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Sample records for controlling transgene expression

  1. Spatial and temporal control of transgene expression in zebrafish.

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    Alexander A Akerberg

    Full Text Available Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.

  2. Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters.

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    L'hostis-Guidet, Anne; Recher, Gaëlle; Guillet, Brigitte; Al-Mohammad, Abdulrahim; Coumailleau, Pascal; Tiaho, François; Boujard, Daniel; Madigou, Thierry

    2009-10-01

    Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.

  3. The methods to generate transgenic animals and to control transgene expression.

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    Houdebine, Louis-Marie

    2002-09-25

    Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

  4. A transgenic approach to control hemipteran insects by expressing insecticidal genes under phloem-specific promoters

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    Javaid, Shaista; Amin, Imran; Jander, Georg; Mukhtar, Zahid; Saeed, Nasir A.; Mansoor, Shahid

    2016-01-01

    The first generation transgenic crops used strong constitutive promoters for transgene expression. However, tissue-specific expression is desirable for more precise targeting of transgenes. Moreover, piercing/sucking insects, which are generally resistant to insecticidal Bacillus thuringiensis (Bt) proteins, have emerged as a major pests since the introduction of transgenic crops expressing these toxins. Phloem-specific promoters isolated from Banana bunchy top virus (BBTV) were used for the expression of two insecticidal proteins, Hadronyche versuta (Blue Mountains funnel-web spider) neurotoxin (Hvt) and onion leaf lectin, in tobacco (Nicotiana tabacum). Here we demonstrate that transgenic plants expressing Hvt alone or in combination with onion leaf lectin are resistant to Phenacoccus solenopsis (cotton mealybug), Myzus persicae (green peach aphids) and Bemisia tabaci (silver leaf whitefly). The expression of both proteins under different phloem-specific promoters resulted in close to 100% mortality and provided more rapid protection than Hvt alone. Our results suggest the employment of the Hvt and onion leaf lectin transgenic constructs at the commercial level will reduce the use of chemical pesticides for control of hemipteran insect pests. PMID:27708374

  5. Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice.

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    DeMayo, F J; Finegold, M J; Hansen, T N; Stanley, L A; Smith, B; Bullock, D W

    1991-08-01

    The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.

  6. Production of pigs expressing a transgene under the control of a tetracycline-inducible system.

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    Yong-Xun Jin

    Full Text Available Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP. At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems.

  7. Cosmetics-triggered percutaneous remote control of transgene expression in mice

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    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin

    2015-01-01

    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies. PMID:25943548

  8. Cosmetics-triggered percutaneous remote control of transgene expression in mice.

    Science.gov (United States)

    Wang, Hui; Ye, Haifeng; Xie, Mingqi; Daoud El-Baba, Marie; Fussenegger, Martin

    2015-08-18

    Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.

  9. Heterologous expression in transgenic mosquitoes

    Institute of Scientific and Technical Information of China (English)

    Santhosh P K; Yu hua Deng; Weidong Gu; Xiaoguang Chen

    2010-01-01

    Arthropod-borne diseases such as malaria and dengue virus afflict billions of people worldwide imposing major economic and social burdens. Control of such pathogens is mainly performed by vector management and treatment of affected individuals with drugs. The failure of these conventional approaches due to emergence of insecticide-resistant insects and drug-resistant parasites demonstrate the need of novel and efficacious control strategies to combat these diseases. Genetic modification(GM) of mosquito vectors to impair their ability to be infected and transmit pathogens has emerged as a new strategy to reduce transmission of many vector-borne diseases and deliver public health gains. Several advances in developing transgenic mosquitoes unable to transmit pathogens have gained support, some of them attempt to manipulate the naturally occurring endogenous refractory mechanisms, while others initiate the identification of an exogenous foreign gene which disrupt the pathogen development in insect vectors. Heterologous expression of transgenes under a native or heterologous promoter is important for the screening and effecting of the transgenic mosquitoes. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this transgenic approach. This review examines these two aspects and describes the basic research work that has been accomplished towards understanding the complex relation between the parasite and its vector and focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to vector-borne disease transmission.

  10. Transgene expression systems in the Triticeae cereals.

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    Hensel, Götz; Himmelbach, Axel; Chen, Wanxin; Douchkov, Dimitar K; Kumlehn, Jochen

    2011-01-01

    The control of transgene expression is vital both for the elucidation of gene function and for the engineering of transgenic crops. Given the dominance of the Triticeae cereals in the agricultural economy of the temperate world, the development of well-performing transgene expression systems of known functionality is of primary importance. Transgenes can be expressed either transiently or stably. Transient expression systems based on direct or virus-mediated gene transfer are particularly useful in situations where the need is to rapidly screen large numbers of genes. However, an unequivocal understanding of gene function generally requires that a transgene functions throughout the plant's life and is transmitted through the sexual cycle, since this alone allows its effect to be decoupled from the plant's response to the generally stressful gene transfer event. Temporal, spatial and quantitative control of a transgene's expression depends on its regulatory environment, which includes both its promoter and certain associated untranslated region sequences. While many transgenic approaches aim to manipulate plant phenotype via ectopic gene expression, a transgene sequence can be also configured to down-regulate the expression of its endogenous counterpart, a strategy which exploits the natural gene silencing machinery of plants. In this review, current technical opportunities for controlling transgene expression in the Triticeae species are described. Apart from protocols for transient and stable gene transfer, the choice of promoters and other untranslated regulatory elements, we also consider signal peptides, as they too govern the abundance and particularly the sub-cellular localization of transgene products.

  11. Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs

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    Ohtaka, Manami; Nakanishi, Mahito

    2016-01-01

    Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3′ untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. PMID:27764162

  12. Combinatorial Control of Transgene Expression by Hypoxia-Responsive Promoter and MicroRNA Regulation for Neural Stem Cell-Based Cancer Therapy

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    Yumei Luo

    2014-01-01

    Full Text Available Owing to their strong migratory capacity, tumor tropism, and tumor inhibitory effect, neural stem cells (NSCs have recently emerged as one of the most attractive gene delivery vectors for cancer therapy. However, further animal studies found that proportional NSC vectors were distributed to nontarget organs after intravenous injection and the nonspecific transgene expression led to significant cytotoxic effects in these organs. Hence, an expression cassette that controls the transgene expression within NSC vectors in a tumor site-specific manner is desired. Considering hypoxia as a hallmark of tumor microenvironment, we have developed a novel NSC vector platform coupling transcriptional targeting with microRNA (miRNA regulation for tumor hypoxia targeting. This combinatorial vector employed a hypoxia-responsive promoter and repeated targeting sequences of an miRNA that is enriched in NSCs but downregulated upon hypoxia induction to control the transgene expression. This resulted in significantly improved hypoxic selectivity over the use of a control vector without miRNA regulation. Thus, incorporating miRNA regulation into a transcriptional targeting vector adds an extra layer of security to prevent off-target transgene expression and should be useful for the development of NSC vectors with high targeting specifcity for cancer therapy.

  13. Positron emission tomography : measurement of transgene expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vaalburg, W

    2002-01-01

    Noninvasive and repetitive imaging of transgene expression can play a pivotal role in the development of gene therapy strategies, as it offers investigators a means to determine the effectiveness of their gene transfection protocols. In the last decade, imaging of transgene expression using positron

  14. Induction of systemic IFITM3 expression does not effectively control foot-and-mouth disease viral infection in transgenic pigs.

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    Zhang, Huawei; Zheng, Haixue; Qian, Ping; Xu, Jinfang; Yang, Xi; Zhou, Rui; Chen, Huanchun; Li, Xiangmin

    2016-08-15

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, and can cause severe economic loss. Interferon-induced transmembrane (IFITM) proteins constitute a family of viral restriction factors that can inhibit the replication of several types of viruses. Our previous study showed that overexpression of swine IFITM3 (sIFITM3) impeded replication of the FMD virus (FMDV) in BHK-21 cells and mice. In this study, sIFITM3-transgenic (TG) pigs were produced by handmade cloning. Results showed that sIFITM3 was highly overexpressed in many organs of sIFITM3-TG pigs compared to wild-type pigs. After a virulent FMDV strain (O/ES/2001) was intramuscularly inoculated, the sIFITM3-TG pigs showed slightly higher susceptibility to FMDV infection than wild-type pigs. Both groups displayed comparable degrees of clinical symptoms throughout the 14-day observation period. Therefore, the induction of systemic sIFITM3 expression does not protect pigs against FMDV infection. Based on these observations, we propose that a combination of interferons and vaccines be used to control FMDV infections and subsequent FMD outbreaks.

  15. MRI of Transgene Expression: Correlation to Therapeutic Gene Expression

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    Tomotsugu Ichikawa

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI can provide highresolution 3D maps of structural and functional information, yet its use of mapping in vivo gene expression has only recently been explored. A potential application for this technology is to noninvasively image transgene expression. The current study explores the latter using a nonregulatable internalizing engineered transferrin receptor (ETR whose expression can be probed for with a superparamagnetic Tf-CLIO probe. Using an HSV-based amplicon vector system for transgene delivery, we demonstrate that: 1 ETR is a sensitive MR marker gene; 2 several transgenes can be efficiently expressed from a single amplicon; 3 expression of each transgene results in functional gene product; and 4 ETR gene expression correlates with expression of therapeutic genes when the latter are contained within the same amplicon. These data, taken together, suggest that MRI of ETR expression can serve as a surrogate for measuring therapeutic transgene expression.

  16. TRANSGENIC PLANTS EXPRESSING BACILLUS THURINGIENSIS DELTA-ENDOTOXINS

    Institute of Scientific and Technical Information of China (English)

    Hua-rong,Li; BrendaOppert; KunYanZhu; RandallA.Higgins; Fang-nengHuang; LawrentL.Buschman

    2003-01-01

    Commercial varieties of transgenic Bacillus thuringiensis (Bt) plants have been developed in many countries to control target pests. Initially, the expression of native Bt genes in plants was low due to mRNA instability, improper splicing, and post-translation modifications. Subsequently, modifications of the native Bt genes greatly enhanced expression levels. This is a review of the developments that made modem high-expression transgenic Bt plants possible, with an emphasis on the reasons for the low-level expression of native Bt genes in plant systems, and the techniques that have been used to improve plant expression of Bt toxin genes.

  17. Fitness of Transgenic Anopheles stephensi Mosquitoes Expressing the SM1 Peptide under the Control of a Vitellogenin Promoter

    OpenAIRE

    Li, Chaoyang; Marrelli, Mauro T.; Yan, Guiyun; Jacobs-Lorena, Marcelo

    2008-01-01

    Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage ex...

  18. Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis.

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    Jia, Hongge; Van Loock, Bram; Liao, Mingjun; Verbelen, Jean-Pierre; Vissenberg, Kris

    2007-07-01

    The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and beta-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY vector conversion system. The method can be extended to other species when enhancer trap lines become available.

  19. Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP.

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    Coon, Charles I; Fiering, Steven; Gaudet, Justin; Wyatt, Colby A; Brinckerhoff, Constance E

    2009-09-01

    Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity. Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ss-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ss-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.

  20. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  1. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  2. Transgenic approaches to western corn rootworm control.

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    Narva, Kenneth E; Siegfried, Blair D; Storer, Nicholas P

    2013-01-01

    The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) is a significant corn pest throughout the United States corn belt. Rootworm larvae feed on corn roots causing yield losses and control expenditures that are estimated to exceed US$1 billion annually. Traditional management practices to control rootworms such as chemical insecticides or crop rotation have suffered reduced effectiveness due to the development of physiological and behavioral resistance. Transgenic maize expressing insecticidal proteins are very successful in protecting against rootworm damage and preserving corn yield potential. However, the high rate of grower adoption and early reliance on hybrids expressing a single mode of action and low-dose traits threatens the durability of commercialized transgenic rootworm technology for rootworm control. A summary of current transgenic approaches for rootworm control and the corresponding insect resistance management practices is included. An overview of potential new modes of action based on insecticidal proteins, and especially RNAi targeting mRNA coding for essential insect proteins is provided.

  3. Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton

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    Luo, Jun-Yu; Zhang, Shuai; Peng, Jun; Zhu, Xiang-Zhen; Lv, Li-Min; Wang, Chun-Yi; Li, Chun-Hua; Zhou, Zhi-Guo; Cui, Jin-Jie

    2017-01-01

    An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels. PMID:28099508

  4. Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

    Science.gov (United States)

    Burkov, I A; Serova, I A; Battulin, N R; Smirnov, A V; Babkin, I V; Andreeva, L E; Dvoryanchikov, G A; Serov, O L

    2013-10-01

    Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

  5. Influence of DNA methylation on transgene expression

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene b-glu- curonidase (GUS) gene (uidA) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.

  6. Efficient expression of transgenes in adult zebrafish by electroporation

    Directory of Open Access Journals (Sweden)

    Rao S Hari

    2005-10-01

    Full Text Available Abstract Background Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish. Results Electroporation parameters such as number of pulses, voltage and amount of plasmid DNA were optimized and it was found that 6 pulses of 40 V·cm-1 at 15 μg of plasmid DNA per fish increased the luciferase expression 10-fold compared to controls. Similar enhancement in transgene expression was also observed in Indian carp (Labeo rohita. To establish the utility of adult zebrafish as a system for transient transfections, the strength of the promoters was compared in A2 cells and adult zebrafish after electroporation. The relative strengths of the promoters were found to be similar in cell lines and in adult zebrafish. GFP fluorescence in tissues after electroporation was also studied by fluorescence microscopy. Conclusion Electroporation after DNA injection enhances gene expression 10-fold in adult zebrafish. Electroporation parameters for optimum transfection of adult zebrafish with tweezer type electrode were presented. Enhanced reporter gene expression upon electroporation allowed comparison of strengths of the promoters in vivo in zebrafish.

  7. RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem.

    Science.gov (United States)

    Arias, Renée S; Dang, Phat M; Sobolev, Victor S

    2015-12-21

    The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p ≤ 0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng · g(-1) of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng · g(-1). This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other

  8. Regulation of transgene expression in genetic immunization

    Directory of Open Access Journals (Sweden)

    Harms J.S.

    1999-01-01

    Full Text Available The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

  9. MAR elements and transposons for improved transgene integration and expression.

    Directory of Open Access Journals (Sweden)

    Déborah Ley

    Full Text Available Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

  10. MAR elements and transposons for improved transgene integration and expression.

    Science.gov (United States)

    Ley, Déborah; Harraghy, Niamh; Le Fourn, Valérie; Bire, Solenne; Girod, Pierre-Alain; Regamey, Alexandre; Rouleux-Bonnin, Florence; Bigot, Yves; Mermod, Nicolas

    2013-01-01

    Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

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

    Science.gov (United States)

    Yan, Quanmei; Yang, Huaqiang; Yang, Dongshan; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Fan, Nana; Ouyang, Hongsheng; Gu, Weiwang; Lai, Liangxue

    2014-01-01

    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.

  12. Expression of the Nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize.

    Science.gov (United States)

    Shou, Huixia; Bordallo, Patricia; Wang, Kan

    2004-05-01

    Drought is one of the most important abiotic stresses affecting the productivity of maize. Previous studies have shown that expression of a mitogen-activated protein kinase kinase kinase (MAPKKK) gene activated an oxidative signal cascade and led to the tolerance of freezing, heat, and salinity stress in transgenic tobacco. To analyse the role of activation of oxidative stress signalling in improving drought tolerance in major crops, a tobacco MAPKKK (NPK1) was expressed constitutively in maize. Results show that NPK1 expression enhanced drought tolerance in transgenic maize. Under drought conditions, transgenic maize plants maintained significantly higher photosynthesis rates than did the non-transgenic control, suggesting that NPK1 induced a mechanism that protected photosynthesis machinery from dehydration damage. In addition, drought-stressed transgenic plants produced kernels with weights similar to those under well-watered conditions, while kernel weights of drought-stressed non-transgenic control plants were significantly reduced when compared with their non-stressed counterparts.

  13. Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation.

    Science.gov (United States)

    Khan, Shaukat; Toyoda, Hidemi; Linehan, Melissa; Iwasaki, Akiko; Nomoto, Akio; Bernhardt, Günter; Cello, Jeronimo; Wimmer, Eckard

    2014-08-01

    An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer's patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer's patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer's patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness.

  14. Heritable retroviral transgenes are highly expressed in chickens.

    OpenAIRE

    Briskin, M J; Hsu, R Y; Boggs, T; Schultz, J. A.; Rishell, W; Bosselman, R A

    1991-01-01

    This report describes expression of heritable reticuloendotheliosis virus (REV) vector ME111 in 20 independent lines of transgenic chickens. The results are strikingly different from studies of Moloney virus in transgenic mice, where restricted expression of inherited proviruses has led to their use primarily as insertional mutagens rather than general agents for gene transfer. In contrast, the REV ME111 provirus is actively transcribed in a variety of tissues from transgenic chickens, is exp...

  15. Expression of transgenes targeted to the Gt(ROSA26Sor locus is orientation dependent.

    Directory of Open Access Journals (Sweden)

    Douglas Strathdee

    Full Text Available BACKGROUND: Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene. METHODOLOGY: In order to develop a reliable system for reproducibly expressing transgenes it was decided to insert constructs at the Gt(ROSA26Sor locus. A cytomegalovirus (CMV promoter was used to drive expression of the Tetracycline (tet transcriptional activator, rtTA2(s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele. CONCLUSIONS: Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.

  16. Transgenic Expression of the Recombinant Phytase in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    LIU Qiao-quan; LI Qian-feng; JIANG Li; ZHANG Da-jiang; WANG Hong-mei; GU Ming-hong; YAO Quan-hong

    2006-01-01

    In most of the cereal crop, phytic acid is the main storage form of phosphorus, which can decrease the bioavailability of phosphate. Transgenic expression of phytase is regarded as an efficient way to release phosphate from phytate in transgenic plants.In this study, a plant expression vector, containing the recombinant phytase gene driven by the maize ubiquitin (Ubi) promoter was constructed and introduced into an elite rice variety via Agrobacterium-mediated transformation. During the experiment, a total of 15 independent transgenic rice lines were regenerated. The results of PCR and Southern blot indicated that the target gene was integrated into the genome of transgenic rice plants. Moreover, the RT-PCR analysis of total RNAs extracted from the immature seeds of several transgenic lines showed that the recombinant phytase gene could be normally expressed. The inorganic phosphorus content, both in the mature seeds and the leaf was significantly higher in the transgenic plants than in the untransformed wild type.

  17. Temporal and spatial patterning of transgene expression by near-infrared irradiation

    Science.gov (United States)

    Gomez, Leyre; Lopez, Daniel; Arruebo, Manuel; Wilson, Christopher G; Franceschi, Renny T.; Voellmy, Richard; Santamaria, Jesus; Vilaboa, Nuria

    2014-01-01

    We investigated whether near-infrared (NIR) light could be employed for patterning transgene expression in plasmonic cell constructs. Hollow gold nanoparticles with a plasmon surface band absorption peaking at ~750 nm, a wavelength within the so called “tissue optical window”, were used as fillers in fibrin-based hydrogels. These composites, which efficiently transduce NIR photon energy into heat, were loaded with genetically-modified cells that harbor a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation in the presence of ligand triggered 3-dimensional patterns of transgene expression faithfully matching the illuminated areas of plasmonic cell constructs. This noninvasive technology was proven useful for remotely controlling in vivo the spatiotemporal bioavailability of transgenic vascular endothelial growth factor. The combination of spatial control by means of NIR irradiation along with safe and timed transgene induction presents a high application potential for engineering tissues in regenerative medicine scenarios. PMID:24957294

  18. Position-independent expression of transgenes in zebrafish.

    Science.gov (United States)

    Caldovic, L; Agalliu, D; Hackett, P B

    1999-10-01

    The variability in expression patterns of transgenes, caused by the influence of neighboring chromatin, is called 'position effect'. Border elements are DNA sequences, which have the ability to alleviate position effects. The abilities of two types of border elements, scs/scs' from the D. melanogaster 87A7 heat shock locus and the A-element from the chicken lysozyme gene, to protect transgenes from position effects were quantified in developing zebrafish embryos. The transgenic construct used was FV3CAT, which consists of the carp beta-actin transcriptional regulatory region, the chloramphenicol acetyltransferase (CAT) gene and the 3'-untranslated region from the Chinook salmon growth hormone gene. FV3CAT constructs flanked by either scs/scs'-elements or A-elements were introduced into zebrafish chromosomes and the spatial and temporal expression patterns of the transgenes were quantified in multiple generations of transgenic zebrafish. Levels of transgene expression were uniform in the pre-differentiated and fully differentiated populations of cells present during embryonic development. Levels of transgene expression were proportional to the numbers of integrated transgenes. Expression of transgenes per cell varied less than two-fold in different transgenic lines. Both types of border elements were able to prevent the influences of neighboring chromatin on transgene expression through three generations of fish. The results are consistent with the ability of border elements to function with equal efficiencies in the many cell types found in vertebrates. Thus, inclusion of border elements in genetic constructs can provide reliable and reproducible levels of gene expression in multiple lines of fish.

  19. Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

    DEFF Research Database (Denmark)

    Huber, Reinhard C.; Remuge, Liliana; Carlisle, Ailsa

    2012-01-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology—animal welfare—has not been approached through systematic assessment...... and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals...... months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs...

  20. Transgenic Anopheles gambiae expressing an antimalarial peptide suffer no significant fitness cost.

    Science.gov (United States)

    McArthur, Clare C; Meredith, Janet M; Eggleston, Paul

    2014-01-01

    Mosquito-borne diseases present some of the greatest health challenges faced by the world today. In many cases, existing control measures are compromised by insecticide resistance, pathogen tolerance to drugs and the lack of effective vaccines. In light of these difficulties, new genetic tools for disease control programmes, based on the deployment of genetically modified mosquitoes, are seen as having great promise. Transgenic strains may be used to control disease transmission either by suppressing vector populations or by replacing susceptible with refractory genotypes. In practice, the fitness of the transgenic strain relative to natural mosquitoes will be a critical determinant of success. We previously described a transgenic strain of Anopheles gambiae expressing the Vida3 peptide into the female midgut following a blood-meal, which exhibited significant protection against malaria parasites. Here, we investigated the fitness of this strain relative to non-transgenic controls through comparisons of various life history traits. Experiments were designed, as far as possible, to equalize genetic backgrounds and heterogeneity such that fitness comparisons focussed on the presence and expression of the transgene cassette. We also employed reciprocal crosses to identify any fitness disturbance associated with inheritance of the transgene from either the male or female parent. We found no evidence that the presence or expression of the effector transgene or associated fluorescence markers caused any significant fitness cost in relation to larval mortality, pupal sex ratio, fecundity, hatch rate or longevity of blood-fed females. In fact, fecundity was increased in transgenic strains. We did, however, observe some fitness disturbances associated with the route of inheritance of the transgene. Maternal inheritance delayed male pupation whilst paternal inheritance increased adult longevity for both males and unfed females. Overall, in comparison to controls, there was

  1. Transgenic Anopheles gambiae expressing an antimalarial peptide suffer no significant fitness cost.

    Directory of Open Access Journals (Sweden)

    Clare C McArthur

    Full Text Available Mosquito-borne diseases present some of the greatest health challenges faced by the world today. In many cases, existing control measures are compromised by insecticide resistance, pathogen tolerance to drugs and the lack of effective vaccines. In light of these difficulties, new genetic tools for disease control programmes, based on the deployment of genetically modified mosquitoes, are seen as having great promise. Transgenic strains may be used to control disease transmission either by suppressing vector populations or by replacing susceptible with refractory genotypes. In practice, the fitness of the transgenic strain relative to natural mosquitoes will be a critical determinant of success. We previously described a transgenic strain of Anopheles gambiae expressing the Vida3 peptide into the female midgut following a blood-meal, which exhibited significant protection against malaria parasites. Here, we investigated the fitness of this strain relative to non-transgenic controls through comparisons of various life history traits. Experiments were designed, as far as possible, to equalize genetic backgrounds and heterogeneity such that fitness comparisons focussed on the presence and expression of the transgene cassette. We also employed reciprocal crosses to identify any fitness disturbance associated with inheritance of the transgene from either the male or female parent. We found no evidence that the presence or expression of the effector transgene or associated fluorescence markers caused any significant fitness cost in relation to larval mortality, pupal sex ratio, fecundity, hatch rate or longevity of blood-fed females. In fact, fecundity was increased in transgenic strains. We did, however, observe some fitness disturbances associated with the route of inheritance of the transgene. Maternal inheritance delayed male pupation whilst paternal inheritance increased adult longevity for both males and unfed females. Overall, in comparison to

  2. Copper-Controllable, Site-Specific DNA Excision in Transgenic Plants

    Institute of Scientific and Technical Information of China (English)

    PENG Xiang-lei; LIANG Bin; CHEN Ming; HU Yuan-lei; LIN Zhong-ping

    2003-01-01

    A copper-inducible, Cre-loxP recombination-mediated DNA excision system has been developed in transgenic tobacco plants. The copper inducible system derived from yeast was used for the control of the expression of the Cre recombinase. Upon copper induction, the GUS reporter gene expression unit flanked by two direct lox sites was excised from the transgenic tobacco genome. Quantitative fluorometric GUS assays,Northern blot and PCR analyses showed a high-efficient, copper-dependent and Cre-loxP mediated DNA recombination in all the tested transgenic lines. The copper inducible foreign gene excision might be of great potential in genetic control of transgenic crops.

  3. Expression systems and species used for transgenic animal bioreactors.

    Science.gov (United States)

    Wang, Yanli; Zhao, Sihai; Bai, Liang; Fan, Jianglin; Liu, Enqi

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

  4. Expression Systems and Species Used for Transgenic Animal Bioreactors

    Directory of Open Access Journals (Sweden)

    Yanli Wang

    2013-01-01

    Full Text Available Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon, the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

  5. Transgenic rice plants expressing cry1Ia5 gene are resistant to stem borer (Chilo agamemnon).

    Science.gov (United States)

    Moghaieb, Reda E A

    2010-01-01

    The stem borer, Chilo agamemnon Bles., is the most serious insect pest in rice fields of the Egyptian Nile Delta. To induce rice plant resistance to Chilo agamemnon, the cry1Ia5 gene was introduced to rice plants (Oryza sativa L.). The integration of the cry1Ia5 gene into the plant genome was confirmed using PCR and Southern blot analyses. The obtained plantlets were transferred to the greenhouse until seeds were collected. Northern blot analysis of the T1 plants confirmed the expression of the cry1Ia5 gene. The insecticidal activity of the transgenic plants against the rice stem borer Chilo agamemnon were tested. The third larval instars were fed on stem cuts from three transgenic lines (L1, L2 and L3) as well as cuts from the control (gfp-transgenic) plants for one week and the mortality percentage was daily recorded. Transgenic line-3 showed the highest mortality percentage after one day (50%) followed by L2 (25%) then L1 (0%). Two days post treatment the mortality percentage increased to 70, 45 and 25% for transgenic lines 1, 2 and 3 respectively. Mortality of 100% was recorded four days post treatment, while those fed on the gfp-transgenic rice (control) showed 0% mortality. Thus, transgenic plants showed high resistance to stem borers and can serve as a novel genetic resource in breeding programs. Transgenic plants expressing BT protein were normal in phenotype with as good seed setting as the nontransgenic control plants.

  6. Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease.

    Science.gov (United States)

    Namukwaya, B; Tripathi, L; Tripathi, J N; Arinaitwe, G; Mukasa, S B; Tushemereirwe, W K

    2012-08-01

    Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.

  7. Transgenic zebrafish recapitulating tbx16 gene early developmental expression.

    Directory of Open Access Journals (Sweden)

    Simon Wells

    Full Text Available We describe the creation of a transgenic zebrafish expressing GFP driven by a 7.5 kb promoter region of the tbx16 gene. This promoter segment is sufficient to recapitulate early embryonic expression of endogenous tbx16 in the presomitic mesoderm, the polster and, subsequently, in the hatching gland. Expression of GFP in the transgenic lines later in development diverges to some extent from endogenous tbx16 expression with the serendipitous result that one line expresses GFP specifically in commissural primary ascending (CoPA interneurons of the developing spinal cord. Using this line we demonstrate that the gene mafba (valentino is expressed in CoPA interneurons.

  8. GH/IGF-I Transgene Expression on Muscle Homeostasis

    Science.gov (United States)

    Schwartz, Robert J.

    1999-01-01

    We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

  9. GH/IGF-I Transgene Expression on Muscle Homeostasis

    Science.gov (United States)

    Schwartz, Robert J.

    1999-01-01

    We propose to test the hypothesis that the growth hormone/ insulin like growth factor-I axis through autocrine/paracrine mechanisms may provide long term muscle homeostasis under conditions of prolonged weightlessness. As a key alternative to hormone replacement therapy, ectopic production of hGH, growth hormone releasing hormone (GHRH), and IGF-I will be studied for its potential on muscle mass impact in transgenic mice under simulated microgravity. Expression of either hGH or IGF-I would provide a chronic source of a growth-promoting protein whose biosynthesis or secretion is shut down in space. Muscle expression of the IGF-I transgene has demonstrated about a 20% increase in hind limb muscle mass over control nontransgenic litter mates. These recent experiments, also establish the utility of hind-limb suspension in mice as a workable model to study atrophy in weight bearing muscles. Thus, transgenic mice will be used in hind-limb suspension models to determine the role of GH/IGF-I on maintenance of muscle mass and whether concentric exercises might act in synergy with hormone treatment. As a means to engineer and ensure long-term protein production that would be workable in humans, gene therapy technology will be used by to monitor muscle mass preservation during hind-limb suspension, after direct intramuscular injection of a genetically engineered muscle-specific vector expressing GHRH. Effects of this gene-based therapy will be assessed in both fast twitch (medial gastrocnemius) and slow twitch muscle (soleus). End-points include muscle size, ultrastructure, fiber type, and contractile function, in normal animals, hind limb suspension, and reambutation.

  10. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  11. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    Science.gov (United States)

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  12. Tetracycline-inducible Expression Systems: New Strategies and Practices in the Transgenic Mouse Modeling

    Institute of Scientific and Technical Information of China (English)

    Yan SUN; Xigu CHEN; Dong XIAO

    2007-01-01

    To accurately analyze the function of transgene(s) of interest in transgenic mice, and to generate credible transgenic animal models for multifarious human diseases to precisely mimic human disease states, it is critical to tightly regulate gene expression in the animals in a conditional manner. The ability to turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedented flexibility in dissecting gene functions in health and disease. Pioneering studies in conditional transgene expression have brought about the development of a wide variety of controlled gene expression systems, which meet this criterion. Among them, the tetracycline-controlled expression systems (e.g. Tet-off system and Tet-on system) have been used extensively in vitro and in vivo. In recent years, some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases. In this review these newly developed strategies are discussed.

  13. Knockdown of myostatin expression by RNAi enhances muscle growth in transgenic sheep.

    Directory of Open Access Journals (Sweden)

    Shengwei Hu

    Full Text Available Myostatin (MSTN has been shown to be a negative regulator of skeletal muscle development and growth. MSTN dysfunction therefore offers a strategy for promoting animal growth performance in livestock production. In this study, we investigated the possibility of using RNAi-based technology to generate transgenic sheep with a double-muscle phenotype. A shRNA expression cassette targeting sheep MSTN was used to generate stable shRNA-expressing fibroblast clones. Transgenic sheep were further produced by somatic cell nuclear transfer (SCNT technology. Five lambs developed to term and three live lambs were obtained. Integration of shRNA expression cassette in three live lambs was confirmed by PCR. RNase protection assay showed that the shRNAs targeting MSTN were expressed in muscle tissues of three transgenic sheep. MSTN expression was significantly inhibited in muscle tissues of transgenic sheep when compared with control sheep. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep. Histological analysis showed that myofiber diameter of transgenic sheep M17 were bigger than that of control sheep. Our findings demonstrate a promising approach to promoting muscle growth in livestock production.

  14. Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Hilary Collver; Katherine Kinken

    2004-01-01

    Genomic research has made a large number of sequences of novel genes or expressed sequence tags available. To investigate functions of these genes, a system for conditional control of gene expression would be a useful tool. Inducible transgene expression that uses green fluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines of cotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir (NOR; Abies nordmanniana Lk.), and rice (RIC; Oryza sativa L. Cv. Radon). Transgenic cell lines were used to test the function of the chemical inducer dexamethasone. Inducible transgene expression was observed with fluorescence and confocal microscopy, and was confirmed by northern blot analyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h after treatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgenic cells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducer for optimum inducible gene expression system varied among transgenic cell lines. The inducible gene expression system described here was very effective and could be valuable in evaluating the function of novel gene.

  15. Mushroom body miscellanea: transgenic Drosophila strains expressing anatomical and physiological sensor proteins in Kenyon cells.

    Science.gov (United States)

    Pech, Ulrike; Dipt, Shubham; Barth, Jonas; Singh, Priyanka; Jauch, Mandy; Thum, Andreas S; Fiala, André; Riemensperger, Thomas

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function.

  16. CHANGES IN ENDOGENOUS GENE TRANSCRIPT AND PROTEIN LEVELS IN MAIZE PLANTS EXPRESSING THE SOYBEAN FERRITIN TRANSGENE

    Directory of Open Access Journals (Sweden)

    Milly N Kanobe

    2013-06-01

    Full Text Available Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, we tested effects of soybean ferritin transgene (SoyFer1, M64337 on transcript and protein levels of endogenous genes in maize. Results showed that the transgene was successfully introduced and expressed in the maize seed endosperm. mRNA abundance of seven tested iron homeostasis genes and seed storage protein genes differed significantly between seed samples positive and negative for the transgene. The PCR negative samples had higher zein and total protein content compared to the positive samples. However, PCR positive samples had significantly higher concentrations of calcium, magnesium and iron. We have shown that the soybean ferritin transgene affected the expression of native iron homeostasis genes in the maize plant. These results underscore the importance of taking a holistic approach to the evaluation of transgenic events in target plants, comparing the transgenic plant to the untransformed controls.

  17. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  18. Calcium electrotransfer for termination of transgene expression in muscle

    DEFF Research Database (Denmark)

    Hojman, Pernille; Spanggaard, Iben; Olsen, Caroline Holkman;

    2011-01-01

    in vivo imaging of infrared fluorescent "Katushka" and erythropoietin evaluated by ELISA and hemoglobin. Histology was performed. Electrotransfer of Katushka and erythropoietin yielded significant expression. Maximal calcium uptake occurred after injection of Ca(2+) before electropulsing using eight high......Gene electrotransfer is expanding in clinical use, thus we have searched for an emergency procedure to stop transgene expression in case of serious adverse events. Calcium is cytotoxic at high intracellular levels, so we tested effects of calcium electrotransfer on transgene expression in muscle....... A clinical grade calcium solution (20 µl, 168 mM) was injected into transfected mouse or rat tibialis cranialis muscle. Ca(2+) uptake was quantified using calcium 45 ((45)Ca), and voltage and time between injection and pulsation were varied. Extinction of transgene expression was investigated by using both...

  19. Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions.

    Science.gov (United States)

    Breitler, Jean Christophe; Vassal, Jean Michel; del Mar Catala, Maria; Meynard, Donaldo; Marfà, Victoria; Melé, Enric; Royer, Monique; Murillo, Isabel; San Segundo, Blanca; Guiderdoni, Emmanuel; Messeguer, Joaquima

    2004-09-01

    Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks

  20. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    Science.gov (United States)

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  1. High-level expressing YAC vector for transgenic animal bioreactors.

    Science.gov (United States)

    Fujiwara, Y; Miwa, M; Takahashi, R; Kodaira, K; Hirabayashi, M; Suzuki, T; Ueda, M

    1999-04-01

    The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

  2. The expression of tga1a gene from tobacco affects the expression of exogenous gene in transgenic plant

    Institute of Scientific and Technical Information of China (English)

    路子显; 常团结; 李旭刚; 徐军望; 李慧芬; 陈宛新; 冯德江; 肖桂芳; 朱祯

    2003-01-01

    The DNA-binding protein TGA1a of tobacco can specially interact with the enhancer sequence as-1 (-83 to -63) of CaMV35S promoter and show the function of transcriptional activation. In order to study the expression of exogenous gene affected by TGA1a, a trans-actingregulation system was formed by tandem connecting tga1a under the control of the phloem-specific promoter rolC with reporter gene under the control of CaMV35S. Then, the system abovewas utilized to construct a plant expression vector. Moreover, two plant expression vectors wereconstructed with the report gene controlled by CaMV35S and rolC promoter respectively as positive controls. Tobacco leaf disc transformed by Agrobacterium-mediated method and transgenic plants were regenerated. It was proved that the reporter gene existed in the genome of transgenic plants by Southern hybridization. The results of GUS activity indicated that the expression of tga1a controlled by rolC remarkably increased the expression of the reporter gene controlled by CaMV35S. GUS activity of transgenic plants containing trans-acting regulation system was higher than that of transgenic plants containing the reporter gene under the control of CaMV35S and rolC respectively, with the highest GUS activity of about tenfolds of two positive controls. Histochemical method demonstrated that GUS staining amassed mainly in phloem tissue of transgenic plantscontaining the trans-acting regulation system. A new model for arising the expression level and tissue-specific expression of exogenous gene in transgenic plant was established in this study.

  3. Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves.

    Science.gov (United States)

    Muñoz-Bertomeu, Jesús; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2008-01-01

    We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

  4. A transgenic rat with ubiquitous expression of firefly luciferase gene

    Science.gov (United States)

    Hakamata, Yoji; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    In vivo imaging strategies provide cellular and molecular events in real time that helps us to understand biological processes in living animals. The development of molecular tags such as green fluorescent proteins and luciferase from the firefly Photinus pyralis has lead to a revolution in the visualization of complex biochemical processes. We developed a novel inbred transgenic rat strain containing firefly luciferase based on the transgenic (Tg) technique in rats. This Tg rat expressed the luciferase gene ubiquitously under control of the ROSA26 promoter. Cellular immune responsiveness against the luciferase protein was evaluated using conventional skin grafting and resulted in the long-term acceptance of Tg rat skin on wild-type rats. Strikingly, organ transplant with heart and small bowel demonstrated organ viability and graft survival, suggesting that cells from luciferase-Tg are transplantable to track their fate. Taking advantage of the less immunogenic luciferase, we also tested the role of hepatocyte-infusion in a liver injury model, and bone marrow-derived cells in a skin defect model. Employed in conjunction with modern advances in optical imaging, this luciferase-Tg rat system provides an innovative animal tool and a new means of facilitating biomedical research such as in the case of regeneration medicine.

  5. Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

    Directory of Open Access Journals (Sweden)

    Yun Seong-Jo

    2012-01-01

    Full Text Available Abstract Background Dimeric human erythropoietin (dHuEPO peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg mice expressing dHuEPO and to investigate the characteristics of these mice. Methods A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile, was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. Results A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47 of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11. We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764. Reverse transcriptase-polymerase chain reaction (RT-PCR and quantitative real-time PCR (qRT-PCR were used for validating the results of the microarray

  6. Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes.

    Science.gov (United States)

    Totten, D C; Vuong, M; Litvinova, O V; Jinwal, U K; Gulia-Nuss, M; Harrell, R A; Beneš, H

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

  7. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    Science.gov (United States)

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  8. Transgenic labeling of parvalbumin-expressing neurons with tdTomato.

    Science.gov (United States)

    Kaiser, T; Ting, J T; Monteiro, P; Feng, G

    2016-05-03

    Parvalbumin (PVALB)-expressing fast-spiking interneurons subserve important roles in many brain regions by modulating circuit function and dysfunction of these neurons is strongly implicated in neuropsychiatric disorders including schizophrenia and autism. To facilitate the study of PVALB neuron function we need to be able to identify PVALB neurons in vivo. We have generated a bacterial artificial chromosome (BAC) transgenic mouse line expressing the red fluorophore tdTomato under the control of endogenous regulatory elements of the Pvalb gene locus (JAX # 027395). We show that the tdTomato transgene is faithfully expressed relative to endogenous PVALB expression throughout the brain. Furthermore, targeted patch clamp recordings confirm that the labeled populations in neocortex, striatum, and hippocampus are fast-spiking interneurons based on intrinsic properties. This new transgenic mouse line provides a useful tool to study PVALB neuron function in the normal brain as well as in mouse models of psychiatric disease.

  9. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    Science.gov (United States)

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  10. [Generation of transgenic mice expressing human lysozyme in mammary gland].

    Science.gov (United States)

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang

    2005-10-01

    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

  11. Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice.

    Science.gov (United States)

    Lafond, R E; Giammalvo, J T; Norkin, L C

    1995-09-01

    The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.

  12. Calcium electrotransfer for termination of transgene expression in muscle

    DEFF Research Database (Denmark)

    Hojman, Pernille; Spanggaard, Iben; Olsen, Caroline Holkman

    2011-01-01

    . A clinical grade calcium solution (20 μl, 168 mM) was injected into transfected mouse or rat tibialis cranialis muscle. Ca(2+) uptake was quantified using calcium 45 ((45)Ca), and voltage and time between injection and pulsation were varied. Extinction of transgene expression was investigated by using both...

  13. Gamma-irradiation enhances transgene expression in leukemic cells.

    Science.gov (United States)

    Vereecque, R; Saudemont, A; Wickham, T J; Gonzalez, R; Hetuin, D; Fenaux, P; Quesnel, B

    2003-02-01

    The majority of immunotherapy-based gene therapy protocols consist of ex vivo gene transfer in tumor cells. To prevent further in vivo growth, modified cells must be irradiated before reinjection into patients. The present study examines the effects of gamma-irradiation on transgene expression in transduced leukemic cells. Human and murine leukemic cells were transfected with retroviral vectors or plasmids carrying beta-galactosidase, GM-CSF or CD80 genes. Fresh leukemic cells from patients with acute myeloid leukemia (AML) were transfected with AdZ.F(pK7) adenoviral vector. gamma-irradiation at various lethal doses enhanced transgene expression in leukemic cell lines and fresh AML cells when the gene of interest was under CMV promoter but not when SV40 promoter was used. Oxidative stress also enhanced transgene expression and both irradiation and oxidative stress effects were inhibited by addition of N-acetyl-L-cysteine, a thiol anti-oxidant, indicating the involvement of reactive oxygen species. Transgene expression was also enhanced in vivo 48 and 120 h after subcutaneous injection of irradiated leukemic cells in syngeneic mice. These results show that a cell vaccine protocol using ex vivo gene transfer of transduced cells might be feasible in acute leukemia even if leukemic cells must be irradiated at lethal doses prior to reinjection to patients.

  14. Transgene expression in the basidiomycete root pathogen Armillaria mellea.

    Science.gov (United States)

    Toward development of a genetic transformation system for Armillaria mellea, we used particle bombardment to identify promoters for driving transgene expression. The plasmid tested was pYES-hph-004iGFP, on which the green fluorescence protein gene, gfp, is linked to the Agaricus bisporus gpdII promo...

  15. [Generation of sugar beet transgenic plants expressing bar gene].

    Science.gov (United States)

    Mishutkina, Ia V; Kamionskaia, A M; Skriabin, K G

    2010-01-01

    The parameters of transformation using Agrobacterium tumefaciens EHA 105 for 5 domestic sorts and lines of sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass) were optimized. The system of transgenic tissue selection based on resistance to phosphinothricin, allowing to avoid the appearing of chimeric shoots among initial transformants was developed. The transgenic plants of sugar beet sorts Ramonskaya single seed 47, L'govskaya single seed 52 and RMS 73, and LBO 17 and LBO 19 lines expressing the gene of phosphinothricin acetyl transferase bar have been obtained. The resistance of these sorts and lines to the effect of phosphinothricin in vitro has been shown.

  16. Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

    Energy Technology Data Exchange (ETDEWEB)

    Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P. [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Eapen, Susan, E-mail: eapenhome@yahoo.com [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085 (India)

    2011-08-15

    Highlights: {yields} Transgenic plants expressing a TvGST gene were tested for tolerance, uptake and degradation of anthracene. {yields} Transgenic plants were more tolerant to anthracene and take up more anthracene from soil and solutions compared to control plants. {yields} Using in vitro T{sub 1} seedlings, we showed that anthracene-a three fused benzene ring compound was phytodegraded to naphthalene derivatives, having two benzene rings. {yields} This is the first time that a transgenic plant was shown to have the potential to phytodegrade anthracene. - Abstract: Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of {sup 14}C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T{sub 0} and T{sub 1}) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

  17. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

    Science.gov (United States)

    Allen, Aron; Islamovic, Emir; Kaur, Jagdeep; Gold, Scott; Shah, Dilip; Smith, Thomas J

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

  18. Over-expression of an Arabidopsis δ-OAT gene enhances salt and drought tolerance in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    WU Liangqi; FAN Zhanmin; GUO Lei; LI Yongqing; ZHANG Wenjing; QU Li-Jia; CHEN Zhangliang

    2003-01-01

    δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsisδ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCl concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenic rice. Furthermore, the over-expression ofδ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%-41% more than that of control lines under 0.1 mol/L NaCl stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.

  19. Enhancive effects of D-glucose and its analogs on expression of d-glucose-unrelated transgenes in mammalian cells.

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    Kimura, Miyuki; Namba, Hikaru; Okubo, Manabu; Ezumi, Mai; Susumu, Nao; Yamada, Masao; Arao, Yujiro

    2011-08-01

    We studied the effects of d-glucose on transgene expression in mammalian cells by a reporter gene assay using CV-1 cells and a CMV promoter-controlled EGFP gene. Treatment of CV-1 cells with 5% D-glucose unchanged the number of fluorescent cells in fluorescence microscopic observation but significantly intensified fluorescence in the fluorometric assay. Furthermore, EGFP itself and mRNA became more abundant in Western blot and quantitative RT-PCR analyses of 5% D-glucose-treated cells, respectively. These results indicate that elevated D-glucose can activate transgene expression via transcriptional stimulation, at least in part. The same concentrations of L-glucose led to only negligible increases in transgene expression, indicating that D-glucose's effect is different from its osmotic effect. The D-glucose-induced augmentation of fluorescence was observed not only in the experiment using the CMV promoter-controlled EGFP gene but also in experiments using the SV40 and RSV promoter-controlled ones, suggesting that elevated D-glucose can enhance transgene expression regulated by various promoters commonly used in transgene expression. The assessment of D-glucose analogs for their enhancive effects on transgene expression revealed that 1,6-anhydro-D-glucose and β-methyl-D-glucoside had stronger effects than D-glucose. From this result, we can expect to find more effective carbohydrates to enhance transgene expression. The α- and β-M-D-glucosides, which are slightly different from each other in three-dimensional structure, exerted largely distinct stimulative effects on transgene expression, suggesting that fundamental rules determine the enhancive effects of saccharides and that the modification of the saccharide by applying such rules will enable us to develop more powerful substances for transgene expression. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

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    David A. Dunn

    2012-01-01

    Full Text Available Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M or wildtype (A6W mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P0.05. This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

  1. The temporal expression pattern of alpha-synuclein modulates olfactory neurogenesis in transgenic mice.

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    Sebastian R Schreglmann

    Full Text Available Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS under the murine Thy1 (mThy1 promoter, a model known to have a particularly high tg expression associated with impaired olfaction.Survival of newly generated neurons (NeuN-positive in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.

  2. Characterization of three loci for homologous gene targeting and transgene expression.

    Science.gov (United States)

    Eyquem, Justin; Poirot, Laurent; Galetto, Roman; Scharenberg, Andrew M; Smith, Julianne

    2013-08-01

    Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare-cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site-specific transgene integration, suitable for a variety of biotechnological applications.

  3. Enhanced Myogenesis in adult skeletal muscle by transgenic expression of Myostatin Propeptide

    Science.gov (United States)

    Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-ß, plays a dominant role in the genetic control of muscle mass. Transgenic expression of myostatin propeptide in skeletal muscle showed ...

  4. Expression of Cry1Ab protein in a marker-free transgenic Bt rice line and its efficacy in controlling a target pest, Chilo suppressalis (Lepidoptera: Crambidae).

    Science.gov (United States)

    Wang, Yanan; Zhang, Lei; Li, Yunhe; Liu, Yanmin; Han, Lanzhi; Zhu, Zhen; Wang, Feng; Peng, Yufa

    2014-04-01

    A marker-free Bt transgenic rice line, mfb-MH86, was recently developed in China, which contains a cry1Ab gene driven by a ubiquitin promoter. This Bt gene confers resistance to a range of lepidopteran species, including the striped stem borer, Chilo suppressalis (Walker). The expression of Cry1Ab protein in mfb-MH86 leaves, stems and leaf sheaths (hereinafter referred to as stems), and roots was evaluated throughout the rice-growing season using an enzyme-linked immunosorbent assay. In addition, mfb-MH86 resistance to C. suppressalis, a major pest of rice, was evaluated in a laboratory bioassay with field-collected rice stems. Cry1Ab protein levels of mfb-MH86 were highest in leaves (9.71-34.09 μg/g dry weight [DW]), intermediate in stems (7.66-18.51 μg/g DW), and lowest in roots (1.95-13.40 μg/g DW). In all tissues, Cry1Ab levels in mfb-MH86 were higher in seedling and tillering stages than in subsequent growth stages. In the laboratory bioassay, mortality of C. suppressalis after 6 d of feeding on mfb-MH86 stems was 100% throughout the rice-growing season; mortality of C. suppressalis when feeding on stems of the nontransformed isoline, MH86, ranged from 15.0 to 38.3%. The results indicate that Cry1Ab protein levels in mfb-MH86 stems are sufficient to protect plants against C. suppressalis throughout the rice-growing season. Although our results are promising, further comprehensive evaluations of mfb-MH86, including field surveys, will be needed before commercial use.

  5. Transgenic expression of the human growth hormone minigene promotes pancreatic β-cell proliferation.

    Science.gov (United States)

    Baan, Mieke; Kibbe, Carly R; Bushkofsky, Justin R; Harris, Ted W; Sherman, Dawn S; Davis, Dawn Belt

    2015-10-01

    Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls.

  6. Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

    Science.gov (United States)

    Yan, Jingbin; Gong, Xiuli; Pan, Shubiao; Guo, Xinbing; Ren, Zhaorui; Zeng, Yitao

    2014-06-01

    Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 μg/ml in milk.

  7. Immunoglobulin gene expression and regulation of rearrangement in kappa transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Ritchie, K.A.

    1986-01-01

    Transgenic mice were produced by microinjection of the functionally rearranged immunoglobulin kappa gene from the myeloma MOPC-21 into the male pronucleus of fertilized mouse eggs, and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were detected by hybridizing tail DNA dots with radioactively labelled pBR322 plasmid DNA, which detects pBR322 sequences left as a tag on the microinjected DNA. Mice that integrated the injected gene (six males) were mated and the DNA, RNA and serum kappa chains of their offspring were analyzed. A rabbit anti-mouse kappa chain antiserum was also produced for use in detection of mouse kappa chains on protein blots. Hybridomas were produced from the spleen cells of these kappa transgenic mice to immortalize representative B cells and to investigate expression of the transgenic kappa gene, its effect on allelic exclusion, and its effect on the control of light chain gene rearrangement and expression. The results show that the microinjected DNA is integrated as concatamers in unique single or, rarely, two separate sites in the genome. The concatamers are composed of several copies (16 to 64) of injected DNA arranged in a head to tail fashion. The transgene is expressed into protein normally and in a tissue specific fashion. For the first time in these transgenic mice, all tissues contain a functionally rearranged and potentially expressible immunoglobulin gene. The transgene is expressed only in B cells and not in hepatocytes, for example. This indicates that rearrangement of immunoglobulin genes is necessary but not sufficient for the tissue specific expression of these genes by B cells.

  8. Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum

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    Han Jigang

    2012-03-01

    Full Text Available Abstract Background The development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L. and barley (Hordeum vulgare L. that reduces both grain yield and quality. Results A construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance. Conclusions Introgression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.

  9. Expression of E. coli heat-labile enterotoxin B subunit in transgenic tobacco plants

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-li; ZHANG Zheng; LI Wen-sheng; ZHENG Jing; KONG Ling-hong; WANG Yi-li; SI Lü-sheng

    2005-01-01

    Objective: To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit (LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L. Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Results: LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3.36-10.56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBILTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the prevention of mucosa-route evading pathogen.

  10. Herbicide resistance of transgenic rice plants expressing human CYP1A1.

    Science.gov (United States)

    Kawahigashi, Hiroyuki; Hirose, Sakiko; Ohkawa, Hideo; Ohkawa, Yasunobu

    2007-01-01

    Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.

  11. Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi.

    Science.gov (United States)

    Carballar-Lejarazú, Rebeca; Jasinskiene, Nijole; James, Anthony A

    2013-04-30

    Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.

  12. Expression Analysis of CB2-GFP BAC Transgenic Mice.

    Science.gov (United States)

    Schmöle, Anne-Caroline; Lundt, Ramona; Gennequin, Benjamin; Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian

    2015-01-01

    The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  13. Expression Analysis of CB2-GFP BAC Transgenic Mice.

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    Anne-Caroline Schmöle

    Full Text Available The endocannabinoid system (ECS is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2. As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

  14. Neoplastic transformation of T lymphocytes through transgenic expression of a virus host modification protein.

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    Sílvia Cristina Paiva Almeida

    Full Text Available Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4(+CD8(+CD69(- lymphoma. The CD4(+CD8(+CD69(- cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4(+CD8(+CD69(- thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4(+CD8(+ CD69(- thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas.

  15. Increased Ubqln2 expression causes neuron death in transgenic rats.

    Science.gov (United States)

    Huang, Bo; Wu, Qinxue; Zhou, Hongxia; Huang, Cao; Xia, Xu-Gang

    2016-10-01

    Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis

  16. Transgenic rabbit that expresses a functional human lipoprotein (a)

    Science.gov (United States)

    Rouy, Didier; Duverger, Nicolas; Emmanuel, Florence; Denefle, Patrice; Houdebine, Louis-Marie; Viglietta, Celine; Rubin, Edward M.; Hughes, Steven D.

    2003-01-01

    A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

  17. Effects of Transgenic Expression of Botulinum Toxins in Drosophila

    OpenAIRE

    Backhaus, Philipp

    2017-01-01

    Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed ...

  18. Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing.

    Science.gov (United States)

    Zou, Xiuping; Jiang, Xueyou; Xu, Lanzhen; Lei, Tiangang; Peng, Aihong; He, Yongrui; Yao, Lixiao; Chen, Shanchun

    2017-03-01

    Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing. Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.

  19. Transgenic mosquitoes expressing a phospholipase A(2 gene have a fitness advantage when fed Plasmodium falciparum-infected blood.

    Directory of Open Access Journals (Sweden)

    Ryan C Smith

    Full Text Available BACKGROUND: Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. RESULTS: We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2 into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. CONCLUSIONS: Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.

  20. Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

    Institute of Scientific and Technical Information of China (English)

    郭岩; 张莉; 肖岗; 曹守云; 谷冬梅; 田文忠; 陈受宜

    1997-01-01

    Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase C BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite, Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived, 13 of which set seeds, and the frequency of seed setting was very low, only 10% . But the controls could not grow under the same condition. The results of BADH ac-tivity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.

  1. Highly phosphorylated functionalized rice starch produced by transgenic rice expressing the potato GWD1 gene

    DEFF Research Database (Denmark)

    Chen, Yaling; Sun, Xiao; Zhou, Xin Mao

    2017-01-01

    . The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P......Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice....... Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges...

  2. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.

    Science.gov (United States)

    Markstein, Michele; Pitsouli, Chrysoula; Villalta, Christians; Celniker, Susan E; Perrimon, Norbert

    2008-04-01

    A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.

  3. Reduced Susceptibility to Xanthomonas citri in Transgenic Citrus Expressing the FLS2 Receptor From Nicotiana benthamiana.

    Science.gov (United States)

    Hao, Guixia; Pitino, Marco; Duan, Yongping; Stover, Ed

    2016-02-01

    Overexpression of plant pattern-recognition receptors by genetic engineering provides a novel approach to enhance plant immunity and broad-spectrum disease resistance. Citrus canker disease associated with Xanthomonas citri is one of the most important diseases damaging citrus production worldwide. In this study, we cloned the FLS2 gene from Nicotiana benthamiana cDNA and inserted it into the binary vector pBinPlus/ARS to transform Hamlin sweet orange and Carrizo citrange. Transgene presence was confirmed by polymerase chain reaction (PCR) and gene expression of NbFLS2 was compared by reverse transcription quantitative PCR. Reactive oxygen species (ROS) production in response to flg22Xcc was detected in transgenic Hamlin but not in nontransformed controls. Low or no ROS production was detected from nontransformed Hamlin seedlings challenged with flg22Xcc. Transgenic plants highly expressing NbFLS2 were selected and were evaluated for resistance to canker incited by X. citri 3213. Our results showed that the integration and expression of the NbFLS2 gene in citrus can increase canker resistance and defense-associated gene expression when challenged with X. citri. These results suggest that canker-susceptible Citrus genotypes lack strong basal defense induced by X. citri flagellin and the resistance of these genotypes can be enhanced by transgenic expression of the flagellin receptor from a resistant species.

  4. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Science.gov (United States)

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P

    1998-03-01

    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  5. Optical modulation of transgene expression in retinal pigment epithelium

    Science.gov (United States)

    Palanker, D.; Lavinsky, D.; Chalberg, T.; Mandel, Y.; Huie, P.; Dalal, R.; Marmor, M.

    2013-03-01

    Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a "biofactory" that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.

  6. Collagenlα1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Osteoblasts participate in bone formation,bone mineralization,osteoclast differentiation and many pathological processes.To study the function of genes in osteoblasts using Cre-LoxP system,we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlal (Coilal) promoter(Coilatl-Cre).Two founders were identified by genomic PCR from 16 offsprings.and the integration efficiency is 12.5%.In order tO determine the tissue distribution and the activity of Cre rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/+mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5.Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice.All these data indicated that the Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

  7. Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

    Institute of Scientific and Technical Information of China (English)

    CUI Xiao-feng; LI Yun-qin; HU Dong-wei; ZHOU Xue-ping

    2005-01-01

    An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.

  8. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

  9. Gene expression of beta carotene genes in transgenic biofortified cassava

    OpenAIRE

    Telengech, P. K.; Maling’a, J. N.; Nyende, A. B.; Gichuki, S. T.; Wanjala, B. W.

    2014-01-01

    Cassava is an important food for millions of people around the world. However, cassava is deficient in protein, iron, zinc, pro-vitamin A and vitamin E. Cassava biofortified with pro-vitamin A can help reduce Vitamin A Deficiency among the undernourished communities that rely upon it for sustenance. BioCassava Plus project has developed transgenic cassava that expresses beta carotene in roots using root specific patatin promoter. This study aimed at confirming expression of nptII, crtB and DX...

  10. Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues.

    Science.gov (United States)

    Strózycki, P M; Karłowski, W M; Dessaux, Y; Petit, A; Legocki, A B

    2000-03-01

    The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the "Class II" hemoglobins.

  11. Transgene expression in plants : Position-induced spatial and temporal variations of luciferase expression

    NARCIS (Netherlands)

    Leeuwen, van W.

    2001-01-01

    In this thesis we have examined the spatial and temporal aspects of gene expression and the position induced differences in transgene expression between individual transformants. For this purpose we imaged luciferase ( luc ) gene expression driven by three different promoters that are active through

  12. Effects of Transgenic Expression of Dopamine Beta Hydroxylase (Dbh) Gene on Blood Pressure in Spontaneously Hypertensive Rats

    Science.gov (United States)

    PRAVENEC, M.; LANDA, V.; ZÍDEK, V.; MLEJNEK, P.; ŠILHAVÝ, J.; MIR, S. A.; VAINGANKAR, S. M.; WANG, J.; KURTZ, T. W.

    2017-01-01

    Summary The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and left ventricular hypertrophy. Catecholamines play an important role in the pathogenesis of both essential hypertension in humans and in the SHR. Recently, we obtained evidence that the SHR harbors a variant in the gene for dopamine beta hydroxylase (Dbh) that is associated with reduced adrenal expression of Dbh mRNA and reduced DBH enzymatic activity which correlated negatively with blood pressure. In the current study, we used a transgenic experiment to test the hypothesis that reduced Dbh expression predisposes the SHR to hypertension and that augmentation of Dbh expression would reduce blood pressure. We derived 2 new transgenic SHR-Dbh lines expressing Dbh cDNA under control of the Brown Norway (BN) wild type promoter. We found modestly increased adrenal expression of Dbh in transgenic rats versus SHR non-transgenic controls that was associated with reduced adrenal levels of dopamine and increased plasma levels of norepinephrine and epinephrine. The observed changes in catecholamine metabolism were associated with increased blood pressure and left ventricular mass in both transgenic lines. We did not observe any consistent changes in brainstem levels of catecholamines or of mRNA levels of Dbh in the transgenic strains. Contrary to our initial expections, these findings are consistent with the possibility that genetically determined decreases in adrenal expression and activity of DBH do not represent primary determinants of increased blood pressure in the SHR model. PMID:27959576

  13. Production of transgenic chickens expressing a tetracycline-inducible GFP gene.

    Science.gov (United States)

    Kwon, Mo Sun; Koo, Bon Chul; Roh, Ji Yeol; Kim, Minjee; Kim, Jin-Hoi; Kim, Teoan

    2011-07-15

    There is much interest in using farm animals as 'bioreactors' to produce large quantities of biopharmaceuticals. However, uncontrolled constitutive expression of foreign genes have been known to cause serious physiological disturbances in transgenic animals. The objective of this study was to test the feasibility of the controllable expression of an exogenous gene in the chicken. A retrovirus vector was designed to express GFP (green fluorescent protein) and rtTA (reverse tetracycline-controlled transactivator) under the control of the tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter, respectively. G0 founder chickens were produced by infecting the blastoderm of freshly laid eggs with concentrated retrovirus vector. Feeding the chickens obtained with doxycycline, a tetracycline derivative, resulted in emission of green body color under fluorescent light, and no apparent significant physiological dysfunctions. Successful germline transmission of the exogenous gene was also confirmed. Expression of the GFP gene reverted to the pre-induction levels when doxycycline was removed from the diet. The results showed that a tetracycline-inducible expression system in transgenic animals might be a promising solution to minimize physiological disturbances caused by the transgene. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Transgenic Paulownia Expressing shiva-1 Gene Has Increased Resistance to Paulownia Witches' Broom Disease

    Institute of Scientific and Technical Information of China (English)

    Tao DU; Yao WANG; Qin-Xue HU; Jie CHEN; Sheng LIU; Wen-Jin HUANG; Mu-Lan LIN

    2005-01-01

    Stem segments from diseased Paulownia tomentosa×P. fortunei and leaves from healthy control were transformed with the expression vector p438PRSI via Agrobacterium tumefaciens. The p438PRSI vector contained shiva-1 gene, which encodes an antibacterial peptide under the control of a CaMV35S promoter. The regenerated plants from transformed explants were planted in a greenhouse and nursery. PCR and Southern blotting analysis showed that the shiva-1 gene was successfully integrated into the Paulownia genome. Transcription of the integrated shiva-1 gene was confirmed by RT-PCR. Bioassay in the green house and phytoplasma DNA-dot blotting demonstrated that resistance to Paulownia witch's broom disease (PWB) increased significantly in shiva-1-transgenic Paulownia. Further investigations indicated that higher Shiva-1 expression correlated with fewer phytoplasma and less symptoms in diseased transgenic Paulownia. Together, our findings strongly suggest that breeding shiva-1-Paulownia is an effective strategy to control PWB disease.

  15. Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.

    Science.gov (United States)

    Kovalchuk, Nataliya; Jia, Wei; Eini, Omid; Morran, Sarah; Pyvovarenko, Tatiana; Fletcher, Stephen; Bazanova, Natalia; Harris, John; Beck-Oldach, Kontanze; Shavrukov, Yuri; Langridge, Peter; Lopato, Sergiy

    2013-08-01

    Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter-driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

  16. Using inositol as a biocompatible ligand for efficient transgene expression.

    Science.gov (United States)

    Zhang, Lei; Bellis, Susan L; Fan, Yiwen; Wu, Yunkun

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

  17. Transgenic CHD1L expression in mouse induces spontaneous tumors.

    Directory of Open Access Journals (Sweden)

    Muhan Chen

    Full Text Available BACKGROUND: Amplification of 1q21 is the most frequent genetic alteration in hepatocellular carcinoma (HCC, which was detected in 58-78% of primary HCC cases by comparative genomic hybridization (CGH. Using chromosome microdissection/hybrid selection approach we recently isolated a candidate oncogene CHD1L from 1q21 region. Our previous study has demonstrated that CHD1L had strong oncogenic ability, which could be effectively suppressed by siRNA against CHD1L. The molecular mechanism of CHD1L in tumorigenesis has been associated with its role in promoting cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: To further investigate the in vivo oncogenic role of CHD1L, CHD1L ubiquitous-expression transgenic mouse model was generated. Spontaneous tumor formations were found in 10/41 (24.4% transgenic mice, including 4 HCCs, but not in their 39 wild-type littermates. In addition, alcohol intoxication was used to induce hepatocyte pathological lesions and results found that overexpression of CHD1L in hepatocytes could promote tumor susceptibility in CHD1L-transgenic mice. To address the mechanism of CHD1L in promoting cell proliferation, DNA content between CHD1L-transgenic and wildtype mouse embryo fibroblasts (MEFs was compared by flow cytometry. Flow cytometry results found that CHD1L could facilitate DNA synthesis and G1/S transition through the up-regulation of Cyclin A, Cyclin D1, Cyclin E, CDK2, and CDK4, and down-regulation of Rb, p27(Kip1, and p53. CONCLUSION/SIGNIFICANCE: Taken together, our data strongly support that CHD1L is a novel oncogene and plays an important role in HCC pathogenesis.

  18. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

    Science.gov (United States)

    Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

    2006-01-23

    Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.

  19. Expression of a defence-related intercellular barley peroxidase in transgenic tobacco

    DEFF Research Database (Denmark)

    Kristensen, B.K.; Brandt, J.; Bojsen, K.

    1997-01-01

    Tobacco plants (Nicotiana benthamiana L.) have been transformed with a T-DNA vector construct carrying the cDNA pBH6-301, encoding the major pathogen induced leaf peroxidase (Prx8) of barley, under control of an enhanced CaMV 35S promoter. Progeny from three independent transformants were analyzed...... genetically, phenotypically and biochemically. The T-DNA was steadily inherited through three generations. The barley peroxidase is expressed and sorted to the intercellular space in the transgenic tobacco plants. The peroxidase can be extracted from the intercellular space in two molecular forms from both...... barley and transgenic tobacco. The tobacco expressed forms are indistinguishable from the barley expressed forms as determined by analytical isoelectric focusing (pI 8.5) and Western-blotting. Staining for N-glycosylation showed that one form only was glycosylated. The N-terminus of purified Prx8 from...

  20. Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo.

    Directory of Open Access Journals (Sweden)

    Susan K Morton

    Full Text Available Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2 with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R protein.

  1. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco.

    Science.gov (United States)

    Higo, K; Saito, Y; Higo, H

    1993-09-01

    Nicotiana tabacum was transformed with a chemically synthesized gene encoding the human epidermal growth factor (hEGF) under control of the CaMV-35S promoter. The hEGF gene sequence was present at one to several copies in the primary transformant plants (R0), and a transcript with the expected length was produced. Slot blot analysis of total RNAs of the progeny (R1) seedlings, originating from self-pollination of the R0 plants, showed that the level of mRNA expression was generally, but not always, heritable. The highest hEGF peptide content per unit of total soluble protein in young (upper) R1 leaves so far examined by an immunological method was about 0.001%. These results suggest that either the hEGF peptide was less stable than the average leaf protein, or the hEGF mRNAs were not efficiently translated.

  2. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  3. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

    Science.gov (United States)

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K

    2014-01-01

    Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  4. Quantitative analysis of tetracycline-inducible expression of the green fluorescent protein gene in transgenic chickens.

    Science.gov (United States)

    Koo, Bon Chul; Kwon, Mo Sun; Roh, Ji Yeol; Kim, Minjee; Kim, Jin-Hoi; Kim, Teoan

    2012-01-01

    The use of transgenic farm animals as "bioreactors" to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side-effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognized limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet. As a proof of principle study, however, quantitative assessment of expression was not possible, as only one G0 and one G1 transgenic chicken was obtained. In the current study, a sufficient number of G2 and G3 transgenic chickens were obtained, and quantification analysis demonstrated up to a 20-fold induction of expression by doxycycline. In addition, stable transmission of the transgene without any apparent genetic modifications was observed through several generations. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene. Importantly, these results also support the use of the retroviral system for generating transgenic animals with minimal risk in terms of biosafety.

  5. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    Science.gov (United States)

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

  6. Transgenic mice expressing constitutive active MAPKAPK5 display gender-dependent differences in exploration and activity

    Directory of Open Access Journals (Sweden)

    Moens Ugo

    2007-11-01

    Full Text Available Abstract Background The mitogen-activated protein kinases, MAPKs for short, constitute cascades of signalling pathways involved in the regulation of several cellular processes that include cell proliferation, differentiation and motility. They also intervene in neurological processes like fear conditioning and memory. Since little remains known about the MAPK-Activated Protein Kinase, MAPKAPK5, we constructed the first MAPKAPK knockin mouse model, using a constitutive active variant of MAPKAPK5 and analyzed the resulting mice for changes in anxiety-related behaviour. Methods We performed primary SHIRPA observations during background breeding into the C57BL/6 background and assessed the behaviour of the background-bred animals on the elevated plus maze and in the light-dark test. Our results were analyzed using Chi-square tests and homo- and heteroscedatic T-tests. Results Female transgenic mice displayed increased amounts of head dips and open arm time on the maze, compared to littermate controls. In addition, they also explored further into the open arm on the elevated plus maze and were less active in the closed arm compared to littermate controls. Male transgenic mice displayed no differences in anxiety, but their locomotor activity increased compared to non-transgenic littermates. Conclusion Our results revealed anxiety-related traits and locomotor differences between transgenic mice expressing constitutive active MAPKAPK5 and control littermates.

  7. Evaluation of transgenic tobacco plants expressing a bacterial Co-Ni transporter for acquisition of cobalt.

    Science.gov (United States)

    Nair, Smitha; Joshi-Saha, Archana; Singh, Sudhir; Ramachandran, V; Singh, Surya; Thorat, Vidya; Kaushik, C P; Eapen, Susan; D'Souza, S F

    2012-11-15

    Phytoremediation is a viable strategy for management of toxic wastes in a large area/volume with low concentrations of toxic elemental pollutants. With increased industrial use of cobalt and its alloys, it has become a major metal contaminant in soils and water bodies surrounding these industries and mining sites with adverse effects on the biota. A bacterial Co-Ni permease was cloned from Rhodopseudomonas palustris and introduced into Nicotiana tabacum to explore its potential for phytoremediation and was found to be specific for cobalt and nickel. The transgenic plants accumulated more cobalt and nickel as compared to control, whereas no significant difference in accumulation of other divalent ions was observed. The transgenic plants were evaluated for cobalt content and showed increased acquisition of cobalt (up to 5 times) as compared to control. The plants were also assessed for accumulation of nickel and found to accumulate up to 2 times more nickel than control. At the same initial concentration of cobalt and nickel, transgenic plant preferentially accumulated cobalt as compared to nickel. The present study is perhaps the first attempt to develop transgenic plants expressing heterologous Co transporter with an improved capacity to uptake cobalt. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene.

    Science.gov (United States)

    Vail, Andrew W; Wang, Ping; Uefuji, Hirotaka; Samac, Deborah A; Vance, Carroll P; Wackett, Lawrence P; Sadowsky, Michael J

    2015-06-01

    The widespread use of atrazine and other s-triazine herbicides to control weeds in agricultural production fields has impacted surface and groundwater in the United States and elsewhere. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradation pathway of Pseudomonas sp. strain ADP, which is initiated by atzA, encoding atrazine chlorohydrolase. Here we explored the use of enhanced expression of a modified bacterial atrazine chlorohydrolase, p-AtzA, in transgenic grasses (tall fescue, perennial ryegrass, and switchgrass) and the legume alfalfa for the biodegradation of atrazine. Enhanced expression of p-AtzA was obtained by using combinations of the badnavirus promoter, the maize alcohol dehydrogenase first intron, and the maize ubiquitin promoter. For alfalfa, we used the first intron of the 5'-untranslated region tobacco alcohol dehydrogenase gene and the cassava vein mosaic virus promoter. Resistance of plants to atrazine in agar-based and hydroponic growth assays was correlated with in vivo levels of gene expression and atrazine degradation. The in planta expression of p-atzA enabled transgenic tall fescue to transform atrazine into hydroxyatrazine and other metabolites. Results of our studies highlight the potential use of transgenic plants for bioremediating atrazine in the environment.

  9. Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.

    Science.gov (United States)

    Alami, R; Greally, J M; Tanimoto, K; Hwang, S; Feng, Y Q; Engel, J D; Fiering, S; Bouhassira, E E

    2000-03-01

    Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.

  10. Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase.

    Science.gov (United States)

    Wang, Fang-Zheng; Wang, Qing-Bin; Kwon, Suk-Yoon; Kwak, Sang-Soo; Su, Wei-Ai

    2005-04-01

    We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.

  11. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    Science.gov (United States)

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A

    1995-01-01

    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  12. Sequential monitoring of transgene expression following Agrobacterium-mediated transformation of rice.

    Science.gov (United States)

    Saika, Hiroaki; Nonaka, Satoko; Osakabe, Keishi; Toki, Seiichi

    2012-11-01

    Although Agrobacterium-mediated transformation technology is now used widely in rice, many varieties of indica-type rice are still recalcitrant to Agrobacterium-mediated transformation. It was reported recently that T-DNA integration into the rice genome could be the limiting step in this method. Here, we attempted to establish an efficient sequential monitoring system for stable transformation events by visualizing stable transgene expression using a non-destructive and highly sensitive visible marker. Our results demonstrate that click beetle luciferase (ELuc) is an excellent marker allowing the observation of transformed cells in rice callus, exhibiting a sensitivity >30-fold higher than that of firefly luciferase. Since we have previously shown that green fluorescent protein (GFP) is a useful visual marker with which to follow transient and/or stable expression of transgenes in rice, we constructed an enhancer trap vector using both the gfbsd2 (GFP fused to the N-terminus of blasticidin S deaminase) and eluc genes. In this vector, the eluc gene is under the control of the Cauliflower mosaic virus 35S minimal promoter, while the gfbsd2 gene is under the control of the full-length rice elongation factor gene promoter. Observation of transformed callus under a dissecting microscope demonstrated that the level of ELuc luminescence reflected exclusively stable transgene expression, and that both transient and stable expression could be monitored by the level of GFP fluorescence. Moreover, we show that our system enables sequential quantification of transgene expression via differential measurement of ELuc luminescence and GFP fluorescence.

  13. 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 in ...

  14. Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora.

    Science.gov (United States)

    Maga, Elizabeth A; Walker, Richard L; Anderson, Gary B; Murray, James D

    2006-08-01

    Lysozyme is a key antimicrobial component of human milk that has several health-promoting functions including the development of a healthy intestinal tract. However, levels of lysozyme in the milk of dairy animals are negligible. We have generated transgenic dairy goats that express human lysozyme (HLZ) in their milk in an attempt to deliver the benefits of human milk in a continual fashion. To test the feasibility of this transgenic approach to achieve a biological impact at the level of the intestine, feeding trials were conducted in two animal models. Pasteurized milk from HLZ transgenic animals was fed to both kid goats (ruminant model) and young pigs (human model), and the numbers of total coliforms and Escherichia coli present in the small intestine were determined. Data from this proof-of-principle study demonstrate that milk from transgenic animals was capable of modulating the bacterial population of the gut in both animal models. Pigs that consumed pasteurized milk from HLZ transgenic goats had fewer numbers of coliforms and E. coli in their intestine than did those receiving milk from non-transgenic control animals. The opposite effect was seen in goats. Milk from these transgenic animals not only represent one of the first transgenic food products with the potential of benefiting human health, but are also a unique model to study the development and role of intestinal microflora on health, well-being and resistance to disease.

  15. Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance

    NARCIS (Netherlands)

    Mlotshwa, S.; Verver, J.; Sithole-Niang, I.; Prins, M.; Kammen, van A.; Wellink, J.

    2002-01-01

    Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed

  16. Development of a novel-type transgenic cotton plant for control of cotton bollworm.

    Science.gov (United States)

    Yue, Zhen; Liu, Xiaoguang; Zhou, Zijing; Hou, Guangming; Hua, Jinping; Zhao, Zhangwu

    2016-08-01

    The transgenic Bt cotton plant has been widely planted throughout the world for the control of cotton budworm Helicoverpa armigera (Hubner). However, a shift towards insect tolerance of Bt cotton is now apparent. In this study, the gene encoding neuropeptide F (NPF) was cloned from cotton budworm H. armigera, an important agricultural pest. The npf gene produces two splicing mRNA variants-npf1 and npf2 (with a 120-bp segment inserted into the npf1 sequence). These are predicted to form the mature NPF1 and NPF2 peptides, and they were found to regulate feeding behaviour. Knock down of larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight, and dsNPF also caused a decrease of glycogen and an increase of trehalose. Moreover, we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expressed dsNPF. Results showed that H. armigera larvae fed on these transgenic plants or leaves had lower food consumption, body size and body weight compared to controls. These results indicate that NPF is important in the control of feeding of H. armigera and valuable for production of potential transgenic cotton.

  17. Comparative transcriptomic analyses of differentially expressed genes in transgenic melatonin biosynthesis ovine HIOMT gene in switchgrass

    Directory of Open Access Journals (Sweden)

    Shan Yuan

    2016-11-01

    Full Text Available Melatonin serves pleiotropic functions in prompting plant growth and resistance to various stresses. The accurate biosynthetic pathway of melatonin remains elusive in plant species, while the N-acetyltransferase and O-methyltransferase were considered to be the last two key enzymes during its biosynthesis. To investigate the biosynthesis and metabolic pathway of melatonin in plants, the RNA-seq profile of overexpression of the ovine HIOMT was analyzed and compared with the previous transcriptome of transgenic oAANAT gene in switchgrass, a model plant for cellulosic ethanol production. A total of 946, 405 and 807 differentially expressed unigenes were observed in AANAT vs. control, HIOMT vs. control, and AANAT vs. HIOMT, respectively. The significantly upregulated (F-box/kelch-repeat protein, zinc finger BED domain-containing protein-3 genes were consistent with enhanced phenotypes of shoot, stem and root growth in transgenic oHIOMT switchgrass. Early flowering in overexpression of oHIOMT switchgrass involved in the regulation of flowering-time genes (APETALA2. Several stress resistant related genes (SPX domain-containing membrane protein, copper transporter 1, late blight resistance protein homolog R1A-6 OS etc. were specifically and significantly upregulated in transgenic oHIOMT only, while metabolism-related genes (phenylalanine-4-hydroxylase, tyrosine decarboxylase 1, protein disulfide-isomerase and galactinol synthase 2 etc. were significantly upregulated in transgenic oAANAT only. These results provide new sights into the biosynthetic and physiological functional networks of melatonin in plants.

  18. Effect of CRC::etr1-1 transgene expression on ethylene production, sex expression, fruit set and fruit ripening in transgenic melon (Cucumis melo L.).

    Science.gov (United States)

    Switzenberg, Jessica A; Beaudry, Randy M; Grumet, Rebecca

    2015-06-01

    Ethylene is a key factor regulating sex expression in cucurbits. Commercial melons (Cucumis melo L.) are typically andromonoecious, producing male and bisexual flowers. Our prior greenhouse studies of transgenic melon plants expressing the dominant negative ethylene perception mutant gene, etr1-1, under control of the carpel- and nectary-primordia targeted CRAB'S CLAW (CRC) promoter showed increased number and earlier appearance of carpel-bearing flowers. To further investigate this phenomenon which could be potentially useful for earlier fruit production, we observed CRC::etr1-1 plants in the field for sex expression, fruit set, fruit development, and ripening. CRC::etr1-1 melon plants showed increased number of carpel-bearing open flowers on the main stem and earlier onset by 7-10 nodes. Additional phenotypes observed in the greenhouse and field were conversion of approximately 50% of bisexual buds to female, and elongated ovaries and fruits. Earlier and greater fruit set occurred on the transgenic plants. However, CRC::etr1-1 plants had greater abscission of young fruit, and smaller fruit, so that final yield (kg/plot) was equivalent to wild type. Earlier fruit set in line M5 was accompanied by earlier appearance of ripe fruit. Fruit from line M15 frequently did not exhibit external ripening processes of rind color change and abscission, but when cut open, the majority showed a ripe or overripe interior accompanied by elevated internal ethylene. The non-ripening external phenotype in M15 fruit corresponded with elevated etr1-1 transgene expression in the exocarp. These results provide insight into the role of ethylene perception in carpel-bearing flower production, fruit set, and ripening.

  19. Modulation of Mammary Gland Development and Milk Production by Growth Hormone Expression in GH Transgenic Goats.

    Science.gov (United States)

    Bao, Zekun; Lin, Jian; Ye, Lulu; Zhang, Qiang; Chen, Jianquan; Yang, Qian; Yu, Qinghua

    2016-01-01

    Mammary gland development during puberty and reconstruction during pregnancy and lactation is under the control of circulating endocrine hormones, such as growth hormone, which are released from the pituitary. In this study, we explored the influence of overexpression of growth hormone in the mammary gland on breast development and milk production in goats. Using transcriptome sequencing, we found that the number of highly expressed genes was greater in GH transgenic goats than non-transgenic goats. Furthermore, KEGG pathway analysis showed that the majority of the genes belonged to the MAPK signaling pathway and the ECM-receptor interaction pathway. The expression of genes related to breast development was further confirmed using qRT-PCR. Interestingly, both milk production and milk quality were increased. The results of these experiments imply that overexpression of growth hormone in the breast may stimulate breast development and enhances milk production by modulating alveolar cell proliferation or branching through the MAPK signaling pathway.

  20. Transgenic banana plants expressing Xanthomonas wilt resistance genes revealed a stable non-target bacterial colonization structure.

    Science.gov (United States)

    Nimusiima, Jean; Köberl, Martina; Tumuhairwe, John Baptist; Kubiriba, Jerome; Staver, Charles; Berg, Gabriele

    2015-12-10

    Africa is among the continents where the battle over genetically modified crops is currently being played out. The impact of GM in Africa could potentially be very positive. In Uganda, researchers have developed transgenic banana lines resistant to banana Xanthomonas wilt. The transgenic lines expressing hrap and pflp can provide a timely solution to the pandemic. However, the impact of the transgenes expression on non-target microorganisms has not yet been investigated. To study this effect, transgenic and control lines were grown under field conditions and their associated microbiome was investigated by 16S rRNA gene profiling combining amplicon sequencing and molecular fingerprinting. Three years after sucker planting, no statistically significant differences between transgenic lines and their non-modified predecessors were detected for their associated bacterial communities. The overall gammaproteobacterial rhizosphere microbiome was highly dominated by Xanthomonadales, while Pseudomonadales and Enterobacteriales were accumulated in the pseudostem. Shannon indices revealed much higher diversity in the rhizosphere than in the pseudostem endosphere. However, the expression of the transgenes did not result in changes in the diversity of Gammaproteobacteria, the closest relatives of the target pathogen. In this field experiment, the expression of the resistance genes appears to have no consequences for non-target rhizobacteria and endophytes.

  1. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    Science.gov (United States)

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  2. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O;

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific tra...... of the transgene was observed in cell types other than beta-islet cells.......Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...

  3. Tetracycline-controlled transcriptional regulation systems:countermeasures to eliminate basal transgene leaks in Tet-based systems

    Institute of Scientific and Technical Information of China (English)

    XIAO Dong; SUN Yan; GU Weiwang; CHEN Xigu

    2007-01-01

    To analyze the function of any given transgene(s) accurately in transgenic mice, and to produce credible transgenic animal models of various human diseases (precisely and realistically mimicking disease states), it is critical to be able to control gene expression in the animals conditionally. The ability to switch gene expression "on" or "off" in the restricted cells or tissue(s) at specific time(s)allows unprecedented flexibility for exploring gene function(s) in both the health and the disease. Pioneering work on inducible transgene expression has led to the development of a wide variety of controlled gene expression systems that meet this criterion. Among them, the tetracycline-inducible systems (e. g. Tet-off and Tet-on) have been widely, frequently and successfully employed in vitro and in vivo.These systems, however, are not always tight but leaky; sometimes the leakage is significant. In some circumstances, the resulting leak is acceptable, but in others, it is more problematic. Though these systems face this disadvantage, i.e. basal transgene leakage in vitro and in vivo, several approaches, including using improved versions (e. g. rtTA2S-M2 and rtTA2S-S2) of rtTA, tetracycline-controlled transcriptional silencer (tTS), an "ideal" minimal promoter in responsive components or combinations thereof, have been developed to avoid this limitation effectively. In this review we discuss the countermeasures available to eliminate basal transgene leakage from Tet-based systems.

  4. Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance

    Directory of Open Access Journals (Sweden)

    Ma Hao

    2011-10-01

    Full Text Available Abstract Background Tomato spotted wilt virus (TSWV has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX in tomato and petunia is related to TSWV resistance. Results The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. Conclusion In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

  5. Comparison of Different MARs(Matrix Attachment Regions)Effect on Transgene Expression

    Institute of Scientific and Technical Information of China (English)

    ZHONG Jin; LIU Shu-jun; YANG Wei; HU Yuan-lei; LIN Zhong-ping

    2004-01-01

    Three MARs(matrix attachment regions)fragments were cloned from tobacco (Nicotiana tabacum)(MAR1),yeast(Saccharomyces cerevisiae) (MAR3)and kidney bean(Phaseolus vulgaris) (MAR5)which ranged 984,822 and 782 bp,respectively.Sequence analysis showed that all the fragments had fairly high A/T content (73,62 and 75%,respectively),harbored different number and different type of some characteristic motifs of MARs,such as A-box and T-box,etc.The results of in vitro binding assay showed that the three MARs fragments derived from different organisms could bind specifically to the matrix extracted from the tobacco nuclei with different strength,which also demonstrated that these MARs fragments are functionally conserved during evolution.By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-barMARs into tobacco through Agrobacteriummediated procedures,the effects of MARs sequences on the expression of transgenes in tobacco were investigated and compared.The GUS activity in individual transformants showed that,comparing to the controls without additional MARs,the overall transgene expression level in transformants with MARs had been greatly increased while the variations in transgene expression among transformants were decreased in different degrees.In accordance with the results of sequence analysis and in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overall expression level.

  6. Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

    Science.gov (United States)

    Young, W S; Iacangelo, A; Luo, X Z; King, C; Duncan, K; Ginns, E I

    1999-12-01

    Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

  7. Analysis of two novel midgut-specific promoters driving transgene expression in Anopheles stephensi mosquitoes.

    Directory of Open Access Journals (Sweden)

    Tony Nolan

    Full Text Available BACKGROUND: Tissue-specific promoters controlling the expression of transgenes in Anopheles mosquitoes represent a valuable tool both for studying the interaction between these malaria vectors and the Plasmodium parasites they transmit and for novel malaria control strategies based on developing Plasmodium-refractory mosquitoes by expressing anti-parasitic genes. With this aim we have studied the promoter regions of two genes from the most important malaria vector, Anopheles gambiae, whose expression is strongly induced upon blood feeding. RESULTS: We analysed the A. gambiae Antryp1 and G12 genes, which we have shown to be midgut-specific and maximally expressed at 24 hours post-bloodmeal (PBM. Antryp1, required for bloodmeal digestion, encodes one member of a family of 7 trypsin genes. The G12 gene, of unknown function, was previously identified in our laboratory in a screen for genes induced in response to a bloodmeal. We fused 1.1 kb of the upstream regions containing the putative promoter of these genes to reporter genes and transformed these into the Indian malaria vector A. stephensi to see if we could recapitulate the expression pattern of the endogenous genes. Both the Antryp1 and G12 upstream regions were able to drive female-predominant, midgut-specific expression in transgenic mosquitoes. Expression of the Antryp1-driven reporter in transgenic A. stephensi lines was low, undetectable by northern blot analysis, and failed to fully match the induction kinetics of the endogenous Antryp1 gene in A. gambiae. This incomplete conservation of expression suggests either subtle differences in the transcriptional machinery between A. stephensi and A. gambiae or that the upstream region chosen lacked all the control elements. In contrast, the G12 upstream region was able to faithfully reproduce the expression profile of the endogenous A. gambiae gene, showing female midgut specificity in the adult mosquito and massive induction PBM, peaking at 24

  8. Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    CHEN XiaoGuang; ZHANG YaJing; ZHENG XueLi; WANG ChunMei

    2007-01-01

    The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles stephensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I digestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively conserved in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.

  9. Bacterial xylanase expression in mammalian cells and transgenic mice.

    Science.gov (United States)

    Fontes, C M; Ali, S; Gilbert, H J; Hazlewood, G P; Hirst, B H; Hall, J

    1999-06-11

    The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

  10. Supplemental control of lepidopterous pests on Bt transgenic sweet corn with biologically-based spray treatments.

    Science.gov (United States)

    Farrar, Robert R; Shepard, B Merle; Shapiro, Martin; Hassell, Richard L; Schaffer, Mark L; Smith, Chad M

    2009-01-01

    Biologically-based spray treatments, including nucleopolyhedroviruses, neem, and spinosad, were evaluated as supplemental controls for the fall armyworm, Spodoptera frugiperda (J. E. Smith), and corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), on transgenic sweet corn, Zea mays (L.) (Poales: Poaceae), expressing a Cry1Ab toxin from Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). Overall, transgenic corn supported lower densities of both pests than did nontransgenic corn. Control of the fall armyworm was improved in both whorl-stage and tassel-stage corn by the use of either a nucleopolyhedrovirus or neem, but the greatest improvement was seen with spinosad. Only spinosad consistently reduced damage to ears, which was caused by both pest species. In general, efficacy of the spray materials did not differ greatly between transgenic and nontransgenic corn.

  11. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    Science.gov (United States)

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

  12. Characterization of expression of Puumala virus nucleocapsid protein in transgenic plants.

    Science.gov (United States)

    Khattak, Shahryar; Darai, Gholamreza; Süle, Sandor; Rösen-Wolff, Angela

    2002-01-01

    Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 microg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest.

  13. Investigating Transgenic Corn Hybrids as a Method for Mycotoxin Control

    Science.gov (United States)

    Transgenic Bt corn hybrids have been available for more than 10 years and are known to control specific insects. More recently, so-called “stacked-gene” hybrids, have been released with multiple insect resistance genes and genes for herbicide resistance, resulting in up to 6 traits per plant. Beca...

  14. Effect of β‑globin MAR characteristic elements on transgene expression.

    Science.gov (United States)

    Li, Qin; Dong, Weihua; Wang, Tianyun; Liu, Zhonghe; Wang, Fang; Wang, Xiaoyin; Zhao, Chunpeng; Zhang, Junhe; Wang, Li

    2013-06-01

    The aim of the present study was to investigate the effect of the characteristic elements of matrix attachment region (MAR) on transgene expression. Human β‑globin MAR was obtained by PCR amplification. A splicing MAR fragment containing all the characteristic elements of β‑globin MAR was artificially synthesized and then cloned into the eukaryotic expression vector. Following digestion and sequence identification, we transfected Chinese hamster ovary (CHO) cells with the two vectors, and then screened for the transformation of stable cells. The transgene expression level was analyzed by ELISA, and the copy numbers of the CAT gene were analyzed by real‑time fluorescent quantitative PCR. β‑globin MAR enhanced CAT reporter gene expression by 2.1489‑fold, whereas the β‑globin MAR characteristic elements did not enhance this expression. The real‑time fluorescent quantitative PCR analysis demonstrated that the relative copy numbers of the CAT gene of the β‑globin MAR expression vector were 1.2‑fold higher compared with those of the non‑MAR expression vector. MAR was able to improve the transgene expression level to a certain extent. The MAR characteristic elements did not improve the transgene expression alone. The transgenic expression levels were not linear with the transgene copy number; however, the enhancement of transgenic expression was relative to the increase in the gene copy number.

  15. AAVPG: A vigilant vector where transgene expression is induced by p53

    Energy Technology Data Exchange (ETDEWEB)

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail: bstrauss@usp.br

    2013-12-15

    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

  16. Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

    Directory of Open Access Journals (Sweden)

    Pons Elsa

    2012-07-01

    trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s of biotechnological interest.

  17. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

    Science.gov (United States)

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-03-05

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.

  18. Transgenic overexpression of cdx1b induces metaplastic changes of gene expression in zebrafish esophageal squamous epithelium.

    Science.gov (United States)

    Hu, Bo; Chen, Hao; Liu, Xiuping; Zhang, Chengjin; Cole, Gregory J; Lee, Ju-Ahng; Chen, Xiaoxin

    2013-06-01

    Cdx2 has been suggested to play an important role in Barrett's esophagus or intestinal metaplasia (IM) in the esophagus. To investigate whether transgenic overexpression of cdx1b, the functional equivalent of mammalian Cdx2 in zebrafish, may lead to IM of zebrafish esophageal squamous epithelium, a transgenic zebrafish system was developed by expressing cdx1b gene under the control of zebrafish keratin 5 promoter (krt5p). Gene expression in the esophageal squamous epithelium of wild-type and transgenic zebrafish was analyzed by Affymetrix microarray and confirmed by in situ hybridization. Morphology, mucin expression, cell proliferation, and apoptosis were analyzed by hematoxylin & eosin (HE) staining, Periodic acid Schiff (PAS) Alcian blue staining, proliferating cell nuclear antigen (PCNA) immunohistochemical staining, and TUNEL assay as well. cdx1b was found to be overexpressed in the nuclei of esophageal squamous epithelial cells of the transgenic zebrafish. Ectopic expression of cdx1b disturbed the development of this epithelium in larval zebrafish and induced metaplastic changes in gene expression in the esophageal squamous epithelial cells of adult zebrafish, that is, up-regulation of intestinal differentiation markers and down-regulation of squamous differentiation markers. However, cdx1b failed to induce histological IM, or to modulate cell proliferation and apoptosis in the squamous epithelium of adult transgenic zebrafish.

  19. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...... of the transgene was observed in cell types other than beta-islet cells....

  20. Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish rod photoreceptors and a gateway-based tet-on toolkit.

    Directory of Open Access Journals (Sweden)

    Leah J Campbell

    Full Text Available The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA, one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.

  1. Enduring toxicity of transgenic Anabaena PCC 7120 expressing mosquito larvicidal genes from Bacillus thuringiensis ssp. israelensis.

    Science.gov (United States)

    Manasherob, Robert; Otieno-Ayayo, Zachariah Ngalo; Ben-Dov, Eitan; Miaskovsky, Rina; Boussiba, Sammy; Zaritsky, Arieh

    2003-10-01

    Persistence of biological control agents against mosquito larvae was tested under simulated field conditions. Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing cry4Aa, cry11Aa and p20 from Bacillus thuringiensis ssp. israelensis was greater than B. thuringiensis ssp. israelensis primary powder (fun 89C06D) or wettable powder (WP) (Bactimos products) when either mixed with silt or exposed to sunlight outdoors. Reduction of Bactimos primary powder toxicity was at least 10-fold higher than Anabaena's after mixing with silt. In outdoors experiments, Bactimos WP remained toxic (over 30% mortality of 3rd instar Aedes aegypti larvae) for 2-4 days only, while transgenic Anabaena's toxicity endured 8-21 days.

  2. Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.

    Directory of Open Access Journals (Sweden)

    San-Ji Gao

    Full Text Available Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP or the β-glucuronidase (GUS reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

  3. Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR.

    Directory of Open Access Journals (Sweden)

    Jin-Qi Zhu

    Full Text Available The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E, predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.

  4. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  5. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    Science.gov (United States)

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    OpenAIRE

    Luchakivska Yu. S.; Komarnytskii I. K.; Kurchenko I. M.; Yurieva O. M.; Zhytkevich N. V.; Kuchuk M. V.

    2015-01-01

    Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the sali...

  7. Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

    OpenAIRE

    Gonzalez-Ruiz Gloriene; Alvarez Derry; Ruiz Oscar N; Torres Cesar

    2011-01-01

    Abstract Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury r...

  8. [Construction of plant expression vectors harboring a peptide antibiotic-apidaecin gene and resistance analysis of the transgenic tobacco].

    Science.gov (United States)

    Wang, H; Sun, C; Peng, X X

    2001-07-01

    Two plant expression vectors(pBinPRHbI and pBinPRSIHbI) were constructed: Firstly, apidaecin gene were fused to the signal peptide coding sequencing of a PR-protein, and cloned into a binary vector pBin438 to form pBinPRHbI. Then, the cassette consisting of 35S promoter, PR signal peptide coding sequencing and apidaecin gene was cut off from pBinPRHbI and inserted into another plant expression vector pBinPRSI to produce a bivalent plant expression vector pBinPRSIHbI. pBinPRSI was constructed previously in our lab and contained PR signal peptide and Shiva-I fusion gene under control of 35S promoter. The three plant expression vectors were introduced into tobacco by Agrobacterium-mediated transformation. The positive rate of PCR was 95% in all putative transgenic plants. Results from Southern blot indicated that foreign genes were integrated into tobacco genome and RT-PCR analysis proved that the foreign gene was transcribed in transgenic tobacco. The transgenic tobacco showed higher resistance to P. syringae pv tabaci, the causal agent of tobacco wild fire disease, than their original cultivars. From the disease index, the transgenic plants carrying apidaecin and Shiva-I genes had highest resistance among three kinds of transgenic plants, and the plants carrying Shiva-I gene alone had lowest resistance.

  9. Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period

    Directory of Open Access Journals (Sweden)

    Aldwinckle Herb S

    2010-06-01

    Full Text Available Abstract Background Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years. Results Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control. Conclusion Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.

  10. Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection.

    Science.gov (United States)

    Lipsky, Alexander; Joshi, Janak Raj; Carmi, Nir; Yedidia, Iris

    2016-11-20

    The genus Ornithogalum includes several ornamental species that suffer substantial losses from bacterial soft rot caused by Pectobacteria. The absence of effective control measures for use against soft rot bacteria led to the initiation of a project in which a small antimicrobial peptide from an Asian horseshoe crab, tachyplesin (tpnI), was introduced into two commercial cultivars: O. dubium and O. thyrsoides. Disease severity and bacterial colonization were examined in transgenic lines expressing this peptide. Disease resistance was evaluated in six lines of each species by measuring bacterial proliferation in the plant tissue. Three transgenic lines of each species were subjected to further analysis in which the expression level of the transgene was evaluated using RT-PCR and qRT-PCR. The development of disease symptoms and bacterial colonization of the plant tissue were also examined using GFP-expressing strain of P. carotovorum subsp. brasiliense Pcb3. Confocal-microscopy imaging revealed significantly reduced quantities of bacterial cells in the transgenic plant lines that had been challenged with the bacterium. The results clearly demonstrate that tpnI expression reduces bacterial proliferation, colonization and disease symptom (reduced by 95-100%) in the transgenic plant tissues. The quantity of tpnI transcripts, as measured by qRT-PCR, was negatively correlated with the protection afforded to the plants, as measured by the reduced severity of disease symptoms in the tissue. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The impact of transgenic wheat expressing GNA (snowdrop lectin) on the aphids Sitobion avenae, Schizaphis graminum, and Rhopalosiphum padi.

    Science.gov (United States)

    Miao, Jin; Wu, Yuqing; Xu, Weigang; Hu, Lin; Yu, Zhenxing; Xu, Qiongfang

    2011-06-01

    This study investigated the impact of transgenic wheat expressing Galanthus nivalis agglutinin (GNA), commonly known as snowdrop lectin, on three wheat aphids: Sitobion avenae (F.), Schizaphis graminum (Rondani), and Rhopalosiphum padi (L.). We compared the feeding behavior and the life-table parameters of aphids reared on GNA transgenic wheat (test group) and those aphids reared on untransformed wheat (control group). The results showed that the feeding behaviors of S. avenae and S. graminum on GNA transgenic wheat were affected. Compared with the control group, they had shorter initial probing period, longer total nonprobing period, shorter initial and total phloem sap ingestion phase (waveform E2), shorter duration of sustained ingestion (E (pd) > 10 min), and lower percentage of phloem phase of the total observation time. Moreover, S. graminum made more probes and had a longer total duration of extracellular stylet pathway (waveform C). The fecundity and intrinsic rate of natural increase (r(m)) of S. avenae and S. graminum on the transgenic wheat were lowered in the first and second generations, however, the survival and lifespan were not affected. The effects of the GNA expressing wheat on S. graminum and S. avenae were not significant in the third generation, suggesting rapid adaptation by the two aphid species. Despite the impact we found on S. avenae and S. graminum, transgenic GNA expressing wheat did not have any effects on R. padi.

  12. Duration and level of transgene expression after gene electrotransfer to skin in mice

    DEFF Research Database (Denmark)

    Gothelf, A; Eriksen, Jens Ole; Hojman, P

    2010-01-01

    . Level and duration of transgene expression after gene electrotransfer to skin is essential and here we present data from two independent quantitative studies. Using in vivo bioimaging of a far-red fluorescent molecule, Katushka, allowing for continuous monitoring of local gene expression, compared...... with measurements of a systemic transgene, that is, serum erythropoietin (EPO) after gene electrotransfer with EPO to skin, we found a significant increase in transgene expression (Pafter transfection. Duration of expression could be 3-4 weeks, which...... electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations...

  13. Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

    Science.gov (United States)

    Butaye, Katleen M J; Goderis, Inge J W M; Wouters, Piet F J; Pues, Jonathan M-T G; Delauré, Stijn L; Broekaert, Willem F; Depicker, Ann; Cammue, Bruno P A; De Bolle, Miguel F C

    2004-08-01

    Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

  14. MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.

    Science.gov (United States)

    Rahman, Jamilur; Karjee, Sumona; Mukherjee, Sunil Kumar

    2012-06-01

    Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.

  15. Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Er-Xia Du; Xiao-Fang Wang; Wu-Chen Yang; Deborah Kaback; Siu-Pok Yee; Chun-Lin Qin; Anne George; Jian-Jun Hao

    2015-01-01

    Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1-and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

  16. Characterization of Fam20C expression in odontogenesis and osteogenesis using transgenic mice.

    Science.gov (United States)

    Du, Er-Xia; Wang, Xiao-Fang; Yang, Wu-Chen; Kaback, Deborah; Yee, Siu-Pok; Qin, Chun-Lin; George, Anne; Hao, Jian-Jun

    2015-06-26

    Our previous studies have demonstrated that Fam20C promotes differentiation and mineralization of odontoblasts, ameloblasts, osteoblasts and osteocytes during tooth and bone development. Ablation of the Fam20C gene inhibits bone and tooth growth by increasing fibroblast growth factor 23 in serum and causing hypophosphatemia in conditional knockout mice. However, control and regulation of the expression of Fam20C are still unknown. In this study, we generated a transgenic reporter model which expresses green fluorescence protein (GFP) driven by the Fam20C promoter. Recombineering was used to insert a 16 kb fragment of the mouse Fam20C gene (containing the 15 kb promoter and 1.1 kb of exon 1) into a pBluescript SK vector with the topaz variant of GFP and a bovine growth hormone polyadenylation sequence. GFP expression was subsequently evaluated by histomorphometry on cryosections from E14 to adult mice. Fluorescence was evident in the bone and teeth as early as E17.5. The GFP signal was maintained stably in odontoblasts and osteoblasts until 4 weeks after birth. The expression of GFP was significantly reduced in teeth, alveolar bone and muscle by 8 weeks of age. We also observed colocalization of the GFP signal with the Fam20C antibody in postnatal 1- and 7-day-old animals. Successful generation of Fam20C-GFP transgenic mice will provide a unique model for studying Fam20C gene expression and the biological function of this gene during odontogenesis and osteogenesis.

  17. Expression of the yeast FRE genes in transgenic tobacco.

    Science.gov (United States)

    Samuelsen, A I; Martin, R C; Mok, D W; Mok, M C

    1998-09-01

    Two yeast genes, FRE1 and FRE2 (encoding Fe(III) reductases) were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation. Homozygous lines containing FRE1, FRE2, or FRE1 plus FRE2 were generated. Northern-blot analyses revealed mRNA of two different sizes in FRE1 lines, whereas all FRE2 lines had mRNA only of the expected length. Fe(III) reduction, chlorophyll contents, and Fe levels were determined in transgenic and control plants under Fe-sufficient and Fe-deficient conditions. In a normal growth environment, the highest root Fe(III) reduction, 4-fold higher than in controls, occurred in the double transformant (FRE1 + FRE2). Elevated Fe(III) reduction was also observed in all FRE2 and some FRE1 lines. The increased Fe(III) reduction occurred along the entire length of the roots and on shoot sections. FRE2 and double transformants were more tolerant to Fe deficiency in hydroponic culture, as shown by higher chlorophyll and Fe concentrations in younger leaves, whereas FRE1 transformants did not differ from the controls. Overall, the beneficial effects of FRE2 were consistent, suggesting that FRE2 may be used to improve Fe efficiency in crop plants.

  18. Transgenic tobacco plants expressing BoRS1 gene from Brassica oleracea var. acephala show enhanced tolerance to water stress

    Indian Academy of Sciences (India)

    Dongqin Tang; Hongmei Qian; Lingxia Zhao; Danfeng Huang; Kexuan Tang

    2005-12-01

    Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through Agrobacterium-mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows that BoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.

  19. A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field.

    Directory of Open Access Journals (Sweden)

    Nata Y S-G Diman

    Full Text Available A transgenic mouse line harbouring a β-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development.

  20. A Retinoic Acid Responsive Hoxa3 Transgene Expressed in Embryonic Pharyngeal Endoderm, Cardiac Neural Crest and a Subdomain of the Second Heart Field

    Science.gov (United States)

    Diman, Nata Y. S.-G.; Remacle, Sophie; Bertrand, Nicolas; Picard, Jacques J.; Zaffran, Stéphane; Rezsohazy, René

    2011-01-01

    A transgenic mouse line harbouring a β-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E) 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA) signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development. PMID:22110697

  1. The Lgr5 transgene is expressed specifically in glycinergic amacrine cells in the mouse retina.

    Science.gov (United States)

    Sukhdeo, Kumar; Koch, Catherine E; Miller, Tyler E; Zhou, Hannah; Rivera, Maricruz; Yan, Kenneth; Cepko, Constance L; Lathia, Justin D; Rich, Jeremy N

    2014-02-01

    Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This "fluorofilling" technique provides a tool to study amacrine morphology and dissect neural networks.

  2. Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter

    Institute of Scientific and Technical Information of China (English)

    VerenaNordhoff; JorgGromoll; LucaFoppiani; C.MarcLuetjens; StefanSchlatt; ElenaKostova; IlpoHuhtaniemi; EberhardNieschlag; ManuelaSimoni

    2003-01-01

    Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5"-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

  3. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Dismuke Adria D

    2009-07-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  4. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Kohn Aimee

    2009-01-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  5. Field Evaluation of the Asian Corn Borer Control in Hybrid of Transgenic Maize Event MON 810

    Institute of Scientific and Technical Information of China (English)

    HE Kang-lai; WANG Zhen-ying; WEN Li-ping; BAI Shu-xiong; ZHOU Da-rong; ZHU Qing-hua

    2003-01-01

    In this study,a transgenic Bt maize hybrid(event MON 810 from Monsanto Company)expressing Cry1Ab protein derived from Bacillus thuringiensis(Bt)and its negative isoline hybrid were evaluatrial. Maize plants were artificially infested with neonate larvae of Asian corn borer at the mid-whorl(firstgeneration),pre-tassel(first-and/or second-generation),and silk(second-generation)growth stages.The transgenic Bt maize hybrid sustained significantly less leaf feeding damage(rating 1.0±0.0)than its negative isoline control(rating 7.3±0.1).With the Bt maize,1.3-6.8%of plants were damaged by corn borer tunneling with<0.5 cm tunneling per stalk under different levels of infestation,compared with 100%of plants damaged with 9.3-25.0 cm tunneling per stalk for the negative isoline control. On average,transgenic Bt maize hybrids had only 0.01-0.05 tunnels per stalk and no stems were broken.In contrast,the negative isoline control had 3.11-8.36 tunnels per stalk and 31.2-73.9% of stems broken.Yields were significantly higher in trahsgenic Bt maize than in the control. These results demonstrate that transgenic Bt maize can significantly minimize yield losses caused by the Asian corn borer through resistance to the first-and second-generation larvae.

  6. Expression of recombinant human lysozyme in the milk of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    YU Zhengquan; FAN Baoliang; DAI Yunping; ZHENG Ming; NIU Huiling; WANG Meili; WANG Lili; FEI Jing; LI Ning

    2003-01-01

    Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal ofimproving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic micewas 0.2 mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.

  7. Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.

    Science.gov (United States)

    Maga, Elizabeth A; Cullor, James S; Smith, Wayne; Anderson, Gary B; Murray, James D

    2006-01-01

    The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.

  8. Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

    Directory of Open Access Journals (Sweden)

    Gonzalez-Ruiz Gloriene

    2011-08-01

    Full Text Available Abstract Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1 and polyphosphate kinase (ppk genes in bacteria in order to provide high mercury resistance and accumulation. Results In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. Conclusion The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and

  9. Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

    Science.gov (United States)

    Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-03-01

    Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection.

  10. 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.

  11. Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening).

    Science.gov (United States)

    Dutt, Manjul; Barthe, Gary; Irey, Michael; Grosser, Jude

    2015-01-01

    Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB), a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars 'Hamlin' and 'Valencia' expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2) promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

  12. Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening.

    Directory of Open Access Journals (Sweden)

    Manjul Dutt

    Full Text Available Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB, a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars 'Hamlin' and 'Valencia' expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2 promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

  13. Hydrogel Macroporosity and the Prolongation of Transgene Expression and the Enhancement of Angiogenesis

    Science.gov (United States)

    Shepard, Jaclyn A.; Virani, Farrukh R.; Goodman, Ashley G.; Gossett, Timothy D.; Shin, Seungjin; Shea, Lonnie D.

    2012-01-01

    The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37°C. The macropores were interconnected, as evidenced by homogeneous cell seeding in vitro and complete cell infiltration in vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications. PMID:22800542

  14. Neural stem cells genetically-modified to express neprilysin reduce pathology in Alzheimer transgenic models.

    Science.gov (United States)

    Blurton-Jones, Mathew; Spencer, Brian; Michael, Sara; Castello, Nicholas A; Agazaryan, Andranik A; Davis, Joy L; Müller, Franz-Josef; Loring, Jeanne F; Masliah, Eliezer; LaFerla, Frank M

    2014-04-16

    Short-term neural stem cell (NSC) transplantation improves cognition in Alzheimer's disease (AD) transgenic mice by enhancing endogenous synaptic connectivity. However, this approach has no effect on the underlying beta-amyloid (Aβ) and neurofibrillary tangle pathology. Long term efficacy of cell based approaches may therefore require combinatorial approaches. To begin to examine this question we genetically-modified NSCs to stably express and secrete the Aβ-degrading enzyme, neprilysin (sNEP). Next, we studied the effects of sNEP expression in vitro by quantifying Aβ-degrading activity, NSC multipotency markers, and Aβ-induced toxicity. To determine whether sNEP-expressing NSCs can also modulate AD-pathogenesis in vivo, control-modified and sNEP-NSCs were transplanted unilaterally into the hippocampus of two independent and well characterized transgenic models of AD: 3xTg-AD and Thy1-APP mice. After three months, stem cell engraftment, neprilysin expression, and AD pathology were examined. Our findings reveal that stem cell-mediated delivery of NEP provides marked and significant reductions in Aβ pathology and increases synaptic density in both 3xTg-AD and Thy1-APP transgenic mice. Remarkably, Aβ plaque loads are reduced not only in the hippocampus and subiculum adjacent to engrafted NSCs, but also within the amygdala and medial septum, areas that receive afferent projections from the engrafted region. Taken together, our data suggest that genetically-modified NSCs could provide a powerful combinatorial approach to not only enhance synaptic plasticity but to also target and modify underlying Alzheimer's disease pathology.

  15. Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.

    Science.gov (United States)

    Ayella, Allan K; Trick, Harold N; Wang, Weiqun

    2007-12-01

    Lignans are phenylpropane dimers that are biosynthesized via the phenylpropanoid pathway, in which pinoresinol lariciresinol reductase (PLR) catalyzes the last steps of lignan production. Our previous studies demonstrated that the contents of lignans in various wheat cultivars were significantly associated with anti-tumor activities in APC(Min) mice. To enhance lignan biosynthesis, this study was conducted to transform wheat cultivars ('Bobwhite', 'Madison', and 'Fielder', respectively) with the Forsythia intermedia PLR gene under the regulatory control of maize ubiquitin promoter. Of 24 putative transgenic wheat lines, we successfully obtained 3 transformants with the inserted ubiquitin-PLR gene as screened by PCR. Southern blot analysis further demonstrated that different copies of the PLR gene up to 5 were carried out in their genomes. Furthermore, a real-time PCR indicated approximately 17% increase of PLR gene expression over the control in 2 of the 3 positive transformants at T(0) generation. The levels of secoisolariciresinol diglucoside, a prominent lignan in wheat as determined by HPLC-MS, were found to be 2.2-times higher in one of the three positive transgenic sub-lines at T(2 )than that in the wild-type (117.9 +/- 4.5 vs. 52.9 +/- 19.8 mug/g, p <0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.

  16. Expression of viral EPS-depolymerase reduces fire blight susceptibility in transgenic pear.

    Science.gov (United States)

    Malnoy, Mickaël; Faize, Mohamed; Venisse, Jean-Stéphane; Geider, Klaus; Chevreau, Elisabeth

    2005-02-01

    Erwinia amylovora is the causal agent of fire blight of Maloideae. One of the main pathogenicity factors of this bacterium is the exopolysaccharide (EPS) of its capsule. In this paper, we used genetic transformation tools to constitutively express an EPS-depolymerase transgene in the pear (Pyrus communis L.) cv. Passe Crassane with the aim of decreasing its high susceptibility to fire blight. Expression of the depolymerase gene in 15 independent transgenic clones led, on average, to low depolymerase activity, although relatively high expression was observed at the transcriptional and translational levels. Only two of the transgenic clones (9X and 10M) consistently showed a decrease in fire blight susceptibility in vitro and in the greenhouse. These clones were also among the highest expressers of depolymerase at the RNA and enzyme activity levels. The correlation observed among all transgenic clones between depolymerase expression and fire blight resistance suggested the potential of this strategy.

  17. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

    Science.gov (United States)

    Kerr, D E; Plaut, K; Bramley, A J; Williamson, C M; Lax, A J; Moore, K; Wells, K D; Wall, R J

    2001-01-01

    Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

  18. New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin.

    Science.gov (United States)

    Gunning, Robin V; Dang, Ho T; Kemp, Fred C; Nicholson, Ian C; Moores, Graham D

    2005-05-01

    In Australia, the cotton bollworm, Helicoverpa armigera, has a long history of resistance to conventional insecticides. Transgenic cotton (expressing the Bacillus thuringiensis toxin Cry1Ac) has been grown for H. armigera control since 1996. It is demonstrated here that a population of Australian H. armigera has developed resistance to Cry1Ac toxin (275-fold). Some 70% of resistant H. armigera larvae were able to survive on Cry1Ac transgenic cotton (Ingard) The resistance phenotype is inherited as an autosomal semidominant trait. Resistance was associated with elevated esterase levels, which cosegregated with resistance. In vitro studies employing surface plasmon resonance technology and other biochemical techniques demonstrated that resistant strain esterase could bind to Cry1Ac protoxin and activated toxin. In vivo studies showed that Cry1Ac-resistant larvae fed Cy1Ac transgenic cotton or Cry1Ac-treated artificial diet had lower esterase activity than non-Cry1Ac-fed larvae. A resistance mechanism in which esterase sequesters Cry1Ac is proposed.

  19. A Stably Transgenic INA Enterobacter cloacae for Control of Insect Pests

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xin-jian; SUN Fu-zai; ZHAO Ting-chang; DING Ai-yun; TANG Chao-rong

    2003-01-01

    Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition.We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1. 2022ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure.The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control).The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at - 5 or - 7℃ for 12 h, the percentages of larvae frozen to death were 85and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability.Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.

  20. A potential and novel type transgenic corn plant for control of the Corn Borer

    Science.gov (United States)

    Yue, Zhen; Li, Xiangrui; Zhang, Enyan; Liu, Xiaoxia; Zhao, Zhangwu

    2017-01-01

    The corn borer is a world-wide agricultural pest. In this study, a full-length neuropeptide F (npf) gene in Ostrinia furnacalis was sequenced and cloned from a cDNA library, in which the npf gene produces two splicing mRNA variants - npf1 and npf2 (with a 120 bp segment inserted into the npf1 sequence to generate npf2). A spatio-temporal expression analysis showed that the highest expression level of npf was in the midgut of 5th instar larvae (the gluttony period), and their npf expression and food consumption were significantly promoted after food deprivation for 6 h. When npf was knocked down by double-stranded RNA for NPF, larval food intake, weight and body size were effectively inhibited through changes of a biosynthesis and metabolism pathway; i.e. gene silencing of NPF causes decreases of total lipid and glycogen and increases of trehalose production. Moreover, we produced transgenic corn plants with stably expressed dsNPF. Results showed that O. furnacalis larvae fed on these transgenic leaves had lower food consumption and smaller body size compared to controls. These results indicate that NPF is important in the feeding control of O. furnacalis and valuable for production of potential transgenic corn. PMID:28290513

  1. Construction and analysis of the transgenic carrot and celery plants expressing the recombinant thaumatin II protein

    Directory of Open Access Journals (Sweden)

    Luchakivska Yu. S.

    2015-08-01

    Full Text Available Aim To obtain the transgenic carrot and celery plants able to express recombinant thaumatin II in order to increase plant stress tolerance. Methods. Agrobacterium-mediated transformation of the carrot and celery seedlings was used for obtaining the transgenic plants. Presence and transcription of the transgene in plant tissues were proved by PCR and RT-PCR analysis. The plants were tested for the biotic stress tolerance by in vitro antifungal and antibacterial activity assays and for the salinity and osmotic stress tolerance by plant survival test in presence of NaCl and PEG in different concentrations. Results. Transgenic plants able to express recombinant thaumatin II gene (transcription proved for 60–100 % were obtained by agrobacterial transformation. The transgenic carrot plant extracts inhibited the growth of the studied phytopathogenic bacteria strains but exhibited no antifungal activity. Survival level of transgenic plants under the salinity and osmotic stress effect was definitely higher comparing to the untransgenic ones. The analysis of the photosynthetic pigment content in the transgenic carrot plants showed no significant difference of this parameter under salinity stress that may indicate a possible protective activity of the recombinant protein. Conclusions. The obtained in our study transgenic carrot and celery plants able to express the recombinant thaumatin II gene were characterized by antibacterial activity and increased tolerance to salinity and osmotic stress factors.

  2. Transgene expression in microalgae – from tools to applications

    Directory of Open Access Journals (Sweden)

    Lior eDoron

    2016-04-01

    Full Text Available Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide

  3. Tissue-specific posttranscriptional downregulation of expression of the S100A4(mts1) gene in transgenic animals

    DEFF Research Database (Denmark)

    Ambartsumian, N; Klingelhöfer, Jörg; Grigorian, M

    1998-01-01

    The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous...... and constitutive 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) gene promoter. In transgenic animals the expression of the transgene RNA was detected in all organs, but only some of the organs showed elevated levels of the protein. Expression of the S100A4(Mts1) protein was downregulated in the organs...... that normally do not express the gene in the wild-type animal. The transgene RNA is detected in the polysomes indicating that it could be translated into the S100A4(Mts1) protein. The specificity of the S100A4(Mts1) protein expression is determined by a complex mechanism including regulation of translation and...

  4. Reduced Mid1 expression and delayed neuromotor development in daDREAM transgenic mice

    Directory of Open Access Journals (Sweden)

    Mara eDierssen

    2012-05-01

    Full Text Available DREAM (downstream regulatory element antagonist modulator is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. Previous work has shown a role for DREAM in cerebellar function regulating the expression of the sodium/calcium exchanger 3 (NCX3 in cerebellar granular neurons to control Ca2+ homeostasis and survival of these neurons. To achieve a global view of the genes regulated by DREAM in the cerebellum, we performed a genome-wide analysis in transgenic cerebellum expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM. Here we show that DREAM regulates the expression of the midline 1 (Mid1 gene early after birth. As a consequence, daDREAM mice exhibit a significant shortening of the rostro-caudal axis of the cerebellum and a severe delay in neuromotor development early after birth. Our results indicate a role for DREAM in cerebellar function.

  5. A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters.

    Science.gov (United States)

    Tchorz, Jan S; Suply, Thomas; Ksiazek, Iwona; Giachino, Claudio; Cloëtta, Dimitri; Danzer, Claus-Peter; Doll, Thierry; Isken, Andrea; Lemaistre, Marianne; Taylor, Verdon; Bettler, Bernhard; Kinzel, Bernd; Mueller, Matthias

    2012-01-01

    Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.

  6. Transgenic chickens expressing human urokinase-type plasminogen activator.

    Science.gov (United States)

    Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

    2013-09-01

    Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

  7. Recurrent selection for transgene expression levels in maize results in proxy selection for a native gene with the same promoter

    Science.gov (United States)

    High expression levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High expression levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurre...

  8. The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana.

    Science.gov (United States)

    Janni, Michela; Sella, Luca; Favaron, Francesco; Blechl, Ann E; De Lorenzo, Giulia; D'Ovidio, Renato

    2008-02-01

    A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.

  9. Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.

    Science.gov (United States)

    Rao, K V; Rathore, K S; Hodges, T K; Fu, X; Stoger, E; Sudhakar, D; Williams, S; Christou, P; Bharathi, M; Bown, D P; Powell, K S; Spence, J; Gatehouse, A M; Gatehouse, J A

    1998-08-01

    Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (Nilaparvata lugens; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes 'hopper burn', as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice (Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.

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

    OpenAIRE

    2014-01-01

    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...

  11. Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

    Science.gov (United States)

    Hasegawa, Yoshinori; Ishikura, Tomoyuki; Hasegawa, Takanori; Watanabe, Takashi; Suzuki, Junpei; Nakayama, Manabu; Okamura, Yoshiaki; Okazaki, Tuneko; Koseki, Haruhiko; Ohara, Osamu; Ikeno, Masashi; Masumoto, Hiroshi

    2015-03-01

    The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

  12. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco

    Energy Technology Data Exchange (ETDEWEB)

    Cahoon, E.B.; Shanklin, J.; Ohlrogge, J.B. (Michigan State Univ., East Lansing (United States))

    1992-12-01

    Little is known about the metabolic origin of petroselinic acid (18:1[Delta][sup 6cis]), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the [Delta][sup 9]-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and [Delta][sup 4]-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase. 27 refs., 5 figs.

  13. Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco.

    Science.gov (United States)

    Cahoon, E B; Shanklin, J; Ohlrogge, J B

    1992-12-01

    Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase.

  14. Food intake and appetite control in a GH-transgenic zebrafish.

    Science.gov (United States)

    Dalmolin, Camila; Almeida, Daniela Volcan; Figueiredo, Marcio Azevedo; Marins, Luis Fernando

    2015-10-01

    The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. The regulatory network for GH is complex and includes many central and peripheral endocrine factors as well as that from the environment. It is known that GH transgenesis results in increased growth, food intake, and consequent metabolic rates in fishes. However, the manner in which GH transgenesis alters the energetic metabolism in fishes has not been well explored. In order to elucidate these consequences, we examined the effect of GH overexpression on appetite control mechanisms in a transgenic zebrafish (Danio rerio) model. To this, we analyzed feeding behavior and the expression of the main appetite-related genes in two different feeding periods (fed and fasting) in non-transgenic (NT) and transgenic (T) zebrafish as well as glycaemic parameters of them. Our initial results have shown that NT males and females present the same feeding behavior and expression of main appetite-controlling genes; therefore, the data of both sexes were properly grouped. Following grouped data analyses, we compared the same parameters in NT and T animals. Feeding behavior results have shown that T animals eat significantly more and faster than NT siblings. Gene expression results pointed out that gastrointestinal (GT) cholecystokinin has a substantial contribution to the communication between peripheral and central control of food intake. Brain genes expression analyses revealed that T animals have a down-regulation of two strong and opposite peptides related to food intake: the anorexigenic proopiomelanocortin (pomc) and the orexigenic neuropeptide Y (npy). The down-regulation of pomc in T when compared with NT is an expected result, since the decrease in an anorexigenic factor might keep the transgenic fish hungry. The down-regulation of npy seemed to be contradictory at first, but if we consider the GH's capacity to

  15. Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice

    Directory of Open Access Journals (Sweden)

    Lambert PF

    2008-11-01

    Full Text Available Abstract Background Although K14E6 transgenic mice develop spontaneous tumors of the skin epithelium, no spontaneous reproductive tract malignancies arise, unless the transgenic mice were treated chronically with 17β-estradiol. These findings suggest that E6 performs critical functions in normal adult cervix and skin, highlighting the need to define E6-controlled transcriptional programs in these tissues. Methods We evaluated the expression profile of 14,000 genes in skin or cervix from young K14E6 transgenic mice compared with nontransgenic. To identify differentially expressed genes a linear model was implemented using R and the LIMMA package. Two criteria were used to select the set of relevant genes. First a set of genes with a Log-odds ≥ 3 were selected. Then, a hierarchical search of genes was based on Log Fold Changes. Results Microarray analysis identified a total of 676 and 1154 genes that were significantly up and down-regulated, respectively, in skin from K14E6 transgenic mice. On the other hand, in the cervix from K14E6 transgenic mice we found that only 97 and 252 genes were significantly up and down-regulated, respectively. One of the most affected processes in the skin from K14E6 transgenic mice was the cell cycle. We also found that skin from transgenic mice showed down-regulation of pro-apoptotic genes and genes related to the immune response. In the cervix of K14E6 transgenic mice, we could not find affected any gene related to the cell cycle and apoptosis pathways but did observe alterations in the expression of immune response genes. Pathways such as angiogenesis, cell junction and epidermis development, also were altered in their gene expression profiles in both tissues. Conclusion Expression of the HPV16 E6 oncoprotein in our model alters expression of genes that fell into several functional groups providing insights into pathways by which E6 deregulate cell cycle progression, apoptosis, the host resistance to infection

  16. Targeted expression of SV40 T antigen in the hair follicle of transgenic mice produces an aberrant hair phenotype.

    Science.gov (United States)

    Keough, R; Powell, B; Rogers, G

    1995-03-01

    Directed expression of SV40 large T antigen (TAg) in transgenic mice can induce tissue-specific tumorigenesis and useful cell lines exhibiting differentiated characteristics can be established from resultant tumor cells. In an attempt to produce an immortalised mouse hair follicle cortical cell line for the study of hair keratin gene control, SV40 TAg expression was targeted to the hair follicles of transgenic mice using a sheep hair gene promoter. Expression of SV40 TAg in the follicle cortex disrupted normal fiber ultrastructure, producing a marked phenotypic effect. Affected hairs were wavy or severely kinked (depending on the severity of the phenotype) producing an appearance ranging from a ruffled coat to a stubble covering the back of the mouse. The transgenic hairs appeared to be weakened at the base of the fibers, leading to premature hair-loss and a thinner pelage, or regions of temporary nudity. No follicle tumors or neoplasia were apparent and immortalisation of cortical cells could not be established in culture. In situ hybridisation studies in the hair follicle using histone H3 as a cell proliferation marker suggested that cell proliferation had ceased prior to commencement of K2.10-TAg expression and was not re-established in the differentiating cortical cells. Hence, TAg was unable to induce cell immortalisation at that stage of cortical cell differentiation. However, transgenic mice developed various other abnormalities including vertebral abnormalities and bladder, liver and intestinal tumors, which resulted in reduced life expectancy.

  17. Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene PvSR2

    Institute of Scientific and Technical Information of China (English)

    CHAI; Tuanyao; (柴团耀); CHEN; Qiong; (陈琼); ZHANG; Yuxiu; (张玉秀); DONG; Juan; (董娟); AN; Chengcai; (安成才)

    2003-01-01

    PvSR2 (Phaseolus vulgaris stress-related gene) has been cloned from French bean and shown to be expressed specifically upon heavy metal treatment. In order to investigate the role of PvSR2 in plant, PvSR2 gene under the control of cauliflower mosaic virus 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens LBA4404. The regenerated plantlets were selected on medium with 100 mg/L kanamycin. PCR and Southern blot analysis showed PvSR2 gene was integrated in tobacco genome. Gus and Northern blot analysis indicated PvSR2 gene was expressed in transgenic seedling. The heavy metal resistance assay showed that the transgenic tobacco seedlings with the PvSR2 coding sequence exhibited higher tolerance to Cd compared with wild-type (WT) under Cd exposure. The Cd content accumulated in root between transgenic and WT seedlings had no obvious difference at lower Cd external concentration (0.05-0.075 mmol/L CdCl2), whereas transgenic plant showed a lower root Cd content than the control at higher external Cd concentration (0.1 mmol/L CdCl2). These results suggested that the expression of PvSR2 can enhance the Cd tolerance, and PvSR2 may be involved in Cd transportation and accumulation at the test concentration of 0.1 mmol/L Cd.

  18. Increase of Expression Levels of Reporter Gene in Transgenic Tobaccos by Matrix Attachment Regions

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The matrix attachment region (MAR) located downstream of Plastocyanin gene was isolated from the genome of pea. To study the effect of MARs on foreign gene expression in transgenic plants, T-DNA vector was constructed in which MARs flanked bothβ-glucuronidase(GUS) gene and selectable marker neomycin phosphotransferase (NPT-II) gene. The plant expression vectors were transferred into leaf discs via Agrobacterium-mediated transformation procedure. The result of GUS measurement showed that pea MAR could increase transgene expression level. The mean expression levels of GUS gene expression in population containing MARs could be increased twofold when compared with that of population without MARs.

  19. Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

    Science.gov (United States)

    François, Isabelle E J A; De Bolle, Miguel F C; Dwyer, Geoff; Goderis, Inge J W M; Woutors, Piet F J; Verhaert, Peter D; Proost, Paul; Schaaper, Wim M M; Cammue, Bruno P A; Broekaert, Willem F

    2002-04-01

    We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

  20. Signal Peptide of Potato PinⅡ Enhances the Expression of Cry1Ac in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    Yun-Jun LIU; Yuan YUAN; Jun ZHENG; Ya-Zhong TAO; Zhi-Gang DONG; Jian-Hua WANG; Guo-Ying WANG

    2004-01-01

    The modified Cry1Ac was expressed in transgenic tobacco plants.To allow secretion of the Cry1Ac protein into the intercellular space,the signal peptide sequence of potato proteinase inhibitor Ⅱ(pinⅡ)was N-terminally fused to the CrylAc encoding region.Expression of Cry1Ac in transgenic tobacco plants was assayed with ELISA.The results showed that pinⅡ signal peptide sequence enhanced the expression of Cry 1 Ac protein and led to the secretion of the Cry 1Ac protein in transgenic tobacco plants.GFP gene was also fused to the signal peptide sequence and transformed to tobacco.The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.

  1. Environmental and transgene expression effects on the barley seed proteome

    DEFF Research Database (Denmark)

    Finnie, Christine; Steenholdt, T.; Noguera, O.R.;

    2004-01-01

    The barley (Hordeum vulgare) cultivar Golden Promise is no longer widely used for malting, but is amenable to transformation and is therefore a valuable experimental cultivar. Its characteristics include high salt tolerance, however it is also susceptible to several fungal pathogens. Proteome....... Eleven of these were identified by mass spectrometric peptide mass mapping, including an abundant chitinase implicated in defence against fungal pathogens and a small heat-shock protein. To enable a comparison with transgenic seed protein patterns, differences in spot patterns between field...... with extra nitrogen. Finally, the fate of transgene products in barley seeds was followed. Spots containing two green fluorescent protein constructs and the herbicide resistance marker phosphinothricin acetyltransferase were observed in 2D-gel patterns of transgenic seeds and identified by mass spectrometry...

  2. Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg

    Institute of Scientific and Technical Information of China (English)

    Yi Gao; Yina Ma; Mei Li; Tonq Chenq; Shao-Wei Li; Jun Zhang; Ning-Shao Xia

    2003-01-01

    AIM: To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests.METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice's serum were examined by ELISA every week.RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo,level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight.In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response.CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.

  3. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.

    Science.gov (United States)

    Kamioka, Yuji; Sumiyama, Kenta; Mizuno, Rei; Sakai, Yoshiharu; Hirata, Eishu; Kiyokawa, Etsuko; Matsuda, Michiyuki

    2012-01-01

    Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

  4. Iron biofortification and homeostasis in transgenic cassava roots expressing an algal iron assimilatory protein, FEA1

    Directory of Open Access Journals (Sweden)

    Uzoma eIhemere

    2012-09-01

    Full Text Available We have engineered the starchy root crop cassava (Manihot esculenta to express the Chlamydomonas reinhardtii iron assimilatory protein, FEA1, in roots to enhance its nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 gm meal. Significantly, the expression of the FEA1 protein did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of iron mediated by the FEA1 protein. Relative to wild-type plants, FEA1 expressing plants had reduced Fe(III chelate reductase activity and gene expression levels consistent with the more efficient uptake of iron in FEA1 transgenic plants. We also show that genes involved in iron homeostasis in cassava have altered tissue-specific patterns of expression in transgenic plants. Steady state transcript levels of the metal-chelate transporter MeYSL1, and the iron storage proteins, MeFER2 and MeFER6, were elevated in various tissues of FEA1 transgenic plants compared to wild-type plants. These results suggest that these gene products play a role in iron translocation and homeostasis in FEA1 transgenic cassava plants. These results are discussed in terms of enhanced strategies for the iron biofortification of plants.

  5. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Callow, M.J.; Stoltzfus, L.J.; Rubin, E.M. [Lawrence Berkeley Lab., CA (United States); Lawn, R.M. [Stanford Univ., CA (United States)

    1994-03-15

    The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vivo analyses partly because it is found in a limited number of experimental animals. Although transgenic mice expressing human apolipoprotein (a) [apo(a)] have previously been described, they failed to assemble Lp(a) particles because of the inability of human apo(a) to associate with mouse apolipoprotein B (apoB). The authors isolated a 90-kilobase P1 phagemid containing the human apoB gene and with this DNA generated 13 lines of transgenic mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. Plasma concentrations of human apoB, as well as low density lipoprotein (LDL), were related to transgene copy number; the transgenic line with the most copies of human apoB had a >4-fold increase in LDL cholesterol compared with nontransgenics and a lipoprotein profile similar to that of humans. When human apoB and apo(a) transgenic mice were bred together, plasma apo(a) in mice expressing both human proteins was tightly associated with lipoproteins in the LDL density region. These studies demonstrate the successful expression of human apoB and the efficient assembly of Lp(a) in mice.

  6. Transgenic organisms expressing genes from Bacillus thuringiensis to combat insect pests.

    Science.gov (United States)

    Zaritsky, Arieh; Ben-Dov, Eitan; Borovsky, Dov; Boussiba, Sammy; Einav, Monica; Gindin, Galina; Horowitz, A Rami; Kolot, Mikhail; Melnikov, Olga; Mendel, Zvi; Yagil, Ezra

    2010-01-01

    Various subspecies (ssp.) of Bacillus thuringiensis (Bt) are considered the best agents known so far to control insects, being highly specific and safe, easily mass produced and with long shelf life.1 The para-crystalline body that is produced during sporulation in the exosporium includes polypeptides named δ-endotoxins, each killing a specific set of insects. The different entomopathogenic toxins of various Bt ssp. can be manipulated genetically in an educated way to construct more efficient transgenic bacteria or plants that express combinations of toxin genes to control pests.2 Joint research projects in our respective laboratories during the last decade demonstrate what can be done by implementing certain ideas using molecular biology with Bt ssp. israelensis (Bti) as a model system. Here, we describe our progress achieved with Gram-negative bacterial species, including cyanobacteria, and some preliminary experiments to form transgenic plants, mainly to control mosquitoes (Diptera), but also a particular Lepidopteran and Coleopteran pest species. In addition, a system is described by which environment-damaging genes can be removed from the recombinants thus alleviating procedures for obtaining permits to release them in nature.

  7. Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

    Institute of Scientific and Technical Information of China (English)

    Jiang-Li DONG; Bo ZHOU; Gang SHENG; Tao WANG

    2005-01-01

    Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). The VP6 and sVp6genes were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP). Then, BALB/c female mice that had been gavaged weekly with 10 mg TSP containing 34 μg VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.

  8. Segregation and expression of transgenes in the progenies of Bt ...

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-04-17

    Apr 17, 2012 ... Therefore, it is feasible using GUS-assisted-selection to preliminarily identify the Bt gene and study the inheritance of transgenes in breeding program. Mendelian ... higher plants including tobacco, tomato and cotton. (Vaeck et .... The size of fragment was estimated using marker ladder as shown on the left.

  9. Expression of bgt gene in transgenic birch (Betula platyphylla Suk.)

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... dispar was verified in feeding bioassays with the insects. ... After the hybridization of the bgt probe, membranes were stripped twice for 15 min ... Southern blot analysis of transgenic birch plant DNA digested with PstI. Lane 1 ...

  10. Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

    Directory of Open Access Journals (Sweden)

    Xiaoming HU,Jing GUO,Chunling BAI,Zhuying WEI,Li GAO,Tingmao HU,Shorgan BOU,Guangpeng LI

    2016-03-01

    Full Text Available In the present study, follistatin (FST gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F0, F1 and F2. Total muscle growth in F0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

  11. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  12. Transgenic rice homozygous lines expressing GNA showed enhanced resistance to rice brown planthopper

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Mature seed-derived calli from two elite Chinese japonica rice (Oryza sativa L.) cultivars Eyi 105 and Ewan 5 were co-transformed with two plasmids, pWRG1515 and pRSSGNA1, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis)lectin gene (gna) via particle bombardment. 61 independent transgenic rice plants were regenerated from 329 bombarded calli. 79% transgenic plants contained all the three genes, revealed by PCR/Southern blot analysis. Western blot analysis revealed that 36 out of 48 gna-containing transgenic plants expressed GNA (75 %) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From the R2 generations whose R1 parent plants showing 3:1 Mendelian segregation patterns,we identified five independent homozygous lines containing and expressing all the three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and declining BPH feeding.These BPH-resistant lines have been incorporated into rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.

  13. Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice.

    Directory of Open Access Journals (Sweden)

    Cissy Chenyi Zhou

    Full Text Available BACKGROUND: Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. PRINCIPAL FINDINGS: By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa and adenosine deaminase (Ada, we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. SIGNIFICANCE: In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.

  14. Expression of cartilage developmental genes in Hoxc8- and Hoxd4-transgenic mice.

    Directory of Open Access Journals (Sweden)

    Claudia Kruger

    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.

  15. Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

    Science.gov (United States)

    Hopp, Ann-Katrin; Saenger, Mélanie; Soichot, Julien; Scholze, Heidi; Boch, Jens; Blandin, Stéphanie A.; Marois, Eric

    2017-01-01

    Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites. PMID:28095489

  16. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等

    1995-01-01

    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  17. Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver.

    Science.gov (United States)

    Zhu, Huan-Zhang; Chen, Jian-Quan; Cheng, Guo-Xiang; Xue, Jing-Lun

    2003-08-01

    To establish transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases. Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6 strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1 generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR. Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant Alb-Cre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice. Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice. Transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase under control of the liver-specific promoter are preliminary established.

  18. Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands.

    Science.gov (United States)

    Sumitani, M; Kasashima, K; Yamamoto, D S; Yagi, K; Yuda, M; Matsuoka, H; Yoshida, S

    2013-02-01

    We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.

  19. The formation of brown adipose tissue induced by transgenic over-expression of PPARγ2.

    Science.gov (United States)

    Zhou, Ying; Yang, Jinzeng; Huang, Jinliang; Li, Ting; Xu, Dequan; Zuo, Bo; Hou, Liming; Wu, Wangjun; Zhang, Lin; Xia, Xiaoliang; Ma, Zhiyuan; Ren, Zhuqing; Xiong, Yuanzhu

    2014-04-18

    Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.

  20. Transgenic Expression of Interferon-γ in Mouse Stomach Leads to Inflammation, Metaplasia, and Dysplasia

    Science.gov (United States)

    Syu, Li-Jyun; El-Zaatari, Mohamad; Eaton, Kathryn A.; Liu, Zhiping; Tetarbe, Manas; Keeley, Theresa M.; Pero, Joanna; Ferris, Jennifer; Wilbert, Dawn; Kaatz, Ashley; Zheng, Xinlei; Qiao, Xiotan; Grachtchouk, Marina; Gumucio, Deborah L.; Merchant, Juanita L.; Samuelson, Linda C.; Dlugosz, Andrzej A.

    2013-01-01

    Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H+/K+ ATPase β promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear β-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion. PMID:23036899

  1. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

    Science.gov (United States)

    Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

    2017-01-12

    Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2(-ΔΔCt) analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g(-1) fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g(-1) fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

  2. Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver.

    Science.gov (United States)

    Argyros, Orestis; Wong, Suet Ping; Fedonidis, Constantinos; Tolmachov, Oleg; Waddington, Simon N; Howe, Steven J; Niceta, Marcello; Coutelle, Charles; Harbottle, Richard P

    2011-05-01

    We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.

  3. Regeneration of transgenic loblolly pine expressing genes for salt tolerance

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Salinity stress is one of the most serious factors limiting the distribution and productivity of crops and forest trees. The detrimental effects of salt on plants are a consequence of both a water deficit resulting in osmotic stress and the effects of excess sodium ions on critical biochemical process. A novel approach to improve salt tolerance has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBIGM which carrying the mannitol-1-phosphate dehydrogenase (Mt1D) and glucitol-6-phosphate dehydrogenase (GutD). Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15mg/L kanamycin and confirmed by Southern blot analysis of genomic DNA. Salt tolerance assays demonstrated that the salt tolerance of transgenic calli and regenerated plantlets were increased. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

  4. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    OpenAIRE

    Yuanxin Miao; Jinzeng Yang; Zhong Xu; Lu Jing; Shuhong Zhao; Xinyun Li

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type li...

  5. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    OpenAIRE

    Yuanxin Miao; Jinzeng Yang; Zhong Xu; Lu Jing; Shuhong Zhao; Xinyun Li

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type li...

  6. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  7. RNA helicase Belle/DDX3 regulates transgene expression in Drosophila.

    Science.gov (United States)

    Lo, Pang-Kuo; Huang, Yi-Chun; Poulton, John S; Leake, Nicholas; Palmer, William H; Vera, Daniel; Xie, Gengqiang; Klusza, Stephen; Deng, Wu-Min

    2016-04-01

    Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we show that genetic inhibition of Bel function leads to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited.

  8. Low levels of the reverse transactivator fail to induce target transgene expression in vascular smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Nikenza Viceconte

    Full Text Available Hutchinson-Gilford progeria syndrome (HGPS is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.

  9. A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

    DEFF Research Database (Denmark)

    Klinck, Rasmus; Füchtbauer, Ernst-Martin; Ahnfelt-Rønne, Jonas;

    2011-01-01

    of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates...

  10. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene

    National Research Council Canada - National Science Library

    Wharram, Bryan L; Goyal, Meera; Wiggins, Jocelyn E; Sanden, Silja K; Hussain, Sabiha; Filipiak, Wanda E; Saunders, Thomas L; Dysko, Robert C; Kohno, Kenji; Holzman, Lawrence B; Wiggins, Roger C

    2005-01-01

    .... For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed...

  11. Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues

    Directory of Open Access Journals (Sweden)

    Seoghyun Lee

    2016-01-01

    Full Text Available Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet-regulated system. Exploiting a Drosophila ecdysone receptor (EcR-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+ and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site. Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

  12. High-level, erythroid specific, expression of the human α-globin gene in transgenic mice and the production of human haemoglobin in murine erythrocytes.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves (David); F.G. Grosveld (Frank)

    1989-01-01

    textabstractUsing the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of

  13. Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.

  14. Over-Expression of ICE1 Gene in Transgenic Rice Improves Cold Tolerance

    Institute of Scientific and Technical Information of China (English)

    XIANG Dian-jun; HU Xiang-yang; ZHANG Yu; YIN Kui-de

    2008-01-01

    ICE1, an Arabidopsis thaliana transcription factor gene, was cloned by RT-PCR and successfully transformed into rice variety Kenjiandao 10 by the Agrobacterium-mediated transformation method. PCR amplification and Southern blot analysis indicated that ICE1 had been integrated into rice genome. Compared with the non-transgenic plants, the transgenic plants exhibited high resistance to hygromycin B and were consistent with the Mendelian inheritance of a single copy of the transgenic ICE1. Under the low temperature stress, the transgenic plants showed the lower mortality rate and the increased proline content. These results suggest that the Arabidopsis ICE1 is functional in rice and the over-expression of ICE1 improves the tolerance to cold stress in rice.

  15. A 28 nt long synthetic 5′UTR (synJ as an enhancer of transgene expression in dicotyledonous plants

    Directory of Open Access Journals (Sweden)

    Kanoria Shaveta

    2012-11-01

    Full Text Available Abstract Background A high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ, which enhances gene expression in tobacco and cotton. Results The influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants. Conclusions synJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in

  16. Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Tamura, Masayuki; Tsuji, Yukiko; Kusunose, Tatsuya; Okazawa, Atsushi; Kamimura, Naofumi; Mori, Tetsuya; Nakabayashi, Ryo; Hishiyama, Shojiro; Fukuhara, Yuki; Hara, Hirofumi; Sato-Izawa, Kanna; Muranaka, Toshiya; Saito, Kazuki; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji; Kajita, Shinya

    2014-10-01

    Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4'-feruloyl ethers. In-depth quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.

  17. Transgenic rice expressing Allium sativum leaf agglutinin (ASAL exhibits high-level resistance against major sap-sucking pests

    Directory of Open Access Journals (Sweden)

    Vudem Dasavantha

    2008-10-01

    Full Text Available Abstract Background Rice (Oryza sativa productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Results Allium sativum leaf lectin gene (asal, coding for mannose binding homodimeric protein (ASAL from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH, green leafhopper (GLH and whitebacked planthopper (WBPH. Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. Conclusion In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice

  18. Transgenic alfalfa plants expressing AtNDPK2 exhibit increased growth and tolerance to abiotic stresses.

    Science.gov (United States)

    Wang, Zhi; Li, Hongbing; Ke, Qingbo; Jeong, Jae Cheol; Lee, Haeng-Soon; Xu, Bingcheng; Deng, Xi-Ping; Lim, Yong Pyo; Kwak, Sang-Soo

    2014-11-01

    In this study, we generated and evaluated transgenic alfalfa plants (Medicago sativa L. cv. Xinjiang Daye) expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SN plants) to develop plants with enhanced tolerance to various abiotic stresses. We selected two SN plants (SN4 and SN7) according to the expression levels of AtNDPK2 and the enzyme activity of NDPK in response to methyl viologen (MV)-mediated oxidative stress treatment using leaf discs for further characterization. SN plants showed enhanced tolerance to high temperature, NaCl, and drought stress on the whole-plant level. When the plants were subjected to high temperature treatment (42 °C for 24 h), the non-transgenic (NT) plants were severely wilted, whereas the SN plants were not affected because they maintained high relative water and chlorophyll contents. The SN plants also showed significantly higher tolerance to 250 mM NaCl and water stress treatment than the NT plants. In addition, the SN plants exhibited better plant growth through increased expression of auxin-related indole acetic acid (IAA) genes (MsIAA3, MsIAA5, MsIAA6, MsIAA7, and MsIAA16) under normal growth conditions compared to NT plants. The results suggest that induced overexpression of AtNDPK2 in alfalfa will be useful for increasing biomass production under various abiotic stress conditions.

  19. Transient Expression of Transgenic IL-12 in Mouse Liver Triggers Unremitting Inflammation Mimicking Human Autoimmune Hepatitis.

    Science.gov (United States)

    Gil-Farina, Irene; Di Scala, Marianna; Salido, Eduardo; López-Franco, Esperanza; Rodríguez-García, Estefania; Blasi, Mercedes; Merino, Juana; Aldabe, Rafael; Prieto, Jesús; Gonzalez-Aseguinolaza, Gloria

    2016-09-15

    The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encoding murine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, anti-nuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-γ remained elevated during the entire study period. IFN-γ was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies.

  20. Alteration of Methamphetamine-induced stereotypic behaviour in transgenic mice expressing HIV-1 envelope protein gp120.

    Science.gov (United States)

    Roberts, Amanda J; Maung, Ricky; Sejbuk, Natalia E; Ake, Christopher; Kaul, Marcus

    2010-02-15

    The use of drugs for recreational purposes, in particular Methamphetamine, is associated with an increased risk of infection with human immunodeficiency virus (HIV)-1. HIV-1 infection in turn can lead to HIV-associated neurological disorders (HAND) that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). Interestingly, post mortem brain specimens from HAD patients and transgenic (tg) mice expressing the viral envelope protein gp120 in the central nervous system display similar neuropathological signs. In HIV patients, the use of Methamphetamine appears to aggravate neurocognitive alterations. In the present study, we injected HIV/gp120tg mice and non-transgenic littermate control animals with Methamphetamine dissolved in Saline or Saline vehicle and assessed locomotion and stereotyped behaviour. We found that HIVgp120-transgenic mice differ significantly from non-transgenic controls in certain domains of their behavioural response to Methamphetamine. Thus this experimental model system may be useful to further study the mechanistic interaction of both the viral envelope protein and the psychostimulant drug in behavioural alterations and neurodegenerative disease.

  1. 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.

  2. Reduction of lesion growth rate of late blight plant disease in transgenic potato expressing harpin protein

    Institute of Scientific and Technical Information of China (English)

    李汝刚; 范云六

    1999-01-01

    Using harpin protein gene from apple fire blight pathogen Erwinia amylavora and potato prp1-1 promoter as main DNA elements, the feasibility of using pathogen infection-induced hypersensitive response was explored as a new strategy of engineering fungal disease resistance. Three plant transformation vectors were constructed and 68 transgenic potato plants were produced through Agrobacterium mediated transformation method. Southern, Northern and Western blot analysis demonstrated the insertion, transcription and protein expression of harpin protein gene in transgenic plants. Disease resistance test using a complex race of Phytophthora infestans as challenging pathogen showed that both constitutive and pathogen infection-induced expression of harpin protein gene in transgenic potato reduced the lesion growth rate of fungus. Among plants where harpin protein gene expression was induced only by fungus infection, two plants were found to be highly resistant to P. infestans infection. Fungal hyphae were not pr

  3. EXPRESSION OF CHITINASE GENE IN TRANSGENIC RAPE PLANTS

    Directory of Open Access Journals (Sweden)

    Lu Longdou

    2005-08-01

    Full Text Available The hypocotyl and cotyledon of Brassica napus L. H165 and Brassica juncea DB3 were transformed with chitinase gene and herbicide-resistance gene by co-culture with Agrobacterium tumefacients LBA4404, and rape plants were obtained which could grow on the medium containing herbicide. The PCR result showed that exotic genes were integrated in the genome of the rape. Further study was performed to determine the impact of temperature on the transgenic rate and the differentiation of explants.

  4. Use of the viral 2A peptide for bicistronic expression in transgenic mice

    Directory of Open Access Journals (Sweden)

    Trichas Georgios

    2008-09-01

    Full Text Available Abstract Background Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. Results To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato and a nuclear localised green fluorescent protein (H2B-GFP, separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating

  5. Expression of tissue inhibitor of matrix metalloproteinase-1 in aging of transgenic mouse liver

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is related to the aging of many organs, but few data are available on the change of TIMP-1 in liver aging. The purpose of this study was to investigate the expression and role of TIMP-1, matrix metalloproteinase-2 (MMP-2) and MMP-9 in the process of natural aging in the livers of normal and transgenic mice, and to detect the effects of TIMP-1 on oxidative level and anti-oxidative ability of the livers of transgenic young mice.Methods Normal and transgenic mice were divided into 3 groups according to their age: 3-month-old group (n=5), 12-month-old group (n=5) and 24-month-old group (n=5). Histopathological changes of the liver were observed after HE and Masson staining. The messenger RNA (mRNA) levels of TIMP-1, MMP-2 and MMP-9 were determined by semi-quantitative reverse transcriptional polymerase chain reaction; protein expression was measured by Western blot in the livers of normal and transgenic mice of various ages. Changes in levels of superoxide dismutase (SOD), monoamine oxidase (MAO), malondialdehyde (MDA) as well as oxidative and anti-oxidative ability were measured.Results Histologically, more fatty degeneration and collagen deposition were found in the aging livers of transgenic mice than in those of the normal mice as their age of months increased. The mRNA and protein expressions of TIMP-1 were significantly high in the oldest animals. The histopathological changes, mRNA and protein expressions of TIMP-1 increased significantly in the liver of transgenic mice as compared with normal mice. The expression of MMP-2 and MMP-9 showed a minor change in the process of aging. Liver change and collagen deposition were not observed in young mice, but the activity of SOD decreased (P<0.05), and the activity of MAO (P<0.01) and the content of MDA increased in the liver of transgenic mice (P<0.01).Conclusions The expression of TIMP-1 is significantly high in the liver of transgenic mouse in the

  6. Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors.

    Science.gov (United States)

    Sgourou, Argyro; Routledge, Samantha; Spathas, Dionysios; Athanassiadou, Aglaia; Antoniou, Michael N

    2009-08-20

    Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

  7. Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.

    Science.gov (United States)

    Wu, Tingquan; Tang, Dingzhong; Chen, Weida; Huang, Hexun; Wang, Rui; Chen, Yongfang

    2013-09-15

    Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed

  8. Resistance to rice blast(Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The gene of trichosanthin has been transferred into rice plants through agrobacterium method.The single copy insertion and the expression of foreign gene have been proved in regenerated plants.In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressing GUS gene as control have been evaluated.The differences such as the time of disease symptom observed,the number of infected plants and damaged leaves,the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant.The transgenic plants with trichosanthin gene grew faster than the plants with GUS gene,even when humidity environment was removed.The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control.In addition,no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.

  9. Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

    Science.gov (United States)

    Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem...

  10. Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout.

    Science.gov (United States)

    Uzbekova, Svetlana; Amoros, Claire; Cauty, Chantale; Mambrini, Muriel; Perrot, Elizabeth; Hew, Choy L; Chourrout, Daniel; Prunet, Patrick

    2003-04-01

    In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5' flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced. sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1-F4 lines harbouring sPRL-lacZ expressed beta-galactosidase (beta-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of beta-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, beta-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells,by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.

  11. Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

    Institute of Scientific and Technical Information of China (English)

    YE Hong-xai; LI Mei; Guo Ze-jian; Shu Qing-yao; xu Xiao-hui; BAO Jin-song; SHEN Sheng-quan

    2008-01-01

    A totaI of 105 transgenic rice lines independently transformed with a pea ferritin gene (Fer)were previously obtained.After seven generations of selfing and β-glucuronidase(GUS)assisted selection,82 transgenic lines with stable agronomic traits were got.Among the 82 transgenic lines,two high-iron transgenic rice lines Fer34 and Fer65,with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11,respectively were identified.In the two transgenic lines,the exogenous Fer gene was highly expressed,and inherited as a single locus.The transgene had no negative effect on the agronomic traits of rice plant,other mineral nutritional components,appearance quailty and eating quailty of the milled rice,indicating that these two lines were elite high-iron breeding lines.Furthermore,the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

  12. Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn

    Science.gov (United States)

    Dively, Galen P.; Finkenbinder, Chad

    2016-01-01

    Background Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. Methodology/Major Findings We present field monitoring data using Cry1Ab (1996–2016) and Cry1A.105+Cry2Ab2 (2010–2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab—event Bt11, and Cry1A.105+Cry2Ab2—event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. Conclusions/Significance After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt

  13. Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn.

    Science.gov (United States)

    Dively, Galen P; Venugopal, P Dilip; Finkenbinder, Chad

    2016-01-01

    Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. We present field monitoring data using Cry1Ab (1996-2016) and Cry1A.105+Cry2Ab2 (2010-2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab-event Bt11, and Cry1A.105+Cry2Ab2-event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H

  14. Transgenic resistance confers effective field level control of bacterial spot disease in tomato.

    Directory of Open Access Journals (Sweden)

    Diana M Horvath

    Full Text Available We investigated whether lines of transgenic tomato (Solanum lycopersicum expressing the Bs2 resistance gene from pepper, a close relative of tomato, demonstrate improved resistance to bacterial spot disease caused by Xanthomonas species in replicated multi-year field trials under commercial type growing conditions. We report that the presence of the Bs2 gene in the highly susceptible VF 36 background reduced disease to extremely low levels, and VF 36-Bs2 plants displayed the lowest disease severity amongst all tomato varieties tested, including commercial and breeding lines with host resistance. Yields of marketable fruit from transgenic lines were typically 2.5 times that of the non-transformed parent line, but varied between 1.5 and 11.5 fold depending on weather conditions and disease pressure. Trials were conducted without application of any copper-based bactericides, presently in wide use despite negative impacts on the environment. This is the first demonstration of effective field resistance in a transgenic genotype based on a plant R gene and provides an opportunity for control of a devastating pathogen while eliminating ineffective copper pesticides.

  15. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J. (CH-PA); (UPENN); (Danforth)

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  16. Green tea polyphenols control dysregulated glutamate dehydrogenase in transgenic mice by hijacking the ADP activation site.

    Science.gov (United States)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A; Smith, Thomas J

    2011-09-30

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic β-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  17. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    Science.gov (United States)

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

  18. In Vivo Recognition of Ovalbumin Expressed by Transgenic Leishmania Is Determined by Its Subcellular Localization1

    Science.gov (United States)

    Prickett, Sara; Gray, Peter M.; Colpitts, Sara L.; Scott, Phillip; Kaye, Paul M.; Smith, Deborah F.

    2009-01-01

    The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity. PMID:16585577

  19. Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

    Institute of Scientific and Technical Information of China (English)

    LI; Yan; (

    2001-01-01

    [1]Napoli, C., Lemieux, C., Jorgensen, R., Introduction of a chimeric chalone synthase gene into petunia results in reversible cosuppession of homologous genes in trans, The Plant Cell, 1990, 2: 279-289.[2]Van der Krol, A.R., Mur, L.A., Beld, M. M. et al., Flavonnoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression, The Plant Cell, 1990, 2: 291-299.[3]Manika, P.B., Bhadra, U., Birchler, J., Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by White-Adh transgene is Polycomb dependent, Cell, 1997, 90: 479-498.[4]de Carvalho Niebel, F., Frendo, P., Van Montagu, M. et al., Post-transcriptional cosuppression of ?-1,3-glucanase transgene expression in homozygous plants, EMBO J., 1992, 11: 2595-2602.[5]Van Blokland, R., Van der Geest, N., Mol, J. N. M. et al., Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover, The Plant Cell, 1994, 6: 861-877.[6]Stam, M., Mol, J. N. M., Kooter, J. M., The silence of genes in transgenic plants, Annals of Bot., 1997, 79: 3-12.[7]Vaucheret, H., Beclin, C., Elmayan, T. et al., Transgene-induced gene silencing in plants, Plant J., 1998, 16(6): 651-659.[8]Shao, L., Li, Y., Yang, M. Z. et al., Transformation of Petunia hybrida with chalcone synthase A (chsA) resulting flower colour alteration and male sterility, Acta Botanica Sinica (in Chinese), 1996, 38(7): 517-524.[9]Koes, R. E., Spelt, C. E., Mol, J. N. M., The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction, Plant Mol. Biol., 1989, 12: 213-225.[10]Drews, G. N., Beals, T. P., Bul, A. Q. et al., Regional and cell-specific expression patterns during petal development, The Plant Cell, 1992, 4: 1383-1404.[11]Martin, C., Gerats, T., Control of pigment biosynthesis genes during petal development, The

  20. Expression of complement system components during aging and amyloid deposition in APP transgenic mice

    Directory of Open Access Journals (Sweden)

    Wiederhold Karl-Heinz

    2009-11-01

    Full Text Available Abstract Background A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components. Methods APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade. Results High mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase. Conclusion Early but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.

  1. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

    Science.gov (United States)

    Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

    2011-01-01

    The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and

  2. Development of transgenic minipigs with expression of antimorphic human cryptochrome 1.

    Directory of Open Access Journals (Sweden)

    Huan Liu

    Full Text Available Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1 of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP. Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.

  3. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  4. Transgenic Expression of a Functional Fragment of Harpin Protein Hpa1 in Wheat Represses English Grain Aphid Infestation

    Institute of Scientific and Technical Information of China (English)

    XU Man-yu; ZHOU Ting; ZHAO Yan-ying; LI Jia-bao; XU Heng; DONG Han-song; ZHANG Chun-ling

    2014-01-01

    The harpin protein Hpa1 produced by the rice bacterial blight pathogen promotes plant growth and induces plant resistance to pathogens and insect pests. The region of 10-42 residues (Hpa110-42) in the Hpa1 sequence is critical as the isolated Hpa110-42 fragment is 1.3-7.5-fold more effective than the full length in inducing plant growth and resistance. Here we report that transgenic expression of Hpa110-42 in wheat induces resistance to English grain aphid, a dominant species of wheat aphids. Hpa110-42-induced resistance is effective to inhibit the aphid behavior in plant preference at the initial colonization stage and repress aphid performances in the reproduction, nymph growth, and instar development on transgenic plants. The resistance characters are correlated with enhanced expression of defense-regulatory genes (EIN2, PP2-A, and GSL10) and consistent with induced expression of defense response genes (Hel, PDF1.2, PR-1b, and PR-2b). As a result, aphid infestations are alleviated in transgenic plants. The level of Hpa110-42-induced resistance in regard to repression of aphid infestations is equivalent to the effect of chemical control provided by an insecticide. These results suggested that the defensive role of Hpa110-42 can be integrated into breeding germplasm of the agriculturally signiifcant crop with a great potential of the agricultural application.

  5. Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×105 cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.

  6. Creation of Transgenic Bananas Expressing Human Lysozyme Gene for Panama Wilt Resistance

    Institute of Scientific and Technical Information of China (English)

    Xin-Wu PEI; Shi-Kai CHEN; Rui-Ming WEN; Shang YE; Jia-Qin HUANG; Yong-Qiang ZHANG; Bing-Shan WANG; Zhi-Xing WANG; Shi-Rong JIA

    2005-01-01

    Human lysozyme (HL) inhibits Fusarium oxysporum (FocR4) growth in vitro. To obtaintransgenic bananas (Musa spp.) that are resistant to Panama wilt (F. oxysporum), we introduced an HL genethat is driven by a constitutive cauliflower mosaic virus 35S promoter into the banana via Agrobacterium-mediated transformation. PCR confirmed that 51 transgenic plants were obtained. The development ofPanama wilt symptoms were examined after the plants had been grown in pots. The non-transgenic plantsdeveloped typical fusarium symptoms 60 d after FocR4 inoculation, whereas 24 of 51 transgenic plants remained healthy. The transgenic banana plants that showed resistance to FocR4 in the pots were then planted in a field that was heavily infected with FocR4 for further investigation. Eleven of 24 plants developed symptoms before bud emergence; another 11 plants showed symptoms after bud emergence and the remaining two plants, H-67 and H-144, remained healthy and were able to fruit. Northern blotting analysisdemonstrated that H-67 and H-144, bearing the strongest resistance to Panama wilt, had the highest level ofHL expression and that the expression of HL was well correlated with the FocR4 resistance of transgenicplants. We conclude that Agrobacterium-mediated transformation, with the assistance of particlebombardment, is a powerful approach for banana transformation and that a transgenic HL gene can causeresistance of the crop to FocR4 in the field.

  7. Bovine PrP expression levels in transgenic mice influence transmission characteristics of atypical bovine spongiform encephalopathy.

    Science.gov (United States)

    Wilson, Rona; Hart, Patricia; Piccardo, Pedro; Hunter, Nora; Casalone, Cristina; Baron, Thierry; Barron, Rona M

    2012-05-01

    Until recently, transmissible spongiform encephalopathy (TSE) disease in cattle was thought to be caused by a single agent strain, bovine spongiform encephalopathy (BSE) (classical BSE or BSE-C). However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. These atypical BSE isolates have been previously transmitted to a range of transgenic mouse models overexpressing PrP from different species at different levels, on a variety of genetic backgrounds. To control for genetic background and expression level in the analysis of these isolates, we performed here a comprehensive comparison of the neuropathological and molecular properties of all three BSE agents (BASE, BSE-C and BSE-H) upon transmission into the same gene-targeted transgenic mouse line expressing the bovine prion protein (Bov6) and a wild-type control of the same genetic background. Significantly, upon challenge with these BSE agents, we found that BASE did not produce shorter survival times in these mice compared with BSE-C, contrary to previous studies using overexpressing bovine transgenic mice. Amyloid plaques were only present in mice challenged with atypical BSE and neuropathological features, including intensity of PrP deposition in the brain and severity of vacuolar degeneration were less pronounced in BASE compared with BSE-C-challenged mice.

  8. Molecular and phenotypic characterization of transgenic wheat and sorghum events expressing the barley alanine aminotransferase.

    Science.gov (United States)

    Peña, Pamela A; Quach, Truyen; Sato, Shirley; Ge, Zhengxiang; Nersesian, Natalya; Dweikat, Ismail M; Soundararajan, Madhavan; Clemente, Tom

    2017-08-11

    The expression of a barley alanine aminotransferase gene impacts agronomic outcomes in a C3 crop, wheat. The use of nitrogen-based fertilizers has become one of the major agronomic inputs in crop production systems. Strategies to enhance nitrogen assimilation and flux in planta are being pursued through the introduction of novel genetic alleles. Here an Agrobacterium-mediated approach was employed to introduce the alanine aminotransferase from barley (Hordeum vulgare), HvAlaAT, into wheat (Triticum aestivum) and sorghum (Sorghum bicolor), regulated by either constitutive or root preferred promoter elements. Plants harboring the transgenic HvAlaAT alleles displayed increased alanine aminotransferase (alt) activity. The enhanced alt activity impacted height, tillering and significantly boosted vegetative biomass relative to controls in wheat evaluated under hydroponic conditions, where the phenotypic outcome across these parameters varied relative to time of year study was conducted. Constitutive expression of HvAlaAT translated to elevation in wheat grain yield under field conditions. In sorghum, expression of HvAlaAT enhanced enzymatic activity, but no changes in phenotypic outcomes were observed. Taken together these results suggest that positive agronomic outcomes can be achieved through enhanced alt activity in a C3 crop, wheat. However, the variability observed across experiments under greenhouse conditions implies the phenotypic outcomes imparted by the HvAlaAT allele in wheat may be impacted by environment.

  9. Neonatal Death and Heart Failure in Mouse with Transgenic HSP60 Expression

    Directory of Open Access Journals (Sweden)

    Tsung-Hsien Chen

    2015-01-01

    Full Text Available Mitochondrial heat shock proteins, such as HSP60, are chaperones responsible for the folding, transport, and quality control of mitochondrial matrix proteins and are essential for maintaining life. Both prosurvival and proapoptotic roles have been proposed for HSP60, and HSP60 is reportedly involved in the initiation of autoimmune, metabolic, and cardiovascular diseases. The role of HSP60 in pathogenesis of these diseases remains unclear, partly because of the lack of mouse models expressing HSP60. In this study we generated HSP60 conditional transgenic mice suitable for investigating in vivo outcomes by expressing HSP60 at the targeted organ in disease models. Ubiquitous HSP60 induction in the embryonic stage caused neonatal death in mice at postnatal day 1. A high incidence of atrial septal defects was observed in HSP60-expressing mice, with increased apoptosis and myocyte degeneration that possibly contributed to massive hemorrhage and sponge-like cardiac muscles. Our results showed that neonatal heart failure through HSP60 induction likely involves developmental defects and excessive apoptosis. The conditional HSP60 mouse model is useful for studying crucial biological questions concerning HSP60.

  10. Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice

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    Liu Zhihui

    2008-08-01

    Full Text Available Abstract Background Interleukin 1 beta (IL-1β plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

  11. Transgenic Expression of ZBP1 in Neurons Suppresses Cocaine-Associated Conditioning

    Science.gov (United States)

    Lapidus, Kyle A. B.; Nwokafor, Chiso; Scott, Daniel; Baroni, Timothy E.; Tenenbaum, Scott A.; Hiroi, Noboru; Singer, Robert H.; Czaplinski, Kevin

    2012-01-01

    To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We…

  12. RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

    Science.gov (United States)

    Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

    2015-04-09

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

  13. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    Directory of Open Access Journals (Sweden)

    Yuanxin Miao

    2015-04-01

    Full Text Available Myostatin (MSTN, a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph, and zinc metallopeptidase STE24 (Zmpste24. In addition, kyphoscoliosis peptidase (Ky, which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA pathways (Dgki, Dgkz, Plcd4 were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

  14. Bovine growth hormone-transgenic mice have major alterations in hepatic expression of metabolic genes.

    Science.gov (United States)

    Olsson, Bob; Bohlooly-Y, Mohammad; Brusehed, Ola; Isaksson, Olle G P; Ahrén, Bo; Olofsson, Sven-Olof; Oscarsson, Jan; Törnell, Jan

    2003-09-01

    Transgenic mice overexpressing growth hormone (GH) have been extensively used to study the chronic effects of elevated serum levels of GH. GH is known to have many acute effects in the liver, but little is known about the chronic effects of GH overexpression on hepatic gene expression. Therefore, we used DNA microarray to compare gene expression in livers from bovine GH (bGH)-transgenic mice and littermates. Hepatic expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) and genes involved in fatty acid activation, peroxisomal and mitochondrial beta-oxidation, and production of ketone bodies was decreased. In line with this expression profile, bGH-transgenic mice had a reduced ability to form ketone bodies in both the fed and fasted states. Although the bGH mice were hyperinsulinemic, the expression of sterol regulatory element-binding protein (SREBP)-1 and most lipogenic enzymes regulated by SREBP-1 was reduced, indicating that these mice are different from other insulin-resistant models with respect to expression of SREBP-1 and its downstream genes. This study also provides several candidate genes for the well-known association between elevated GH levels and cardiovascular disease, e.g., decreased expression of scavenger receptor class B type I, hepatic lipase, and serum paraoxonase and increased expression of serum amyloid A-3 protein. We conclude that bGH-transgenic mice display marked changes in hepatic genes coding for metabolic enzymes and suggest that GH directly or indirectly regulates many of these hepatic genes via decreased expression of PPARalpha and SREBP-1.

  15. Expression of a complete soybean leghemoglobin gene in root nodules of transgenic Lotus corniculatus.

    Science.gov (United States)

    Stougaard, J; Petersen, T E; Marcker, K A

    1987-08-01

    The complete soybean leghemoglobin lbc(3) gene was transferred into the legume Lotus corniculatus using an Agrobacterium rhizogenes vector system. Organ-specific expression of the soybean gene was observed in root nodules formed on regenerated transgenic plants after infection with Rhizobium loti. The primary transcript was processed in the same way as in soybean nodules and the resulting mRNA was translated into Lbc(3) protein. Quantitative determination of the Lbc(3) protein in nodules of transgenic plants indicated that the steady-state level of the soybean protein is comparable to that of endogenous Lotus leghemoglobin.

  16. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    Science.gov (United States)

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  17. Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene.

    Science.gov (United States)

    Zhang, Xiuchun; Sato, Shirley; Ye, Xiaohong; Dorrance, Anne E; Morris, T Jack; Clemente, Thomas E; Qu, Feng

    2011-11-01

    Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

  18. Production and characterization of transgenic mice systemically expressing endo-beta-galactosidase C.

    Science.gov (United States)

    Watanabe, Satoshi; Misawa, Masako; Matsuzaki, Takashi; Sakurai, Takayuki; Muramatsu, Takashi; Yokomine, Taka-Aki; Sato, Masahiro

    2008-01-01

    The alphaGal epitope (Galalpha1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alphaGal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alphaGal epitope by cleaving the Galbeta1-4GlcNAc linkage in the Galalpha1-3Galbeta1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alphaGal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta1,4-galactosyltransferase 1 (beta4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.

  19. Integration of biological control and transgenic insect protection for mitigation of mycotoxins in corn

    Science.gov (United States)

    Biological control is known to be effective in reducing aflatoxin contamination of corn and some transgenic corn hybrids incur greatly reduced damage from corn earworm (Helicoverpa zea). We conducted seven field trials over two years to test the hypothesis that transgenic insect protection and biol...

  20. The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants.

    Science.gov (United States)

    De Bolle, Miguel F C; Butaye, Katleen M J; Goderis, Inge J W M; Wouters, Piet F J; Jacobs, Anni; Delauré, Stijn L; Depicker, Ann; Cammue, Bruno P A

    2007-03-01

    Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a beta-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chi-MARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chi-MARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression

  1. Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ (pinⅡ) in Transgenic Rice

    Institute of Scientific and Technical Information of China (English)

    CHENG Zhong-yi; XUE Qing-zhong

    2003-01-01

    The inheritance and expression of bar gene and pinⅡ gene were studied in three transgenic ricelines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S.By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bargene and pinⅡ gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but afew plants in F2 have not sufficiently expressed. The wound inducible pin Ⅱ gene has an expression regulatedspatially and temporally, and the signal transduction pathway is not only upward, but also downward. The in-ducible expression of pinⅡ in different rice transgenic lines is not completely coincident.

  2. Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems

    Science.gov (United States)

    Maksimova, E. E.; Popova, L. Yu.

    Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types.

  3. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  4. EXPRESSION OF PORPHYRA YEZOENSIS TPS GENE IN TRANSGENIC RICE ENHANCED THE SALT TOLENRANCE

    Directory of Open Access Journals (Sweden)

    Bao-Tai Guo

    2014-04-01

    Full Text Available The trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS was isolated and cloned into a plant gene expression vector pCAMBIA2300-35S-OCS, and the resulting construct pCAMBIA2300-PyTPS was transformed into Agrobacterium tumefaciens (A. tumefaciens strain AGL1. Genetic transformation of rice variety TP309 was performed with the A. tumefaciens containing pCAMBIA2300-PyTPS. After antibiotic G418 screening and PCR analysis, one hundred T0 transgenic plants were seclected and transplanted into the trial field in the greenhouse and used for further study. Ninety-five of these 100 T0 transgenic cultivaries produced their seeds, which were harvested and stored separately. All of the 95 potential T1 transgenic lines were re-identified by PCR analysis, and their salt-tolerance was tested with 3‰ and 5‰ NaCl solutions. Results indicated that 78 of the 95 T1 transgenic lines were PCR- positive and resistant to 5‰ NaCl solution. Salt-tolerance of these 78 T1 transgenic lines was further tested with higher concentration of NaCl solutions. Of which, three lines (H155, H191 and Y308 showed resistance to 8‰ NaCl in the test. These 3 lines were comprehensively analyzed by PCR, Southern hybridization, northern hybridization and RT-PCR analyses. In addition, trehalose content measurement and preliminary yield evaluation were carried out, results indicated that the PyTPS gene was integrated into the genomic DNA sequences of these 3 transgenic lines and expressed indeed in the transgenic plants. Detection of the transformed PyTPS gene in these 3 transgenic lines was performed in plants from T1 to T6 generations; results indicated that the transformed PyTPS gene was present in transgenic plants from T1 to T6 generations.

  5. Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

    Directory of Open Access Journals (Sweden)

    B.A.A. Santana

    2006-05-01

    Full Text Available Acute promyelocytic leukemia (APL is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa and the promyelocytic leukemia (PML genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase. In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12 developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05. Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.

  6. Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

    Institute of Scientific and Technical Information of China (English)

    李艳; 惠有为; 张仲凯; 黄兴奇; 李毅

    2001-01-01

    Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression of chsA gene in transgenic Petunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal of chsA gene, and transferred the fusion gene into Petunia hybrida via Agrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNA in situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.

  7. Iron biofortification and homeostasis in transgenic cassava roots expressing an algal iron assimilatory protein, FEA1

    OpenAIRE

    2012-01-01

    We have engineered the starchy root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory protein, FEA1, in roots to enhance its nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 gm meal. Significantly, the expression of the FEA1 protein did not alter iron levels in l...

  8. Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics

    Directory of Open Access Journals (Sweden)

    Shidlovsky Konstantin

    2010-11-01

    Full Text Available Abstract Background KillerRed (KR is a novel photosensitizer that efficiently generates reactive oxygen species (ROS in KR-expressing cells upon intense green or white light illumination in vitro, resulting in damage to their plasma membrane and cell death. Results We report an in vivo modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR-expressing transgenic zebrafish. We generated several stable zebrafish Tol2 transposon-mediated enhancer-trap (ET transgenic lines expressing mem-KR (SqKR series, and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos. Conclusions An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.

  9. Transgenic mice expressing human glucocerebrosidase variants: utility for the study of Gaucher disease.

    Science.gov (United States)

    Sanders, Angela; Hemmelgarn, Harmony; Melrose, Heather L; Hein, Leanne; Fuller, Maria; Clarke, Lorne A

    2013-08-01

    Gaucher disease is an autosomal recessively inherited storage disorder caused by deficiency of the lysosomal hydrolase, acid β-glucosidase. The disease manifestations seen in Gaucher patients are highly heterogeneous as is the responsiveness to therapy. The elucidation of the precise factors responsible for this heterogeneity has been challenging as the development of clinically relevant animal models of Gaucher disease has been problematic. Although numerous murine models for Gaucher disease have been described each has limitations in their specific utility. We describe here, transgenic murine models of Gaucher disease that will be particularly useful for the study of pharmacological chaperones. We have produced stable transgenic mouse strains that individually express wild type, N370S and L444P containing human acid β-glucosidase and show that each of these transgenic lines rescues the lethal phenotype characteristic of acid β-glucosidase null mice. Both the N370S and L444P transgenic models show early and progressive elevations of tissue sphingolipids with L444P mice developing progressive splenic Gaucher cell infiltration. We demonstrate the potential utility of these new transgenic models for the study of Gaucher disease pathogenesis. In addition, since these mice produce only human enzyme, they are particularly relevant for the study of pharmacological chaperones that are specifically targeted to human acid β-glucosidase and the common mutations underlying Gaucher disease.

  10. Expression of chimeric P450 genes encoding flavonoid-3', 5'-hydroxylase in transgenic tobacco and petunia plants(1).

    Science.gov (United States)

    Shimada, Y; Nakano-Shimada, R; Ohbayashi, M; Okinaka, Y; Kiyokawa, S; Kikuchi, Y

    1999-11-19

    Flavonoid-3',5'-hydroxylase (F3'5'H), a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3', 5'-hydroxylated anthocyanins, which are generally required for blue or purple flowers. A full-length cDNA, TG1, was isolated from prairie gentian by heterologous hybridization with a petunia cDNA, AK14, which encodes F3'5'H. To investigate the in vivo function of TG1 and AK14, they were subcloned into a plant expression vector and expressed under the control of the CaMV35S promoter in transgenic tobacco or petunia, both of which originally lack the enzyme. Transgenic petunia plants had a dramatic change in flower color from pink to magenta with a high content of 3',5'-hydroxylated anthocyanins. In contrast, transgenic tobacco plants had minimal color change with at most 35% 3',5'-hydroxylated anthocyanin content. These results indicate that the products of TG1 and AK14 have F3'5'H activity in planta and that interspecific gene transfer alters anthocyanin pigment synthesis. The difference in apparent F3'5'H activity between tobacco and petunia is discussed.

  11. Impact of age and vector construct on striatal and nigral transgene expression

    Directory of Open Access Journals (Sweden)

    Nicole K Polinski

    2016-01-01

    Full Text Available Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson's disease (PD but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2, 2/5 (rAAV2/5, 2/9 (rAAV2/9, and lentivirus (LV expressing green fluorescent protein (GFP in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.

  12. Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zhang, Hui; Zhao, Lingxia; Chen, Yuhui; Cui, Lijie; Ren, Weiwei; Tang, Kexuan

    2007-10-01

    In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

  13. STUDY ON SERUM GH EXPRESSION OF TRANSGENIC AND CONTROL COMMON CARP UNDER HUNGRY CONDITION%饥饿状态下转基因鲤鱼和对照鲤鱼血清生长激素的表达研究

    Institute of Scientific and Technical Information of China (English)

    钟山; 罗大极; 汪亚平; 陈竹; 管波; 廖兰杰; 朱作言

    2009-01-01

    This study comparatively analyzed the change regulation of serum growth hormone of transgenic fish and control under starvation and feeding condition by using the Enzyme-linked immunosorbent assays, ELISA technology, and discussed its possible mechanism. The result showed that there was no obviously change in serum growth hormone level of both transgenic fish and the control in feeding experiment, but serum growth hormone level of the former was extremely higher than the latter, which were (142.0±4.9)ng/mL and (1.6±0.2) ng/mL respectively, body weight growth rate of the former was also higher than the latter. In starvation experiment, the concentration of serum growth hormone in transgenic fish descended from (142.0±4.9) ng/mL to (46.0±3.2) ng/mL rapidly, and then kept at this lower level, while the concentration of serum growth hormone in the control rose up from (1.6±0.2) ng/mL to (10.9±1.4) ng/mL continuously, there was no significant difference in body weight minus growth rate between transgenic fish and the control. The study result revealed that the regulatory mechanism of serum growth hormone of transgenic common carp was different with that of the control, because its expression level was not impacted by pituitary feedback inhibition mechanism but related to the control mode of β-actin gene promoter in the transgene.%研究采用酶联免疫吸附测定(Enzyme-linked immunosorbent assays,ELISA)技术,比较分析了转GH基因鲤鱼和对照鲤鱼在饥饿和饱食状态下血清生长激素水平的变化规律,并探讨其可能机制.实验结果表明,在投喂实验中,转基因鲤鱼和对照鲤鱼血清生长激素水平均无明显变化,但转基因鲤鱼血清生长激素远高于对照鲤鱼,分别为(142.0±4.9)ng/mL和(1.6±0.2)ng/mL,转基因鲤鱼体重增长速率显著高于对照鱼.在饥饿实验中,转基因鲤鱼的血清生长激素迅速下降,从(142.0±4.9)ng/mL降至(46.0±3.2)ng/mL,而后稳定在这一较低水平;对照

  14. Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.

  15. GFAP expression and social deficits in transgenic mice overexpressing human sAPPα

    Science.gov (United States)

    Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun

    2013-01-01

    Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007

  16. Regional gene expression of LOX-1, VCAM-1, and ICAM-1 in aorta of HIV-1 transgenic rats.

    Directory of Open Access Journals (Sweden)

    Anne Mette Fisker Hag

    Full Text Available BACKGROUND: Increased prevalence of atherosclerotic cardiovascular disease in HIV-infected patients has been observed. The cause of this accelerated atherosclerosis is a matter of controversy. As clinical studies are complicated by a multiplicity of risk-factors and a low incidence of hard endpoints, studies in animal models could be attractive alternatives. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1, vascular cell adhesion molecule-1 (VCAM-1, and intercellular adhesion molecule-1 (ICAM-1 in HIV-1 transgenic (HIV-1Tg rats; these genes are all thought to play important roles in early atherogenesis. Furthermore, the plasma level of sICAM-1 was measured. We found that gene expressions of LOX-1 and VCAM-1 were higher in the aortic arch of HIV-1Tg rats compared to controls. Also, the level of sICAM-1 was elevated in the HIV-1Tg rats compared to controls, but the ICAM-1 gene expression profile did not show any differences between the groups. CONCLUSIONS/SIGNIFICANCE: HIV-1Tg rats have gene expression patterns indicating endothelial dysfunction and accelerated atherosclerosis in aorta, suggesting that HIV-infection per se may cause atherosclerosis. This transgenic rat model may be a very promising model for further studies of the pathophysiology behind HIV-associated cardiovascular disease.

  17. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

    NARCIS (Netherlands)

    Dingjan, GM; Maas, A; Nawijn, MC; Smit, L; Voerman, JSA; Grosveld, F; Hendriks, RW

    1998-01-01

    To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially

  18. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase.

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); A. Maas (Alex); M.C. Nawijn (Martijn); L. Smit (Linda); J.S. Voerman (Jane); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)

    1998-01-01

    textabstractTo identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse ef

  19. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase.

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); A. Maas (Alex); M.C. Nawijn (Martijn); L. Smit (Linda); J.S. Voerman (Jane); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)

    1998-01-01

    textabstractTo identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse

  20. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

    NARCIS (Netherlands)

    Dingjan, GM; Maas, A; Nawijn, MC; Smit, Linda; Voerman, JSA; Grosveld, F; Hendriks, RW

    1998-01-01

    To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially

  1. Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera).

    Science.gov (United States)

    Nagadhara, D; Ramesh, S; Pasalu, I C; Rao, Y Kondala; Sarma, N P; Reddy, V D; Rao, K V

    2004-11-01

    Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper.

  2. Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

    Science.gov (United States)

    de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

    2016-01-01

    Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

  3. Transgenic cotton plants expressing Cry1Ia12 toxin confer resistance to fall armyworm (Spodoptera frugiperda and cotton boll weevil (Anthonomus grandis

    Directory of Open Access Journals (Sweden)

    Raquel Sampaio Oliveira

    2016-02-01

    Full Text Available Gossypium hirsutum (commercial cooton is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized with PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold. Also, a significant reduction of Anthonomus grandis emerging adults (up to 60% was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda and the Coleopteran (A. grandis insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests.

  4. 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.

  5. Hepatitis B virus HBx protein impairs liver regeneration through enhanced expression of IL-6 in transgenic mice.

    Science.gov (United States)

    Quétier, Ivan; Brezillon, Nicolas; Duriez, Marion; Massinet, Hélène; Giang, Eric; Ahodantin, James; Lamant, Céline; Brunelle, Marie-Noëlle; Soussan, Patrick; Kremsdorf, Dina

    2013-08-01

    Conflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH. PH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration. Cell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor. HBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  6. Transgenic silencing of neurons in the mammalian brain by expression of the allatostatin receptor (AlstR).

    Science.gov (United States)

    Wehr, M; Hostick, U; Kyweriga, M; Tan, A; Weible, A P; Wu, H; Wu, W; Callaway, E M; Kentros, C

    2009-10-01

    The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

  7. Transgenic Silencing of Neurons in the Mammalian Brain by Expression of the Allatostatin Receptor (AlstR)

    Science.gov (United States)

    Wehr, M.; Hostick, U.; Kyweriga, M.; Tan, A.; Weible, A. P.; Wu, H.; Wu, W.; Callaway, E. M.

    2009-01-01

    The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting “silencer” transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K+ channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits. PMID:19692509

  8. Function of resveratrol de- rived from transgenic plant expressing resveratrol synthase gene

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. An Escherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-W fragment. PCR-positive transgenic tobacco plants were obtained after transformation with Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resvera-trol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G0 and G1 phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.

  9. Augmentation of transgenic expression by ultrasound‑mediated liposome microbubble destruction.

    Science.gov (United States)

    Chen, Zhi-Yi; Sun, Xiao-Fang; Liu, Jian-Qiao; Si-Tu, Bing; Qiu, Ri-Xiang; Liang, Kun; Liu, Jian-Hua; Liang, Wei-Xiang; Zhou, Xin-Xin; Zhang, Hua; Yu, Jiang-Xiu

    2012-04-01

    Non-invasive, efficient and tissue-specific transgenic technologies could be valuable in gene therapy. Although non-viral carriers may be safer and cheaper, they have a much lower transfection efficiency than viral gene carriers. The present study was designed to test the transgenic expression and safety of red fluorescent protein (RFP) in HeLa cells in vitro and in transplanted tumors of nude mice in vivo under ultrasound-mediated liposome microbubble destruction (UMLMD) conditions. Plasmids containing RFP were gently mixed with liposome microbubbles (LMs). The mixture was added to HeLa cells or injected into BALB/c mice by the tail vein under various ultrasound exposure and LM parameters, and then the transfection efficiencies were examined. The results in vivo and in vitro demonstrated that, following a comparison of the plasmid group, the ultrasound + plasmid group and the LM + plasmid group, UMLMD significantly increased the transgenic expression (P<0.01) without causing any apparent detrimental effect. From the study, we concluded that UMLMD could be a non-invasive, effective and promising non-viral technique for gene therapy and transgenic research.

  10. High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

    Science.gov (United States)

    Banguela, Alexander; Arrieta, Juan G; Rodríguez, Raisa; Trujillo, Luis E; Menéndez, Carmen; Hernández, Lázaro

    2011-06-10

    Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

  11. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus dragline silk protein.

    Directory of Open Access Journals (Sweden)

    Yoshihiko Kuwana

    Full Text Available Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol% native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

  12. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

    Science.gov (United States)

    Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

    2014-01-01

    Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

  13. RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.

    Directory of Open Access Journals (Sweden)

    Christina Rauschhuber

    Full Text Available BACKGROUND: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. PRINCIPAL FINDINGS: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.

  14. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    Science.gov (United States)

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species.

  15. Synaptophysin expression in motor neurons of transgenic mice with amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Juan Liu; Dawei Zang; Surindar Cheema

    2006-01-01

    BACKGROUND: Affected signal convection of synaptophysin on motor neurons may Cause injury of motor neurons and then induce neurodegeneration and cell death in the end.OBJECTTVE: To investigate the number and density of synaptophysin on motor neurons in the anterior horn of lumbar spinal cord and sensorimotor cortex of the transgenic mouse model of amyotrophic lateral sclerosis(ALS).DESIGN: Randomized controlled animal study.SETTTNG: Brain Injury and Repair Group, HFI Institute of Melbourne University.MATERIALS: Transgenic mice expressing a mutated human superoxide dismutase 1 (SOD-1) were taken as ALS group (n =36), while those dedved from the B6SJL-TgN gene line were taken as control group (n =36),according to the difference of gender and three postnatal time points (postnatal 60, 90 and 120 days), twelve mice of either gender were allocated in each subgroup.METHODS: The experiment was carried out in Brain Injury and Repair Group, HFI Institute of Melbourne University from November 2003 to June 2004. ① Fluorogold labeling was used for the motor neurons in the lumbar and sensorimotor cortex. ② Immunofluorescence was applied for the labeling of synaptophysin; positive control sections were represented by adding the synaptophysin antibody and the staining, showing a positive result. For negative controls, the synaptophysin antibody was omitted. ③ Stereological counting system was adopted in the statistical analysis.MAIN OUTCOME MEASURES: ① Fluorogold labeling of motor neurons; ② number of synaptophysin on the motor neurons.RESULTS: ① Fluorogold labeling of motor neurons: The motor neurons in the lumbar and sensorimotor cortex were clearly labeled by fluorogold under the detection of fluorescent microscope. ② The number of synaptophysin on the motor neurons: The number statistically decreased at the mid stage (postnatal 90 days)and late stage (postnatal 120 days) [motor neuron somas at lumbar spinal cord: (0.75±0.06), (0.59±0.09)/μm;motor neuron

  16. Comparison of factor VIII transgenes bioengineered for improved expression in gene therapy of hemophilia A.

    Science.gov (United States)

    Dooriss, Kerry L; Denning, Gabriela; Gangadharan, Bagirath; Javazon, Elisabeth H; McCarty, David A; Spencer, H Trent; Doering, Christopher B

    2009-05-01

    Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/10(6) cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.

  17. A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available Recent progress in neural stem cell- (NSC- based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is “switched off” by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is “switched on” by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

  18. Transgenic bioreactors.

    Science.gov (United States)

    Jänne, J; Alhonen, L; Hyttinen, J M; Peura, T; Tolvanen, M; Korhonen, V P

    1998-01-01

    Since the generation of the first transgenic mice in 1980, transgene technology has also been successfully applied to large farm animals. Although this technology can be employed to improve certain production traits of livestock, this approach has not been very successful so far owing to unwanted effects encountered in the production animals. However, by using tissue-specific targeting of the transgene expression, it is possible to produce heterologous proteins in the extracellular space of large transgenic farm animals. Even though some recombinant proteins, such as human hemoglobin, have been produced in the blood of transgenic pigs, in the majority of the cases mammary gland targeted expression of the transgene has been employed. Using production genes driven by regulatory sequences of milk protein genes a number of valuable therapeutic proteins have been produced in the milk of transgenic bioreactors, ranging from rabbits to dairy cattle. Unlike bacterial fermentors, the mammary gland of transgenic bioreactors appear to carry out proper postsynthetic modifications of human proteins required for full biological activity. In comparison with mammalian cell bioreactors, transgenic livestock with mammary gland targeted expression seems to be able to produce valuable human therapeutic proteins at very low cost. Although not one transgenically produced therapeutic protein is yet on the market, the first such proteins have recently entered or even completed clinical trials required for their approval.

  19. An efficient and reproducible protocol for the production of salt tolerant transgenic wheat plants expressing the Arabidopsis AtNHX1 gene.

    Science.gov (United States)

    Moghaieb, Reda E A; Sharaf, Ahmed N; Soliman, Mohamed H; El-Arabi, Nagwa I; Momtaz, Osama A

    2014-01-01

    We present an efficient method for the production of transgenic salt tolerant hexaploid wheat plants expressing the Arabidopsis AtNHX1 gene. Wheat mature zygotic embryos were isolated from two hexaploid bread wheat (Triticum aestivum) cultivars (namely: Gemmeiza 9 and Gemmeiza 10) and were transformed with the A. tumefaciens LBA4404 harboring the pBI-121 vector containing the AtNHX1 gene. Transgenic wheat lines that express the gus intron was obtained and used as control. The results confirmed that npt-II gene could be transmitted and expressed in the T2 following 3:1 Mendelian segregation while the control plant couldn't. The data indicate that, the AtNHX1 gene was integrated in a stable manner into the wheat genome and the corresponding transcripts were expressed. The transformation efficiency was 5.7 and 7.5% for cultivars Gemmeiza 10 and Gemmeiza 9, respectively. A greenhouse experiment was conducted to investigate the effect of AtNHX1 gene in wheat salt tolerance. The transgenic wheat lines could maintain high growth rate under salt stress condition (350 mM NaCl) while the control plant couldn't. The results confirmed that Na(+)/H(+) antiporter gene AtNHX1 increased salt tolerance by increasing Na(+) accumulation and keeping K+/Na(+) balance. Thus, transgenic plants showed high tolerance to salt stress and can be considered as a new genetic resource in breeding programs.

  20. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2011-01-01

    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  1. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

    OpenAIRE

    Sanderson, N.; Factor, V; Nagy, P; Kopp, J; Kondaiah, P; WAKEFIELD, L.; Roberts, A B; Sporn, M B; Thorgeirsson, S S

    1995-01-01

    Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of th...

  2. Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae).

    Science.gov (United States)

    Allen, Margaret L; Christensen, Bruce M

    2004-12-01

    A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.

  3. Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology.

    Science.gov (United States)

    Agrawal, Aditi; Rajamani, Vijayalakshmi; Reddy, Vanga Siva; Mukherjee, Sunil Kumar; Bhatnagar, Raj K

    2015-10-01

    The success of Bt transgenics in controlling predation of crops has been tempered by sporadic emergence of resistance in targeted insect larvae. Such emerging threats have prompted the search for novel insecticidal molecules that are specific and could be expressed through plants. We have resorted to small RNA-based technology for an investigative search and focused our attention to an insect-specific miRNA that interferes with the insect molting process resulting in the death of the larvae. In this study, we report the designing of a vector that produces artificial microRNA (amiR), namely amiR-24, which targets the chitinase gene of Helicoverpa armigera. This vector was used as transgene in tobacco. Northern blot and real-time analysis revealed the high level expression of amiR-24 in transgenic tobacco plants. Larvae feeding on the transgenic plants ceased to molt further and eventually died. Our results demonstrate that transgenic tobacco plants can express amiR-24 insectice specific to H. armigera.

  4. Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector.

    Science.gov (United States)

    Koo, Bon Chul; Kwon, Mo Sun; Choi, Bok Ryul; Kim, Jin-Hoi; Cho, Seong-Keun; Sohn, Sea Hwan; Cho, Eun Jung; Lee, Hoon Taek; Chang, Wonkyung; Jeon, Iksoo; Park, Jin-Ki; Park, Jae Bok; Kim, Teoan

    2006-11-01

    The Moloney murine leukemia virus (MoMLV) -based retrovirus vector system has been used most often in gene transfer work, but has been known to cause silencing of the imported gene in transgenic animals. In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of enhanced green fluorescent protein (eGFP). The level of eGFP expression was conserved after germline transmission and as much as 100 microg of eGFP could be detected per 1 mg of tissue protein. DNA sequencing showed that the transgene had been integrated at chromosome 26 of the G1 and G2 generation transgenic chickens. Owing to the stable integration of the transgene, it is now feasible to produce G3 generation of homozygous eGFP transgenic chickens that will provide 100% transgenic eggs. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors.

  5. Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Kuklin, Alexander [ORNL; Mynatt, Randall [ORNL; Klebig, Mitch [ORNL; Kiefer, Laura [Glaxo Wellcome, Research Triangle Park, NC; Wilkison, William O [Glaxo Wellcome, Research Triangle Park, NC; Woychik, Richard P [Jackson Laboratory, The, Bar Harbor, ME; Michaud III, Edward J [ORNL

    2004-01-01

    Background: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse agouti gene that cause the wild- ype protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the -melanocyte stimulating hormone ( MSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded. Results: The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver with an increased number

  6. Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes

    Directory of Open Access Journals (Sweden)

    Kiefer Laura L

    2004-06-01

    Full Text Available Abstract Background The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r on melanocytes. Lethal yellow (Ay and viable yellow (Avy are dominant regulatory mutations in the mouse agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the α-melanocyte stimulating hormone (αMSH to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg of the hepatocellular carcinogen, diethylnitrosamine (DEN, at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded. Results The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver

  7. Expression of the intact C4 type pepc gene cloned from maize in transgenic winter wheat

    Institute of Scientific and Technical Information of China (English)

    CHEN Xuqing; ZHANG Xiaodong; LIANG Rongqi; ZHANG Liquan; YANG Fengping; CAO Mingqing

    2004-01-01

    Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as Pbac214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of Mrna sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two Mrna, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector Pbac214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4-pepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.

  8. GmACP expression is decreased in GmNORK knockdown transgenic soybean roots

    Institute of Scientific and Technical Information of China (English)

    Lijun Wang; Lingwei Deng

    2016-01-01

    NORK and soybean acyl carrier protein (ACP) both play important roles in nodulation. However, the relationship between Nod factor signaling and fatty acid (FA) biosynthesis during symbiotic development is unknown. In this study, an RNAi plasmid of GmNORK was constructed and transformed into soybean roots by Agrobacterium rhizogene-mediated hairy-root transformation. The nodule number decreased substantially in GmNORK knock-down soybean transgenic roots. To investigate the relationship between GmACP and Nod factor signaling, we measured GmACP expression levels in GmNORK RNAi soybean transgenic roots and found that rhizobia inoculation led to substantially reduced GmACP expression. Thus, FA biosynthesis was affected by Nod factor signaling during nodule development in soybean, a finding that provides valuable information that improves our understanding of the functions of GmNORK and GmACP in symbiotic signaling and nodule development.

  9. Antisense expression of a rice cellular apoptosis susceptibility gene (OsCAS) alters the height of transgenic rice

    Institute of Scientific and Technical Information of China (English)

    XU Chunxiao; HE Chaozu

    2007-01-01

    Cellular apoptosis susceptibility (CAS) gene plays important roles in mitosis, development and export of importin αfrom the nucleus, but its function in plant is unknown. In this study, a rice CAS ortholog (OsCAS), which encodes a predicted protein of 983 amino acids with 62% similarity to human CAS, was identified. DNA gel blot analysis revealed a single copy of OsCAS in the rice genome. A 973 bp fragment at the 3' end of OsCAS cDNA was cloned from rice cDNA library and transferred into rice in the antisense direction under the control of CaMV 35S promoter via Agrobacterium-mediated transformation method, 105 transgenic lines were obtained. Expression of OsCAS was suppressed in the antisense transgenic lines as revealed by semi-quantitative RT-PCR. The antisense transgenic lines showed dwarf phenotypes. The results indicated that OsCAS was involved in culm development of rice.

  10. Expression of Human Papillomavirus Type 16 L1 Protein in Transgenic Tobacco Plants

    Institute of Scientific and Technical Information of China (English)

    Hong-Li LIU; Wen-Sheng LI; Ting LEI; Jing ZHENG; Zheng ZHANG; Xiao-Fei YAN; Zhe-Zhi WANG; Yi-Li WANG; Lü-Sheng SI

    2005-01-01

    To develop a plant expression system for the production of the human papillomavirus type 16(HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55 nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV 16L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.

  11. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    OpenAIRE

    Natalia V. Permyakova; Zagorskaya, Alla A.; Belavin, Pavel A.; Elena A. Uvarova; Nosareva, Olesya V.; Nesterov, Andrey E.; Novikovskaya, Anna A.; Evgeniy L. Zav’yalov; Mikhail P Moshkin; Deineko, Elena V.

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This co...

  12. Inheritance and expression of multiple disease and insect re- sistance genes in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    2-3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6-7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plan-ts carrying 6-7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consis-tent with Mendel's 3︰1 segregation law. 8 homozygous R1 transgenic plants harboring 2-3 anti-fungal and 2 insectici-dal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.

  13. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

    Science.gov (United States)

    Katter, Katharina; Geurts, Aron M; Hoffmann, Orsolya; Mátés, Lajos; Landa, Vladimir; Hiripi, László; Moreno, Carol; Lazar, Jozef; Bashir, Sanum; Zidek, Vaclav; Popova, Elena; Jerchow, Boris; Becker, Katja; Devaraj, Anantharam; Walter, Ingrid; Grzybowksi, Michael; Corbett, Molly; Filho, Artur Rangel; Hodges, Matthew R; Bader, Michael; Ivics, Zoltán; Jacob, Howard J; Pravenec, Michal; Bosze, Zsuzsanna; Rülicke, Thomas; Izsvák, Zsuzsanna

    2013-03-01

    Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

  14. Expression of GhNAC2 from G. herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis.

    Science.gov (United States)

    Gunapati, Samatha; Naresh, Ram; Ranjan, Sanjay; Nigam, Deepti; Hans, Aradhana; Verma, Praveen C; Gadre, Rekha; Pathre, Uday V; Sane, Aniruddha P; Sane, Vidhu A

    2016-04-26

    NAC proteins are plant-specific transcription factors that play essential roles in regulating development and responses to abiotic and biotic stresses. We show that over-expression of the cotton GhNAC2 under the CaMV35S promoter increases root growth in both Arabidopsis and cotton under unstressed conditions. Transgenic Arabidopsis plants also show improved root growth in presence of mannitol and NaCl while transgenic cotton expressing GhNAC2 show reduced leaf abscission and wilting upon water stress compared to control plants. Transgenic Arabidopsis plants also have larger leaves, higher seed number and size under well watered conditions, reduced transpiration and higher relative leaf water content. Micro-array analysis of transgenic plants over-expressing GhNAC2 reveals activation of the ABA/JA pathways and a suppression of the ethylene pathway at several levels to reduce expression of ERF6/ERF1/WRKY33/ MPK3/MKK9/ACS6 and their targets. This probably suppresses the ethylene-mediated inhibition of organ expansion, leading to larger leaves, better root growth and higher yields under unstressed conditions. Suppression of the ethylene pathway and activation of the ABA/JA pathways also primes the plant for improved stress tolerance by reduction in transpiration, greater stomatal control and suppression of growth retarding factors.

  15. Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

    Institute of Scientific and Technical Information of China (English)

    WU; Aimin; LIU; Jinyuan

    2006-01-01

    β-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) β-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, sequence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.

  16. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

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    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  17. New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

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    Ana Isabel Garcia Diaz

    2016-04-01

    Full Text Available The Wistar Kyoto (WKY rat and the spontaneously hypertensive (SHR rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN and metabolic syndrome, respectively. Novel transgenic (Tg strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP under the rat elongation factor 1 alpha (EF1a promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

  18. DISTRIBUTION AND EXPRESSION OF STRIPED CATFISH (Pangasionodon hypophtalmus GROWTH HORMONE GENE (PhGH IN THE ORGAN OF AFRICAN CATFISH (Clarias gariepinus TRANSGENIC FOUNDER

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    Huria Marnis

    2012-06-01

    Full Text Available Faster growing African catfish can be produced by transgenesis. This study was conducted to investigate the distribution and expression of growth hormone gene (PhGH in various organs of the transgenic African catfish (Clarias gariepinus founder (F0. Transgene was detected using the PCR method in various organs, namely pituitary, brain, liver, heart, spleen, kidney, intestine, stomach, muscle, caudal fin, gill and eye. Transgene expression levels were analyzed using the method of reverse transcriptase-polymerase chain reaction (RT-PCR, -actin gene used as internal controls. The results showed that the PhGH was detected and expressed in all organs of the transgenic African catfish founder. The high level of PhGH expression was found in the liver, pituitary, intestine and brain; smaller amounts were detectable in muscle, spleen, kidneys, heart, and stomach, caudal fin, gill and eyes, range from 0.02-0.75 PhGH/-actin mRNA. The expression levels of PhGH had positive correlation with tissue and body size (P<0.05.

  19. [Transgenic Belarussian-bred potato plants expressing genes for antimicrobial peptides of the cecropin-melittin type].

    Science.gov (United States)

    Vutto, N L; Gapeeva, T A; Pundik, A N; Tret'iakova, T G; Volotovskiĭ, I D

    2010-12-01

    Binary vectors for Agrobacterium-mediated transformation were constructed to express the genes for antimicrobial peptides (APs) of the cectropin-melittin type under the control of the cauliflower mosaic virus 35S RNA promoter in plants. It was shown with Escherichia coli and Agrobacterium tumefaciens cells that the cassettes could be cloned in pB1121-based vectors with deletion of the 3-D-glycuronidase gene only in the orientation opposite to that of the original vector. Transgenic potato plants were obtained using the Belarussian varieties Odyssey, Vetraz, and Scarb. Their cells expressed the MsrA1 or CEMA peptides of the cecropin-melittin type. The expression was shown to confer higher resistance to bacterial (Erwinia carotovora) infection and extremely high resistance to fungal (Phytophtora infestans and Alternarla solani) infections.

  20. Effect of UTRs from TMV-RNA on the expression of foreign gene in transgenic plants

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To investigate the effects of 5′ and 3′ untranslated region (UTR) from tobacco mosaic virus (TMV) on expression of foreign genes, four plant-expression vectors pBG437, pBG438, pBG440 and pBG440△NOS containing expression cassettes of GUS-NOS; Ω-GUS-NOS, Ω-GUS-3′UTR-NOS and Ω-GUS-3′UTR downstream of CaMV 35S promoter with double enhancer sequences respectively have been constructed. Results from a large number of transgenic tobacco plants show that the GUS activity of pBG440 transformed plants is the highest, being 5-fold that of pBG437 and 1.6-fold that of pBG438. Similar results have been obtained in transient expression by injection of Agrobacterium tumefaciens harbouring these constructs into N. benthamiana leaves. These results obtained at the whole plant level confirm the conclusion drawn from the transient expression studies in protoplast system that TMV-Ω fragment can be a translation enhancer and the 3′UTR has a coordinate effect with Ω fragment to enhance foreign gene expression, but unlike the situation in transient expression in protoplast system, the 3′UTR of TMV cannot act as a poly(A) tail nor as a transcription terminator in transgenic plants. The coordinate effect of 3′ UTR with Ω fragment needs the presence of a normal plant transcriptional terminator.

  1. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

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    Jacob Barbara A

    2009-02-01

    Full Text Available Abstract Background HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

  2. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  3. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  4. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV.

  5. Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.

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    Young-Hee Jeong

    Full Text Available The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC compared with the human umbilical vein endothelial cells (HUVEC. Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative

  6. Gene transfer to hepatocellular carcinoma: transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors.

    Science.gov (United States)

    Gerolami, R; Uch, R; Jordier, F; Chapel, S; Bagnis, C; Bréchot, C; Mannoni, P

    2000-09-01

    Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

  7. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

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    Mazahir T Hasan

    2004-06-01

    Full Text Available Genetically encoded fluorescent calcium indicator proteins (FCIPs are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes-for the measurement of population activity, in particular-have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs and synaptic and sensory stimulation can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

  8. Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

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    Antoniou Michael

    2007-09-01

    Full Text Available Abstract Background A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox inducible rtTA2S-M2 Tet-transactivator in transgenic mice. Results An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 transgenic mouse line demonstrated Dox-inducible GFP transgene expression in many tissues. Using this line we also show highly sensitive quantitative induction with low doses of Dox of an assayable plasma protein transgene under the control of a Tet Responsive Element (TRE. The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 Tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. Conclusion The ubiquitously expressing rtTA2S-M2 transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice.

  9. Effects of Metformin on Tissue Oxidative and Dicarbonyl Stress in Transgenic Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein.

    Science.gov (United States)

    Malínská, Hana; Oliyarnyk, Olena; Škop, Vojtěch; Šilhavý, Jan; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šimáková, Miroslava; Strnad, Hynek; Kazdová, Ludmila; Pravenec, Michal

    2016-01-01

    Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its "pleiotropic" effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action-studied by gene expression profiling in the liver-revealed deregulated genes from inflammatory and insulin signaling, AMP

  10. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. (Cincinnati College of Medicine, OH (USA))

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  11. Heterology expression of the sweet pepper CBF3 gene confers elevated tolerance to chilling stress in transgenic tobacco.

    Science.gov (United States)

    Yang, Sha; Tang, Xian-Feng; Ma, Na-Na; Wang, Li-Yan; Meng, Qing-Wei

    2011-10-15

    Various studies have confirmed that the CBF (C-repeat binding factor) family of transcription factors has a key role in regulating many plants' responses to cold stress. Here we isolated CBF3 from sweet pepper (Capsicum frutescens). Green fluorescent protein (GFP) fusion protein of CfCBF3 was targeted to the nucleus of the onion epidermis cell. RNA gel blot analysis indicated that CfCBF3 was expressed in leaves of sweet pepper and the expression was induced by low temperature, drought and salinity stresses but not by ABA. Overexpression of CfCBF3 under the control of the CaMV35S promoter in tobacco induced expression of orthologs of CBF3-targeted genes and increased chilling tolerance without a dwarf phenotype. Indeed it also led to multiple biochemical and physiological changes associated with chilling stress. Higher levels of proline (Pro) and soluble sugars and lower content of reactive oxygen species (ROS) were observed in transgenic plants. Our results demonstrated that the increase in total unsaturated fatty acids, especially in phosphatidylglycerol (PG) was detected by overexpression of CfCBF3. During exposure to chilling stress, the transgenic lines were less susceptible to chilling-induced photoinhibition than wild-type (WT) plants. These results suggest that overexpression of CfCBF3 led to modification of the fatty acid unsaturation and alleviated the injuries under chilling stress.

  12. Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

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    Yu-Guo Yuan

    2014-01-01

    Full Text Available To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC containing a hybrid goat β-lactoglobulin (BLG promoter/cytomegalovirus (CMV enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT. Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2% from the non-transfected line and five recipients delivered six kids (1.8% from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P<0.05. Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

  13. Establishment of La-tPA/G-CSF dual transgenic mice and expression in their mammary gland

    Institute of Scientific and Technical Information of China (English)

    卢一凡; 田靫; 邓继先; 程萱; 黄培堂

    1999-01-01

    Expression vectors of human granulocyte colony stimulating factor (G-CSG) and long acting tissue plasminogen activator (La-tPA) in mammary gland were constructed using promoters of mouse whey acid protein gene (WAP) and sheep β-lactoglobulin gene (BLG) with sizes of 2.6 and 5 kb respectively. Two kinds of transgenic mice of G-CSF and La-tPA were produced with microinjection. The expression of G-CSF and La-tPA was achieved in mammary glands of transgenic mice, respectively. In order to establish dual transgenic mice of La-tPA/G-CSF, transgenic mice carrying G-CSF and La-tPA gene characterized with specific expression in mammary gland were mated. La-tPA/G-CSF dual transgenic mice were screened out from the hybrid offspring by Once-PCR. The co-expression of La-tPA and G-CSF in mammary gland of the dual transgenic mice was confirmed by the milk assayed and Northern blot analysis. Some parameters about the dual transgenic mice indicated that there were fewer litters than that of normal mice. The ratio of du

  14. Growth, fecundity and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura expressing a sunflower seed albumin gene.

    Science.gov (United States)

    Godfree, Robert C; Woods, Matthew J; Young, Andrew G; Burdon, Jeremy J; Higgins, T J V

    2004-01-01

    Ecological risk assessment is an important step in the production and commercialisation of transgenic plants. To date, however, most risk assessment studies have been performed on crop plants, and few have considered the ecological consequences associated with genetic modification of pasture species. In this study we compared the growth, yield, population dynamics and competitive ability of transgenic Trifolium subterraneum subsp. subterraneum cv. Leura (subclover) expressing a nutritive sunflower seed albumin (ssa) gene with the equivalent non-transgenic commercial line in a glasshouse competition trial. Plants were grown in low-fertility soil typical of unimproved native southeastern Australian grasslands. We measured survivorship, seed production rate, seed germination rate, seed weight, dry weight yield and the intrinsic rate of population increase (lambda) of plants grown in mixtures and monocultures over a range of densities (250 to 2000 plants m(-2)), and also determined intragenotypic and intergenotypic competition coefficients for each line. There were no significant differences between transgenic and non-transgenic plants in any of the measured variables except survivorship; transgenic plants had a significantly lower survival rate than non-transgenic plants when grown at high densities (pdensity-dependent effects were observed for all measured variables, and in all models plant density affected the response variables more than the presence of the transgene. Based on these results, we conclude that the ssa gene construct appears to confer no advantage to transgenic T. s. subterraneum cv. Leura growing in mixed or pure swards under the fertility and density regimes examined in the trial. Our data also suggest that transgenic subterranean clover expressing the ssa gene is unlikely to exhibit a competitive advantage over associated non-transgenic commercial cultivars when grown in dense swards in low-fertility pastures.

  15. Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress.

    Science.gov (United States)

    Checker, Vibha G; Chhibbar, Anju K; Khurana, Paramjit

    2012-10-01

    Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.

  16. Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

    Directory of Open Access Journals (Sweden)

    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

  17. bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Furong Wang; Yongsheng Jiang; Yan Liu; Wenwu Xiao; Suming Zhang

    2008-01-01

    BACKGROUND: Basal cell lymphoma-extra large (bcl-xl) can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE: This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/dreperfusion model by middle cerebral artery occlusion (MCAO) in transgenic and wild-type mice, and observed changes in the number and distribution of apoptotic neural cells, differences in cerebral infarct volume, in neurological function score, and in cytochrome C expression in the ischemic cerebral cortex, at different time points, DESIGN AND SETTING: The present gene engineering and cell biology experiment was performed at the Laboratory of Biology, Hubei Academy of Agricultural Sciences and at the Laboratory of Immunology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Male bcl-xl over-expression Kunming mice aged 8 weeks and age-matched male wild-type mice were used for this study. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) kits were purchased from Boliman, France. Cytochrome C antibody and Bcl-x immunohistochemical kit were purchased from PharMingen, USA and Santa Cruz Biotechnology, USA, respectively. METHODS: Following MCAO and reperfusion, apoptosis in the ischemic cerebral cortex was detected by the TUNEL assay. Prior to MCAO and 3 hours after reperfusion, the Bcl-xl protein level in the ischemic cerebral cortex was measured by immunohistochemistry. At 3, 6, 12 and 24 hours after reperfusion, the level of cytochrome C in the ischemic cerebral cortex was examined by western blot analysis. Subsequent to MCAO, cerebral infarct volume measurement and neurological examination were performed. MAIN OUTCOME MEASURES: Neural cell apoptosis and cytochrome C expression in the ischemic cerebral cortex; cerebral infarct volume and neurological function score. RESULTS: Twenty-four hours after

  18. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    Science.gov (United States)

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  19. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

    Directory of Open Access Journals (Sweden)

    Stefanie Besser

    Full Text Available GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65. TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

  20. Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1

    OpenAIRE

    2012-01-01

    We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene...

  1. Gene expression profiling of R6/2 transgenic mice with different CAG repeat lengths reveals genes associated with disease onset and progression in Huntington's disease.

    Science.gov (United States)

    Tang, Bin; Seredenina, Tamara; Coppola, Giovanni; Kuhn, Alexandre; Geschwind, Daniel H; Luthi-Carter, Ruth; Thomas, Elizabeth A

    2011-06-01

    R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice

  2. Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress.

    Science.gov (United States)

    Singh, Rajiv Kumar; Singh, Vivek Kumar; Raghavendrarao, Sanagala; Phanindra, Mullapudi Lakshmi Venkata; Venkat Raman, K; Solanke, Amolkumar U; Kumar, Polumetla Ananda; Sharma, Tilak Raj

    2015-09-01

    One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops.

  3. Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production

    Directory of Open Access Journals (Sweden)

    Zhou Xue-Rong

    2010-03-01

    Full Text Available Abstract Background Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. Results In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2 gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. Conclusions The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

  4. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein.

    Science.gov (United States)

    Ge, Jiachun; Dong, Zhangji; Li, Jingyun; Xu, Zhiqiang; Song, Wei; Bao, Jie; Liang, Dong; Li, Junbo; Li, Kui; Jia, Wenshuang; Zhao, Muzi; Cai, Yongxiang; Yang, Jiaxin; Pan, Jianlin; Zhao, Qingshun

    2012-10-01

    Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

  5. The MAR-Mediated Reduction in Position Effect Can Be Uncoupled from Copy Number-Dependent Expression in Transgenic Plants

    NARCIS (Netherlands)

    Mlynárová, Ľudmila; Jansen, Ritsert C.; Conner, Anthony J.; Stiekema, Willem J.; Nap, Jan-Peter

    To study the role of matrix-associated regions (MARs) in establishing independent chromatin domains in plants, two transgenes were cloned between chicken lysozyme A elements. These transgenes were the neomycin phosphotransferase (NPTII) gene under control of the nopaline synthase (nos) promoter and

  6. Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

    Science.gov (United States)

    Permyakova, Natalia V; Zagorskaya, Alla A; Belavin, Pavel A; Uvarova, Elena A; Nosareva, Olesya V; Nesterov, Andrey E; Novikovskaya, Anna A; Zav'yalov, Evgeniy L; Moshkin, Mikhail P; Deineko, Elena V

    2015-01-01

    Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  7. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

    Full Text Available Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L. genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  8. Expression of a Modified Crylle Gene in E.Coli and in Transgenic Tobacco Confers Resistance to Corn Borer

    Institute of Scientific and Technical Information of China (English)

    Yun-Jun LIU; Fu-Ping SONG; Kang-Lai HE; Yuan YUAN; Xiao-Xia ZHANG; Peng GAO; Jian-Hua WANG; Guo-Ying WANG

    2004-01-01

    The wild-type Crylle gene from Bacillus thuringiensis was modified for its efficient expression in transgenic plants. Modified Cry1 Ie gene (designated as Cry1 Iem) was cloned into prokaryotic expression vector pET28b and its expression in E. coli was confirmed by SDS-PAGE analysis. Bioassays using crude expression products in E. coli revealed that Cry1 Iem protein had a similar toxicity to corn borer as wild-type Cry1 Ie. Cry1 Iem gene was then inserted downstream of the maize ubiquitin-1 promoter in plant expression vector p3301. Transgenic tobacco plants carrying Cry1 Iem showed insecticidal activity against com borer.

  9. Transgenic expression of BRCA1 disturbs hematopoietic stem and progenitor cells quiescence and function

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Lin; Shi, Guiying; Zhang, Xu; Dong, Wei; Zhang, Lianfeng, E-mail: zhanglf@cnilas.org

    2013-10-15

    The balance between quiescence and proliferation of HSCs is an important regulator of hematopoiesis. Loss of quiescence frequently results in HSCs exhaustion, which underscores the importance of tight regulation of proliferation in these cells. Studies have indicated that cyclin-dependent kinases are involved in the regulation of quiescence in HSCs. BRCA1 plays an important role in the repair of DNA double-stranded breaks, cell cycle, apoptosis and transcription. BRCA1 is expressed in the bone marrow. However, the function of BRCA1 in HSCs is unknown. In our study, we generated BRCA1 transgenic mice to investigate the effects of BRCA1 on the mechanisms of quiescence and differentiation in HSCs. The results demonstrate that over-expression of BRCA1 in the bone marrow impairs the development of B lymphocytes. Furthermore, BRCA1 induced an increase in the number of LSKs, LT-HSCs, ST-HSCs and MPPs. A competitive transplantation assay found that BRCA1 transgenic mice failed to reconstitute hematopoiesis. Moreover, BRCA1 regulates the expression of p21{sup waf1}/cip1 and p57{sup kip2}, which results in a loss of quiescence in LSKs. Together, over-expression of BRCA1 in bone marrow disrupted the quiescent of LSKs, induced excessive accumulation of LSKs, and disrupted differentiation of the HSCs, which acts through the down-regulated of p21{sup waf1}/cip1 and p57{sup kip2}. - Highlights: • Over-expression of BRCA1 results in impaired B lymphocyte development. • BRCA1 transgenic mice disrupted the quiescent of LSKs, induced excessive accumulation of LSKs. • BRCA1 impairs the function of HSCs through the down-regulated of p21{sup waf1/cip1} and p57{sup kip2}.

  10. Transgenic overexpression of BAFF regulates the expression of immune-related genes in zebrafish, Danio rerio

    Indian Academy of Sciences (India)

    LI ZHANG; CHAO LIU; XIN ZHOU; YING XIE; LIBO SU; QI GENG; BINGHUI LIU; SHUFENG LIU

    2016-12-01

    The B-cell activating factor (BAFF) is a member of tumour necrosis factor (TNF) superfamily that specifically regulates B lymphocyte proliferation and survival. Excess BAFF leads to overproduction of antibodies for secretion, anti-dsDNA antibodies and a lupus-like syndrome in mice. To investigate whether transgenic overexpression of the zebrafish BAFF leads to immunoglobulin changes and/or early maturing of the immune system, a Tol2-GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences. Functional GFP and BAFF proteins were expressed separately and confirmed in HeLa cells. The relative expression of immune-related genes (IgLC-1, IgLC-2, IgLC-3, IgD, IgM and IL-4), early lymphoid markers (Ikaros, Rag-1 and TCRAC), and the protooncogene Bcl-2 were evaluated by quantitative polymerase chain reaction (PCR) in F0 founder of transgenic zebrafish juveniles and adults. Ectopic expression of BAFF in adults was confirmed using Western blots and was shown to upregulate IgLC-1, IgLC- 2, IgD, IgM, IgZ/T, Ikaros, Rag-1, TCRAC, IL-4 and Bcl-2 expression in juveniles on day 21 and IgLC-1, IgLC-2, IgD, IgM, IgZ/T, Rag-1, TCRAC and Bcl-2 expression in zebrafish three months postfertilization. The relative titers of specific IgM against Edwardsiella tarda WED were assessed using modified enzyme-linked immunosorbent assay (ELISA) with the whole body homogenate of zebrafish and demonstrated a significant increase in BAFF-transgenic group. Therefore, our findings provided novel insight into further exploration of modulating adaptive immunity and studying autoimmune diseases caused by regulating BAFF.

  11. Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

    Institute of Scientific and Technical Information of China (English)

    MAO Shengji; GU Hongya; QU Lijia; CHEN Zhangliang

    2003-01-01

    The strategy of the two-component system, composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.

  12. Gene expression analysis of pancreatic cystic neoplasm in SV40Tag transgenic mice model

    Institute of Scientific and Technical Information of China (English)

    Jie Feng; Qiang Sun; Cheng Gao; Juan Dong; Xiao-Luan Wei; Hua Xing; Hou-Da Li

    2007-01-01

    AIM: To study the gene expression changes in pancreatic cystic neoplasm in SV40Tag transgenic mice model and to provide information about the prevention,clinical diagnosis and therapy of pancreatic cancer.METHODS: Using the pBC-SV40Tag transgenic mice model of pancreatic cystic neoplasm, we studied the gene expression changes by applying high-density microarrays. Validation of part gene expression profiling data was performed using real-time PCR.RESULTS: By using high-density oligonucleotide microarray, of 14113 genes, 453 were increased and 760 decreased in pancreatic cystic neoplasm, including oncogenes, cell-cycle-related genes, signal transduction-related genes, skeleton-related genes and metabolism-related genes. Among these, we confirmed the changes in Igf, Shh and Wnt signal pathways with real-time PCR.The results of real-time PCR showed similar expression changes in gene chip.CONCLUSION: all the altered expression genes are associated with cell cycle, DNA damage and repair, signal pathway, and metabolism. SV40Tag may cooperate with several proteins in promoting tumorigenesis.

  13. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

    Science.gov (United States)

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

    2014-04-01

    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

  14. INFLUENCE OF CHEMOTHERAPEUTANTS AND CYTOKINES ON GROWTH AND TRANSGENE EXPRESSION OF BONE MARROW CELLS FROM MT/P210bcr-ab1 TRANSGENIC MICE

    Institute of Scientific and Technical Information of China (English)

    CHEN Hanchun; Andrew Pierce; Tony Whetton

    1999-01-01

    Objective: To investigate the influence of chemotherapeutic agents and cytokines on growth of bone marrow cells from MT/p210 bcr-ab1 transgenic mice.Methods: The bone marrow cells of transgenic chronic myelogenous leukemia (CML) model mice carrying metallothionein (MT) promoter/enhancer, bcr-abl (p210)cDNA and SV40 splicing/poly (A) signal sequences were cultured in liquid and soft agar with hydroxyurea (Hu),5-fluorouracil (5-Fu), mouse stem cell factor (mSCF)and mouse interleukin-3 (mIL-3) independently or collectively. The cells and colonies were counted. The levels of transgene expression were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).Results: The cell proliferation, colony formation and transgene expression of the bone marrow cells were stimulated with mSCF and mIL-3, but there was little growth without any growth factors, or when mSCF,mIL-3 and Hu or 5-Fu were added. Conclusion: The combined utilization of chemotherapeutants and cytokines is a potentially effective strategy of clinical treatment for CML.

  15. Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Balaji, Vasudevan; Smart, Christine D

    2012-02-01

    Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T(0) transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T(0) lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T(0) lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T(1) plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100-10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.

  16. Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat (Triticum aestivum L.) Varieties Transferred from Transgenic Wheat through Conventional Crossing

    Institute of Scientific and Technical Information of China (English)

    Sanhe LI; Ju LI; Nali WANG; Yuesheng WANG; Guangxiao YANG; Jingye FANG; Guangyuan HE

    2007-01-01

    To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Emai 18 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Emai18×B72-8-11b, and F1 plants were selfed or backcrossed to obtain different generation populations.Protein analysis in grains of F1 and F2 and backcross progenies of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2 and BC2F3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes 1Dx5 and 1Ax1 were expressed and segregated in the target wheat according to Mendelian laws. A range of 1Dx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai18×B72-8-11b, but the expression levels of 1Ax1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign 1Dx5 gene of 5-10 copies had the more complicated expression mechanism than the 1Ax1 gene of 4-5 copies.

  17. Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

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    Maria Arantzazu Sanchez-Fernandez

    Full Text Available BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK, a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

  18. Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits.

    Science.gov (United States)

    Chen, Yuhui; Wang, Aoxue; Zhao, Lingxia; Shen, Guoan; Cui, Lijie; Tang, Kexuan

    2009-04-01

    Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.

  19. Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance.

    Science.gov (United States)

    Romero, C; Bellés, J M; Vayá, J L; Serrano, R; Culiáñez-Macià, F A

    1997-03-01

    The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehaloseaccumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance.

  20. Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

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    Richard W Daniels

    Full Text Available Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A and Thosea asigna virus (T2A worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

  1. Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

    Science.gov (United States)

    Nayak, P; Basu, D; Das, S; Basu, A; Ghosh, D; Ramakrishnan, N A; Ghosh, M; Sen, S K

    1997-03-18

    Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

  2. Construction of plant expression vectors carrying glnA gene encoding glutamine synthetase and regeneration of transgenic rice plants

    Institute of Scientific and Technical Information of China (English)

    苏金; 张雪琴; 颜秋生; 陈章良; 尤崇杓

    1995-01-01

    The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilenseSp7 with PCR technique.The amplified 1.4-kb DNA fragment flanked with a BamH Ⅰ site at each end wascloned into EcoR V site of Bluescript-SK vector.A recombinant plasmid pGSJ1 containing this 1.4-kb DNA frag-ment was selected by restriction digestion analysis.The sequencing data also confirmed that the amplified 1.4-kbDNA fragment was undoubtedly the glnA gene of A.brasilense Sp7.Then the 1.4-kb BamH Ⅰ fragment was ex-cised from pGSJ1.A glnA plant expression vector pAGNB92 with rice actin 1 (Act1) promoter was constructedby using colony in situ hybridization to screen positive clones,and 3 rounds of ligation and transformation wereperformed.Protoplasts isolated from rice (Oryza sativa,L.Japonica) cell suspension line (cv.T986) weretransformed with the glnA plant expression vector pAGNB92 carrying neomycin phosphotransferase Ⅱ (NPT Ⅱ)gene by PEG fusion or electroporation.G418~ calli were used to detect NPT Ⅱ enzyme activity.The resultsshow that G418~ calli possess high positive hybridization signal with the frequency of 37%.The regeneratedG418~NPTII~+ rice plants were used for PCR amplification of glnA gene,and a 1.4-kb DNA fragment was ampli-fied from glnA-transgenic rice plants (R0 generation).The results of Southern blot hybridization prove that the1.4-kb DNA fragment amplified from the total DNA of glnA transgenic rice plants is indeed the glnA gene of A.brasilense Sp7.Northern blot hybridization was carried out using the same glnA gene as probe.The glnAgene was expressed in the transgenic rice plants.Bioassays also confirmed that the glnA transgenic rice plantsgrew much better than that of the control plants under a condition with nitrogen poor source (0.75 mmol/L).

  3. Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

    Science.gov (United States)

    Chan, Hui-Ting; Chia, Min-Yuan; Pang, Victor Fei; Jeng, Chian-Ren; Do, Yi-Yin; Huang, Pung-Ling

    2013-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.

  4. Brain beta-amyloid accumulation in transgenic mice expressing mutant superoxide dismutase 1.

    Science.gov (United States)

    Turner, Bradley J; Li, Qiao-Xin; Laughton, Katrina M; Masters, Colin L; Lopes, Elizabeth C; Atkin, Julie D; Cheema, Surindar S

    2004-12-01

    Oxidative stress is implicated in both the deposition and pathogenesis of beta-amyloid (Abeta) protein in Alzheimer's disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces Abeta toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates Abeta toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound Abeta peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated Abeta-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1G93A mice using enzyme-linked immunosorbent assay of acid-guanidine extracts revealed age-dependent elevations in Abeta levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither Abeta toxicity nor brain accumulation in these ALS models.

  5. Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human β1 and α2 integrin.

    Directory of Open Access Journals (Sweden)

    Nicklas Heine Staunstrup

    Full Text Available Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to

  6. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

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    Guidot David M

    2008-04-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 infection and the consequent acquired immunodeficiency syndrome (AIDS has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef. Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1 and Transforming Growth Factor-β1 (TGFβ1, three factors associated with muscle catabolism. Results Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss, the oxidized form of the anti-oxidant cysteine (Cys, and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold and TGFβ1 (~5-fold and ~3-fold in heart and

  7. Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco.

    Science.gov (United States)

    Yevtushenko, Dmytro P; Romero, Rafael; Forward, Benjamin S; Hancock, Robert E; Kay, William W; Misra, Santosh

    2005-06-01

    Expression of defensive genes from a promoter that is specifically activated in response to pathogen invasion is highly desirable for engineering disease-resistant plants. A plant transformation vector was constructed with transcriptional fusion between the pathogen-responsive win3.12T promoter from poplar and the gene encoding the novel cecropin A-melittin hybrid peptide (CEMA) with strong antimicrobial activity. This promoter-transgene combination was evaluated in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) for enhanced plant resistance against a highly virulent pathogenic fungus Fusarium solani. Transgene expression in leaves was strongly increased after fungal infection or mechanical wounding, and the accumulation of CEMA transcripts was found to be systemic and positively correlated with the number of transgene insertions. A simple and efficient in vitro regeneration bioassay for preliminary screening of transgenic lines against pathogenic fungi was developed. CEMA had strong antifungal activity in vitro, inhibiting conidia germination at concentrations that were non-toxic to tobacco protoplasts. Most importantly, the expression level of the CEMA peptide in vivo, regulated by the win3.12T promoter, was sufficient to confer resistance against F. solani in transgenic tobacco. The antifungal resistance of plants with high CEMA expression was strong and reproducible. In addition, leaf tissue extracts from transgenic plants significantly reduced the number of fungal colonies arising from germinated conidia. Accumulation of CEMA peptide in transgenic tobacco had no deleterious effect on plant growth and development. This is the first report showing the application of a heterologous pathogen-inducible promoter to direct the expression of an antimicrobial peptide in plants, and the feasibility of this approach to provide disease resistance in tobacco and, possibly, other crops.

  8. Transgenic GNA expressing potato plants augment the beneficial biocontrol of Lacanobia oleracea (Lepidoptera; Noctuidae) by the parasitoid Eulophus pennicornis (Hymenoptera; Eulophidae).

    Science.gov (United States)

    Bell, H A; Fitches, E C; Marris, G C; Bell, J; Edwards, J P; Gatehouse, J A; Gatehouse, A M

    2001-01-01

    The effect of expressing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin, GNA) in transgenic potato plants, on parasitism of the phytophagous insect pest Lacanobia oleracea by the gregarious ectoparasitoid Eulophus pennicornis, was investigated in glasshouse trials. Expression of GNA (approx. 1.0% total soluble protein) by transgenic plants significantly reduced the level of pest damage, thus confirming previous studies. Furthermore, the presence of the parasitoid significantly reduced the levels of damage incurred either by the transgenic or control plants when compared to those plants grown in the absence of the parasitoid. For the GNA expressing plants the presence of the parasitoid resulted in further reductions (ca. 21%) in the level of damage caused by the pest species. The ability of the wasp to parasitise and subsequently develop on the pest larvae was not altered by the presence of GNA in the diet of the host. E. pennicornis progeny that developed on L. oleracea reared on GNA expressing plants showed no significant alteration in fecundity when compared with wasps that had developed on hosts fed on control potato plants, although mean size and longevity of female parasitoids was significantly reduced. The number of F2 progeny produced by parasitoids derived from hosts fed on GNA expressing plants was not significantly different to those produced by parasitoids from hosts fed control plants. Results from the present study demonstrate that the use of transgenic plants expressing insecticidal proteins can be compatible with the deployment of beneficial insects and that the two factors may interact in a positive manner.

  9. Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia.

    Science.gov (United States)

    Ratliff, Eric P; Gutierrez, Alejandra; Davis, Roger A

    2006-07-01

    Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.

  10. Immune-mediated loss of transgene expression from virally transduced brain cells is irreversible, mediated by IFNγ, perforin, and TNFα, and due to the elimination of transduced cells.

    Science.gov (United States)

    Zirger, Jeffrey M; Puntel, Mariana; Bergeron, Josee; Wibowo, Mia; Moridzadeh, Rameen; Bondale, Niyati; Barcia, Carlos; Kroeger, Kurt M; Liu, Chunyan; Castro, Maria G; Lowenstein, Pedro R

    2012-04-01

    The adaptive immune response to viral vectors reduces vector-mediated transgene expression from the brain. It is unknown, however, whether this loss is caused by functional downregulation of transgene expression or death of transduced cells. Herein, we demonstrate that during the elimination of transgene expression, the brain becomes infiltrated with CD4(+) and CD8(+) T cells and that these T cells are necessary for transgene elimination. Further, the loss of transgene-expressing brain cells fails to occur in the absence of IFNγ, perforin, and TNFα receptor. Two methods to induce severe immune suppression in immunized animals also fail to restitute transgene expression, demonstrating the irreversibility of this process. The need for cytotoxic molecules and the irreversibility of the reduction in transgene expression suggested to us that elimination of transduced cells is responsible for the loss of transgene expression. A new experimental paradigm that discriminates between downregulation of transgene expression and the elimination of transduced cells demonstrates that transduced cells are lost from the brain upon the induction of a specific antiviral immune response. We conclude that the anti-adenoviral immune response reduces transgene expression in the brain through loss of transduced cells.

  11. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    Science.gov (United States)

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  12. Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus.

    Science.gov (United States)

    Miao, G H; Hirel, B; Marsolier, M C; Ridge, R W; Verma, D P

    1991-01-01

    A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

  13. Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.

    Science.gov (United States)

    Bruewer, Ashlee R; Mowat, Freya M; Bartoe, Joshua T; Boye, Sanford L; Hauswirth, William W; Petersen-Jones, Simon M

    2013-01-01

    Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these

  14. Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.

    Directory of Open Access Journals (Sweden)

    Ashlee R Bruewer

    Full Text Available Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F capsid mutant, self-complementary (sc AAV2/8 (single Y-F capsid mutant and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain

  15. Expression activity of the CpTI gene in transgenic rice plants

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Plant harboured protease inhibitor is a part of the natural plant defense system against insect predation. Plants transformed with foreign plant protease inhibitor genes can enhance resistance to insect pests. So far, at least 20 kinds of plants, including tobacco, rice, tomato, cotton et al., have been transformed with various plant protease inhibitor genes. We have transformed rice with CpTI (cowpea trypsin inhibitor) gene. To assess the range and stability of expression of the CpTI gene, CpTI protein activities were determined in various tissues and at different development stages of transgenic inbred lines.

  16. Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression.

    Science.gov (United States)

    Nadolska-Orczyk, Anna; Pietrusinska, Aleksandra; Binka-Wyrwa, Agnieszka; Kuc, Dominik; Orczyk, Wacław

    2007-01-01

    This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.

  17. MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

    Science.gov (United States)

    Kostyrko, Kaja; Neuenschwander, Samuel; Junier, Thomas; Regamey, Alexandre; Iseli, Christian; Schmid-Siegert, Emanuel; Bosshard, Sandra; Majocchi, Stefano; Le Fourn, Valérie; Girod, Pierre-Alain; Xenarios, Ioannis; Mermod, Nicolas

    2017-02-01

    Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

  18. Doxycycline-Induced Expression of Transgenic Human Tumor Necrosis Factor α in Adult Mice Results in Psoriasis-like Arthritis

    Science.gov (United States)

    Retser, Eugen; Schied, Tanja; Skryabin, Boris V; Vogl, Thomas; Kanczler, Janos M; Hamann, Nina; Niehoff, Anja; Hermann, Sven; Eisenblätter, Michel; Wachsmuth, Lydia; Pap, Thomas; van Lent, Peter L E M; Loser, Karin; Roth, Johannes; Zaucke, Frank; Ludwig, Stephan; Wixler, Viktor

    2013-01-01

    Objective To generate doxycycline-inducible human tumor necrosis factor α (TNFα)–transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice. Methods A transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases. Results Doxycycline-inducible human TNFα–transgenic mice developed an inflammatory arthritis– and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of “sausage digits” with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3–6 weeks later, only minimal signs of disease were visible. Conclusion Upon doxycycline administration, the doxycycline-inducible human TNFα–transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα–transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα. PMID:23740547

  19. Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period

    OpenAIRE

    Aldwinckle Herb S; Norelli John L; Borejsza-Wysocka Ewa; Malnoy Mickael

    2010-01-01

    Abstract Background Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight dise...

  20. Expression of a novel piscine growth hormone gene results in growth enhancement in transgenic tilapia (Oreochromis niloticus).

    Science.gov (United States)

    Rahman, M A; Mak, R; Ayad, H; Smith, A; Maclean, N

    1998-09-01

    Several lines of transgenic G1 and G2 tilapia fish (Oreochromis niloticus) have been produced following egg injection with gene constructs carrying growth hormone coding sequences of fish origin. Using a construct in which an ocean pout antifreeze promoter drives a chinook salmon growth hormone gene, dramatic growth enhancement has been demonstrated, in which the mean weight of the 7 month old G2 transgenic fish is more than three fold that of their non transgenic siblings. Somewhat surprisingly G1 fish transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement, although salmon transgenic for this construct do show greatly enhanced growth. The growth enhanced transgenic lines were also strongly positive in a radio-immuno assay for the specific hormone in their serum, whereas the non growth enhanced lines were negative. Attempts to induce expression from the metallothionein promoter by exposing fish to increased levels of zinc were also unsuccessful. Homozygous transgenic fish have been produced from the ocean pout antifreeze/chinook salmon GH construct and preliminary trials suggest that their growth performance is similar to that of the hemizygous transgenics. No abnormalities were apparent in the growth enhanced fish, although minor changes to skull shape and reduced fertility were noted in some fish. There is also preliminary evidence for improved food conversion ratios when growth enhanced transgenic tilapia are compared to their non-transgenic siblings. The long term objective of this study is to produce lines of tilapia which are both growth enhanced and sterile, so offering improved strains of this important food fish for aquaculture.

  1. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  2. Ectopic Terpene Synthase Expression Enhances Sesquiterpene Emission in Nicotiana attenuata without Altering Defense or Development of Transgenic Plants or Neighbors1[W

    Science.gov (United States)

    Schuman, Meredith C.; Palmer-Young, Evan C.; Schmidt, Axel; Gershenzon, Jonathan; Baldwin, Ian T.

    2014-01-01

    Sesquiterpenoids, with approximately 5,000 structures, are the most diverse class of plant volatiles with manifold hypothesized functions in defense, stress tolerance, and signaling between and within plants. These hypotheses have often been tested by transforming plants with sesquiterpene synthases expressed behind the constitutively active 35S promoter, which may have physiological costs measured as inhibited growth and reduced reproduction or may require augmentation of substrate pools to achieve enhanced emission, complicating the interpretation of data from affected transgenic lines. Here, we expressed maize (Zea mays) terpene synthase10 (ZmTPS10), which produces (E)-α-bergamotene and (E)-β-farnesene, or a point mutant ZmTPS10M, which produces primarily (E)-β-farnesene, under control of the 35S promoter in the ecological model plant Nicotiana attenuata. Transgenic N. attenuata plants had specifically enhanced emission of target sesquiterpene(s) with no changes detected in their emission of any other volatiles. Treatment with herbivore or jasmonate elicitors induces emission of (E)-α-bergamotene in wild-type plants and also tended to increase emission of (E)-α-bergamotene and (E)-β-farnesene in transgenics. However, transgenics did not differ from the wild type in defense signaling or chemistry and did not alter defense chemistry in neighboring wild-type plants. These data are inconsistent with within-plant and between-plant signaling functions of (E)-β-farnesene and (E)-α-bergamotene in N. attenuata. Ectopic sesquiterpene emission was apparently not costly for transgenics, which were similar to wild-type plants in their growth and reproduction, even when forced to compete for common resources. These transgenics would be well suited for field experiments to investigate indirect ecological effects of sesquiterpenes for a wild plant in its native habitat. PMID:25187528

  3. Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Soitamo Arto J

    2012-11-01

    Full Text Available Abstract Background RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum leaves and in flowers. Results Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. Conclusions AC2 RSS in

  4. Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants.

    Science.gov (United States)

    Soitamo, Arto J; Jada, Balaji; Lehto, Kirsi

    2012-11-07

    RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers. Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. AC2 RSS in transgenic tobacco plants interferes with the silencing

  5. Duration and level of transgene expression after gene electrotransfer to skin in mice

    DEFF Research Database (Denmark)

    Gothelf, A; Eriksen, Jens Ole; Hojman, P

    2010-01-01

    In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene...... is a suitable time frame for vaccinations and is applicable, for example, in gene therapy for wound healing or treatment of cancer.......In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene...... electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations...

  6. Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encoding human erythropoietin (hEPO) and governed by regulatory sequences of mouse whey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%. hEPO was expressed in the milk of 16 mice of 39 mice measured by hEPO ELISA kit .The expression level gets over 15 m g/mL.

  7. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Tomioka, Yukiko, E-mail: ytomi@muses.tottori-u.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Morimatsu, Masami, E-mail: mmorimat@vetmed.hokudai.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Nishijima, Ken-ichi, E-mail: nishijma@nubio.nagoya-u.ac.jp [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Nagoya 464-8603 (Japan); Usui, Tatsufumi, E-mail: usutatsu@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Yamamoto, Sayo, E-mail: ysayo@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Suyama, Haruka, E-mail: sharuka@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ozaki, Kinuyo, E-mail: k-ozaki@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ito, Toshihiro, E-mail: toshiito@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); and others

    2014-07-18

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

  8. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

    Directory of Open Access Journals (Sweden)

    Quatromoni Jon G

    2011-11-01

    Full Text Available Abstract Regulatory T cells (Treg that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg or induced Treg (iTreg converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL and splenocytes (SPL in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA. Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR and exposure to transforming growth factor β (TGFβ. B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.

  9. Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China); Liu, Junhong, E-mail: liujh@qust.edu.cn [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China)

    2011-05-15

    Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione S-transferase (GST) were generated from hypocotyl segments by the use of an Agrobacterium transformation system for the phytoremediation of the mixed contaminated soil with heavy metals and organic pollutants. The transgenic alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST and CYP2E1 activity and Western blot analysis. The capabilities of mixed contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic alfalfa plants became markedly increased compared with the transgenic alfalfa plants expressing single gene (GST or CYP2E1) and the non-transgenic control plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the introduced GST and CYP2E1 in combination. Our results show that the pKHCG transgenic alfalfa plants have good potential for phytoremediation because they have cross-tolerance towards the complex contaminants of heavy metals and organic pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and human P450 CDNAs may have a great potential for phytoremediation of mixed environmental contaminants.

  10. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  11. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages.

  12. Pest control and resistance management through release of insects carrying a male-selecting transgene

    OpenAIRE

    2015-01-01

    Background Development and evaluation of new insect pest management tools is critical for overcoming over-reliance upon, and growing resistance to, synthetic, biological and plant-expressed insecticides. For transgenic crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis (‘Bt crops’) emergence of resistance is slowed by maintaining a proportion of the crop as non-Bt varieties, which produce pest insects unselected for resistance. While this strategy has been largel...

  13. Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models.

    Science.gov (United States)

    Amaral, Andreia J; Brito, Francisco F; Chobanyan, Tamar; Yoshikawa, Seiko; Yokokura, Takakazu; Van Vactor, David; Gama-Carvalho, Margarida

    2014-01-01

    RNA-sequencing (RNA-seq) is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order to achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of "biological quality" are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system (CNS) of a Drosophila transgenic strain with neuronal-specific RNAi of an ubiquitous gene was profiled using RNA-seq. After observing the existence of an unusual variance in our dataset, we showed that the expression profile of a small panel of marker genes, including GAL4 under control of a tissue specific driver, can identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We further analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we showed that trimming 5' ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes with a potential positive impact in the incurrence of systematic

  14. Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models

    Directory of Open Access Journals (Sweden)

    Andreia J Amaral

    2014-03-01

    Full Text Available RNA-sequencing (RNA-seq is rapidly emerging as the technology of choice for whole-transcriptome studies. However, RNA-seq is not a bias free technique. It requires large amounts of RNA and library preparation can introduce multiple artifacts, compounded by problems from later stages in the process. Nevertheless, RNA-seq is increasingly used in multiple studies, including the characterization of tissue-specific transcriptomes from invertebrate models of human disease. The generation of samples in this context is complex, involving the establishment of mutant strains and the delicate contamination prone process of dissecting the target tissue. Moreover, in order achieve the required amount of RNA, multiple samples need to be pooled. Such datasets pose extra challenges due to the large variability that may occur between similar pools, mostly due to the presence of cells from surrounding tissues. Therefore, in addition to standard quality control of RNA-seq data, analytical procedures for control of 'biological quality’ are critical for successful comparison of gene expression profiles. In this study, the transcriptome of the central nervous system of a Drosophila transgenic strain with neuronal-specific RNAi of an ubiquitous gene was profiled using RNA-seq. After observing the existence of an unusual variance in our dataset, we showed that the expression profile of a small panel of marker genes, including GAL4 under control of a tissue specific driver, can identify libraries with low levels of contamination from neighboring tissues, enabling the selection of a robust dataset for differential expression analysis. We further analyzed the potential of profiling a complex tissue to identify cell-type specific changes in response to target gene down-regulation. Finally, we showed that trimming 5’ ends of reads decreases nucleotide frequency biases, increasing the coverage of protein coding genes and decreasing the occurrence of systematic technical

  15. Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3

    Directory of Open Access Journals (Sweden)

    Yokota Takashi

    2008-05-01

    Full Text Available Abstract Background The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. Results Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B was shown to be restricted to the inner cell mass (ICM of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. Conclusion Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent

  16. Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit (HMW-GS) genes in winter wheat

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic