. A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics. © 2014 American Heart Association, Inc.

  5. Overexpression of allene oxide cyclase improves the biosynthesis of artemisinin in Artemisia annua L.

    Directory of Open Access Journals (Sweden)

    Xu Lu

    Full Text Available Jasmonates (JAs are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase, CYP71AV1 (cytochrome P450 dependent hydroxylase and DBR2 (double bond reductase 2 were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A. annua.

  6. CCK Response Deficiency in Synphilin-1 Transgenic Mice.

    Science.gov (United States)

    Smith, Wanli W; Smith, Megan; Yang, Dejun; Choi, Pique P; Moghadam, Alexander; Li, Tianxia; Moran, Timothy H

    2015-01-01

    Previously, we have identified a novel role for the cytoplasmic protein, synphilin-1(SP1), in the controls of food intake and body weight in both mice and Drosophila. Ubiquitous overexpression of human SP1 in brain neurons in transgenic mice results in hyperphagia expressed as an increase in meal size. However, the mechanisms underlying this action of SP1 remain to be determined. Here we investigate a potential role for altered gut feedback signaling in the effects of SP1 on food intake. We examined responses to peripheral administration of cholecytokinin (CCK), amylin, and the glucagon like peptide-1 (GLP-1) receptor agonist, exendin-4. Intraperitoneal administration of CCK at doses ranging from 1-10 nmol/kg significantly reduced glucose intake in wild type (WT) mice, but failed to affect intake in SP1 transgenic mice. Moreover, there was a significant attenuation of CCK-induced c-Fos expression in the dorsal vagal complex in SP1 transgenic mice. In contrast, WT and SP1 transgenic mice were similarly responsive to both amylin and exendin-4 treatment. These studies demonstrate that SP1 results in a CCK response deficiency that may contribute to the increased meal size and overall hyperphagia in synphillin-1 transgenic mice.

  7. Leaf proteome profiling of transgenic mint infected with Alternaria alternata.

    Science.gov (United States)

    Sinha, Ragini; Bhattacharyya, Dipto; Majumdar, Aparupa Bose; Datta, Riddhi; Hazra, Saptarshi; Chattopadhyay, Sharmila

    2013-11-20

    The genus Mentha has been widely used in food, flavor, culinary, cosmetic and pharmaceutical industries. Substantial damage to this crop happened regularly due to environmental stresses like metal toxicity and pathogen attack. Here, an approach has been taken to raise transgenic mint over-expressing γ-glutamyl-cysteine synthetase (γ-ECS), the rate-limiting enzyme of GSH biosynthesis, resulted enhanced GSH content and its in planta expression confers significant tolerance towards abiotic/biotic stresses viz. metal toxicity - Cd, Zn as well as against infection of Alternaria alternata and Rhizoctonia solani. A differential proteomic analysis through 2-DE and MALDI TOF-TOF MSMS was performed to focus on the altered abundance of functionally important protein species in control and infected transgenic mint. Results showed a significant variation in the protein profile of the infected transgenic plant as compared to the wild/control transgenic counterpart. In addition to protein species related to stress and defense, redox regulation, transcription factors and energy & metabolism, protein species related to signaling and gene regulation as well as cell division also showed differential accumulation in infected transgenic. Hence, proteomics can be used as a tool to decipher the mechanism of action of GSH in providing tolerance against a necrotrophic fungus, A. alternata in transgenic mint. The reported work describes a comparative proteomics of non-model unsequenced plants like Mentha. There is a comparative protein profile between transgenic and its wild counterparts under control and infected condition. The work has an impact in crop proteomics and also tries to explain the application of proteomic approach to decipher the mechanism by which a foreign metabolite mediates stress tolerance in plant under control and infected condition. This article is part of a Special Issue entitled: Translational Plant Proteomics. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Cardiovascular end-organ damage in Ren-2 transgenic rats compared to spontaneously hypertensive rats

    NARCIS (Netherlands)

    Pinto, YM; Buikema, H; vanGilst, WH; vanGeel, PP; deGraeff, PA; Wagner, J; Paul, M

    To compare hypertensive end-organ damage in two genetic forms of hypertension we assessed cardiovascular function in two rat strains of generic hypertension: transgenic rats overexpressing the mouse Ren-2 gene [(TGR(mREN2)27]) and blood pressure matched spontaneously hypertensive rats (SHR). Despite

  9. MusaWRKY71 overexpression in banana plants leads to altered abiotic and biotic stress responses.

    Directory of Open Access Journals (Sweden)

    Upendra K S Shekhawat

    Full Text Available WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR protein genes, non-expressor of pathogenesis-related genes 1 (NPR1 and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana.

  10. MusaWRKY71 overexpression in banana plants leads to altered abiotic and biotic stress responses.

    Science.gov (United States)

    Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2013-01-01

    WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana.

  11. Overexpression of SmMYC2 Increases the Production of Phenolic Acids in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Na Yang

    2017-10-01

    Full Text Available MYC2 is a core transcription factor in the plant response to jasmonates. It also functions in secondary metabolism and various processes for growth and development. However, the knowledge about its role in Salvia miltiorrhiza is still very limited. We determined that the biosynthesis of salvianolic acid B (Sal B was strongly induced in 2-month-old transgenic plants that over-expressed SmMYC2. In the roots of transgenic line 12 that over-expressed SmMYC2 (OEM-12, the Sal B concentration was as high as 5.95 ± 0.07 mg g-1, a level that was 1.88-fold higher than that in control plants that had been transformed with an empty vector. Neither tanshinone IIA nor cryptotanshinone was detected by high-performance liquid chromatography in any of the genotypes. Global transcriptomic analysis using RNA sequencing revealed that most enzyme-encoding genes for the phenylpropanoid biosynthesis pathway were up-regulated in the overexpression lines. Furthermore, both the phenylalanine and tyrosine biosynthesis pathways were activated in those transgenics. Our data demonstrate that overexpression of SmMYC2 promotes the production of phenolic acids by simultaneously activating both primary and secondary pathways for metabolism in S. miltiorrhiza.

  12. Behavioral characterization of a mouse model overexpressing DSCR1/ RCAN1.

    Science.gov (United States)

    Dierssen, Mara; Arqué, Gloria; McDonald, Jerome; Andreu, Nuria; Martínez-Cué, Carmen; Flórez, Jesús; Fillat, Cristina

    2011-02-25

    DSCR1/ RCAN1 is a chromosome 21 gene found to be overexpressed in the brains of Down syndrome (DS) and postulated as a good candidate to contribute to mental disability. However, even though Rcan1 knockout mice have pronounced spatial learning and memory deficits, the possible deleterious effects of its overexpression in DS are not well understood. We have generated a transgenic mouse model overexpressing DSCR1/RCAN1 in the brain and analyzed the effect of RCAN1 overexpression on cognitive function. TgRCAN1 mice present a marked disruption of the learning process in a visuo-spatial learning task. However, no significant differences were observed in the performance of the memory phase of the test (removal session) nor in a step-down passive avoidance task, thus suggesting that once learning has been established, the animals are able to consolidate the information in the longer term.

  13. Behavioral characterization of a mouse model overexpressing DSCR1/ RCAN1.

    Directory of Open Access Journals (Sweden)

    Mara Dierssen

    Full Text Available DSCR1/ RCAN1 is a chromosome 21 gene found to be overexpressed in the brains of Down syndrome (DS and postulated as a good candidate to contribute to mental disability. However, even though Rcan1 knockout mice have pronounced spatial learning and memory deficits, the possible deleterious effects of its overexpression in DS are not well understood. We have generated a transgenic mouse model overexpressing DSCR1/RCAN1 in the brain and analyzed the effect of RCAN1 overexpression on cognitive function. TgRCAN1 mice present a marked disruption of the learning process in a visuo-spatial learning task. However, no significant differences were observed in the performance of the memory phase of the test (removal session nor in a step-down passive avoidance task, thus suggesting that once learning has been established, the animals are able to consolidate the information in the longer term.

  14. Vldlr overexpression causes hyperactivity in rats

    Directory of Open Access Journals (Sweden)

    Iwata Keiko

    2012-10-01

    Full Text Available Abstract Background Reelin regulates neuronal positioning in cortical brain structures and neuronal migration via binding to the lipoprotein receptors Vldlr and Lrp8. Reeler mutant mice display severe brain morphological defects and behavioral abnormalities. Several reports have implicated reelin signaling in the etiology of neurodevelopmental and psychiatric disorders, including autism, schizophrenia, bipolar disorder, and depression. Moreover, it has been reported that VLDLR mRNA levels are increased in the post-mortem brain of autistic patients. Methods We generated transgenic (Tg rats overexpressing Vldlr, and examined their histological and behavioral features. Results Spontaneous locomotor activity was significantly increased in Tg rats, without detectable changes in brain histology. Additionally, Tg rats tended to show performance deficits in the radial maze task, suggesting that their spatial working memory was slightly impaired. Thus, Vldlr levels may be involved in determining locomotor activity and memory function. Conclusions Unlike reeler mice, patients with neurodevelopmental or psychiatric disorders do not show striking neuroanatomical aberrations. Therefore, it is notable, from a clinical point of view, that we observed behavioral phenotypes in Vldlr-Tg rats in the absence of neuroanatomical abnormalities.

  15. Dimethylarginine Dimethylaminohydrolase Overexpression enhances Insulin Sensitivity

    Science.gov (United States)

    Sydow, Karsten; Mondon, Carl E.; Schrader, Joerg; Konishi, Hakuoh; Cooke, John P.

    2011-01-01

    Objective Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. Methods and Results To test this hypothesis we employed a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22–70 weeks) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistence index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2μM) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. Conclusions These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity. PMID:18239148

  16. Prothymosin α overexpression contributes to the development of pulmonary emphysema

    Science.gov (United States)

    Su, Bing-Hua; Tseng, Yau-Lin; Shieh, Gia-Shing; Chen, Yi-Cheng; Shiang, Ya-Chieh; Wu, Pensee; Li, Kuo-Jung; Yen, Te-Hsin; Shiau, Ai-Li; Wu, Chao-Liang

    2013-01-01

    Emphysema is one of the disease conditions that comprise chronic obstructive pulmonary disease. Prothymosin α transgenic mice exhibit an emphysema phenotype, but the pathophysiological role of prothymosin α in emphysema remains unclear. Here we show that prothymosin α contributes to the pathogenesis of emphysema by increasing acetylation of histones and nuclear factor-kappaB, particularly upon cigarette smoke exposure. We find a positive correlation between prothymosin α levels and the severity of emphysema in prothymosin α transgenic mice and emphysema patients. Prothymosin α overexpression increases susceptibility to cigarette smoke-induced emphysema, and cigarette smoke exposure further enhances prothymosin α expression. We show that prothymosin α inhibits the association of histone deacetylases with histones and nuclear factor-kappaB, and that prothymosin α overexpression increases expression of nuclear factor-kappaB-dependent matrix metalloproteinase 2 and matrix metalloproteinase 9, which are found in the lungs of patients with chronic obstructive pulmonary disease. These results demonstrate the clinical relevance of prothymosin α in regulating acetylation events during the pathogenesis of emphysema. PMID:23695700

  17. Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene.

    Science.gov (United States)

    Fu, Jianming; Ristic, Zoran

    2010-06-01

    We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

  18. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    Science.gov (United States)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  19. The use of transgenic animals to study lipoprotein metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, E.M.; Plump, A.S.

    1993-12-01

    The application of transgenic technology to lipoprotein metabolism and atherosclerosis was first reported in 1988. Today, a large percentage of the genes involved in lipoprotein metabolism have been overexpressed in mice, and a substantial number of these same genes have been disrupted by homologous recombination in embryonic stem (ES) cells. The utility of animal models of lipoprotein metabolism and atherosclerosis is far-reaching given the complex nature of these systems. There are at least 17 known genes directly involved in lipoprotein metabolism and likely dozens more may be involved. This massive network of interacting factors has necessitated the development of in vivo systems which can be subject to genetic manipulation. The power of overexpression is obvious: elucidating function in a relatively controlled genetic environment in which the whole system is present and operational. The not-so-obvious problem with transgenics is ``background,`` or for purposes of the current discussion, the mouse`s own lipoprotein system. With the advent of gene knockout, we have been given the ability to overcome ``background.`` By recreating the genetic complement of the mouse we can alter a system in essentially any manner desired. As unique tools, and in combination with one another, the overexpression of foreign genes and the targeted disruption or alteration of endogenous genes has already and will continue to offer a wealth of information on the biology of lipoprotein metabolism and its effect on atherosclerosis susceptibility.

  20. Transgenic algae engineered for higher performance

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  1. Effect of glycogen synthase overexpression on insulin-stimulated muscle glucose uptake and storage.

    Science.gov (United States)

    Fogt, Donovan L; Pan, Shujia; Lee, Sukho; Ding, Zhenping; Scrimgeour, Angus; Lawrence, John C; Ivy, John L

    2004-03-01

    Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in noncontracted and postcontracted muscle of GSL3-transgenic and wild-type mice. GSL3-transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3-transgenic and wild-type mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty minutes of contraction significantly reduced muscle glycogen concentration in wild-type (49%) and transgenic (27%) mice, although transgenic mice retained 168.8 +/- 20.5 micromol/g glycogen compared with 17.7 +/- 2.6 micromol/g glycogen for wild-type mice. Muscle of transgenic and wild-type mice demonstrated similar pre- (3.6 +/- 0.3 and 3.9 +/- 0.6 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) and postcontraction (7.9 +/- 0.4 and 7.0 +/- 0.4 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) insulin-stimulated glucose uptakes. However, the [14C]glucose incorporated into glycogen was greater in noncontracted (151%) and postcontracted (157%) transgenic muscle vs. muscle of corresponding wild-type mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.

  2. Plant biotechnology: transgenic crops.

    Science.gov (United States)

    Shewry, Peter R; Jones, Huw D; Halford, Nigel G

    2008-01-01

    Transgenesis is an important adjunct to classical plant breeding, in that it allows the targeted manipulation of specific characters using genes from a range of sources. The current status of crop transformation is reviewed, including methods of gene transfer, the selection of transformed plants and control of transgene expression. The application of genetic modification technology to specific traits is then discussed, including input traits relating to crop production (herbicide tolerance and resistance to insects, pathogens and abiotic stresses) and output traits relating to the composition and quality of the harvested organs. The latter include improving the nutritional quality for consumers as well as the improvement of functional properties for food processing.

  3. Transgenic Forest Trees

    Directory of Open Access Journals (Sweden)

    Ertuğrul Filiz

    2011-11-01

    Full Text Available Biotechnological methods are used in many areas nowadays and one of these areas is applications of biotechnology in forest trees. Biotechnological methods are used frequently on vital issues such as gaining resistance against diseases and herbicide of forest trees, increasing tree growth rates and development of resistance against environmental stresses (drought, salinity, climate change etc.. Also, for improving the quality of wood that reducing lignin content and increasing the amount of cellulose draws attention. This together with applications, positive and negative effects of transgenic trees to the environment is discussed and it was tried to be provided on the auditing legal regulations concerned with these studies.

  4. Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes

    DEFF Research Database (Denmark)

    Brouwers, Olaf; Niessen, Petra M G; Miyata, Toshio

    2014-01-01

    AIMS/HYPOTHESIS: In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed ...

  5. Overexpression of AtSTO1 leads to improved salt tolerance in Populus tremula × P. alba.

    Science.gov (United States)

    Lawson, Shaneka S; Michler, Charles H

    2014-10-01

    One of the major abiotic stress conditions limiting healthy growth of trees is salinity stress. The use of gene manipulation for increased tolerance to abiotic stress has been successful in many plant species. Overexpression of the Arabidopsis SALT TOLERANT1 (STO1) gene leads to increased concentrations of 9-cis-epoxycarotenoid dioxygenase3, a vital enzyme in Arabidopsis abscisic acid biosynthesis. In the present work, the Arabidopsis STO1 gene (AtSTO1) was overexpressed in poplar to determine if the transgene would confer enhanced salt tolerance to the generated transgenics. The results of multiple greenhouse trials indicated that the transgenic poplar lines had greater levels of resistance to NaCl than wild-type plants. Analysis using RT-PCR indicated a variation in the relative abundance of the STO1 transcript in the transgenics that coincided with tolerance to salt. Several physiological and morphological changes such as greater overall biomass, greater root biomass, improved photosynthesis, and greater pith size were observed in the transgenics when compared to controls undergoing salt stress. These results indicated overexpression of AtSTO1 improved salt tolerance in poplar.

  6. Growth hormone (GH)/STAT5 signaling during the growth period in liver of mice overexpressing GH

    Science.gov (United States)

    Martinez, Carolina S; Piazza, Verónica G; Díaz, María E; Boparai, Ravneet K; Arum, Oge; Ramírez, María C; González, Lorena; Becú-Villalobos, Damasia; Bartke, Andrzej; Turyn, Daniel; Miquet, Johanna G; Sotelo, Ana I

    2016-01-01

    Growth hormone (GH)/STAT5 signaling is desensitized in liver of adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess if the STAT5 pathway is active during the growth period in liver of these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus but presents higher basal phosphorylation in liver of growing GH-overexpressing mice. GH receptor and positive modulators GR and HNF1 display greater abundance in transgenic animals, supporting STAT5 activity. Negative modulators CIS and PTP1B are increased in GH-overexpressing mice. Suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, suggesting they are being actively degraded. Therefore, STAT5 signaling is increased in liver of GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in an accelerated but controlled growth. PMID:25691498

  7. Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

    Directory of Open Access Journals (Sweden)

    Hourinaz Behesti

    2013-01-01

    BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

  8. Behavioral decline and premature lethality upon pan-neuronal ferritin overexpression in Drosophila infected with a virulent form of Wolbachia.

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    Stylianos eKosmidis

    2014-04-01

    Full Text Available Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host’s innate immune response to various pathogens, for example in Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.

  9. [TSA improve transgenic porcine cloned embryo development and transgene expression].

    Science.gov (United States)

    Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

    2011-07-01

    Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

  10. Novel Transgenic Mouse Model of Polycystic Kidney Disease.

    Science.gov (United States)

    Kito, Yusuke; Saigo, Chiemi; Takeuchi, Tamotsu

    2017-09-01

    Transmembrane protein 207 (TMEM207) is characterized as an important molecule for invasiveness of gastric signet-ring cell carcinoma cells. To clarify the pathobiological effects of TMEM207, we generated 13 transgenic mouse strains, designated C57BL/6-transgenic (Tg) (ITF-TMEM207), where the mouse Tmem207 is ectopically expressed under the proximal promoter of the murine intestinal trefoil factor gene. A C57BL/6-Tg (ITF-TMEM207) mouse strain unexpectedly exhibited a high incidence of spontaneous kidney cysts with histopathological features resembling human polycystic kidney disease, which were found in approximately all mice within 1 year. TMEM207 immunoreactivity was found in noncystic kidney tubules and in renal cysts of the transgenic mice. The ITF-TMEM207 construct was inserted into Mitf at chromosome 6. Cystic kidney was not observed in other C57BL/6-Tg (ITF-TMEM207) transgenic mouse strains. Although several genetically manipulated animal models exist, this mouse strain harboring a genetic mutation in Mitf and overexpression of Tmem207 protein was not reported as a model of polycystic kidney disease until now. This study demonstrates that the C57BL/6-Tg (ITF-TMEM207) mouse may be a suitable model for understanding human polycystic kidney disease. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  11. Overexpression of a Vesicle Trafficking Gene, OsRab7, Enhances Salt Tolerance in Rice

    Directory of Open Access Journals (Sweden)

    Xiaojue Peng

    2014-01-01

    Full Text Available High soils salinity is a main factor affecting agricultural production. Studying the function of salt-tolerance-related genes is essential to enhance crop tolerance to stress. Rab7 is a small GTP-binding protein that is distributed widely among eukaryotes. Endocytic trafficking mediated by Rab7 plays an important role in animal and yeast cells, but the current understanding of Rab7 in plants is still very limited. Herein, we isolated a vesicle trafficking gene, OsRab7, from rice. Transgenic rice over-expressing OsRab7 exhibited enhanced seedling growth and increased proline content under salt-treated conditions. Moreover, an increased number of vesicles was observed in the root tip of OsRab7 transgenic rice. The OsRab7 over-expression plants showed enhanced tolerance to salt stress, suggesting that vacuolar trafficking is important for salt tolerance in plants.

  12. Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

    Science.gov (United States)

    Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

  13. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation.

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    Agustín Sola Carvajal

    Full Text Available Hutchinson-Gilford progeria syndrome (HGPS is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.

  14. Impact of Adiponectin Overexpression on Allergic Airways Responses in Mice

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    Norah G. Verbout

    2013-01-01

    Full Text Available Obesity is an important risk factor for asthma. Obese individuals have decreased circulating adiponectin, an adipose-derived hormone with anti-inflammatory properties. We hypothesized that transgenic overexpression of adiponectin would attenuate allergic airways inflammation and mucous hyperplasia in mice. To test this hypothesis, we used mice overexpressing adiponectin (Adipo Tg. Adipo Tg mice had marked increases in both serum adiponectin and bronchoalveolar lavage (BAL fluid adiponectin. Both acute and chronic ovalbumin (OVA sensitization and challenge protocols were used. In both protocols, OVA-induced increases in total BAL cells were attenuated in Adipo Tg versus WT mice. In the acute protocol, OVA-induced increases in several IL-13 dependent genes were attenuated in Adipo Tg versus WT mice, even though IL-13 per se was not affected. With chronic exposure, though OVA-induced increases in goblet cells numbers per millimeter of basement membrane were greater in Adipo Tg versus WT mice, mRNA abundance of mucous genes in lungs was not different. Also, adiponectin overexpression did not induce M2 polarization in alveolar macrophages. Our results indicate that adiponectin protects against allergen-induced inflammatory cell recruitment to the airspaces, but not development of goblet cell hyperplasia.

  15. Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation.

    Science.gov (United States)

    Sola Carvajal, Agustín; McKenna, Tomás; Wallén Arzt, Emelie; Eriksson, Maria

    2015-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the LMNA gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS LMNA mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.

  16. Epigenetic silencing in transgenic plants.

    Science.gov (United States)

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review.

  17. TNF-overexpression in Borna disease virus-infected mouse brains triggers inflammatory reaction and epileptic seizures.

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    Katharina Kramer

    Full Text Available Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic, TNF-receptor (TNFR1, TNFR2, IL-1 and N-methyl-D-aspartate (NMDA-receptor subunit 2B (NR2B mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated

  18. TNF-overexpression in Borna disease virus-infected mouse brains triggers inflammatory reaction and epileptic seizures.

    Science.gov (United States)

    Kramer, Katharina; Schaudien, Dirk; Eisel, Ulrich L M; Herzog, Sibylle; Richt, Jürgen A; Baumgärtner, Wolfgang; Herden, Christiane

    2012-01-01

    Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV)-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF) was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic), TNF-receptor (TNFR1, TNFR2), IL-1 and N-methyl-D-aspartate (NMDA)-receptor subunit 2B (NR2B) mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated cytokine

  19. Salivary Gland Derived BDNF Overexpression in Mice Exerts an Anxiolytic Effect

    OpenAIRE

    Saruta, Juri; To, Masahiro; Sugimoto, Masahiro; Yamamoto, Yuko; Shimizu, Tomoko; Nakagawa, Yusuke; Inoue, Hiroko; Saito, Ichiro; Tsukinoki, Keiichi

    2017-01-01

    Brain-derived neurotrophic factor (BDNF) is abundant in the hippocampus and plays critical roles in memory and synapse formation, as well as exerting antidepressant-like effects in psychiatric disorders. We previously reported that BDNF is expressed in salivary glands and affects blood BDNF content. However, the function of salivary BDNF remains unclear. The aim of this study was to generate transgenic mice overexpressing BDNF in the salivary glands. Hence, we used the Lama construct (hemaggl...

  20. Anosmin-1 over-expression regulates oligodendrocyte precursor cell proliferation, migration and myelin sheath thickness.

    Science.gov (United States)

    Murcia-Belmonte, Verónica; Esteban, Pedro F; Martínez-Hernández, José; Gruart, Agnès; Luján, Rafael; Delgado-García, José María; de Castro, Fernando

    2016-04-01

    During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination.

  1. Towards efficient photosynthesis: overexpression of Zea mays phosphoenolpyruvate carboxylase in Arabidopsis thaliana.

    Science.gov (United States)

    Kandoi, Deepika; Mohanty, Sasmita; Govindjee; Tripathy, Baishnab C

    2016-12-01

    Plants with C4 photosynthesis are efficient in carbon assimilation and have an advantage over C3 photosynthesis. In C4 photosynthesis, the primary CO2 fixation is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Here, we show that overexpression of Zea mays PEPC cDNA, under the control of (35)S promoter, in Arabidopsis thaliana resulted in ~7-10 fold higher protein abundance and ~7-10 fold increase in PEPC activity in the transgenic lines than that in the vector control. We suggest that overexpression of PEPC played an anaplerotic role to increase the supply of 4-carbon carboxylic acids, which provided carbon skeletons for increased amino acid and protein synthesis. Higher protein content must have been responsible for increased metabolic processes including chlorophyll biosynthesis, photosynthesis, and respiration. Consequently, the PEPC-overexpressed transgenic plants had higher chlorophyll content, enhanced electron transport rate (ETR), lower non-photochemical quenching (NPQ) of chlorophyll a fluorescence, and a higher performance index (PI) than the vector control. Consistent with these observations, the rate of CO2 assimilation, the starch content, and the dry weight of PEPC-overexpressed plants increased by 14-18 %, 10-18 %, and 6.5-16 %, respectively. Significantly, transgenics were tolerant to salt stress as they had increased ability to synthesize amino acids, including the osmolyte proline. NaCl (150 mM)-treated transgenic plants had higher variable to maximum Chl a fluorescence (F v/F m) ratio, higher PI, higher ETR, and lower NPQ than the salt-treated vector controls. These results suggest that expression of C4 photosynthesis enzyme(s) in a C3 plant can improve its photosynthetic capacity with enhanced tolerance to salinity stress.

  2. Mitogenic signaling pathways in the liver of growth hormone (GH)-overexpressing mice during the growth period.

    Science.gov (United States)

    Martinez, Carolina S; Piazza, Verónica G; González, Lorena; Fang, Yimin; Bartke, Andrzej; Turynl, Danie; Miquet, Johanna G; Sotelo, Ana I

    2016-01-01

    Growth hormone (GH) is a pleiotropic hormone that triggers STATs, ERK1/2 and Akt signaling, related to cell growth and proliferation. Transgenic mice overexpressing GH present increased body size, with a disproportionate liver enlargement due to hypertrophy and hyperplasia of the hepatocytes. We had described enhanced mitogenic signaling in liver of young adult transgenic mice. We now evaluate the activation of these signaling cascades during the growth period and relate them to the morphological alterations found. Signaling mediators, cell cycle regulators and transcription factors involved in cellular growth in the liver of GH-overexpressing growing mice were assessed by immunoblotting, RT-qPCR and immunohistochemistry. Hepatocyte enlargement can be seen as early as 2-weeks of age in GH-overexpressing animals, although it is more pronounced in young adults. Levels of cell cycle mediators PCNA and cyclin D1, and transcription factor c-Jun increase with age in transgenic mice with no changes in normal mice, whereas c-Myc levels are higher in 2-week-old transgenic animals and cyclin E levels decline with age for both genotypes. STAT3, Akt and GSK3 present higher activation in the adult transgenic mice than in the growing animals, while for c-Src and mTOR, phosphorylation in GH-overexpressing mice is higher than in control siblings at 4 and 9 weeks of age. No significant changes are observed for ERK1/2, neither by age or genotype. Thus, the majority of the mitogenic signaling pathways are gradually up-regulated in the liver of GH-transgenic mice, giving rise to the hepatic morphological changes these mice exhibit.

  3. 5-HT2A Receptor Binding in the Frontal Cortex of Parkinson's Disease Patients and Alpha-Synuclein Overexpressing Mice

    DEFF Research Database (Denmark)

    Rasmussen, Nadja Bredo; Olesen, Mikkel Vestergaard; Brudek, Tomasz

    2016-01-01

    by increased receptor binding in PD brains. In a separate study, we looked for changes in receptors in the prefrontal cortex in 52-week-old transgenic mice overexpressing human AS. We performed region-specific receptor binding measurements followed by gene expression analysis. The transgenic mice showed lower......) overexpression, could be associated with alterations. Binding density for the -specific radioligand [3H]-MDL 100.907 was measured in membrane suspensions of frontal cortex tissue from PD patients. Protein levels of AS were further measured using western blotting. Results showed higher AS levels accompanied...... binding in the frontal association cortex that was not accompanied by changes in gene expression levels. This study is one of the first to look at differences in serotonin receptor levels in PD and in relation to AS overexpression....

  4. A novel α/β-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato.

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    Degao Liu

    Full Text Available Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L. Lam. line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19 plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.

  5. genetic overexpression of NR2B subunit enhances social recognition memory for different strains and species.

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    Stephanie A Jacobs

    Full Text Available The ability to learn and remember conspecifics is essential for the establishment and maintenance of social groups. Many animals, including humans, primates and rodents, depend on stable social relationships for survival. Social learning and social recognition have become emerging areas of interest for neuroscientists but are still not well understood. It has been established that several hormones play a role in the modulation of social recognition including estrogen, oxytocin and arginine vasopression. Relatively few studies have investigated how social recognition might be improved or enhanced. In this study, we investigate the role of the NMDA receptor in social recognition memory, specifically the consequences of altering the ratio of the NR2B:NR2A subunits in the forebrain regions in social behavior. We produced transgenic mice in which the NR2B subunit of the NMDA receptor was overexpressed postnatally in the excitatory neurons of the forebrain areas including the cortex, amygdala and hippocampus. We investigated the ability of both our transgenic animals and their wild-type littermate to learn and remember juvenile conspecifics using both 1-hr and 24-hr memory tests. Our experiments show that the wild-type animals and NR2B transgenic mice preformed similarly in the 1-hr test. However, transgenic mice showed better performances in 24-hr tests of recognizing animals of a different strain or animals of a different species. We conclude that NR2B overexpression in the forebrain enhances social recognition memory for different strains and animal species.

  6. Overexpression of DMP1 accelerates mineralization and alters cortical bone biomechanical properties in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Bhatia A.; Miller L.; Albazza, M.; Espinoza Orias, A.A.; Inoue, N.; Acerbo, A.; George, A.; Sumner, D.R.

    2011-09-29

    Dentin matrix protein-1 (DMP1) is a key regulator of biomineralization. Here, we examine changes in structural, geometric, and material properties of cortical bone in a transgenic mouse model overexpressing DMP1. Micro-computed tomography and three-point bending were performed on 90 femora of wild type and transgenic mice at 1, 2, 4, and 6 months. Fourier transform infrared imaging was performed at 2 months. We found that the transgenic femurs were longer (p < 0.01), more robust in cross-section (p < 0.05), stronger (p < 0.05), but had less post-yield strain and displacement (p < 0.01), and higher tissue mineral density (p < 0.01) than the wild type femurs at 1 and 2 months. At 2 months, the transgenic femurs also had a higher mineral-to-matrix ratio (p < 0.05) and lower carbonate substitution (p < 0.05) compared to wild type femurs. These findings indicate that increased mineralization caused by overexpressing DMP1 led to increased structural cortical bone properties associated with decreased ductility during the early post-natal period.

  7. Proteomic analysis of phytase transgenic and non-transgenic maize seeds.

    Science.gov (United States)

    Tan, Yanhua; Tong, Zheng; Yang, Qian; Sun, Yong; Jin, Xiang; Peng, Cunzhi; Guo, Anping; Wang, Xuchu

    2017-08-23

    Proteomics has become a powerful technique for investigating unintended effects in genetically modified crops. In this study, we performed a comparative proteomics of the seeds of phytase-transgenic (PT) and non-transgenic (NT) maize using 2-DE and iTRAQ techniques. A total of 148 differentially expressed proteins (DEPs), including 106 down-regulated and 42 up-regulated proteins in PT, were identified. Of these proteins, 32 were identified through 2-DE and 116 were generated by iTRAQ. It is noteworthy that only three proteins could be detected via both iTRAQ and 2-DE, and most of the identified DEPs were not newly produced proteins but proteins with altered abundance. These results indicated that many DEPs could be detected in the proteome of PT maize seeds and the corresponding wild type after overexpression of the target gene, but the changes in these proteins were not substantial. Functional classification revealed many DEPs involved in posttranscriptional modifications and some ribosomal proteins and heat-shock proteins that may generate adaptive effects in response to the insertion of exogenous genes. Protein-protein interaction analysis demonstrated that the detected interacting proteins were mainly ribosomal proteins and heat-shock proteins. Our data provided new information on such unintended effects through a proteomic analysis of maize seeds.

  8. Cloning and functional characterization of MusaVND1 using transgenic banana plants.

    Science.gov (United States)

    Negi, Sanjana; Tak, Himanshu; Ganapathi, T R

    2015-06-01

    Vascular related NAC (NAM, ATAF and CUC) domain-containing genes regulate secondary wall deposition and differentiation of xylem vessel elements. MusaVND1 is an ortholog of Arabidopsis VND1 and contains the highly conserved NAC domain. The expression of MusaVND1 is highest in developing corm and during lignification conditions, the increase in expression of MusaVND1 coincides with the expression of PAL, COMT and C4H genes. MusaVND1 encodes a nuclear localized protein as MusaVND1-GFP fusion protein gets localized to nucleus. Transient overexpression of MusaVND1 converts banana embryogenic cells to xylem vessel elements, with a final differentiation frequency of 33.54% at the end of tenth day. Transgenic banana plants overexpressing MusaVND1 showed stunted growth and were characterized by PCR and Southern blot analysis. Transgenic banana plants showed transdifferentiation of various types of cells into xylem vessel elements and ectopic deposition of lignin in cells of various plant organs such as leaf and corm. Tracheary element formation was seen in the cortical region of transgenic corm as well as in epidermal cells of leaves. Biochemical analysis indicates significantly higher levels of lignin and cellulose content in transgenic banana lines than control plants. MusaVND1 overexpressing transgenic banana plants showed elevated expression levels of genes involved in lignin and cellulose biosynthesis pathway. Further expression of different MYB transcription factors positively regulating secondary wall deposition was also up regulated in MusaVND1 transgenic lines.

  9. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    Li-Fen Huang

    2015-08-01

    Full Text Available Ferredoxins (FDX are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC; power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

  10. Overexpression of Jatropha Gibberellin 2-oxidase 6 (JcGA2ox6 Induces Dwarfism and Smaller Leaves, Flowers and Fruits in Arabidopsis and Jatropha

    Directory of Open Access Journals (Sweden)

    Ying-Xiong Hu

    2017-12-01

    Full Text Available Gibberellins (GAs are plant hormones that play fundamental roles in plant growth and development. Gibberellin 2-oxidase (GA2ox plays a direct role in determining the levels of bioactive GAs by catalyzing bioactive GAs or their immediate precursors to inactive forms. In this study, a GA2ox gene, designated JcGA2ox6, was isolated from Jatropha curcas. JcGA2ox6 is expressed in all tissues of adult Jatropha, with the highest expression level in male flowers and the lowest expression level in young leaves. Overexpression of JcGA2ox6 in Arabidopsis resulted in a typical dwarf phenotype, along with late flowering, smaller leaves and flowers, shorter siliques and smaller seeds. Similarly, when JcGA2ox6 was overexpressed in Jatropha, the transgenic plants exhibited a dwarf phenotype with dark-green leaves and smaller inflorescences, flowers, fruits and seeds. However, the flowering time of Jatropha was not affected by overexpression of JcGA2ox6, unlike that in the transgenic Arabidopsis. Moreover, the number of flowers per inflorescence, the weight of 10 seeds and the seed oil content were significantly decreased in transgenic Jatropha. The results indicated that overexpression of JcGA2ox6 had a great impact on the vegetative and reproductive growth of transgenic Jatropha. Furthermore, we found that the dwarf phenotype of transgenic Jatropha was caused by a decrease in endogenous bioactive GA4, which was correlated with the degree of dwarfism.

  11. Odontoblast-targeted Bcl-2 Overexpression Impairs Dentin Formation

    Science.gov (United States)

    Zhang, Wenjian; Ju, Jun; Gronowicz, Gloria.

    2010-01-01

    Apoptosis has been described extensively in tooth development, which is under tight control of multiple apoptosis regulators, including anti-apoptotic protein Bcl-2. However, it is totally unclear how Bcl-2 is related to odontogenesis, especially dentinogenesis. Using a transgenic mouse Col2.3Bcl-2 in which human Bcl-2 was overexpressed in odontoblasts, the effect of Bcl-2 on dentinogenesis was investigated. Overexpression of Bcl-2 was detected by immunohistochemistry and Western blot. Odontoblast apoptosis was evaluated by TUNEL and Western blot detection of cleaved caspase-3. Odontoblast differentiation was assessed by real-time PCR detection of dentin matrix expression. Dentin mineralization was evaluated by micro-CT in vivo, and alizarin red S staining and calcium content analysis in vitro. Bcl-2 was found to be overexpressed in odontoblasts and prevent their apoptosis. Odontoblast differentiation and mineralization was inhibited by Bcl-2, as evidenced by lower expressions of DMP-1, OC, and DSPP, and decreased odontoblast mineralization in vitro, as well as decreased dentin thickness and mineral density in vivo when compared to the wild type animals. Inhibition of odontoblast differentiation by Bcl-2 occurs, at least partially, via a suppression of MEK-ERK1/2 signaling pathway. In conclusion, Bcl-2 overexpression prevents odontoblast apoptosis and impairs dentin formation, partially via an inhibition of odontoblast differentiation. This study revealed some novel functions of Bcl-2 in dentinogenesis in addition to its anti-apoptotic effect, which shed some light on the genetic complexity of tooth development. PMID:20518070

  12. Transgenics, agroindustry and food sovereignty

    Directory of Open Access Journals (Sweden)

    Xavier Alejandro León Vega

    2014-10-01

    Full Text Available Food sovereignty has been implemented constitutionally in Ecuador; however, many of the actions and policies are designed to benefit the dominant model of food production, based in agroindustry, intensive monocultures, agrochemicals and transgenics. This article reflects upon the role of family farming as a generator of food sovereignty, and secondly the threat to them by agroindustry agriculture based in transgenic. The role played by food aid in the introduction of transgenic in Latin America and other regions of the world is also analyzed.

  13. Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

    DEFF Research Database (Denmark)

    Olesen, Mikkel V; Christiansen, Søren Hofman Oliveira; Gøtzsche, Casper René

    2012-01-01

    -like effect in rodents. The present study explored an alternative and more specific approach: overexpression of Y1 receptors. Using a recombinant adeno-associated viral vector (rAAV) encoding the Y1 gene (rAAV-Y1), we, for the first time, induced overexpression of functional transgene Y1 receptors...... in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene...... overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data...

  14. Over-expression of AQUA1 in Populus alba Villafranca clone increases relative growth rate and water use efficiency, under Zn excess condition.

    Science.gov (United States)

    Ariani, Andrea; Francini, Alessandra; Andreucci, Andrea; Sebastiani, Luca

    2016-02-01

    Transgenic Populus alba over-expressing a TIP aquaporin ( aqua1) showed a higher growth rate under Zn excess, suggesting that aqua1 could be involved in water homeostasis, rather than in Zn homeostasis. Populus is the internationally accepted model for physiological and developmental studies of tree traits under stress. In plants, aquaporins facilitate and regulate the diffusion of water, however, few poplar aquaporins have been characterized to date. In this study, we reported for the first time an in vivo characterization of Populus alba clone Villafranca transgenic plants over-expressing a TIP aquaporin (aqua1) of P. x euramericana clone I-214. An AQUA1:GFP chimeric construct, over-expressed in P. alba Villafranca clones, shows a cytoplasmic localization in roots, and it localizes in guard cells in leaves. When over-expressed in transgenic plants, aqua1 confers a higher growth rate compared to wild-type (wt) plants, without affecting chlorophyll accumulation, relative water content (RWC), and fluorescence performances, but increasing the intrinsic Transpiration Efficiency. In response to Zn (1 mM), transgenic lines did not show a significant increase in Zn accumulation as compared to wt plants, even though the over-expression of this gene confers higher tolerance in root tissues. These results suggest that, in poplar plants, this gene could be principally involved in regulation of water homeostasis and biomass production, rather than in Zn homeostasis.

  15. Transgenic mouse models of metabolic bone disease.

    Science.gov (United States)

    McCauley, L K

    2001-07-01

    The approach of gene-targeted animal models is likely the most important experimental tool contributing to recent advances in skeletal biology. Modifying the expression of a gene in vivo, and the analysis of the consequences of the mutation, are central to the understanding of gene function during development and physiology, and therefore to our understanding of the gene's role in disease states. Researchers had been limited to animal models primarily involving pharmaceutical manipulations and spontaneous mutations. With the advent of gene targeting, however, animal models that impact our understanding of metabolic bone disease have evolved dramatically. Interestingly, some genes that were expected to yield dramatic phenotypes in bone, such as estrogen receptor-alpha or osteopontin, proved to have subtle phenotypes, whereas other genes, such as interleukin-5 or osteoprotegerin, were initially identified as having a role in bone metabolism via the analysis of their phenotype after gene ablation or overexpression. Particularly important has been the advance in knowledge of osteoblast and osteoclast independent and dependent roles via the selective targeting of genes and the consequent disruption of bone formation, bone resorption, or both. Our understanding of interactions of the skeletal system with other systems, ie, the vascular system and homeostatic controls of adipogenesis, has evolved via animal models such as the matrix gla protein, knock-out, and the targeted overexpression of Delta FosB. Challenging transgenic models such as the osteopontin-deficient mice with mediators of bone remodeling like parathyroid hormone and mechanical stimuli and extending phenotype characterization to mechanistic in vitro studies of primary bone cells is providing additional insight into the mechanisms involved in pathologic states and their potentials for therapeutic strategies. This review segregates characterization of transgenic models based on the category of gene altered

  16. Effects of mineralocorticoid receptor overexpression on anxiety and memory after early life stress in female mice

    Directory of Open Access Journals (Sweden)

    Sofia eKanatsou

    2016-01-01

    Full Text Available Early-life stress is a risk factor for the development of psychopathology, particularly in women. Human studies have shown that certain haplotypes of NR3C2, encoding the mineralocorticoid receptor (MR, that result in gain of function, may protect against the consequences of stress exposure, including childhood trauma. Here, we tested the hypothesis that forebrain-specific overexpression of MR in female mice would ameliorate the effects of early-life stress on anxiety and memory in adulthood. We found that early-life stress increased anxiety, did not alter spatial discrimination and reduced contextual fear memory in adult female mice. Transgenic overexpression of MR did not alter anxiety but affected spatial memory performance and enhanced contextual fear memory formation. The effects of early life stress on anxiety and contextual fear were not affected by transgenic overexpression of MR. Thus MR overexpression in the forebrain does not represent a major resilience factor to early life adversity in female mice.

  17. Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions

    OpenAIRE

    Lee, Young Pyo; Baek, Kwang-Hyun; Lee, Haeng-Soon; Kwak, Sang-Soo; Bang, Jae-Woog; Kwon, Suk-Yoon

    2010-01-01

    Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal gr...

  18. Quantitative assessment of aortic atherosclerosis in APOE*3 leiden transgenic mice and its relationship to serum cholesterol exposure

    NARCIS (Netherlands)

    Groot, P.H.E.; Vlijmen, B.J.M. van; Benson, G.M.; Hofker, M.H.; Schiffelers, R.; Vidgeon-Hart, M.; Havekes, L.M.

    1996-01-01

    Transgenic mice overexpressing the human dysfunctional apolipoprotein E variant, APOE*3 Leiden, develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. In the present study, we investigated the effects of diet composition and feeding period on serum cholesterol exposure

  19. Blood-brain barrier disruption in CCL2 transgenic mice during pertussis toxin-induced brain inflammation

    DEFF Research Database (Denmark)

    Schellenberg, Angela E; Buist, Richard; Del Bigio, Marc R

    2012-01-01

    ABSTRACT: BACKGROUND: The chemokine CCL2 has an important role in the recruitment of inflammatory cells into the central nervous system (CNS). A transgenic mouse model that overexpresses CCL2 in the CNS shows an accumulation of leukocytes within the perivascular space surrounding vessels, which...

  20. Enhancement of production of eugenol and its glycosides in transgenic aspen plants via genetic engineering.

    Science.gov (United States)

    Koeduka, Takao; Suzuki, Shiro; Iijima, Yoko; Ohnishi, Toshiyuki; Suzuki, Hideyuki; Watanabe, Bunta; Shibata, Daisuke; Umezawa, Toshiaki; Pichersky, Eran; Hiratake, Jun

    2013-06-21

    Eugenol, a volatile phenylpropene found in many plant species, exhibits antibacterial and acaricidal activities. This study attempted to modify the production of eugenol and its glycosides by introducing petunia coniferyl alcohol acetyltransferase (PhCFAT) and eugenol synthase (PhEGS) into hybrid aspen. Gas chromatography analyses revealed that wild-type hybrid aspen produced small amount of eugenol in leaves. The heterologous overexpression of PhCFAT alone resulted in up to 7-fold higher eugenol levels and up to 22-fold eugenol glycoside levels in leaves of transgenic aspen plants. The overexpression of PhEGS alone resulted in a subtle increase in either eugenol or eugenol glycosides, and the overexpression of both PhCFAT and PhEGS resulted in significant increases in the levels of both eugenol and eugenol glycosides which were nonetheless lower than the increases seen with overexpression of PhCFAT alone. On the other hand, overexpression of PhCFAT in transgenic Arabidopsis and tobacco did not cause any synthesis of eugenol. These results indicate that aspen leaves, but not Arabidopsis and tobacco leaves, have a partially active pathway to eugenol that is limited by the level of CFAT activity and thus the flux of this pathway can be increased by the introduction of a single heterologous gene. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Tβ4-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics.

    Science.gov (United States)

    Shi, Bingbo; Ding, Qiang; He, Xiaolin; Zhu, Haijing; Niu, Yiyuan; Cai, Bei; Cai, Jiao; Lei, Anming; Kang, Danju; Yan, Hailong; Ma, Baohua; Wang, Xiaolong; Qu, Lei; Chen, Yulin

    2017-02-01

    Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tβ4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.

  2. Flagella Overexpression Attenuates Salmonella Pathogenesis

    OpenAIRE

    Xinghong Yang; Theresa Thornburg; Zhiyong Suo; SangMu Jun; Amanda Robison; Jinquan Li; Timothy Lim; Ling Cao; Teri Hoyt; Recep Avci; Pascual, David W.

    2012-01-01

    Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to...

  3. Flagella overexpression attenuates Salmonella pathogenesis.

    Directory of Open Access Journals (Sweden)

    Xinghong Yang

    Full Text Available Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE, was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co-expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC's adjuvant effect and conferred robust protection against wild-type Salmonella challenge.

  4. Flagella Overexpression Attenuates Salmonella Pathogenesis

    Science.gov (United States)

    Yang, Xinghong; Thornburg, Theresa; Suo, Zhiyong; Jun, SangMu; Robison, Amanda; Li, Jinquan; Lim, Timothy; Cao, Ling; Hoyt, Teri; Avci, Recep; Pascual, David W.

    2012-01-01

    Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co-expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC’s adjuvant effect and conferred robust protection against wild-type Salmonella challenge. PMID:23056473

  5. Progressive inflammatory pathology in the retina of aluminum-fed 5xFAD transgenic mice

    OpenAIRE

    Pogue, AI; Dua, P; Hill, JM; Lukiw, WJ

    2015-01-01

    At least 57 murine transgenic models for Alzheimer's disease (Tg-AD) have been developed to overexpress the 42 amino acid amyloid-beta (Aβ42) peptide in the central nervous system (CNS). These ‘humanized murine Tg-AD models’ have greatly expanded our understanding of the contribution of Aβ42 peptide-mediated pro-inflammatory neuropathology to the AD process. A number of independent laboratories using different amyloid-overexpressing Tg-AD models have shown that supplementation of murine Tg-AD...

  6. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Directory of Open Access Journals (Sweden)

    Tengale Dipak Bhauso

    2014-01-01

    Full Text Available In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L. crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

  7. Overexpression of a Modified Plant Thionin Enhances Disease Resistance to Citrus Canker and Huanglongbing (HLB).

    Science.gov (United States)

    Hao, Guixia; Stover, Ed; Gupta, Goutam

    2016-01-01

    Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. Since no HLB-resistant citrus cultivars have been identified, we attempted to develop citrus resistant to both HLB and citrus canker through overexpression of a modified plant thionin. To improve effectiveness for disease resistance, we modified and synthesized the sequence encoding a plant thionin and cloned into the binary vector pBinPlus/ARS. The construct was then introduced into Agrobacterium strain EHA105 for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were generated by Agrobacterium-mediated transformation. Successful transformation and transgene gene expression was confirmed by molecular analysis. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at a range of concentrations, and a significant reduction in canker symptoms and a decrease in bacterial growth were demonstrated compared to nontransgenic plants. Furthermore, the transgenic citrus plants were challenged with HLB via graft inoculation. Our results showed significant Las titer reduction in roots of transgenic Carrizo compared with control plants and reduced scion Las titer 12 months after graft inoculation. These data provide promise for engineering citrus disease resistance against HLB and canker.

  8. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Science.gov (United States)

    Bhauso, Tengale Dipak; Radhakrishnan, Thankappan; Kumar, Abhay; Mishra, Gyan Prakash; Dobaria, Jentilal Ramjibhai; Patel, Kirankumar; Rajam, Manchikatla Venkat

    2014-01-01

    In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated. PMID:25436223

  9. Distinct effects of ubiquitin overexpression on NMJ structure and motor performance in mice expressing catalytically inactive USP14

    Directory of Open Access Journals (Sweden)

    Jada H Vaden

    2015-04-01

    Full Text Available Ubiquitin-specific protease 14 (USP14 is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (axJ mice and demonstrated that transgenic ubiquitin overexpression partially rescues the axJ neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the axJ deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically-inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the axJ mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway.

  10. Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato

    Directory of Open Access Journals (Sweden)

    Ayed M. Al-Abdallat

    2014-10-01

    Full Text Available Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant.

  11. Tumorigenic potential of pituitary tumor transforming gene (PTTG) in vivo investigated using a transgenic mouse model, and effects of cross breeding with p53 (+/-) transgenic mice.

    Science.gov (United States)

    Fong, Miranda Y; Farghaly, Hanan; Kakar, Sham S

    2012-11-20

    Pituitary tumor-transforming gene (PTTG) is an oncogene that is overexpressed in variety of tumors and exhibits characteristics of a transforming gene. Previous transgenic mouse models to access the tumorigenic potential in the pituitary and ovary have resulted in dysplasia without formation of visible tumors, possibly due to the insufficient expression of PTTG. PTTG expression level is critical for ovarian tumorigenesis in a xenograft model. Therefore, the tumorigenic function of PTTG in vivo remains unclear. We generated a transgenic mouse that overexpresses PTTG driven by the CMV promoter to determine whether PTTG functions as a transforming oncogene that is capable of initiating tumorigenesis. Transgenic animals were generated by microinjection of PTTG transgene into the male pronucleus of FVB 0.5 day old embryos. Expression levels of PTTG in tissues of transgenic animals were analyzed using an immunohistochemical analysis. H&E staining and immunohistostaining were performed to examine the type of tumor in transgenic and PTTG transgenic/p53+/- animals. PTTG transgenic offspring (TgPTTG) were monitored for tumor development at various ages. H&E analysis was performed to identify the presence of cancer and hyperplastic conditions verified with the proliferation marker PCNA and the microvessel marker CD31. Immunohistochemistry was performed to determine transgene expression, revealing localization to the epithelium of the fallopian tube, with more generalized expression in the liver, lung, kidney, and spleen. At eight months of age, 2 out of 15 TgPTTG developed ovarian cancer, 2 out of 15 developed benign tumors, 2 out of 15 developed cervical dysplasia, and 3 out of 15 developed adenomyosis of the uterus. At ten months of age, 2 out of 10 TgPTTG developed adenocarcinoma of the ovary, 1 out of 10 developed a papillary serous adenocarcinoma, and 2 out of 10 presented with atypia of ovarian epithelial cells. Tumorigenesis is a multi-step process, often requiring

  12. Tumorigenic potential of pituitary tumor transforming gene (PTTG in vivo investigated using a transgenic mouse model, and effects of cross breeding with p53 (+/− transgenic mice

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    Fong Miranda Y

    2012-11-01

    Full Text Available Abstract Background Pituitary tumor-transforming gene (PTTG is an oncogene that is overexpressed in variety of tumors and exhibits characteristics of a transforming gene. Previous transgenic mouse models to access the tumorigenic potential in the pituitary and ovary have resulted in dysplasia without formation of visible tumors, possibly due to the insufficient expression of PTTG. PTTG expression level is critical for ovarian tumorigenesis in a xenograft model. Therefore, the tumorigenic function of PTTG in vivo remains unclear. We generated a transgenic mouse that overexpresses PTTG driven by the CMV promoter to determine whether PTTG functions as a transforming oncogene that is capable of initiating tumorigenesis. Methods Transgenic animals were generated by microinjection of PTTG transgene into the male pronucleus of FVB 0.5 day old embryos. Expression levels of PTTG in tissues of transgenic animals were analyzed using an immunohistochemical analysis. H&E staining and immunohistostaining were performed to examine the type of tumor in transgenic and PTTG transgenic/p53+/- animals. Results PTTG transgenic offspring (TgPTTG were monitored for tumor development at various ages. H&E analysis was performed to identify the presence of cancer and hyperplastic conditions verified with the proliferation marker PCNA and the microvessel marker CD31. Immunohistochemistry was performed to determine transgene expression, revealing localization to the epithelium of the fallopian tube, with more generalized expression in the liver, lung, kidney, and spleen. At eight months of age, 2 out of 15 TgPTTG developed ovarian cancer, 2 out of 15 developed benign tumors, 2 out of 15 developed cervical dysplasia, and 3 out of 15 developed adenomyosis of the uterus. At ten months of age, 2 out of 10 TgPTTG developed adenocarcinoma of the ovary, 1 out of 10 developed a papillary serous adenocarcinoma, and 2 out of 10 presented with atypia of ovarian epithelial cells

  13. A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

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    Pierce, Anson, E-mail: piercea2@uthscsa.edu [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); The Department of Veteran' s Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284 (United States); Wei, Rochelle; Halade, Dipti [Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Yoo, Si-Eun [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Ran, Qitao; Richardson, Arlan [Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229 (United States); The Department of Veteran' s Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284 (United States)

    2010-11-05

    Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

  14. Overexpression of PRL7D1 in Leydig Cells Causes Male Reproductive Dysfunction in Mice.

    Science.gov (United States)

    Liu, Yaping; Su, Xingyu; Hao, Jie; Chen, Maoxin; Liu, Weijia; Liao, Xiaogang; Li, Gang

    2016-01-13

    Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice.

  15. Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides.

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    Maximova, Siela N; Marelli, Jean-Philippe; Young, Ann; Pishak, Sharon; Verica, Joseph A; Guiltinan, Mark J

    2006-09-01

    Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.

  16. Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice.

    Science.gov (United States)

    Karmakar, Subhasis; Molla, Kutubuddin A; Das, Kaushik; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2017-08-11

    Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

  17. Overexpression of RCAN1 isoform 4 in mouse neurons leads to a moderate behavioral impairment.

    Science.gov (United States)

    Bhoiwala, Devang L; Koleilat, Issam; Qian, Jiang; Beyer, Barbara; Hushmendy, Shazaan F; Mathew, Alex; Bhoiwala, Dipti L; Ferland, Russell J; Crawford, Dana R

    2013-01-01

    Recent evidence supports the involvement of RCAN1 in Down syndrome and Alzheimer's disease. To better assess this, we generated and analyzed transgenic mice overexpressing human RCAN1 isoform 4 in neurons. Cognitive behavioral (Morris water maze, open field, zero maze, elevated plus maze assays); cognitive-associated proteins (CREB, ERK and Tau Western immunoblotting); motor coordination (Rotarod assay); structural abnormalities (immunohistological analyses), and proinflammatory cytokines (cytometric bead assay) were measured in young (2 month) and old (18 month) transgenics and compared with wild type controls. In old mice, male but not female transgenics exhibited a significant decrease in anxiety as compared with wild type controls, whereas female but not male transgenic mice exhibited significantly less motor coordination. No differences were observed in the Morris water maze (spatial learning). pERK levels were reduced in transgenic males but not females, while no differences were observed between genotypes for pCREB and pTau. In young mice, a modest learning and exploratory behavior was observed in transgenic mice using a limited number of mice, and at higher N values, pCREB and pERK (but not pTau) levels were reduced in transgenics. No macro- and micro-scopic structural abnormalities or proinflammatory cytokine level differences were observed. These results indicate that elevated RCAN1 isoform 4 in neurons leads to a modest cognition-related impairment that is overall stronger at 2 months, suggesting a compensatory adaptation over time. These RCAN1 isoform 4 effects may contribute to at least some of the observed phenotypes in individuals with Down syndrome and Alzheimer's.

  18. Overexpression of the AtSHI gene in poinsettia, Euphorbia pulcherrima, results in compact plants.

    Science.gov (United States)

    Islam, M Ashraful; Lütken, Henrik; Haugslien, Sissel; Blystad, Dag-Ragnar; Torre, Sissel; Rolcik, Jakub; Rasmussen, Søren K; Olsen, Jorunn E; Clarke, Jihong Liu

    2013-01-01

    Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21-52%) and internode lengths (31-49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11-31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.

  19. Overexpression of the AtSHI gene in poinsettia, Euphorbia pulcherrima, results in compact plants.

    Directory of Open Access Journals (Sweden)

    M Ashraful Islam

    Full Text Available Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21-52% and internode lengths (31-49% were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1. The indole-3-acetic acid (IAA content appeared lower (11-31% reduction in the transgenic lines compared to the wild type (WT controls, with the lowest level (31% reduction in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.

  20. Transgenic plants for enhanced biodegradation and phytoremediation of organic xenobiotics.

    Science.gov (United States)

    Abhilash, P C; Jamil, Sarah; Singh, Nandita

    2009-01-01

    Phytoremediation--the use of plants to clean up polluted soil and water resources--has received much attention in the last few years. Although plants have the inherent ability to detoxify xenobiotics, they generally lack the catabolic pathway for the complete degradation of these compounds compared to microorganisms. There are also concerns over the potential for the introduction of contaminants into the food chain. The question of how to dispose of plants that accumulate xenobiotics is also a serious concern. Hence the feasibility of phytoremediation as an approach to remediate environmental contamination is still somewhat in question. For these reasons, researchers have endeavored to engineer plants with genes that can bestow superior degradation abilities. A direct method for enhancing the efficacy of phytoremediation is to overexpress in plants the genes involved in metabolism, uptake, or transport of specific pollutants. Furthermore, the expression of suitable genes in root system enhances the rhizodegradation of highly recalcitrant compounds like PAHs, PCBs etc. Hence, the idea to amplify plant biodegradation of xenobiotics by genetic manipulation was developed, following a strategy similar to that used to develop transgenic crops. Genes from human, microbes, plants, and animals are being used successfully for this venture. The introduction of these genes can be readily achieved for many plant species using Agrobacterium tumefaciens-mediated plant transformation or direct DNA methods of gene transfer. One of the promising developments in transgenic technology is the insertion of multiple genes (for phase 1 metabolism (cytochrome P450s) and phase 2 metabolism (GSH, GT etc.) for the complete degradation of the xenobiotics within the plant system. In addition to the use of transgenic plants overexpressed with P450 and GST genes, various transgenic plants expressing bacterial genes can be used for the enhanced degradation and remediation of herbicides, explosives

  1. Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection.

    Science.gov (United States)

    Distefano, Gaetano; La Malfa, Stefano; Vitale, Alessandro; Lorito, Matteo; Deng, Ziniu; Gentile, Alessandra

    2008-10-01

    Constitutive over-expression of antifungal genes from microorganisms involved in plant defence mechanisms represents a promising strategy for conferring genetic resistance against a broad range of plant pathogenic fungi. In the present work, two transgenic lemon clones with the chit42 gene from Trichoderma harzianum were tested for resistance to fungal disease and expression level of defence-related genes was evaluated. Different resistance-related processes, such as production of reactive oxygen species (ROS), systemic acquired resistance (SAR) and induced systemic resistance (ISR), were monitored in transgenic and wild type lemon clones inoculated with Botrytis cinerea, the causal agent of grey mould in citrus. Expression of genes that encode gluthatione peroxidase (GPX), a producer of ROS, chitinases, glucanases (SAR), PAL, HPL, and AOS (ISR) was measured by quantitative PCR during the first 24 h after leaf inoculation. Leaves of transgenic lemon plants inoculated with B. cinerea showed significantly less lesion development than wild type leaves. Tissues from detached leaves of different transgenic lemon clones showed a significant correlation between resistance and transgene expression. On the other hand, the over-expression of the transgenic fungal gene enhanced by two-three folds transcript levels of genes associated with enhanced ROS production and ISR establishment, while the expression of native chitinase and glucanase genes involved in SAR was down-regulated.

  2. Changes in fatty acid content and composition between wild type and CsHMA3 overexpressing Camelina sativa under heavy-metal stress.

    Science.gov (United States)

    Park, Won; Feng, Yufeng; Kim, Hyojin; Suh, Mi Chung; Ahn, Sung-Ju

    2015-09-01

    Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids-especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)-in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content-factors that are responsible for the fuel properties of biodiesel-and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.

  3. Overexpression of MIC-3 indicates a direct role for the MIC gene family in mediating Upland cotton (Gossypium hirsutum) resistance to root-knot nematode (Meloidogyne incognita).

    Science.gov (United States)

    Wubben, Martin J; Callahan, Franklin E; Velten, Jeff; Burke, John J; Jenkins, Johnie N

    2015-02-01

    Transgene-based analysis of the MIC-3 gene provides the first report of a cotton gene having a direct role in mediating cotton resistance to root-knot nematode. Major quantitative trait loci have been mapped to Upland cotton (Gossypium hirsutum L.) chromosomes 11 and 14 that govern the highly resistant phenotype in response to infection by root-knot nematode (RKN; Meloidogyne incognita); however, nearly nothing is known regarding the underlying molecular determinants of this RKN-resistant phenotype. Multiple lines of circumstantial evidence have strongly suggested that the MIC (Meloidogyne Induced Cotton) gene family plays an integral role in mediating cotton resistance to RKN. In this report, we demonstrate that overexpression of MIC-3 in the RKN-susceptible genetic background Coker 312 reduces RKN egg production by ca. 60-75 % compared to non-transgenic controls and transgene-null sibling lines. MIC-3 transcript and protein overexpression were confirmed in root tissues of multiple independent transgenic lines with each line showing a similar level of increased resistance to RKN. In contrast to RKN fecundity, transgenic lines showed RKN-induced root galling similar to the susceptible controls. In addition, we determined that this effect of MIC-3 overexpression was specific to RKN as no effect was observed on reniform nematode (Rotylenchulus reniformis) reproduction. Transgenic lines did not show obvious alterations in growth, morphology, flowering, or fiber quality traits. Gene expression analyses showed that MIC-3 transcript levels in uninfected transgenic roots exceeded levels observed in RKN-infected roots of naturally resistant plants and that overexpression did not alter the regulation of native MIC genes in the genome. These results are the first report describing a direct role for a specific gene family in mediating cotton resistance to a plant-parasitic nematode.

  4. Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

    Science.gov (United States)

    Park, Ki Youl; Kim, Eun Yu; Seo, Young Sam; Kim, Woo Taek

    2016-03-01

    Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

  5. Expression of cartilage developmental genes in Hoxc8- and Hoxd4-transgenic mice.

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    Claudia Kruger

    2010-02-01

    Full Text Available Hox genes encode transcription factors, which regulate skeletal patterning and chondrocyte differentiation during the development of cartilage, the precursor to mature bone. Overexpression of the homeobox transcription factors Hoxc8 and Hoxd4 causes severe cartilage defects due to delay in cartilage maturation. Matrix metalloproteinases (MMPs, bone morphogenetic proteins (BMPs and fibroblastic growth factors (FGFs are known to play important roles in skeletal development and endochondral bone formation and remodeling. In order to investigate whether these molecules are aberrantly expressed in Hoxc8- and/or Hoxd4-transgenic cartilage, we performed quantitative RT-PCR on chondrocytes from Hox-transgenic mice. Gene expression levels of Bmp4, Fgf8, Fgf10, Mmp9, Mmp13, Nos3, Timp3, Wnt3a and Wnt5a were altered in Hoxc8-transgenic chondrocytes, and Fgfr3, Ihh, Mmp8, and Wnt3a expression levels were altered in Hoxd4-transgenic chondrocytes, respectively. Notably, Wnt3a expression was elevated in Hoxc8- and reduced in Hoxd4-transgenic cartilage. These results suggest that both transcription factors affect cartilage maturation through different molecular mechanisms, and provide the basis for future studies into the role of these genes and possible interactions in pathogenesis of cartilage defects in Hoxc8- and Hoxd4-transgenic mice.

  6. Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

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    Postina Rolf

    2009-02-01

    Full Text Available Abstract Background In a transgenic mouse model of Alzheimer disease (AD, cleavage of the amyloid precursor protein (APP by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant. Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice. Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. Conclusion In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of

  7. Overexpression of Human and Fly Frataxins in Drosophila Provokes Deleterious Effects at Biochemical, Physiological and Developmental Levels

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    Soriano, Sirena; Botella, José A.; Schneuwly, Stephan; Martínez-Sebastián, María J.; Moltó, María D.

    2011-01-01

    Background Friedreich's ataxia (FA), the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN) and fly (FH) frataxins in Drosophila. Methodology/Principal Findings We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant. Conclusion/Significance Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels. PMID:21779322

  8. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    Directory of Open Access Journals (Sweden)

    Chunxiang Yao

    Full Text Available Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2, react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat, an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  9. Matrix metalloproteinase-20 over-expression is detrimental to enamel development: a Mus musculus model.

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    Masashi Shin

    Full Text Available BACKGROUND: Matrix metalloproteinase-20 (Mmp20 ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20(-/- or Mmp20(+/+ in which the transgenes were expressed. METHODOLOGY/PRINCIPAL FINDINGS: Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. CONCLUSIONS/SIGNIFICANCE: Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It

  10. Cartilage-specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/CTGF stimulates insulin-like growth factor expression and bone growth.

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    Nao Tomita

    Full Text Available Previously we showed that CCN family member 2/connective tissue growth factor (CCN2 promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage-related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2

  11. Effect of BBX-B8 overexpression on development, body weight, silk protein synthesis and egg diapause of Bombyx mori.

    Science.gov (United States)

    Zheng, Xiaojian; Gong, Yongchang; Kumar, Dhiraj; Chen, Fei; Kuan, Sulan; Liang, Zi; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2016-08-01

    Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5-3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56-14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.

  12. Overexpression of the transporters AtZIP1 and AtMTP1 in cassava changes zinc accumulation and partitioning

    Directory of Open Access Journals (Sweden)

    Eliana eGaitan-Solis

    2015-07-01

    Full Text Available Zinc deficiency in humans is a serious problem worldwide with an estimated one third of populations at risk for insufficient zinc in diet which leads to impairment of cognitive abilities and immune system function. The goal of this research was to increase the bioavailable zinc in the edible portion of cassava roots to improve the overall zinc nutrition of populations that rely on cassava as a dietary staple. To increase zinc concentrations, two A. thaliana genes coding for ZIP1 and MTP1 were overexpressed with a tuber-specific or constitutive promoter. Eighteen transgenic events from four constructs, out of a total of 73 events generated, showed significantly higher zinc concentrations in the edible portion of the storage root compared to the non-transgenic controls. The zinc content in the transgenic lines ranged from 4 - 73 mg/Kg Dry Weight (DW as compared to the non-transgenic control which contained 8 mg/Kg. Striking changes in whole plant phenotype such as smaller plant size and chlorotic leaves were observed in transgenic lines that over accumulated zinc. In a confined field trial five transgenic events grown for 12 months showed a range of zinc concentrations from 18 – 217 mg/Kg DW. Although the overexpression of zinc transporters was successful in increasing the zinc concentrations in 25% of the transgenic lines generated, it also resulted in a decrease in plant and tuber size and overall yield due to what appears to be zinc deficiency in the aerial parts of the plant.

  13. Smad2 overexpression enhances adhesion of gingival epithelial cells.

    Science.gov (United States)

    Hongo, Shoichi; Yamamoto, Tadashi; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Ugawa, Yuki; Kochi, Shinsuke; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

    2016-11-01

    Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. By using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Vasomotor control in mice overexpressing human endothelial nitric oxide synthase.

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    van Deel, Elza D; Merkus, Daphne; van Haperen, Rien; de Waard, Monique C; de Crom, Rini; Duncker, Dirk J

    2007-08-01

    Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.

  15. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ying [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Adachi, Hiroaki, E-mail: hadachi-ns@umin.org [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Katsuno, Masahisa [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Huang, Zhe [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Funakoshi, Hiroshi [Center for Advanced Research and Education, Asahikawa Medical University, 1-1-1- Higashinijo Midorigaoka, Asahikawa 078-8510 (Japan); Nakamura, Toshikazu [Neurogen Inc., 1-1-52-201 Nakahozumi, Ibaraki 567-0034 (Japan); Sobue, Gen, E-mail: sobueg@med.nagoya-u.ac.jp [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Research Division of Dementia and Neurodegenerative Disease, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan)

    2015-12-25

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. - Highlights: • HGF overexpression ameliorates the motor phenotypes of the SBMA mouse model. • HGF overexpression induces Akt phosphorylation in the SBMA mouse model. • This is the first report of combination therapy in a mouse model of polyQ diseases.

  16. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

    Science.gov (United States)

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-04-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop-weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop-weed hybrids produced 48-125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  17. A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide

    Science.gov (United States)

    Wang, Wei; Xia, Hui; Yang, Xiao; Xu, Ting; Si, Hong Jiang; Cai, Xing Xing; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2014-01-01

    Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization. Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene. Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls. Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression. PMID:23905647

  18. LOTUS overexpression accelerates neuronal plasticity after focal brain ischemia in mice.

    Science.gov (United States)

    Takase, Hajime; Kurihara, Yuji; Yokoyama, Taka-Akira; Kawahara, Nobutaka; Takei, Kohtaro

    2017-01-01

    Nogo receptor-1 (NgR1) and its ligands inhibit neuronal plasticity and limit functional recovery after brain damage such as ischemic stroke. We have previously shown that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated signaling. Here, we investigated whether LOTUS enhances neuronal plasticity and functional recovery after brain focal ischemia in adult mice. Focal ischemic infarcts were induced in wild-type and LOTUS-overexpressing transgenic mice via middle cerebral artery occlusion. Endogenous LOTUS expression was increased in brain and cervical spinal cord of the contralateral side of ischemia in the chronic phase after brain ischemia. LOTUS overexpression accelerated midline-crossing axonal sprouting from the contralateral side to the ipsilateral side of ischemia in the medullar reticular formation and gray matter of denervated cervical spinal cord. Importantly, LOTUS overexpression improved neurological score highly correlated with laterality ratio of corticoreticular fibers of the medulla oblongata, indicating that LOTUS overexpression may overcome the inhibitory environment induced by NgR1 signaling for damaged motor pathway reconstruction after ischemic stroke. Thus, our data suggest that LOTUS overexpression accelerates neuronal plasticity in the brainstem and cervical spinal cord after stroke and LOTUS administration is useful for future therapeutic strategies.

  19. [Transgenic animals and animal welfare

    Science.gov (United States)

    Reinhardt, Christoph

    1998-01-01

    Under the pressure of a public vote in Switzerland (7 June 1998) on an initiative to ban the production, use and patenting of transgenic animals, their value for biomedical research and development is intensely debated. In addition, the Swiss legislation has adopted (1992) a constitutional obligation to "take into account the dignity of creatures". The term "dignity of creatures", however, can be interpreted in anthropocentric or biocentric ways. The government has now formulated the legal implications of this term for transgenic animals and plants in various laws including the animal and environmental protection laws. This paper gives arguments for a fair evaluation of trangenic animals from an animal welfare point of view where not only the costs of animal suffering must be considered but also the probability of potential benefit for man. A self-confident research community should allow such an evaluation procedure even in view of an outcome which could ban many uses of transgenic animals

  20. Inducible gene manipulations in brain serotonergic neurons of transgenic rats.

    Directory of Open Access Journals (Sweden)

    Tillmann Weber

    Full Text Available The serotonergic (5-HT system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP, in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.

  1. Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation

    NARCIS (Netherlands)

    Creemers, L. B.; Meng, X.; den Ouden, K.; van Pelt, A. M. M.; Izadyar, F.; Santoro, M.; Sariola, H.; de rooij, D. G.

    2002-01-01

    With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals.

  2. Selective endothelial overexpression of arginase II induces endothelial dysfunction and hypertension and enhances atherosclerosis in mice.

    Directory of Open Access Journals (Sweden)

    Boris L Vaisman

    Full Text Available Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis.Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE -/- mice, hArgII mice had increased aortic atherosclerotic lesions.We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.

  3. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene confers drought tolerance in maize (Zea mays L.).

    Science.gov (United States)

    Lu, Yao; Li, Yajun; Zhang, Jiachang; Xiao, Yitao; Yue, Yuesen; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2013-01-01

    Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H(2)O(2) content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.

  4. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene confers drought tolerance in maize (Zea mays L..

    Directory of Open Access Journals (Sweden)

    Yao Lu

    Full Text Available Abscisic acid (ABA is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5 in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L. exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA and H(2O(2 content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.

  5. Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene.

    Science.gov (United States)

    Folta, Adam; Bargsten, Joachim W; Bisseling, Ton; Nap, Jan-Peter; Mlynarova, Ludmila

    2016-02-01

    Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Overexpression of a Pathogenesis-Related Protein 10 Enhances Biotic and Abiotic Stress Tolerance in Rice

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    Jingni Wu

    2016-12-01

    Full Text Available Pathogenesis-related proteins play multiple roles in plant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related gene named “jasmonic acid inducible pathogenesis-related class 10” (JIOsPR10 to gain an insight into its functional properties. Semi-quantitative RT-PCR analysis showed up-regulation of JIOsPR10 under salt and drought stress conditions. Constitutive over-expression JIOsPR10 in rice promoted shoot and root development in transgenic plants, however, their productivity was unaltered. Further experiments exhibited that the transgenic plants showed reduced susceptibility to rice blast fungus, and enhanced salt and drought stress tolerance as compared to the wild type. A comparative proteomic profiling of wild type and transgenic plants showed that overexpression of JIOsPR10 led to the differential modulation of several proteins mainly related with oxidative stresses, carbohydrate metabolism, and plant defense. Taken together, our findings suggest that JIOsPR10 plays important roles in biotic and abiotic stresses tolerance probably by activation of stress related proteins.

  7. Reprogramming fibroblasts to neural-precursor-like cells by structured overexpression of pallial patterning genes.

    Science.gov (United States)

    Raciti, Marilena; Granzotto, Marilena; Duc, Minh Do; Fimiani, Cristina; Cellot, Giada; Cherubini, Enrico; Mallamaci, Antonello

    2013-11-01

    In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogramme mouse embryonic fibroblasts towards neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even after switching off the reprogramming transgenes. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP positive cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cell output was achieved by inhibition of the BMP and TGFβ pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype. © 2013.

  8. A Mutation of the Prdm9 Mouse Hybrid Sterility Gene Carried by a Transgene.

    Science.gov (United States)

    Mihola, O; Trachtulec, Z

    2017-01-01

    PRDM9 is a protein with histone-3-methyltransferase activity, which specifies the sites of meiotic recombination in mammals. Deficiency of the Prdm9 gene in the laboratory mouse results in complete arrest of the meiotic prophase of both sexes. Moreover, the combination of certain PRDM9 alleles from different mouse subspecies causes hybrid sterility, e.g., the male-specific meiotic arrest found in the (PWD/Ph × C57BL/6J)F1 animals. The fertility of all these mice can be rescued using a Prdm9-containing transgene. Here we characterized a transgene made from the clone RP24-346I22 that was expected to encompass the entire Prdm9 gene. Both (PWD/Ph × C57BL/6J)F1 intersubspecific hybrid males and Prdm9-deficient laboratory mice of both sexes carrying this transgene remained sterile, suggesting that Prdm9 inactivation occurred in the Tg(RP24-346I22) transgenics. Indeed, comparative qRT-PCR analysis of testicular RNAs from transgene-positive versus negative animals revealed similar expression levels of Prdm9 mRNAs from the exons encoding the C-terminal part of the protein but elevated expression from the regions coding for the N-terminus of PRDM9, indicating that the transgenic carries a new null Prdm9 allele. Two naturally occurring alternative Prdm9 mRNA isoforms were overexpressed in Tg(RP24-346I22), one formed via splicing to a 3'-terminal exon consisting of short interspersed element B2 and one isoform including an alternative internal exon of 28 base pairs. However, the overexpression of these alternative transcripts was apparently insufficient for Prdm9 function or for increasing the fertility of the hybrid males.

  9. Overexpression of homogentisate phytyltransferase in lettuce results ...

    African Journals Online (AJOL)

    In this study, HPT gene isolated from Lactuca sativa L. (LsHPT) was transferred into L. sativa (lettuce) via Agrobacterium-mediated method and 15 transgenic plants were obtained. The expression of LsHPT gene and the total content of and -tocopherol in transgenic plants were increased up to 4 and 2.61 folds, respectively, ...

  10. Effect of Rhizobium and arbuscular mycorrhizal fungi inoculation on electrolyte leakage in Phaseolus vulgaris roots overexpressing RbohB

    Science.gov (United States)

    Arthikala, Manoj-Kumar; Nava, Noreide; Quinto, Carmen

    2015-01-01

    Respiratory oxidative burst homolog (RBOH)-mediated reactive oxygen species (ROS) regulate a wide range of biological functions in plants. They play a critical role in the symbiosis between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. For instance, overexpression of PvRbohB enhances nodule numbers, but reduces mycorrhizal colonization in Phaseolus vulgaris hairy roots and downregulation has the opposite effect. In the present study, we assessed the effect of both rhizobia and AM fungi on electrolyte leakage in transgenic P. vulgaris roots overexpressing (OE) PvRbohB. We demonstrate that elevated levels of electrolyte leakage in uninoculated PvRbohB-OE transgenic roots were alleviated by either Rhizobium or AM fungi symbiosis, with the latter interaction having the greater effect. These results suggest that symbiont colonization reduces ROS elevated electrolyte leakage in P. vulgaris root cells. PMID:25946118

  11. Effect of Rhizobium and arbuscular mycorrhizal fungi inoculation on electrolyte leakage in Phaseolus vulgaris roots overexpressing RbohB.

    Science.gov (United States)

    Arthikala, Manoj-Kumar; Nava, Noreide; Quinto, Carmen

    2015-01-01

    Respiratory oxidative burst homolog (RBOH)-mediated reactive oxygen species (ROS) regulate a wide range of biological functions in plants. They play a critical role in the symbiosis between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. For instance, overexpression of PvRbohB enhances nodule numbers, but reduces mycorrhizal colonization in Phaseolus vulgaris hairy roots and downregulation has the opposite effect. In the present study, we assessed the effect of both rhizobia and AM fungi on electrolyte leakage in transgenic P. vulgaris roots overexpressing (OE) PvRbohB. We demonstrate that elevated levels of electrolyte leakage in uninoculated PvRbohB-OE transgenic roots were alleviated by either Rhizobium or AM fungi symbiosis, with the latter interaction having the greater effect. These results suggest that symbiont colonization reduces ROS elevated electrolyte leakage in P. vulgaris root cells.

  12. The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum.

    Science.gov (United States)

    An, Juan; Song, Aiping; Guan, Zhiyong; Jiang, Jiafu; Chen, Fadi; Lou, Wanghuai; Fang, Weimin; Liu, Zhaolei; Chen, Sumei

    2014-06-01

    Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na(+)/H(+) antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum 'Jinba'. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na(+) into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K(+)/Na(+) ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na(+) accumulation. This study is the first to report the transgenic expression of a Na(+) efflux carrier in chrysanthemum.

  13. Overexpression of an Arabidopsis heterogeneous nuclear ribonucleoprotein gene, AtRNP1, affects plant growth and reduces plant tolerance to drought and salt stresses

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhenyu, E-mail: wzy72609@163.com [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Zhao, Xiuyang, E-mail: xiuzh@psb.vib-ugent.be [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Wang, Bing, E-mail: wangbing@ibcas.ac.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Liu, Erlong, E-mail: liuel14@lzu.edu.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Chen, Ni, E-mail: 63710156@qq.com [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Zhang, Wei, E-mail: wzhang1216@yahoo.com [Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444 (China); Liu, Heng, E-mail: hengliu@lzu.edu.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China)

    2016-04-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) participate in diverse regulations of plant growth and environmental stress responses. In this work, an Arabidopsis hnRNP of unknown function, AtRNP1, was investigated. We found that AtRNP1 gene is highly expressed in rosette and cauline leaves, and slightly induced under drought, salt, osmotic and ABA stresses. AtRNP1 protein is localized to both the nucleus and cytoplasm. We performed homologous overexpression of AtRNP1 and found that the transgenic plants showed shortened root length and plant height, and accelerated flowering. In addition, the transgenic plants also showed reduced tolerance to drought, salt, osmotic and ABA stresses. Further studies revealed that under both normal and stress conditions, the proline contents in the transgenic plants are markedly decreased, associated with reduced expression levels of a proline synthase gene and several stress-responsive genes. These results suggested that the overexpression of AtRNP1 negatively affects plant growth and abiotic stress tolerance. - Highlights: • AtRNP1 is a widely expressed gene and its expression is slightly induced under abiotic stresses. • AtRNP1 protein is localized to both the nucleus and cytoplasm. • Overexpression of AtRNP1 affects plant growth. • Overexpression of AtRNP1 reduces plant tolerance to drought and salt stresses. • AtRNP1 overexpression plants show decreased proline accumulation and stress-responsive gene expressions.

  14. Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit.

    Science.gov (United States)

    Leclercq, J; Martin, F; Sanier, C; Clément-Vidal, A; Fabre, D; Oliver, G; Lardet, L; Ayar, A; Peyramard, M; Montoro, P

    2012-10-01

    Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.

  15. Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds.

    Science.gov (United States)

    Liu, Wen Xian; Liu, Hua Liang; Qu, Le Qing

    2013-09-01

    Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.

  16. Effects of transgenic expression of Brevibacterium linens methionine gamma lyase (MGL) on accumulation of Tylenchulus semipenetrans and key aminoacid contents in Carrizo citrange.

    Science.gov (United States)

    Castillo, Elenor; Martinelli, Federico; Zakharov-Negre, Florence; Ebeler, Susan E; Buzo, Tom R; McKenry, Michael V; Dandekar, Abhaya M

    2017-11-01

    Carrizo transgenic plants overexpressing methionine-gamma-lyase produced dimethyl sulfide. The transgenic plants displayed more resistance to nematode attacks (Tylenculus semipenetrans) and may represent an innovative strategy for nematode control. Tylenchulus semipenetrans is a nematode pest of many citrus varieties that causes extensive damage to commercial crops worldwide. Carrizo citrange vr. (Citrus sinensis L. Usb × Poncirus trifoliate L. Raf) plants overexpressing Brevibacterium linens methionine-gamma-lyase (BlMGL) produced the sulfur volatile compound dimethyl sulfide (DMS). The aim of this work was to determine if transgenic citrus plants expressing BlMGL showed increased tolerance to T. semipenetrans infestation and to determine the effect on the content of key amino acids. While transgenic lines emitted dimethyl sulfide from leaves and roots, no sulfur-containing volatiles were detectable in wild-type Carrizo in the same tissues. Significant changes detected some key amino acids from leaves of transgenic plants such as aspartate, lysine, glycine, leucine and threonine with no changes in the amounts of methionine and α-ketobutyrate. In roots only glycine showed significant changes across all transgenic lines in comparison to wild-type plants. Transgenic plants expressing BlMGL and emitting DMS had less T. semipenetrans aggregation and more biomass than infected WT control plants, indicating that they may represent an innovative management alternative to pesticide/nematicide-based remedies.

  17. Vascular endothelium-specific overexpression of human catalase in cloned pigs.

    Science.gov (United States)

    Whyte, J J; Samuel, M; Mahan, E; Padilla, J; Simmons, G H; Arce-Esquivel, A A; Bender, S B; Whitworth, K M; Hao, Y H; Murphy, C N; Walters, E M; Prather, R S; Laughlin, M H

    2011-10-01

    The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

  18. NUCKS overexpression in breast cancer

    Directory of Open Access Journals (Sweden)

    Kittas Christos

    2009-08-01

    Full Text Available Abstract Background NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that NUCKS is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR, real-time PCR (qRT-PCR and Western immunoblot analyses in the primary cell cultures developed from the same biopsies. Results The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas in situ (DCIS. It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of NUCKS expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures. Conclusion The results show overexpression of the NUCKS protein in a number of non

  19. Overexpression of a New Chitinase Gene EuCHIT2 Enhances Resistance to Erysiphe cichoracearum DC. in Tobacco Plants

    OpenAIRE

    Xuan Dong; Yichen Zhao; Xin Ran; Linxia Guo; De-Gang Zhao

    2017-01-01

    In this study, we cloned a new chitinase gene, EuCHIT2, from Eucommia ulmoides Oliver (E. ulmoides) using rapid amplification of cDNA ends (RACE) technology and constructed an overexpression vector, pSH-35S-EuCHIT2, to introduce it into tobacco (Nicotiana tabacum cv. Xanthi). Resistance to Erysiphe cichoracearum de Candolle (E.cichoracearum DC) and molecular mechanisms in the transgenic tobacco were determined by drop inoculation, spore counting, determination of physicochemical indicators, a...

  20. Over-expression of AtPAP2 in Camelina sativa leads to faster plant growth and higher seed yield.

    Science.gov (United States)

    Zhang, Youjun; Yu, Laura; Yung, Ka-Fu; Leung, Dennis Yc; Sun, Feng; Lim, Boon L

    2012-04-02

    Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa. Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants. Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels.