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Sample records for t1 transgenic lines

  1. No direct effects of two transgenic Bt rice lines, T1C-19 and T2A-1, on the arthropod communities.

    PubMed

    Lu, Z B; Tian, J C; Han, N S; Hu, C; Peng, Y F; Stanley, David; Ye, G Y

    2014-10-01

    A 2-yr field trial was conducted to assess the impacts of two new transgenic Bt rice lines, T1C-19 expressing Cry1C protein and T2A-1 expressing Cry2A protein, on the arthropod community sampled via vacuum. All the arthropods were classified into five guilds, including herbivores, parasitoids, predators, detritivores, and others. The seasonal density and dominance distribution of each guild and community-level indices (species richness, Shannon-Wiener diversity index, Simpson diversity index, and evenness index) were compared among rice types. Principal response curves were used to investigate the differences of entire arthropod community of Bt rice plots relative to non-Bt rice plots. The results showed no significant difference was detected in the community-level indices and dominance distribution of guilds between Bt and non-Bt rice plots. The seasonal density of herbivores, detritivores, and others as well as density of the arthropod overall community were also not significantly affected by rice types in either year, although the density of predators and parasitoids in Bt rice plots was significantly lower than those in non-Bt rice plots. The lower abundances of Braconidae, Eulophidae, Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), and Theridiidae in Bt rice plots are likely attributed to the lower abundances of prey species or hosts. Principal response curves revealed that arthropod community in Bt was similar with that in non-Bt rice plots. In conclusion, our findings indicate that these two tested Bt rice lines had no marked negative effects on the arthropod community in the paddy fields. PMID:25203669

  2. Transgenic cry1C(?) gene rough rice line T1C-19 does not change the host preferences of the non-target stored product pest, Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae), and its parasitoid wasp, Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae).

    PubMed

    Sun, Xiao; Yan, Miao-Jun; Zhang, Aijun; Wang, Man-Qun

    2015-10-01

    Rough rice grains are often stored for extended periods before they are used or consumed. However, during storage, the rough rice is vulnerable to insect infestation, resulting in significant economic loss. Previous studies have shown that volatiles cues, physical characteristics, and taste chemicals on the grains could be the important key behavior factors for storage insect pests to locate the hosts and select oviposition sites. It is also well known that the transgenic Bt rough rice line T1C-19, which expresses a cry1C(?) gene has a high resistance to Lepidoptera pests. However, there were no evidences to show the consequences of host preference for non-target insect pests after growing Bt transgenic rice. In this study, the potential key factors of Bt rough rice were investigated for their impacts on the behaviors of non-target pest lesser grain borer Rhyzopertha dominica, the main weevil pest of grain and its parasitic wasps Anisopteromalus calandrae, the natural enemy of the beetle. Both electronic nose and electronic tongue analyses showed that the parameters of Bt rough rice were analogous to those of the non-Bt rough rice. The volatile profiles of Bt and non-Bt rough rice examined by gas chromatographic mass spectrometry (GC-MS) were similar. For most volatile compounds, there were no significantly quantitative differences in compound quantities between Bt and non-Bt rough rice. The densities of sclereids and trichomes on the rough rice husk surface were statistically equal in Bt and non-Bt rough rice. The non-target pest, R. dominica, and its parasitoid wasp, A. calandrae, were attracted to both rough rice and could not distinguish the transgenic T1C-19 from the isogenic rough rice. These results demonstrated that Bt rough rice has no negative impacts on the host preference behaviors of non-target stored product pest R. dominica and its parasitoid A. calandrae. PMID:26150137

  3. 2008 FHB Analysis of Transgenic Barley Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic lines have been developed with the goal of reducing FHB and DON in barley. Replicated field trials for FHB reaction of 48 Conlon transgenic lines were conducted in 2008 in Langdon, ND and Rosemount, MN. The Langdon trials consisted of three replicates in hill plots in an inoculated misted...

  4. Abstract Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombard-

    E-print Network

    Pawlowski, Wojtek

    Abstract Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A heterochromatic C ge- nome and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes

  5. Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny.

    PubMed

    Newhouse, Andrew E; Polin-McGuigan, Linda D; Baier, Kathleen A; Valletta, Kristia E R; Rottmann, William H; Tschaplinski, Timothy J; Maynard, Charles A; Powell, William A

    2014-11-01

    American chestnut (Castanea dentata) is a classic example of a native keystone species that was nearly eradicated by an introduced fungal pathogen. This report describes progress made toward producing a fully American chestnut tree with enhanced resistance to the blight fungus (Cryphonectria parasitica). The transgenic American chestnut 'Darling4,' produced through an Agrobacterium co-transformation procedure to express a wheat oxalate oxidase gene driven by the VspB vascular promoter, shows enhanced blight resistance at a level intermediate between susceptible American chestnut and resistant Chinese chestnut (Castanea mollissima). Enhanced resistance was identified first with a leaf-inoculation assay using young chestnuts grown indoors, and confirmed with traditional stem inoculations on 3- and 4-year-old field-grown trees. Pollen from 'Darling4' and other events was used to produce transgenic T1 seedlings, which also expressed the enhanced resistance trait in leaf assays. Outcrossed transgenic seedlings have several advantages over tissue-cultured plantlets, including increased genetic diversity and faster initial growth. This represents a major step toward the restoration of the majestic American chestnut. PMID:25438789

  6. Field tests of transgenic barley lines in North Dakota

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Testing transgenic barley lines for FHB in the greenhouse does not necessarily give the same results as field tests. The objective of this project was to test 18 transgenic lines in replicated trials in an inoculated FHB nursery. Several programs have developed barley lines expressing anti-fungal a...

  7. [Obtaining the transgenic lines of finger millet Eleusine coracana (L.) Gaertn. With dinitroaniline resistance].

    PubMed

    Baer, G Ia; Emets, A I; Blium, Ia B

    2014-01-01

    The current data is dedicated to the study of bioballistic and Agrobacterium-mediated transformation of finger millet with the constructs carrying the mutant alpha-tubulin gene (TUAm 1), isolated from R-biotype goosegrass (Eleusine indica L.), for the decision of problem of dinitroaniline-resistance. It was found that 10 microM of trifluralin is optimal for the selection of transgene plants of finger millet. PCR analysis of transformed lines confirmed the transgene nature of plants. The analysis of seed of T1 oftransgene lines confirmed heterozygous character of inheritance of the resistance. PMID:25016822

  8. Case Study: Polycystic Livers in a Transgenic Mouse Line

    SciTech Connect

    Lovaglio, Jamie A.; Artwohl, James E.; Ward, Christopher J.; Diekwisch, Thomas G. H.; Ito, Yoshihiro; Fortman, Jeffrey D.

    2014-04-01

    Three mice (2 male, 1 female; age, 5 to 16 mo) from a mouse line transgenic for keratin 14 (K14)-driven LacZ expression and on an outbred Crl:CD1(ICR) background, were identified as having distended abdomens and livers that were diffusely enlarged by numerous cysts (diameter, 0.1 to 2.0 cm). Histopathology revealed hepatic cysts lined by biliary type epithelium and mild chronic inflammation, and confirmed the absence of parasites. Among 21 related mice, 5 additional affected mice were identified via laparotomy. Breeding of these 5 mice (after 5 mo of age) did not result in any offspring; the K14 mice with olycystic livers failed to reproduce. Affected male mice had degenerative testicular lesions, and their sperm was immotile. Nonpolycystic K14 control male mice bred well, had no testicular lesions, and had appropriate sperm motility. Genetic analysis did not identify an association of this phenotype with the transgene or insertion site.

  9. Generation and characterization of transgenic plum lines expressing gafp-1 with the bul409 promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Gastrodia anti fungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously-generated transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were create...

  10. Skin Fibroblasts from Patients with Type 1 Diabetes (T1D) Can Be Chemically Transdifferentiated into Insulin-Expressing Clusters: A Transgene-Free Approach

    PubMed Central

    Pereyra-Bonnet, Federico; Gimeno, María L.; Argumedo, Nelson R.; Ielpi, Marcelo; Cardozo, Johana A.; Giménez, Carla A.; Hyon, Sung-Ho; Balzaretti, Marta; Loresi, Mónica; Fainstein-Day, Patricia; Litwak, León E.; Argibay, Pablo F.

    2014-01-01

    The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D. PMID:24963634

  11. T1-11 and JMF1907 ameliorate polyglutamine-expanded ataxin-3-induced neurodegeneration, transcriptional dysregulation and ataxic symptom in the SCA3 transgenic mouse.

    PubMed

    Chou, An-Hsun; Chen, Ying-Ling; Chiu, Ching-Chi; Yuan, Shin-Je; Weng, Yi-Hsin; Yeh, Tu-Hsueh; Lin, Yun-Lian; Fang, Jim-Min; Wang, Hung-Li

    2015-12-01

    More studies are required to develop therapeutic agents for treating spinocerebellar ataxia type 3 (SCA3), which is caused by mutant polyglutamine-expanded ataxin-3 and is the most prevalent subtype of spinocerebellar ataxias. T1-11 [N(6)-(4-Hydroxybenzyl) adenosine], isolated from a Chinese medicinal herb Gastordia elata, is an adenosine A2A receptor agonist. SCA3 and Huntington's disease (HD) belong to a family of polyglutamine neurodegenerative diseases. T1-11 exerted a therapeutic effect on HD transgenic mouse by decreasing protein level of polyglutamine-expanded huntingtin in the striatum. In the present study, we test the possibility that T1-11 or JMF1907 [N(6)-(3-Indolylethyl) adenosine], a synthetic analog of T1-11, alleviates pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom in the SCA3 transgenic mouse expressing HA-tagged polyglutamine-expanded ataxin-3-Q79 (ataxin-3-Q79HA). Daily oral administration of T1-11 or JMF1907 prevented neuronal death of pontine nuclei in the SCA3 mouse with a dose-dependent manner. Oral application of T1-11 or JMF1907 reversed mutant ataxin-3-Q79-induced cerebellar transcriptional repression in the SCA3 transgenic mouse. T1-11 or JMF1907 ameliorated the symptom of motor incoordination displayed by SCA3 mouse. Oral administration of T1-11 or JMF1907 significantly decreased protein level of ataxin-3-Q79HA in the pontine nuclei or cerebellum of SCA3 mouse. T1-11 or JMF1907 significantly augmented the chymotrypsin-like activity of proteasome in the pontine nuclei or cerebellum of SCA3 mouse. Our results suggests that T1-11 and JMF1907 alleviate pontine neuronal death, cerebellar transcriptional downregulation and ataxic symptom of SCA3 transgenic mouse by augmenting the proteasome activity and reducing the protein level of polyglutamine-expanded ataxin-3-Q79 in the pontine nuclei and cerebellum. PMID:26254860

  12. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

  13. Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

    PubMed Central

    Nunna, Hariprasad Rao; Puligundla, Sateesh Kumar; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2013-01-01

    Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects. PMID:24023750

  14. Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests.

    PubMed

    Vajhala, Chakravarthy S K; Sadumpati, Vijaya Kumar; Nunna, Hariprasad Rao; Puligundla, Sateesh Kumar; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2013-01-01

    Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1-2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects. PMID:24023750

  15. Response of sensitive human ataxia and resistant T-1 cell lines to accelerated heavy ions

    SciTech Connect

    Tobias, C.A.; Blakely, E.A.; Chang, P.Y.; Lommel, L.; Roots, R.

    1983-07-01

    The radiation dose responses of fibroblast from a patient with Ataxia telangiectasis (AT-2SF) and an established line of human T-1 cells were studied. Nearly monoenergetic accelerated neon and argon ions were used at the Berkeley Bevalac with various residual range values. The LET of the particles varied from 30 keV/..mu..m to over 1000 keV/..mu..m. All Ataxia survival curves were exponential functions of the dose. Their radiosensitivity reached peak values at 100 to 200 keV/..mu..m. Human T-1 cells have effective sublethal damage repair as has been evidenced by split dose experiments, and they are much more resistant to low LET than to high LET radiation. The repair-misrepair model has been used to interpret these results. We have obtained mathematical expressions that describe the cross sections and inactivation coefficients for both human cell lines as a function of the LET and the type of particle used. The results suggest either that high-LET particles induce a greater number of radiolesions per track or that heavy-ions at high LET induce lesions that kill cells more effectively and that are different from those produced at low LET. We assume that the lesions induced in T-1 and Ataxia cells are qualitatively similar and that each cell line attempts to repair these lesions. The result in most irradiated Ataxia cells, however, is either lethal misrepair or incomplete repair leading to cell death. 63 references, 10 figures, 1 table.

  16. Stable expression of a bacterial GUS gene in vegetatively propagated transgenic pear lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stability of a transgene in the genomes of in vitro propagated transgenic pear lines was assessed. A bacterial GUS reporter gene under the control of an Arabidopsis sucrose transporter gene promoter was introduced into pear cultivar ‘Old Home’ through Agrobacterium-mediated leaf-explant transfo...

  17. Calcium signaling properties of a thyrotroph cell line, mouse T?T1 cells.

    PubMed

    Tomi?, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    T?T1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in T?T1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. T?T1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that T?T1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. PMID:26453278

  18. In vivo immunomodulatory effects of Antrodia camphorata polysaccharides in a T1/T2 doubly transgenic mouse model for inhibiting infection of Schistosoma mansoni

    SciTech Connect

    Cheng, P.-C.; Hsu, C.-Y.; Chen, C.-C.; Lee, K.-M.

    2008-03-01

    Antrodia camphorata (A. camphorata) is a fungus commonly used for treatment of viral hepatitis and cancer in Chinese folk medicine. Extract of A. camphorate is reported to possess anti-inflammatory, antihepatitis B virus and anticancer activities. In this study, we tested the in vivo effects of polysaccharides derived from A. camphorata (AC-PS) on immune function by detection of cytokine expression and evaluation of the immune phenotype in a T1/T2 doubly transgenic mouse model. The protective effect of AC-PS in mice was tested by infection with Schistosoma mansoni. The induction of large amounts of IFN-{gamma}, IL-2 and TNF-a mRNA were detected after 2 and 4 weeks of oral AC-PS administration in BALB/c and C57BL/6 mice. In transgenic mice, 3 to 6 weeks of oral AC-PS administration increased the proportion of CD4{sup +} T cells and B cells within the spleen. More specifically, there was an increase of Th1 CD4{sup +} T cells and Be1 cells among spleen cells as observed by detection the of Type1/Type2 marker molecules. By using a disease model of parasitic infection, we found that AC-PS treatment inhibited infection with S. mansoni in BALB/C and C57BL/6 mice. AC-PS appears to influence the immune system of mice into developing Th1 responses and have potential for preventing infection with S. mansoni.

  19. Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids

    E-print Network

    Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids Amir, USA 3 Department of Agronomy & Horticulture, Center for Biotechnology, Center for Plant Science. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized

  20. Field Assessment of AtCBF1 Transgenic Potato Lines (Solanum tuberosum) for Drought Tolerance

    E-print Network

    Douches, David S.

    Field Assessment of AtCBF1 Transgenic Potato Lines (Solanum tuberosum) for Drought Tolerance Nicole 2014 Abstract Drought prone areas have been increasing around the world and it is expected that these areas will continue to expand and become more severe due to climate change. Increasing the drought

  1. RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

    NASA Astrophysics Data System (ADS)

    Wang, Tiegu; Huang, Qunce; Feng, Weisen

    2007-10-01

    Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

  2. Evaluation of the Agronomic Performance of Atrazine-Tolerant Transgenic japonica Rice Parental Lines for Utilization in Hybrid Seed Production

    PubMed Central

    Li, Yanlan; Li, Yanan; Wang, Shengjun; Su, Jinping; Liu, Xuejun; Chen, Defu; Chen, Xiwen

    2014-01-01

    Currently, the purity of hybrid seed is a crucial limiting factor when developing hybrid japonica rice (Oryza sativa L.). To chemically control hybrid seed purity, we transferred an improved atrazine chlorohydrolase gene (atzA) from Pseudomonas ADP into hybrid japonica parental lines (two maintainers, one restorer), and Nipponbare, by using Agrobacterium-mediated transformation. We subsequently selected several transgenic lines from each genotype by using PCR, RT-PCR, and germination analysis. In the presence of the investigated atrazine concentrations, particularly 150 µM atrazine, almost all of the transgenic lines produced significantly larger seedlings, with similar or higher germination percentages, than did the respective controls. Although the seedlings of transgenic lines were taller and gained more root biomass compared to the respective control plants, their growth was nevertheless inhibited by atrazine treatment compared to that without treatment. When grown in soil containing 2 mg/kg or 5 mg/kg atrazine, the transgenic lines were taller, and had higher total chlorophyll contents than did the respective controls; moreover, three of the strongest transgenic lines completely recovered after 45 days of growth. After treatment with 2 mg/kg or 5 mg/kg of atrazine, the atrazine residue remaining in the soil was 2.9–7.0% or 0.8–8.7% respectively, for transgenic lines, and 44.0–59.2% or 28.1–30.8%, respectively, for control plants. Spraying plants at the vegetative growth stage with 0.15% atrazine effectively killed control plants, but not transgenic lines. Our results indicate that transgenic atzA rice plants show tolerance to atrazine, and may be used as parental lines in future hybrid seed production. PMID:25275554

  3. Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines.

    PubMed

    Klocke, Rainer; Gómez-Lechón, M José; Ehrhardt, Anja; Mendoza-Figueroa, Tomas; Donato, M Teresa; López-Revilla, Rubén; Castell, José V; Paul, Dieter

    2002-06-21

    The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions. PMID:12061787

  4. Transgenic tomato line expressing modified Bacillus thuringiensis cry1Ab gene showing complete resistance to two lepidopteran pests.

    PubMed

    Koul, Bhupendra; Srivastava, Sugandha; Sanyal, Indraneel; Tripathi, Bhuminath; Sharma, Vinay; Amla, Devindra Vijay

    2014-01-01

    The modified truncated Bt-cry1Ab gene of Bacillus thuringiensis has been used for the development and selection of over expressing transgenic events in a commercially important variety of tomato (Solanum lycopersicum L.) by Agrobacterium-mediated leaf-disc transformation procedure. The integration and inheritance of cry1Ab gene in T0 transgenic plants and their progenies were determined by PCR, RT-PCR and Southern blot hybridization analysis. The toxin expression was monitored by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The transgenic line Ab25 E, expressing 0.47?±?0.01% Cry1Ab toxin of total soluble protein (TSP) was finally selected in the T4 generation from the segregating population, showing 100% mortality to the second instar larvae of H. armigera and S. litura and minimal damages to leaves and fruits. Southern blot analysis data revealed single copy introgression of cry1Ab gene in highly-expressing Ab25 E transgenic line and expression of Cry1Ab toxin of molecular mass ~65 kDa was evident in Western blot analyses in transgenic plants of T4, T5 and T6 generation. Receptor binding assay performed with partially purified Cry1Ab protein from Ab25 E transgenic tomato line, confirmed efficient protein-protein interaction of Cry1Ab toxin with receptor(s) of both the insects. The higher level of Cry1Ab toxin (? 0.47?±?0.01% TSP) did not affect the normal in vitro regeneration, plant development and fruit yield in this transgenic line. This high expressing Cry1Ab homozygous transgenic line can be a useful candidate in tomato breeding programmes for introgression of important agronomical traits. PMID:24600542

  5. Establishment of Gal4 transgenic zebrafish lines for analysis of development of cerebellar neural circuitry.

    PubMed

    Takeuchi, Miki; Matsuda, Koji; Yamaguchi, Shingo; Asakawa, Kazuhide; Miyasaka, Nobuhiko; Lal, Pradeep; Yoshihara, Yoshihiro; Koga, Akihiko; Kawakami, Koichi; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    The cerebellum is involved in some forms of motor coordination and motor learning. Here we isolated transgenic (Tg) zebrafish lines that express a modified version of Gal4-VP16 (GFF) in the cerebellar neural circuits: granule, Purkinje, or eurydendroid cells, Bergmann glia, or the neurons in the inferior olive nuclei (IO) which send climbing fibers to Purkinje cells, with the transposon Tol2 system. By combining GFF lines with Tg lines carrying a reporter gene located downstream of Gal4 binding sequences (upstream activating sequence: UAS), we investigated the anatomy and developmental processes of the cerebellar neural circuitry. Combining an IO-specific Gal4 line with a UAS reporter line expressing the photoconvertible fluorescent protein Kaede demonstrated the contralateral projections of climbing fibers. Combining a granule cell-specific Gal4 line with a UAS reporter line expressing wheat germ agglutinin (WGA) confirmed direct and/or indirect connections of granule cells with Purkinje cells, eurydendroid cells, and IO neurons in zebrafish. Time-lapse analysis of a granule cell-specific Gal4 line revealed initial random movements and ventral migration of granule cell nuclei. Transgenesis of a reporter gene with another transposon Tol1 system visualized neuronal structure at a single cell resolution. Our findings indicate the usefulness of these zebrafish Gal4 Tg lines for studying the development and function of cerebellar neural circuits. PMID:25300581

  6. Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line.

    PubMed Central

    Blechinger, Scott R; Warren, James T; Kuwada, John Y; Krone, Patrick H

    2002-01-01

    The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development. PMID:12361930

  7. Stripe rust resistance and dough quality of new wheat - Dasypyrum villosum translocation lines T1DL•1V#3S and T1DS•1V#3L and the location of HMW-GS genes.

    PubMed

    Zhao, W C; Gao, X; Dong, J; Zhao, Z J; Chen, Q G; Chen, L G; Shi, Y G; Li, X Y

    2015-01-01

    The transfer of agronomically useful genes from wild wheat species into cultivated wheat is one of the most effective approaches to improvement of wheat varieties. To evaluate the transfer of genes from Dasypyrum villosum into Triticum aestivum, wheat quality and disease resistance was evaluated in two new translocation lines, T1DL•1V#3S and T1DS•1V#3L. We examined the levels of stripe rust resistance and dough quality in the two lines, and identified and located the stripe rust resistant genes and high molecular weight glutenin subunit (HMW-GS) genes Glu-V1 of D. villosum. Compared to the Chinese Spring (CS) variety, T1DL•1V#3S plants showed moderate resistance to moderate susceptibility to the stripe rust races CYR33 and Su11-4. However, T1DS•1V#3L plants showed high resistance or immunity to these stripe rusts. The genes for resistance to stripe rust were located on 1VL of D. villosum. In comparison to CS, the dough from T1DS•1V#3L had a significantly shorter developing time (1.45 min) and stable time (1.0 min), a higher weakness in gluten strength (208.5 FU), and a lower farinograph quality index (18). T1DL•1V#3S had a significantly longer developing time (4.2 min) and stable time (5.25 min), a lower weakness in gluten strength (53 FU) and a higher farinograph quality index (78.5). We also found that T1DS•1V#3L had reduced gluten strength and dough quality compared to CS, but T1DL•1V#3S had increased gluten strength and dough quality. The results of SDS-PAGE analysis indicated that Glu-V1 of D. villosum was located on short arm 1VS and long arm 1VL. These results prove that the new translocation lines, T1DS•1V#3L and T1DS•1V#3L, have valuable stripe rust resistance and dough quality traits that will be important for improving wheat quality and resistance in future wheat breeding programs. PMID:26214490

  8. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies.

    PubMed

    Marquart, Gregory D; Tabor, Kathryn M; Brown, Mary; Strykowski, Jennifer L; Varshney, Gaurav K; LaFave, Matthew C; Mueller, Thomas; Burgess, Shawn M; Higashijima, Shin-Ichi; Burgess, Harold A

    2015-01-01

    Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3' untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish. PMID:26635538

  9. A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies

    PubMed Central

    Marquart, Gregory D.; Tabor, Kathryn M.; Brown, Mary; Strykowski, Jennifer L.; Varshney, Gaurav K.; LaFave, Matthew C.; Mueller, Thomas; Burgess, Shawn M.; Higashijima, Shin-ichi; Burgess, Harold A.

    2015-01-01

    Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3? untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish. PMID:26635538

  10. RE-ESTABLISHMENT OF A TRANSGENIC CHICKEN LINE 0-ALV6 AT THE AVIAN DISEASE AND ONCOLOGY LABORATORY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first transgenic chicken line, 0.ALV6, was produced at the Avian Disease and Oncology Laboratory (ADOL) in 1989. Blastoderms of fertile freshly laid line 0 eggs were injected with the long terminal repeats of the endogenous Rous-associated virus (RAV-0) and the envelope gene (env) of a subgroup ...

  11. Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

    PubMed Central

    Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.

    2013-01-01

    Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096

  12. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    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. PMID:26076353

  13. Beneficial ‘unintended effects’ of a cereal cystatin in transgenic lines of potato, Solanum tuberosum

    PubMed Central

    2012-01-01

    Background Studies reported unintended pleiotropic effects for a number of pesticidal proteins ectopically expressed in transgenic crops, but the nature and significance of such effects in planta remain poorly understood. Here we assessed the effects of corn cystatin II (CCII), a potent inhibitor of C1A cysteine (Cys) proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein. Results The leaf proteome of lines accumulating CCII at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control (parental) line. Out of ca. 700 proteins monitored on 2-D gels, 23 were significantly up- or downregulated in CCII-expressing leaves, including 14 proteins detected de novo or up-regulated by more than five-fold compared to the control. Most up-regulated proteins were abiotic or biotic stress-responsive proteins, including different secretory peroxidases, wound inducible protease inhibitors and pathogenesis-related proteins. Accordingly, infection of leaf tissues by the fungal necrotroph Botryris cinerea was prevented in CCII-expressing plants, despite a null impact of CCII on growth of this pathogen and the absence of extracellular Cys protease targets for the inhibitor. Conclusions These data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. They also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance. PMID:23116303

  14. Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™.

    PubMed

    Forsberg, Cecil W; Meidinger, Roy G; Liu, Mingfu; Cottrill, Michael; Golovan, Serguei; Phillips, John P

    2013-04-01

    The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig™, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3' to the other copies with a 6 kbp deletion in the 5' promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4-6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal. PMID:22948309

  15. Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

    PubMed

    Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

    2015-01-01

    The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation. PMID:25515246

  16. Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth--Application to the Eif3

    E-print Network

    Copenhaver, Gregory P.

    Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant

  17. Transgenic Indian Mustard Overexpressing Selenocysteine

    E-print Network

    Transgenic Indian Mustard Overexpressing Selenocysteine Lyase or Selenocysteine Methyltransferase new transgenic Indian mustard [Brassica juncea (L.) Czern.] lines were tested under field conditions mustard. Further, this study confirms the importance of field testing for evaluating future transgenic

  18. A retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene.

    PubMed

    Kato, K; Ishiguro, S; Yamamoto, H; Yanai, N; Obinata, M; Tamai, M

    1996-12-01

    We established a retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 T-antigen gene (tsSV40T) and examined its characteristics. We enucleated both eyes from a 2-month-old transgenic mouse and removed the retinal pigment epithelial (RPE) cells and neuroretinal cells. After cloning the RPE cells, we obtained a cell line (RPET). RPET cells grew well at 33 degrees C but not at 37 degrees C, expressing on the temperature-sensitive character of tsSV40T, and maintained characters of RPE cells such as T1-tyrosinase production, phagocytosis of rod outer segments, and presence of cytokeratin, microvilli on the cell surface and lysosome-like granules around the Golgi apparatus in the cytoplasm. Conditioned medium (CM) from a culture of neuroretinal cells harboring tsSV40T was essentially required for growth. The factor(s) in CM was heat-and acid labile, but was resistant to trypsin digestion. In the presence of 3% CM, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and strong effects on RPET cells, whereas insulin, insulin-like growth factor I (IGF-I), and IGF-II had moderate growth effects. Interestingly, none of these growth factors stimulated the RPET cells in the absence of CM. EHS-Matrix had growth effect, whereas laminin, collagen types I and IV, and fibronectin had no marked growth effects on RPET cells. RPET cells were morphologically changed on a laminin-coated dish. They could not spread on the coated dish, and the majority of the cells floated. But when the floating cells were transferred to non-coated dishes, they immediately attached themselves. These results suggest that RPET cells are a good model for for finding novel growth factor(s) and for investigating the mechanism of cell-laminin attachment. PMID:9078403

  19. Comparison of the Rhizosphere Bacterial Communities of Zigongdongdou Soybean and a High-Methionine Transgenic Line of This Cultivar

    PubMed Central

    Ji, Jun; Wu, Haiying; Meng, Fang; Zhang, Mingrong; Zheng, Xiaobo; Wu, Cunxiang; Zhang, Zhengguang

    2014-01-01

    Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine ?-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars. PMID:25079947

  20. Developing Fiber Specific Promoter-Reporter Transgenic Lines to Study the Effect of Abiotic Stresses on Fiber Development in Cotton

    PubMed Central

    Chen, Junping; Burke, John J.

    2015-01-01

    Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality. PMID:26030401

  1. Comparison of the physiological characteristics of transgenic insect-resistant cotton and conventional lines

    PubMed Central

    Li, Xiaogang; Ding, Changfeng; Wang, Xingxiang; Liu, Biao

    2015-01-01

    The introduction of transgenic insect-resistant cotton into agricultural ecosystems has raised concerns regarding its ecological effects. Many studies have been conducted to compare the differences in characteristics between transgenic cotton and conventional counterparts. However, few studies have focused on the different responses of transgenic cotton to stress conditions, especially to the challenges of pathogens. The aim of this work is to determine the extent of variation in physiological characteristics between transgenic insect-resistant cotton and the conventional counterpart infected by cotton soil-borne pathogens. The results showed that the difference in genetic backgrounds is the main factor responsible for the effects on biochemical characteristics of transgenic cotton when incubating with cotton Fusarium oxysporum. However, genetic modification had a significantly greater influence on the stomatal structure of transgenic cotton than the effects of cotton genotypes. Our results highlight that the differences in genetic background and/or genetic modifications may introduce variations in physiological characteristics and should be considered to explore the potential unexpected ecological effects of transgenic cotton. PMID:25737015

  2. Impact of six transgenic Bacillus thuringiensis rice lines on four nontarget thrips species attacking rice panicles in the paddy field.

    PubMed

    Akhtar, Z R; Tian, J C; Chen, Y; Fang, Q; Hu, C; Peng, Y F; Ye, G Y

    2013-02-01

    As a key component of ecological risk assessments, nontarget effects of Bacillus thuringiensis (Bt) rice have been tested under laboratory and field conditions for various organisms. A 2-yr field experiment was conducted to observe the nontarget effects of six transgenic rice lines (expressing the Cry1Ab or fused protein of Cry1Ab and Cry1Ac) on four nontarget thrips species including Frankliniella intonsa (Trybom), F. tenuicornis (Uzel), Haplothrips aculeatus (F.), and H. tritici (Kurd), as compared with their rice parental control lines. Two sampling methods including the beat plate and plastic bag method were used to monitor the population densities of the four thrips species for 2 yr. The results showed that the seasonal average densities of four tested thrips species in Bt rice plots were significantly lower than or very similar to those in the non-Bt rice plots depending on rice genotypes, sampling methods, and years. Among all six tested Bt rice lines, transgenic B1 and KMD2 lines suppressed the population of these tested thrips species the most. Our results indicate that the tested Bt rice lines are unlikely to result in high population pressure of thrips species in comparison with non-Bt rice. In some cases, Bt rice lines could significantly suppress thrips populations in the rice ecosystem. In addition, compatibility of Bt rice, with rice host plant resistance to nontarget sucking pests is also discussed within an overall integrated pest management program for rice. PMID:23339799

  3. Two Types of Tet-On Transgenic Lines for Doxycycline-Inducible Gene Expression in Zebrafish Rod Photoreceptors and a Gateway-Based Tet-On Toolkit

    PubMed Central

    Jensen, Abbie M.

    2012-01-01

    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. PMID:23251476

  4. Diversity of Reporter Expression Patterns in Transgenic Mouse Lines Targeting Corticotropin-Releasing Hormone-Expressing Neurons.

    PubMed

    Chen, Yuncai; Molet, Jenny; Gunn, Benjamin G; Ressler, Kerry; Baram, Tallie Z

    2015-12-01

    Transgenic mice, including lines targeting corticotropin-releasing factor (CRF or CRH), have been extensively employed to study stress neurobiology. These powerful tools are poised to revolutionize our understanding of the localization and connectivity of CRH-expressing neurons, and the crucial roles of CRH in normal and pathological conditions. Accurate interpretation of studies using cell type-specific transgenic mice vitally depends on congruence between expression of the endogenous peptide and reporter. If reporter expression does not faithfully reproduce native gene expression, then effects of manipulating unintentionally targeted cells may be misattributed. Here, we studied CRH and reporter expression patterns in 3 adult transgenic mice: Crh-IRES-Cre;Ai14 (tdTomato mouse), Crfp3.0CreGFP, and Crh-GFP BAC. We employed the CRH antiserum generated by Vale after validating its specificity using CRH-null mice. We focused the analyses on stress-salient regions, including hypothalamus, amygdala, bed nucleus of the stria terminalis, and hippocampus. Expression patterns of endogenous CRH were consistent among wild-type and transgenic mice. In tdTomato mice, most CRH-expressing neurons coexpressed the reporter, yet the reporter identified a few non-CRH-expressing pyramidal-like cells in hippocampal CA1 and CA3. In Crfp3.0CreGFP mice, coexpression of CRH and the reporter was found in central amygdala and, less commonly, in other evaluated regions. In Crh-GFP BAC mice, the large majority of neurons expressed either CRH or reporter, with little overlap. These data highlight significant diversity in concordant expression of reporter and endogenous CRH among 3 available transgenic mice. These findings should be instrumental in interpreting important scientific findings emerging from the use of these potent neurobiological tools. PMID:26402844

  5. A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo

    PubMed Central

    Phiwpan, Krung; Guo, Jie; Zhang, Wei; Hu, Tanyu; Boruah, Bhargavi M.; Zhang, Jianhua; Zhou, Xuyu

    2015-01-01

    MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level. PMID:26030404

  6. Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

    PubMed Central

    Gfrerer, Lisa; Dougherty, Max; Liao, Eric C.

    2013-01-01

    Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants. PMID:24121214

  7. Generation of an ABCG2{sup GFPn-puro} transgenic line - A tool to study ABCG2 expression in mice

    SciTech Connect

    Orford, Michael; Mean, Richard; Lapathitis, George; Genethliou, Nicholas; Panayiotou, Elena; Panayi, Helen; Malas, Stavros; Department of Biological Sciences, University of Cyprus, P.O. Box 20537, 1678 Nicosia

    2009-06-26

    The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.

  8. Characterization of Multiple Bistratified Retinal Ganglion Cells in A Purkinje Cell Protein 2-Cre Transgenic Mouse Line

    PubMed Central

    Ivanova, Elena; Lee, Patrick; Pan, Zhuo-Hua

    2012-01-01

    Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional-selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in a cell type-specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type-specific expression of Cre recombinase. In this study, we characterized the Cre-positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)-cre mouse line. We found that all of the Cre-positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On- and Off-dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn-3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On-Off and On-DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice. PMID:23224947

  9. Generation and characterization of transgenic plum lines expressing the Gastrodia anti-fungal protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Gastrodia anti-fungal protein (GAFP) is a monocot mannose-binding plant lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has provided documented disease resistance in transgenic tobacco and cotton against several root diseases, but it's potential to confer disease resistance...

  10. Derivation and Characterization of Embryonic Stem Cells Lines Derived from Transgenic Fischer 344 and Dark Agouti Rats

    PubMed Central

    Hong, James; He, Hong

    2012-01-01

    Rat embryonic stem cell (ESC) lines are not widely available, and there are only 2 lines available for distribution. Here, ESC lines were derived and characterized from Fischer 344 (F344) rats that express marker transgenes either ?-galactosidase or human placental alkaline phosphatase (AP), nontransgenic F344 rats, and from Dark Agouti (DA) rats. The ESC lines were maintained in an undifferentiated state as characterized by colony morphology, expression of Oct4, Nanog, Sox-2, Cdx2, and Stella, staining for AP, and stage-specific embryonic antigen-1. Pluripotency was demonstrated in vitro by differentiation to embryoid bodies, followed by embryonic monsters. The Cdx2 expression by ESCs was unexpected and was confirmed via reverse transcriptase-polymerase chain reaction, immunocytochemistry. Pluripotency of ESCs was demonstrated in vivo by production of teratoma after an injection into F344 nontransgenic rats, and by an injection of male DA ESCs into F344 or Sprague-Dawley rat blastocysts and the generation of chimeric rats and germline contribution. ESCs from both F344 and DA contributed to chimeric rats, and one DA ESC line was proved to be germline competent. ESC sublines were created by transfection with a plasmid expressing enhanced green fluorescent protein (eGFP) under the control of a beta actin promoter and cytomegalovirus enhancer (pCX-eGFP) or by transfection with a plasmid expressing GFP under the control of a 3.1?kb portion of the rat Oct4 promoter (pN1-Oct4-GFP). In pN1-Oct4-GFP sublines, GFP gene expression and fluorescence were shown to be correlated with endogenous Oct4 gene expression. Therefore, these new ESC lines may be useful for tissue engineering and transplantation studies or for optimizing culture conditions required for self-renewal and differentiation of rat ESCs. While they made chimeric rats, further work is needed to confirm whether the transgenic F344 rat ESCs described here are germline-competent ESCs. PMID:21995453

  11. Development of transgenic lines of Eimeria tenella expressing M2e-enhanced yellow fluorescent protein (M2e-EYFP).

    PubMed

    Liu, Xianyong; Zou, Jun; Yin, Guangwen; Su, Huali; Huang, Xiaoxi; Li, Jianan; Xie, Li; Cao, Yingqiong; Cui, Yujuan; Suo, Xun

    2013-03-31

    Eimeria parasites are obligate intracellular apicomplexan protists that can cause coccidiosis, resulting in substantial economic losses in the poultry industry annually. As the component of anticoccidial vaccines, seven Eimeria spp. of chickens are characterized with potent immunogenicity. Whether genetically modified Eimeria spp. maintains this property or not needs to be verified. In this study, two identical transgenic lines of Eimeria tenella were developed by virtue of single sporocyst isolation from a stably transfected population expressing fused protein of M2 ectodomain of avian influenza virus (M2e) and enhanced yellow fluorescent protein (EYFP). The chromosomal integration and expression of M2e-EYFP were confirmed by Southern blot, plasmid rescue and Western blot analysis. We found that the reproduction of transgenic parasites was higher than that of the parental strain. Chickens challenged with wild type E. tenella after immunization with 200 oocysts of transgenic parasites had similar performance compared to those in non-immunized and non-challenged group. In another trial, the performance of transgenic parasite-immunized birds was also comparable to that of the Decoquinate Premix-treated chickens. These results suggest that this transgenic line of E. tenella is capable of inducing potent protection against homologous challenge as a live anticoccidial vaccine. Taking together, our study indicates that transgenic eimerian parasites have the potential to be developed as a vaccine vehicle for animal use in the future. PMID:23298569

  12. Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

    PubMed

    Wilhelm, Márta; Lawrence, J Josh; Gábriel, Robert

    2015-02-01

    We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons. PMID:25592616

  13. Megaesophagus in a line of transgenic rats: a model of achalasia.

    PubMed

    Pang, J; Borjeson, T M; Muthupalani, S; Ducore, R M; Carr, C A; Feng, Y; Sullivan, M P; Cristofaro, V; Luo, J; Lindstrom, J M; Fox, J G

    2014-11-01

    Megaesophagus is defined as the abnormal enlargement or dilatation of the esophagus, characterized by a lack of normal contraction of the esophageal walls. This is called achalasia when associated with reduced or no relaxation of the lower esophageal sphincter (LES). To date, there are few naturally occurring models for this disease. A colony of transgenic (Pvrl3-Cre) rats presented with megaesophagus at 3 to 4 months of age; further breeding studies revealed a prevalence of 90% of transgene-positive animals having megaesophagus. Affected rats could be maintained on a total liquid diet long term and were shown to display the classic features of dilated esophagus, closed lower esophageal sphincter, and abnormal contractions on contrast radiography and fluoroscopy. Histologically, the findings of muscle degeneration, inflammation, and a reduced number of myenteric ganglia in the esophagus combined with ultrastructural lesions of muscle fiber disarray and mitochondrial changes in the striated muscle of these animals closely mimic that seen in the human condition. Muscle contractile studies looking at the response of the lower esophageal sphincter and fundus to electrical field stimulation, sodium nitroprusside, and L-nitro-L-arginine methyl ester also demonstrate the similarity between megaesophagus in the transgenic rats and patients with achalasia. No primary cause for megaesophagus was found, but the close parallel to the human form of the disease, as well as ease of care and manipulation of these rats, makes this a suitable model to better understand the etiology of achalasia as well as study new management and treatment options for this incurable condition. PMID:24457157

  14. Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice

    PubMed Central

    2013-01-01

    Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice. PMID:23822604

  15. Comparative transcriptional and proteomic profiling of bread wheat cultivar and its derived transgenic line over-expressing a low molecular weight glutenin subunit gene in the endosperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that over-expresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding non-transformed genotype. Proteomic analyses showed that, during seed develop...

  16. Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

    PubMed Central

    2011-01-01

    Background The recovery of high performing transgenic lines in clonal crops is limited by the occurrence of somaclonal variation during the tissue culture phase of transformation. This is usually circumvented by developing large populations of transgenic lines, each derived from the first shoot to regenerate from each transformation event. This study investigates a new strategy of assessing multiple shoots independently regenerated from different transformed cell colonies of potato (Solanum tuberosum L.). Results A modified cry9Aa2 gene, under the transcriptional control of the CaMV 35S promoter, was transformed into four potato cultivars using Agrobacterium-mediated gene transfer using a nptII gene conferring kanamycin resistance as a selectable marker gene. Following gene transfer, 291 transgenic lines were grown in greenhouse experiments to assess somaclonal variation and resistance to potato tuber moth (PTM), Phthorimaea operculella (Zeller). Independently regenerated lines were recovered from many transformed cell colonies and Southern analysis confirmed whether they were derived from the same transformed cell. Multiple lines regenerated from the same transformed cell exhibited a similar response to PTM, but frequently exhibited a markedly different spectrum of somaclonal variation. Conclusions A new strategy for the genetic improvement of clonal crops involves the regeneration and evaluation of multiple shoots from each transformation event to facilitate the recovery of phenotypically normal transgenic lines. Most importantly, regenerated lines exhibiting the phenotypic appearance most similar to the parental cultivar are not necessarily derived from the first shoot regenerated from a transformed cell colony, but can frequently be a later regeneration event. PMID:21995716

  17. Transgenic barley lines prove the involvement of TaCBF14 and TaCBF15 in the cold acclimation process and in frost tolerance

    PubMed Central

    Soltész, Alexandra; Smedley, Mark; Vashegyi, Ildikó; Galiba, Gábor; Harwood, Wendy; Vágújfalvi, Attila

    2013-01-01

    The enhancement of winter hardiness is one of the most important tasks facing breeders of winter cereals. For this reason, the examination of those regulatory genes involved in the cold acclimation processes is of central importance. The aim of the present work was the functional analysis of two wheat CBF transcription factors, namely TaCBF14 and TaCBF15, shown by previous experiments to play a role in the development of frost tolerance. These genes were isolated from winter wheat and then transformed into spring barley, after which the effect of the transgenes on low temperature stress tolerance was examined. Two different types of frost tests were applied; plants were hardened at low temperature before freezing, or plants were subjected to frost without a hardening period. The analysis showed that TaCBF14 and TaCBF15 transgenes improve the frost tolerance to such an extent that the transgenic lines were able to survive freezing temperatures several degrees lower than that which proved lethal for the wild-type spring barley. After freezing, lower ion leakage was measured in transgenic leaves, showing that these plants were less damaged by the frost. Additionally, a higher Fv/Fm parameter was determined, indicating that photosystem II worked more efficiently in the transgenics. Gene expression studies showed that HvCOR14b, HvDHN5, and HvDHN8 genes were up-regulated by TaCBF14 and TaCBF15. Beyond that, transgenic lines exhibited moderate retarded development, slower growth, and minor late flowering compared with the wild type, with enhanced transcript level of the gibberellin catabolic HvGA2ox5 gene. PMID:23567863

  18. Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

    PubMed

    Kato, M; Liu, W; Akhand, A A; Dai, Y; Ohbayashi, M; Tuzuki, T; Suzuki, H; Isobe, K; Takahashi, M; Nakashima, I

    1999-01-21

    We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma. PMID:9989837

  19. A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry

    PubMed Central

    Ikeda, Keiko; Takahashi, Masanori; Sato, Shigeru; Igarashi, Hiroyuki; Ishizuka, Toru; Yawo, Hiromu; Arata, Satoru; Southard-Smith, E. Michelle; Kawakami, Kiyoshi; Onimaru, Hiroshi

    2015-01-01

    The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems. PMID:26147470

  20. Detection of feral GT73 transgenic oilseed rape (Brassica napus) along railway lines on entry routes to oilseed factories in Switzerland.

    PubMed

    Hecht, Mirco; Oehen, Bernadette; Schulze, Jürg; Brodmann, Peter; Bagutti, Claudia

    2014-01-01

    To obtain a reference status prior to cultivation of genetically modified oilseed rape (OSR, Brassica napus L.) in Switzerland, the occurrence of feral OSR was monitored along transportation routes and at processing sites. The focus was set on the detection of (transgenic) OSR along railway lines from the Swiss borders with Italy and France to the respective oilseed processing factories in Southern and Northern Switzerland (Ticino and region of Basel). A monitoring concept was developed to identify sites of largest risk of escape of genetically modified plants into the environment in Switzerland. Transport spillage of OSR seeds from railway goods cars particularly at risk hot spots such as switch yards and (un)loading points but also incidental and continuous spillage were considered. All OSR plants, including their hybridization partners which were collected at the respective monitoring sites were analyzed for the presence of transgenes by real-time PCR. On sampling lengths each of 4.2 and 5.7 km, respectively, 461 and 1,574 plants were sampled in Ticino and the region of Basel. OSR plants were found most frequently along the routes to the oilseed facilities, and in larger amounts on risk hot spots compared to sites of random sampling. At three locations in both monitored regions, transgenic B. napus line GT73 carrying the glyphosate resistance transgenes gox and CP4 epsps were detected (Ticino, 22 plants; in the region of Basel, 159). PMID:23917737

  1. Plasmodium falciparum: Development of a Transgenic Line for Screening Antimalarials Using Firefly Luciferase as the Reporter

    PubMed Central

    Cui, Long; Miao, Jun; Wang, Jiaxu; Li, Qianjun; Cui, Liwang

    2008-01-01

    High-throughput screening (HTS) of small-molecule libraries against pharmacological targets is a key strategy of contemporary drug discovery. This study reports a simple, robust, and cell-based luminescent method for assaying antimalarial drugs. Using transfection technology, we generated a stable Plasmodium falciparum line with high levels of firefly luciferase expression. A luciferase assay based on this parasite line was optimized in a 96-well plate format and used to compare with the standard [3H] hypoxanthine radioisotope method. The 50% inhibitory concentrations (IC50s) of chloroquine, artesunate, artemether, dihydroartemisinin and curcumin obtained by these two methods were not significantly different (P > 0.05, ANOVA). In addition, this assay could be performed conveniently with a luminescence plate reader using unsynchronized stages within as early as 12 h. Furthermore, the luciferase assay is robust with a Z? score of 0.77 to 0.92, which suggests the feasibility for further miniaturization and automation. PMID:18579134

  2. Immunolocalization of human alpha-synuclein in the Thy1-aSyn ("Line 61") transgenic mouse line.

    PubMed

    Delenclos, M; Carrascal, L; Jensen, K; Romero-Ramos, M

    2014-09-26

    Alpha-synuclein (a-syn) is the major component of the intracytoplasmic inclusions known as Lewy bodies (LB), which constitute the hallmark of Parkinson's disease (PD). Mice overexpressing human a-syn under the Thy-1 promoter (ASO) show slow neurodegeneration and some behavioral deficits similar to those seen in human PD patients. Here, we describe a whole-brain distribution of human a-syn in adult ASO mice. We find that the human a-syn is ubiquitously distributed in the brain including the cerebellar cortex, but the intensity and sub-cellular localization of the staining differed in the various regions of the central nervous system. Among particular CNS areas with human a-syn immunoreactivity, we describe staining patterns in the olfactory bulb, cortex, hippocampus, thalamic region, brainstem nuclei and cerebellar cortex. This immunohistochemical study provides an anatomical map of the human a-syn distribution in ASO mice. Our data show that human a-syn, although not present at levels that were detectable by immunostaining in dopaminergic neurons of substantia nigra or noradrenergic neurons of locus coeruleus, was highly expressed in other PD relevant regions of the brain in different neuronal subtypes. These data will help to relate a-syn expression to the phenotypic manifestations observed in this widely used mouse line. PMID:25090921

  3. Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

    PubMed

    Gao, Xiaoqing; Zhou, Jie; Li, Jun; Zou, Xiaowei; Zhao, Jianhua; Li, Qingliang; Xia, Ran; Yang, Ruifang; Wang, Dekai; Zuo, Zhaoxue; Tu, Jumin; Tao, Yuezhi; Chen, Xiaoyun; Xie, Qi; Zhu, Zengrong; Qu, Shaohong

    2015-01-01

    Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) ?-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops. PMID:25371551

  4. Transgenic pearl millet male fertility restorer line (ICMP451) and hybrid (ICMH451) expressing Brassica juncea Nonexpressor of pathogenesis related genes 1 (BjNPR1) exhibit resistance to downy mildew disease.

    PubMed

    Ramineni, Ramadevi; Sadumpati, Vijayakumar; Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

    2014-01-01

    Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids. PMID:24603762

  5. Transgenic Pearl Millet Male Fertility Restorer Line (ICMP451) and Hybrid (ICMH451) Expressing Brassica juncea Nonexpressor of Pathogenesis Related Genes 1 (BjNPR1) Exhibit Resistance to Downy Mildew Disease

    PubMed Central

    Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

    2014-01-01

    Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids. PMID:24603762

  6. Transgenic mouse lines expressing rat AH receptor variants - A new animal model for research on AH receptor function and dioxin toxicity mechanisms

    SciTech Connect

    Pohjanvirta, Raimo

    2009-04-15

    Han/Wistar (Kuopio; H/W) rats are exceptionally resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity mainly because of their mutated aryl hydrocarbon receptor (AHR) gene. In H/W rats, altered splicing of the AHR mRNA generates two AHR proteins: deletion (DEL) and insertion (INS) variants, with the INS isoform being predominantly expressed. To gain further insight into their functional properties, cDNAs of these and rat wild-type (rWT) isoform were transferred into C57BL/6J-derived mice by microinjection. The endogenous mouse AHR was eliminated by selective crossing with Ahr-null mice. A single mouse line was obtained for each of the three constructs. The AHR mRNA levels in tissues were generally close to those of C57BL/6 mice in INS and DEL mice and somewhat higher in rWT mice; in testis, however, all 3 constructs exhibited marked overexpression. The transgenic mouse lines were phenotypically normal except for increased testis weight. Induction of drug-metabolizing enzymes by TCDD occurred similarly to that in C57BL/6 mice, but there tended to be a correlation with AHR concentrations, especially in testis. In contrast to C57BL/6 mice, the transgenics did not display any major gender difference in susceptibility to the acute lethality and hepatotoxicity of TCDD; rWT mice were highly sensitive, DEL mice moderately resistant and INS mice highly resistant. Co-expression of mouse AHR and rWT resulted in augmented sensitivity to TCDD and abolished the natural resistance of female C57BL/6 mice, whereas mice co-expressing mouse AHR and INS were resistant. Thus, these transgenic mouse lines provide a novel promising tool for molecular studies on dioxin toxicity and AHR function.

  7. [Overexpression of the glutathione S-transferase gene from Pyrus pyrifolia fruit improves tolerance to abiotic stress in transgenic tobacco plants].

    PubMed

    Liu, D; Liu, Y; Rao, J; Wang, G; Li, H; Ge, F; Chen, C

    2013-01-01

    Glutathione S-transferases (GSTs) are ubiquitous enzymes in animals and plants, and they are multifunctional proteins encoded by a large gene family. GSTs are involved in response to the oxidative stress including drought, salt, heavy metals, and so on. Under oxidative stress, the excessive reactive oxygen species (ROS) induce an increase in GST levels, and then the GSTs metabolize the toxic products of lipid peroxidation, damaged DNA and other molecules. Previously, a full-length cDNA of a novel zeta GST gene, PpGST, was characterized from fruit of Pyrus pyrifolia Nakai cv Huobali. In the present study, a constitutive plant expression vector of PpGSTwas constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi) to verify the function of PpGST. As a result, the PpGSTgene was successfully integrated into the genome of the transgenic tobacco lines and expressed as expected in the transformants through Southern blotting and quantitative reverse transcription-polymerase chain reaction analysis. Growth of T1 generation plants of PpGST transgenic lines and WT under non-stressful conditions was similar, however, the transgenic tobacco lines showed relatively normal growth under drought, NaCl, and cadmium (Cd) stresses. Furthermore, the T1 transgenic tobacco lines showed significantly slower superoxide anion production rate than the WT under abiotic stress. Simultaneously, the MDA content of each T1 transgenic tobacco plant was only slightly increased and significantly lower than that of the WT under drought, salt and Cd stress. Together with the GST activity of the transgenic tobacco lines, which was significantly increased under stressful conditions, as compared with that in WT, overexpression of PpGSTin tobacco enhanced the tolerance of transgenic tobacco lines to oxidative damage caused by drought, NaCl, and Cd stresses. PMID:24466748

  8. Testing Transgenic Spring Wheat and Barley Lines for Reaction to Fusarium Head Blight: 2008 Field Nursery Report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2008 field screening nursery, with 64 wheat and 208 barley plots was located at UMore Park, Rosemount MN. Trial entries were submitted by Rutgers University (5 wheat), University of North Texas (2 wheat) and USDA (48 barley). In addition to the submitted transgenic entries, untransformed contr...

  9. Study of heat and radiation response of a malignant, melanin-producing cell line derived from C3H 10T1/2 cells transformed in culture by radiation

    SciTech Connect

    Raaphorst, G.P.; Vadasz, J.; Azzam, E.I.

    1986-12-01

    The mouse C3H 10T1/2 cell line was transformed to the malignant state using ionizing radiation. One of the transformed lines (R25) that was isolated, displayed some properties similar to malignant melanoma cells. The cells became dark and pigmented after prolonged time in culture and this cell line produced tumors in C3H mice. The radiation survival curve of R25 had a large shoulder which was also observed for human melanoma cell lines. R25 was more resistant to heating at 45.0 degrees C than the normal cell line. Heating at 45.0 degrees C before irradiation resulted in a reduction of the survival curve shoulder. The heat and radiation sensitivity of R25 did not appear to be related to the melanin content of these cells.

  10. Transgenic sorghum plants via microprojectile bombardment.

    PubMed Central

    Casas, A M; Kononowicz, A K; Zehr, U B; Tomes, D T; Axtell, J D; Butler, L G; Bressan, R A; Hasegawa, P M

    1993-01-01

    Transgenic sorghum plants have been obtained after microprojectile bombardment of immature zygotic embryos of a drought-resistant sorghum cultivar, P898012. DNA delivery parameters were optimized based on transient expression of R and C1 maize anthocyanin regulatory elements in scutellar cells. The protocol for obtaining transgenic plants consists of the delivery of the bar gene to immature zygotic embryos and the imposition of bialaphos selection pressure at various stages during culture, from induction of somatic embryogenesis to rooting of regenerated plantlets. One in about every 350 embryos produced embryogenic tissues that survived bialaphos treatment; six transformed callus lines were obtained from three of the eight sorghum cultivars used in this research. Transgenic (T0) plants were obtained from cultivar P898012 (two independent transformation events). The presence of the bar and uidA genes in the T0 plants was confirmed by Southern blot analysis of genomic DNA. Phosphinothricin acetyltransferase activity was detected in extracts of the T0 plants. These plants were resistant to local application of the herbicide Ignite/Basta, and the resistance was inherited in T1 plants as a single dominant locus. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8248230

  11. DISEASE RESISTANCE IN TRANSGENIC COTTONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic Upland cottons (Gossypium hirsutum L.) expressing the antifungal peptide D4E1 were evaluated for tolerance to Fusarium wilt caused by Fusarium oxysporum f.sp. vasinfectum (FOV) race 1 in a sandy soil field, also infected with root-knot nematodes (Meloidogyne incognita). A transgenic line...

  12. Response of PiCypA tobacco T2 transgenic matured plant to potential tolerance to salinity stress

    PubMed Central

    Trivedi, Dipesh Kumar; Ansari, Mohammad Wahid; Bhavesh, Neel Sarovar; Johri, Atul Kumar; Tuteja, Narendra

    2014-01-01

    Cyclophilins are molecular chaperone act as peptidyl prolyl cis-trans isomerase responsible for protein folding and assembly in many normal cellular processes, stabilize proteins and membranes under stress conditions. Recently, we report on the role cyclophilin A-like gene from Piriformospora indica (PiCypA) in salinity stress tolerance in T1 transgenic and up to seedling stage of T2 transgenic of tobacco plants. Here, PiCypA T2 generation matured tobacco plants were evaluated under salt (200 mM NaCl) up to flowering and seed set stages. We found that PiCypA T2 tobacco lines showed comparatively better survival and exhibited higher root growth and fresh weight as compared with wild type and vector control. This study provides further direct evidence that PiCypA transgene maintained the sustainability in providing salinity stress tolerance in T2 generation of transgenic tobacco plants. PMID:24394360

  13. Response of PiCypA tobacco T2 transgenic matured plant to potential tolerance to salinity stress.

    PubMed

    Trivedi, Dipesh Kumar; Ansari, Mohammad Wahid; Bhavesh, Neel Sarovar; Johri, Atul Kumar; Tuteja, Narendra

    2014-01-01

    Cyclophilins are molecular chaperone act as peptidyl prolyl cis-trans isomerase responsible for protein folding and assembly in many normal cellular processes, stabilize proteins and membranes under stress conditions. Recently, we report on the role cyclophilin A-like gene from Piriformospora indica (PiCypA) in salinity stress tolerance in T1 transgenic and up to seedling stage of T2 transgenic of tobacco plants. Here, PiCypA T2 generation matured tobacco plants were evaluated under salt (200 mM NaCl) up to flowering and seed set stages. We found that PiCypA T2 tobacco lines showed comparatively better survival and exhibited higher root growth and fresh weight as compared with wild type and vector control. This study provides further direct evidence that PiCypA transgene maintained the sustainability in providing salinity stress tolerance in T2 generation of transgenic tobacco plants. PMID:24394360

  14. Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway.

    PubMed

    Miesfeld, Joel B; Link, Brian A

    2014-08-01

    To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output. PMID:24560909

  15. A transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against porcine reproductive and respiratory syndrome virus.

    PubMed

    Zhou, Fang; Liang, Shuang; Chen, An-hui; Singh, Chabungbam Orville; Bhaskar, Roy; Niu, Yan-shan; Miao, Yun-gen

    2012-06-01

    Porcine reproductive and respiratory syndrome (PRRS) is now considered to be one of the most important diseases in countries with intensive swine industries. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In this study, we designed 5 of the small hairpin RNAs (shRNAs) targeting the GP5 and M gene of PRRSV respectively, and investigated their inhibition to the production of PRRSV. The highest activity displayed in shRNAs of the ORF6e sequence (nts 261-279), which the inhibition rate reached was 99.09%. The result suggests that RNAi technology might serve as a potential molecular strategy for PRRSV therapy. Furthermore, the transgenic Marc-145 cell line of piggyBac transposon-derived targeting shRNA interference against PRRS virus was established. It presented stable inhibition to the replication and amplification of PRRS. The work implied that shRNAs targeting the GP5 and M gene of PRRSV may be used as potential RNA vaccines in vivo, and supplied the screening methods of transformed pig embryonic fibroblast which are prerequisite for the disease-resistant transgenic pigs to PRRS. PMID:22297554

  16. Generation of Pet1210-Cre Transgenic Mouse Line Reveals Non-Serotonergic Expression Domains of Pet1 Both in CNS and Periphery

    PubMed Central

    Pelosi, Barbara; Migliarini, Sara; Pacini, Giulia; Pratelli, Marta; Pasqualetti, Massimo

    2014-01-01

    Neurons producing serotonin (5-hydroxytryptamine, 5-HT) constitute one of the most widely distributed neuronal networks in the mammalian central nervous system (CNS) and exhibit a profuse innervation throughout the CNS already at early stages of development. Serotonergic neuron specification is controlled by a combination of secreted molecules and transcription factors such as Shh, Fgf4/8, Nkx2.2, Lmx1b and Pet1. In the mouse, Pet1 mRNA expression appears between 10 and 11 days post coitum (dpc) in serotonergic post-mitotic precursors and persists in serotonergic neurons up to adulthood, where it promotes the expression of genes defining the mature serotonergic phenotype such as tryptophan hydroxylase 2 (Tph2) and serotonin transporter (SERT). Hence, the generation of genetic tools based on Pet1 specific expression represents a valuable approach to study the development and function of the serotonergic system. Here, we report the generation of a Pet1210-Cre transgenic mouse line in which the Cre recombinase is expressed under the control of a 210 kb fragment from the Pet1 genetic locus to ensure a reliable and faithful control of somatic recombination in Pet1 cell lineage. Besides Cre-mediated recombination accurately occurred in the serotonergic system as expected and according to previous studies, Pet1210-Cre transgenic mouse line allowed us to identify novel, so far uncharacterized, Pet1 expression domains. Indeed, we showed that in the raphe Pet1 is expressed also in a non-serotonergic neuronal population intermingled with Tph2-expressing cells and mostly localized in the B8 and B9 nuclei. Moreover, we detected Cre-mediated recombination also in the developing pancreas and in the ureteric bud derivatives of the kidney, where it reflected a specific Pet1 expression. Thus, Pet1210-Cre transgenic mouse line faithfully drives Cre-mediated recombination in all Pet1 expression domains representing a valuable tool to genetically manipulate serotonergic and non-serotonergic Pet1 cell lineages. PMID:25098329

  17. Localization of mutant ubiquitin in the brain of a transgenic mouse line with proteasomal inhibition and its validation at specific sites in Alzheimer's disease

    PubMed Central

    Gentier, Romina J. G.; Verheijen, Bert M.; Zamboni, Margherita; Stroeken, Maartje M. A.; Hermes, Denise J. H. P.; Küsters, Benno; Steinbusch, Harry W. M.; Hopkins, David A.; Van Leeuwen, Fred W.

    2015-01-01

    Loss of protein quality control by the ubiquitin-proteasome system (UPS) during aging is one of the processes putatively contributing to cellular stress and Alzheimer's disease (AD) pathogenesis. Recently, pooled Genome Wide Association Studies (GWAS), pathway analysis and proteomics identified protein ubiquitination as one of the key modulators of AD. Mutations in ubiquitin B mRNA that result in UBB+1 dose-dependently cause an impaired UPS, subsequent accumulation of UBB+1 and most probably depositions of other aberrant proteins present in plaques and neurofibrillary tangles. We used specific immunohistochemical probes for a comprehensive topographic mapping of the UBB+1 distribution in the brains of transgenic mouse line 3413 overexpressing UBB+1. We also mapped the expression of UBB+1 in brain areas of AD patients selected based upon the distribution of UBB+1 in line 3413. Therefore, we focused on the olfactory bulb, basal ganglia, nucleus basalis of Meynert, inferior colliculus and raphe nuclei. UBB+1 distribution was compared with established probes for pre-tangles and tangles and A? plaques. UBB+1 distribution found in line 3413 is partly mirrored in the AD brain. Specifically, nuclei with substantial accumulations of tangle-bearing neurons, such as the nucleus basalis of Meynert and raphe nuclei also present high densities of UBB+1 positive tangles. Line 3413 is useful for studying the contribution of proteasomal dysfunction in AD. The findings are consistent with evidence that areas outside the forebrain are also affected in AD. Line 3413 may also be predictive for other conformational diseases, including related tauopathies and polyglutamine diseases, in which UBB+1 accumulates in their cellular hallmarks. PMID:25852488

  18. Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression.

    PubMed

    Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

    2014-01-01

    Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17?-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants. PMID:24626481

  19. Digestive utilization of phosphorus from plant-based diets in the Cassie line of transgenic Yorkshire pigs that secrete phytase in the saliva.

    PubMed

    Meidinger, R G; Ajakaiye, A; Fan, M Z; Zhang, J; Phillips, J P; Forsberg, C W

    2013-03-01

    A line of transgenic Yorkshire pigs referred to as the Cassie (CA) line was generated, which possessed a stable, low copy number phytase transgene insertion that enabled phytase secretion in the saliva. This study was conducted to assess growth and efficacy for improving P, Ca, and other macromineral utilization in the CA pigs receiving diets typical of those used for commercial swine production. In Exp. 1, 12 CA boars and 12 CA gilts fed diets without supplemental P gained weight and exhibited feed efficiency similar to conventional age-matched 12 Yorkshire boars and 12 Yorkshire gilts raised on similar diets with supplemental P. Serum concentrations of P and Ca were similar for CA and Yorkshire pigs during the growing and finishing phases, indicating that the CA pigs were not P limited. In Exp. 2, 6 CA (13.1 kg BW) and 6 Yorkshire barrows (8.8 kg BW) were fed 3 diets (control; low in Ca and P; and low in Ca, P, and CP) over 3 phases. The CA barrows fed the diet without supplemental P retained 25 to 40% (P < 0.001), 77 to 91% (P < 0.001), and 27 to 56% (P < 0.001) more P during the weaning, growing, and finishing phases, respectively, than conventional Yorkshire barrows fed similar diets without supplemental P. In Exp. 3, CA and Yorkshire barrows of similar ages weighing 66.2 ± 1.7 kg (n = 10) and 50.0 ± 1.0 kg (n = 10), respectively, were used. The P retention of CA finisher barrows fed a diet without supplemental P was 34% greater (P < 0.001) than conventional Yorkshire barrows fed the same diet with 750 units of exogenous phytase/kg diet. Urinary Ca to P ratio in the CA pigs was 0.27, whereas that for the Yorkshire barrows was 30, thereby, indicating that the Yorkshire barrows suffered a P deficiency. Furthermore, digestive utilization of major electrolyte macrominerals, K and Na, was improved (P < 0.05) by 18 and 16%, respectively, in the CA finisher pigs compared with the conventional Yorkshire finisher pigs fed phytase; however, only K exhibited enhanced retention. In conclusion, the CA line pigs secrete sufficient phytase from the salivary glands to enable efficient digestion of plant P, Ca, and major electrolyte macrominerals. PMID:23296825

  20. Transgenic Animals.

    ERIC Educational Resources Information Center

    Jaenisch, Rudolf

    1988-01-01

    Describes three methods and their advantages and disadvantages for introducing genes into animals. Discusses the predictability and tissue-specificity of the injected genes. Outlines the applications of transgenic technology for studying gene expression, the early stages of mammalian development, mutations, and the molecular nature of chromosomes.…

  1. Testing Transgenic Spring Wheat and Barley Lines for Reaction to Fusarium Head Blight: 2010 Field Nursery Report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2010 field screening nursery, with 88 barley plots was located at UMore Park, Rosemount MN. Trial entries (n=18) and an the untransformed 2-row control Conlon (susceptible) were submitted by USDA-ARS, RRVARC Fargo. Barley lines with known reactions to Fusarium head blight (FHB) were also incl...

  2. Testing Transgenic Spring Wheat and Barley Lines for Reaction to Fusarium Head Blight: 2009 Field Nursery Report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2009 field screening nursery, with 128 wheat and 208 barley plots was located at UMore Park, Rosemount MN. Trial entries and untransformed controls were submitted by the University of Minnesota (19+1 wheat), the Donald Danforth Plant Science Center (4+2 wheat) and USDA (48+1 barley). Lines wit...

  3. Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

    PubMed

    Gutiérrez, Jorge; García-Villa, Enrique; Ocadiz-Delgado, Rodolfo; Cortés-Malagón, Enoc M; Vázquez, Juan; Roman-Rosales, Alejandra; Alvarez-Rios, Elizabeth; Celik, Haydar; Romano, Marta C; Üren, Aykut; Lambert, Paul F; Gariglio, Patricio

    2015-10-01

    Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein. PMID:26173416

  4. Transgenic mice in developmental toxicology

    SciTech Connect

    Woychik, R.P.

    1992-01-01

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.

  5. Transgenic mice in developmental toxicology

    SciTech Connect

    Woychik, R.P.

    1992-12-31

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.

  6. Transgenic Resistance to Citrus tristeza virus in Grapefruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grapefruit (Citrus paradisi) transgenic plants transformed with a variety of constructs derived from the Citrus tristeza virus (CTV) genome were tested for their resistance to the virus. Most transgenic lines were susceptible (27 lines), a few were partially resistant (6 lines) and only one line, tr...

  7. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    PubMed Central

    Zhu-Ge, Jian; Yu, Ying-Nian; Qian, Yu-Li; Li, Xin

    2002-01-01

    AIM: The human cytochrome P-450 2C18 (CYP2C18) has been characterized. However, the protein has not been purified from liver and very little is known regarding the specific substrate of CYP2C18. In order to study its enzymatic activity for drug metabolism, the CYP2C18 cDNA was cloned and a stable CHL cell line expressing recombinant CYP 2C18 was established. METHODS: The human CYP2C18 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2C18 to Chinese hamster lung (CHL) cell. The enzyme activity of CYP2C18 catalyzing oxidation of tolbutamide to hydroxytolbutamide in postmitochondrial supernant (S9) fraction of the cell was determined by high performance liquid chromatography (HPLC). RESULTS: The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al[3] (GenBank accession number: M61856, J05326). The S9 fraction of the established cell line metabolizes tolbutamide to hydroxytolbutamide. Tolbutamide hydroxylase activity was found to be 0.509 ± 0.052 ?mol·min-1·g-1 S9 protein or 8.82 ± 0.90 mol·min-1·mol-1 CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing. CONCLUSION: The cDNA of human CYP2C18 was successfully cloned and a cell line, CHL-CYP2C18, efficiently expressing the protein of CYP2C18, was established. A spliced variant of CYP2C18 with exon 5 missing was identified in the cloning process. PMID:12378636

  8. Transgenic mouse model for skin malignant melanoma.

    PubMed

    Kato, M; Takahashi, M; Akhand, A A; Liu, W; Dai, Y; Shimizu, S; Iwamoto, T; Suzuki, H; Nakashima, I

    1998-10-01

    We report here on a novel metallothionein-I (MT)/ret transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma metastasizing to distant organs develop stepwise. The process of tumor development and its malignant transformation in this line may resemble that of the human giant congenital melanocytic nevus that is present at birth and that frequently gives rise to malignant melanoma during aging. We observed an increase in the expression level and activity of the ret transgene during the disease progression. That increase in transgene expression accompanied an activation of mitogen-activated protein kinases (MAPKs) and c-Jun as well as matrix metalloproteinases. These results suggest that progressive dysregulation of the expression level of the ret transgene might play a crucial role in the malignant transformation of melanocytic tumors developed in the MT/ret transgenic mouse line. PMID:9778055

  9. Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBL Targeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines

    PubMed Central

    Cerbini, Trevor; Funahashi, Ray; Luo, Yongquan; Liu, Chengyu; Park, Kyeyoon; Rao, Mahendra; Malik, Nasir; Zou, Jizhong

    2015-01-01

    Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%–58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells. PMID:25587899

  10. A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.

    PubMed

    Apuschkin, Mia; Stilling, Sara; Rahbek-Clemmensen, Troels; Sřrensen, Gunnar; Fortin, Guillaume; Herborg Hansen, Freja; Eriksen, Jacob; Trudeau, Louis-Eric; Egerod, Kristoffer; Gether, Ulrik; Rickhag, Mattias

    2015-10-01

    Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000-30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology. PMID:26286107

  11. T-1 Training Area

    SciTech Connect

    2014-11-07

    Another valuable homeland security asset at the NNSS is the T-1 training area, which covers more than 10 acres and includes more than 20 separate training venues. Local, County, and State first responders who train here encounter a variety of realistic disaster scenarios. A crashed 737 airliner lying in pieces across the desert, a helicopter and other small aircraft, trucks, buses, and derailed train cars are all part of the mock incident scene. After formal classroom education, first responders are trained to take immediate decisive action to prevent or mitigate the use of radiological or nuclear devices by terrorists. The Counterterrorism Operations Support Center for Radiological Nuclear Training conducts the courses and exercises providing first responders from across the nation with the tools they need to protect their communities. All of these elements provide a training experience that cannot be duplicated anywhere else in the country.

  12. T-1 Training Area

    ScienceCinema

    None

    2015-01-09

    Another valuable homeland security asset at the NNSS is the T-1 training area, which covers more than 10 acres and includes more than 20 separate training venues. Local, County, and State first responders who train here encounter a variety of realistic disaster scenarios. A crashed 737 airliner lying in pieces across the desert, a helicopter and other small aircraft, trucks, buses, and derailed train cars are all part of the mock incident scene. After formal classroom education, first responders are trained to take immediate decisive action to prevent or mitigate the use of radiological or nuclear devices by terrorists. The Counterterrorism Operations Support Center for Radiological Nuclear Training conducts the courses and exercises providing first responders from across the nation with the tools they need to protect their communities. All of these elements provide a training experience that cannot be duplicated anywhere else in the country.

  13. Strategies for Designing Transgenic DNA Constructs

    PubMed Central

    Liu, Chengyu

    2013-01-01

    Generation and characterization of transgenic mice are important elements of biomedical research. In recent years, transgenic technology has become more versatile and sophisticated, mainly because of the incorporation of recombinase-mediated conditional expression and targeted insertion, site-specific endonuclease-mediated genome editing, siRNA-mediated gene knockdown, various inducible gene expression systems, and fluorescent protein marking and tracking techniques. Site-specific recombinases (such as PhiC31) and engineered endonucleases (such as ZFN and Talen) have significantly enhanced our ability to target transgenes into specific genomic loci, but currently a great majority of transgenic mouse lines are continuingly being created using the conventional random insertion method. A major challenge for using this conventional method is that the genomic environment at the integration site has a substantial influence on the expression of the transgene. Although our understanding of such chromosomal position effects and our means to combat them are still primitive, adhering to some general guidelines can significantly increase the odds of successful transgene expression. This chapter first discusses the major problems associated with transgene expression, and then describes some of the principles for using plasmid and bacterial artificial chromosomes (BACs) for generating transgenic constructs. Finally, the strategies for conducting each of the major types of transgenic research are discussed, including gene overexpression, promoter characterization, cell-lineage tracing, mutant complementation, expression of double or multiple transgenes, siRNA knockdown, and conditional and inducible systems. PMID:23912987

  14. 2013 North Dakota Transgenic Barley Research and FHB Nursery Report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Research continues to develop and test new transgenic plants using genes provided by collaborators. As lines are developed in Golden Promise, they are crossed to Conlon for field testing. Transgenic lines developed in Conlon are being crossed to resistant lines developed by the breeding programs. ...

  15. Herbicide sensitivity of transgenic multiple herbicide-tolerant oilseed rape.

    PubMed

    Senior, Ian J; Moyes, Catherine; Dale, Philip J

    2002-04-01

    Glyphosate and glufosinate-ammonium herbicide tolerance traits were combined into both winter and spring lines of Brassica napus L. This allowed the study of possible interactions between these transgenes in two genetic backgrounds when treated with a variety of herbicides. Selective herbicides that are commonly used within Brassica crops showed no adverse effects on the transgenic plants or their null controls. Lines containing both glyphosate and glufosinate transgenes remained tolerant to their respective herbicides, regardless of the presence of the second tolerance transgene. Lines containing only a single transgene retained tolerance to the encoded trait and did not show cross-tolerance to the second. Null lines were killed by either herbicide. All plant lines, regardless of their transgene content, were found to be equally susceptible to three herbicides (paraquat, metsulfuronmethyl and mecoprop), commonly used to remove volunteer B napus from succeeding crops and set-a-side land. PMID:11975190

  16. Assessment of peanut quality and compositional characteristics among transgenic sclerotinia blight-resistant and non-transgenic susceptible cultivars.

    PubMed

    Hu, Jiahuai; Telenko, Darcy E P; Phipps, Patrick M; Grabau, Elizabeth A

    2014-08-01

    This study presents the results of a comparison that includes an analysis of variance and a canonical discriminant analysis to determine compositional equivalence and similarity between transgenic, sclerotinia blight-resistant and non-transgenic, susceptible cultivars of peanut in 3 years of field trials. Three Virginia-type cultivars (NC 7, Wilson, and Perry) and their corresponding transgenic lines (N70, W73, and P39) with a barley oxalate oxidase gene were analyzed for differences in key mineral nutrients, fatty acid components, hay constituents, and grade characteristics. Results from both analyses demonstrated that transgenic lines were compositionally similar to their non-transgenic parent cultivar in all factors as well as market-grade characteristics and nutritional value. Transgenic lines expressing oxalate oxidase for resistance to sclerotinia blight were substantially equivalent to their non-transgenic parent cultivar in quality and compositional characteristics. PMID:24972023

  17. Every silver lining has a cloud: the scientific and animal welfare issues surrounding a new approach to the production of transgenic animals.

    PubMed

    Combes, Robert D; Balls, Michael

    2014-05-01

    The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged. PMID:24901907

  18. Transgene translatability increases effectiveness of replicase-mediated resistance to cucumber mosaic virus.

    PubMed

    Wintermantel, W M; Zaitlin, M

    2000-03-01

    Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of Cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted virus movement when inoculated mechanically or by aphid vectors. Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full-length or truncated replicase proteins. In contrast, among plants which contained nontranslatable transgene constructs, only one of 61 lines examined exhibited delays or resistance. Once infected, plants never recovered, regardless of transgene translatability. Transgenic plants exhibiting a range of resistance levels were examined for transgene copy number, mRNA and protein levels. Although ribonuclease protection assays demonstrated that transgene mRNA levels were very low, resistant lines had consistently more steady-state transgene mRNA than susceptible lines. Furthermore, chlorotic or necrotic local lesions developed on the inoculated leaves of transgenic lines containing translatable transgenes, but not on inoculated leaves of lines containing nontranslatable transgenes. These results demonstrate that translatability of the transgene and possibly expression of the transgene protein itself facilitates replicase-mediated resistance to CMV in tobacco. PMID:10675396

  19. Transgenic apple (Malus x domestica) shoot showing low browning potential.

    PubMed

    Murata, M; Haruta, M; Murai, N; Tanikawa, N; Nishimura, M; Homma, S; Itoh, Y

    2000-11-01

    Transgenic apple shoots were prepared from leaf disks by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistance gene and antisense polyphenol oxidase (PPO) DNA. Four transgenic apple lines that grew on the medium containing 50 microgram/mL KM were obtained. They contained the KM resistance gene and grew stably on the medium for >3 years. Two transgenic shoot lines containing antisense PPO DNA in which PPO activity was repressed showed a lower browning potential than a control shoot. PMID:11087467

  20. Transcription-dependent silencing of inducible convergent transgenes in transgenic mice

    E-print Network

    Calero-Nieto, Fernando J.; Bert, Andrew G.; Cockerill, Peter N.

    2010-01-19

    in a specific subset of lines in T cells that had encountered a previous episode of activation. Transgene silencing appeared to be both transcription-dependent and mediated by epigenetic mechanisms. Silencing was accompanied by loss of DNase I...

  1. EXPRESSION OF ENDOGLUCANASE E1 IN TRANSGENIC DUCKWEED LEMNA MINOR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic duckweed (Lemna minor) that expresses Acidothermus cellulolyticus E1 endoglucanase was generated using Agrobacterium-mediated transformation. Out of 15 independent transgenic lines, 1 line with the highest CMC-degrading activity was selected for further studies. The 2-week-old transgeni...

  2. Efficient Generation of Mice with Consistent Transgene Expression by FEEST

    PubMed Central

    Gao, Lei; Jiang, Yonghua; Mu, Libing; Liu, Yanbin; Wang, Fengchao; Wang, Peng; Zhang, Aiqun; Tang, Nan; Chen, Ting; Luo, Minmin; Yu, Lei; Gao, Shaorong; Chen, Liang

    2015-01-01

    Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale. PMID:26573149

  3. Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond

    E-print Network

    Feng, Guoping

    The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic ...

  4. Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.).

    PubMed

    Keshavareddy, G; Rohini, S; Ramu, S V; Sundaresha, S; Kumar, A R V; Kumar, P Ananda; Udayakumar, M

    2013-07-01

    Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T2 generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 ?g/g FW. Further, Southern blot analysis of ten T2 transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n?=?369) and 26.0 (n?=?80) were recorded in T1 and T2 generations, respectively. Segregation analysis of the selected lines in the T3 generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T2 generation, the cry1AcF gene was effective against S. litura only to some extent. PMID:24431503

  5. {open_quotes}Horizontal{close_quotes} gene transfer from a transgenic potato line to a bacterial pathogen (Erwinia chrysanthemi) occurs - if at all - at an extremely low frequency

    SciTech Connect

    Schlueter, K.; Fuetterer, J.; Potrykus, I.

    1995-10-01

    The frequency of possible {open_quotes}horizontal{close_quotes} gene transfer between a plant and a tightly associated bacterial pathogen was studied in a model system consisting of transgenic Solanum tuberosum, containing a {beta}-lactamase gene linked to a pBR322 origin of replication, and Erwinia chrysanthemi. This experimental system offers optimal conditions for the detection of possible horizontal gene transfer events, even when they occur at very low frequency. Horizontal gene transfer was not detected under conditions mimicking a {open_quotes}natural{close_quotes} infection. The gradual, stepwise alteration of artificial, positive control conditions to idealized natural conditions, however, allowed the characterization of factors that affected gene transfer, and revealed a gradual decrease of the gene transfer frequency from 6.3 x 10{sup -2} under optimal control conditions to a calculated 2.0 x 10{sub -17} under idealized natural conditions. These data, in combination with other published studies, argue that horizontal gene transfer is so rare as to be essentially irrelevant to any realistic assessment of the risk involved in release experiments involving transgenic plants. 22 refs., 3 figs., 2 tabs.

  6. Influence of a nonfragile FHIT transgene on murine tumor susceptibility.

    PubMed

    McCorkell, K A; Mancini, R; Siprashvili, Z; Barnoski, B L; Iliopoulos, D; Siracusa, L D; Zanesi, N; Croce, C M; Fong, L Y Y; Druck, T; Huebner, K

    2007-01-01

    FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction. PMID:18000371

  7. Molecular Analyses of Transgenic Plants.

    PubMed

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

    2016-01-01

    One of the major challenges in plant molecular biology is to generate transgenic plants that express transgenes stably over generations. Here, we describe some routine methods to study transgene locus structure and to analyze transgene expression in plants: Southern hybridization using DIG chemiluminescent technology for characterization of transgenic locus, SYBR Green-based real-time RT-PCR to measure transgene transcript level, and protein immunoblot analysis to evaluate accumulation and stability of transgenic protein product in the target tissue. PMID:26614292

  8. Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression.

    PubMed Central

    Bruggeman, L A; Dikman, S; Meng, C; Quaggin, S E; Coffman, T M; Klotman, P E

    1997-01-01

    HIV-associated nephropathy (HIVAN) is a progressive glomerular and tubular disease that is increasingly common in AIDS patients and one of the leading causes of end stage renal disease in African Americans. A major unresolved issue in the pathogenesis of HIVAN is whether the kidney disease is due to renal cell infection or a "bystander" phenomenon mediated by systemically dysregulated cytokines. To address this issue, we have used two different experimental approaches and an HIV-1 transgenic mouse line that develops a progressive renal disease histologically similar to HIVAN in humans. In the murine model, kidney tissue expresses the transgene and in heterozygous adults, renal disease develops shortly thereafter. We demonstrate by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling assay that similar to the disease in humans, apoptosis of renal tubular epithelial cells is a component of the molecular pathogenesis. To determine whether apoptosis is due to transgene expression or environmental factors, we treated fetal kidney explants (normal and transgenic) with UV light to induce transgene expression. Apoptosis occurred in transgenic but not normal littermates after stimulation of transgene expression. To confirm a direct effect of HIV expression on the production of HIVAN, we transplanted kidneys between normal and transgenic mice. HIVAN developed in transgenic kidneys transplanted into nontransgenic littermates. Normal kidneys remained disease free when transplanted into transgenic littermates. Thus, the renal disease in the murine model is intrinsic to the kidney. Using two different experimental approaches, we demonstrate a direct effect of transgene expression on the development of HIVAN in the mouse. These studies suggest that in humans, a direct effect of HIV-1 expression is likely the essential cause of HIVAN, rather than an indirect effect of cytokine dysregulation. PMID:9202060

  9. [Study on the transgenic tobacco expressing truncated wheat cold shock protein gene TA3-13 and analysis of disease resistance].

    PubMed

    Li, Na; DU, Xiu-Zhen; Pan, Xiao-Mei; Wang, Jin-Sheng; Song, Cong-Feng

    2011-05-01

    TA3-13 is a truncated gene coding for the fragment of wheat (Triticum aestivum L.) cold shock protein WCP1. It has been shown previously that the procaryotically expressed TA3-13 can induce resistance to Tobacco mosaic virus (TMV) when sprayed onto plant leaves. In this study, we constructed an expression vector pB-3-13 by cloning TA3-13 into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciense strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was performed using the leaf disc infection method. After screening on MS medium containing kanamycin and PCR analysis, 33 T0 plantlets were identified as transgenic. Seeds from twenty T0 plants were collected and planted as T1 lines. Two T1 lines were selected for further characterization. PCR and GUS staining analysis showed that TA3-13 was integrated into the T1 tobacco genome and expressed. When inoculated on leaves, the transgenic tobacco showed significant resistance against TMV and rot pathogen Pectobactrium carotovorum subsp. carotovorum. These results suggest that the expression of TA3-13 in tobacco can induce defense responses to pathogen infection. PMID:21586399

  10. CMV-driven expression of green uorescent protein (GFP) in male germ cells of transgenic mice

    E-print Network

    Museo Nacional de Ciencias Naturales

    CMV-driven expression of green Żuorescent protein (GFP) in male germ cells of transgenic mice if the expression of green Żuorescent protein (GFP) during sperma- togenesis can compromise the fertility of transgenic animals, we have produced mouse transgenic lines expressing GFP in the testis under the control

  11. Competitive Performance of Transgenic Wheat Resistant to Powdery Mildew

    PubMed Central

    Kalinina, Olena; Zeller, Simon L.; Schmid, Bernhard

    2011-01-01

    Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes. PMID:22132219

  12. WEEDING IN TRANSGENES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenes promise to reduce insecticide and fungicide use, but relatively little has been done to significantly reduce herbicide use through genetic engineering. Three strategies for transgene utilization are discussed which have the potential to change this: 1) improvement of weed-specific biocon...

  13. TRANSGENIC COMPARISONS OF PINK BOLLWORM EFFICACY AND RESPONSE TO HEAT STRESS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fifteen lines from 3 different cotton families were compared. Each family had a conventional, non-transgenic standard, as well as 4 other transgenic lines. Each Bt line was evaluated for this trait's efficacy in controlling pink bollworm under high pressure, artificial infestations. Various agronomi...

  14. Pathology waste includes: Transgenic animals.

    E-print Network

    Mease, Kenneth D.

    Pathology waste includes: · Transgenic animals. · Potentially transgenic animals including, "no-takes" in the production of transgenic animals, and off-spring of transgenic animals. · Recognizable human anatomical parts specimens. · Human tissues that have been fixed in formaldehyde or other fixatives*. · Animal carcasses

  15. Heterologous expression of taro cystatin protects transgenic tomato against Meloidogyne incognita infection by means of interfering sex determination and suppressing gall formation.

    PubMed

    Chan, Yuan-Li; Yang, Ai-Hwa; Chen, Jen-Tzu; Yeh, Kai-Wun; Chan, Ming-Tsair

    2010-03-01

    Plant-parasitic nematodes are a major pest of many plant species and cause global economic loss. A phytocystatin gene, Colocasia esculenta cysteine proteinase inhibitor (CeCPI), isolated from a local taro Kaosiang No. 1, and driven by a CaMV35S promoter was delivered into CLN2468D, a heat-tolerant cultivar of tomato (Solanum lycopersicum). When infected with Meloidogyne incognita, one of root-knot nematode (RKN) species, transgenic T1 lines overexpressing CeCPI suppressed gall formation as evidenced by a pronounced reduction in gall numbers. In comparison with wild-type plants, a much lower proportion of female nematodes without growth retardation was observed in transgenic plants. A decrease of RKN egg mass in transgenic plants indicated seriously impaired fecundity. Overexpression of CeCPI in transgenic tomato has inhibitory functions not only in the early RKN infection stage but also in the production of offspring, which may result from intervention in sex determination. PMID:20054551

  16. Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision

    PubMed Central

    Woo, Hee-Jong; Qin, Yang; Park, Soo-Yun; Park, Soon Ki; Cho, Yong-Gu; Shin, Kong-Sik; Lim, Myung-Ho; Cho, Hyun-Suk

    2015-01-01

    Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, ?-, ?-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice. PMID:26172549

  17. Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime.

    PubMed

    López, Carmelo; Cervera, Magdalena; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peńa, Leandro

    2010-01-01

    Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs. PMID:20078774

  18. Split-Cre Complementation Restores Combination Activity on Transgene Excision in Hair Roots of Transgenic Tobacco

    PubMed Central

    Wen, Mengling; Gao, Yuan; Wang, Lijun; Ran, Lingyu; Li, Jiahui; Luo, Keming

    2014-01-01

    The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ‘‘split-Cre’’ fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants. PMID:25329460

  19. A dominant-negative mutation within AtMYB90 blocks flower pigment production in transgenic tobacco.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During de novo shoot induction in cultured transgenic tobacco callus a spontaneous mutation within the coding region of a AtMYB90 transgene produced a plant line in which the original transgene-induced over-pigmented phenotype (dark red/purple from anthocyanin overproduction in most tissues) was los...

  20. Gene flow from transgenic common beans expressing the bar gene.

    PubMed

    Faria, Josias C; Carneiro, Geraldo E S; Aragăo, Francisco J L

    2010-01-01

    Gene flow is a common phenomenon even in self-pollinated plant species. With the advent of genetically modified plants this subject has become of the utmost importance due to the need for controlling the spread of transgenes. This study was conducted to determine the occurrence and intensity of outcrossing in transgenic common beans. In order to evaluate the outcross rates, four experiments were conducted in Santo Antonio de Goiás (GO, Brazil) and one in Londrina (PR, Brazil), using transgenic cultivars resistant to the herbicide glufosinate ammonium and their conventional counterparts as recipients of the transgene. Experiments with cv. Olathe Pinto and the transgenic line Olathe M1/4 were conducted in a completely randomized design with ten replications for three years in one location, whereas the experiments with cv. Pérola and the transgenic line Pérola M1/4 were conducted at two locations for one year, with the transgenic cultivar surrounded on all sides by the conventional counterpart. The outcross occurred at a negligible rate of 0.00741% in cv. Pérola, while none was observed (0.0%) in cv. Olathe Pinto. The frequency of gene flow was cultivar dependent and most of the observed outcross was within 2.5 m from the edge of the pollen source. Index terms: Phaseolus vulgaris, outcross, glufosinate ammonium. PMID:21865877

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

    PubMed

    Morton, Susan K; Chaston, Daniel J; Baillie, Brett K; Hill, Caryl E; Matthaei, Klaus I

    2014-01-01

    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. PMID:24755679

  2. Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

    PubMed Central

    Baillie, Brett K.; Hill, Caryl E.; Matthaei, Klaus I.

    2014-01-01

    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 LacIR, 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 LacIR protein. PMID:24755679

  3. Transgenic horticultural crops in Asia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modern biotechnology applications, including genetic engineering, are a powerful tool to complement the conventional methods of crop improvement. Asia currently has three countries cultivating biotech/transgenic crops – China, India, and the Philippines, but only China commercially grows a transgen...

  4. Introgression of the SbASR-1 Gene Cloned from a Halophyte Salicornia brachiata Enhances Salinity and Drought Endurance in Transgenic Groundnut (Arachis hypogaea) and Acts as a Transcription Factor

    PubMed Central

    Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

    2015-01-01

    The SbASR-1 gene, cloned from a halophyte Salicornia brachiata, encodes a plant-specific hydrophilic and stress responsive protein. The genome of S. brachiata has two paralogs of the SbASR-1 gene (2549 bp), which is comprised of a single intron of 1611 bp, the largest intron of the  abscisic acid stress ripening [ASR] gene family yet reported. In silico analysis of the 843-bp putative promoter revealed the presence of ABA, biotic stress, dehydration, phytohormone, salinity, and sugar responsive cis-regulatory motifs. The SbASR-1 protein belongs to Group 7 LEA protein family with different amino acid composition compared to their glycophytic homologs. Bipartite Nuclear Localization Signal (NLS) was found on the C-terminal end of protein and localization study confirmed that SbASR-1 is a nuclear protein. Furthermore, transgenic groundnut (Arachis hypogaea) plants over-expressing the SbASR-1 gene constitutively showed enhanced salinity and drought stress tolerance in the T1 generation. Leaves of transgenic lines exhibited higher chlorophyll and relative water contents and lower electrolyte leakage, malondialdehyde content, proline, sugars, and starch accumulation under stress treatments than wild-type (Wt) plants. Also, lower accumulation of H2O2 and O2.- radicals was detected in transgenic lines compared to Wt plants under stress conditions. Transcript expression of APX (ascorbate peroxidase) and CAT (catalase) genes were higher in Wt plants, whereas the SOD (superoxide dismutase) transcripts were higher in transgenic lines under stress. Electrophoretic mobility shift assay (EMSA) confirmed that the SbASR-1 protein binds at the consensus sequence (C/G/A)(G/T)CC(C/G)(C/G/A)(A/T). Based on results of the present study, it may be concluded that SbASR-1 enhances the salinity and drought stress tolerance in transgenic groundnut by functioning as a LEA (late embryogenesis abundant) protein and a transcription factor. PMID:26158616

  5. Limited Fitness Advantages of Crop-Weed Hybrid Progeny Containing Insect-Resistant Transgenes (Bt/CpTI) in Transgenic Rice Field

    PubMed Central

    Yang, Xiao; Wang, Feng; Su, Jun; Lu, Bao-Rong

    2012-01-01

    Background The spread of insect-resistance transgenes from genetically engineered (GE) rice to its coexisting weedy rice (O. sativa f. spontanea) populations via gene flow creates a major concern for commercial GE rice cultivation. Transgene flow to weedy rice seems unavoidable. Therefore, characterization of potential fitness effect brought by the transgenes is essential to assess environmental consequences caused by crop-weed transgene flow. Methodology/Principal Findings Field performance of fitness-related traits was assessed in advanced hybrid progeny of F4 generation derived from a cross between an insect-resistant transgenic (Bt/CpTI) rice line and a weedy strain. The performance of transgene-positive hybrid progeny was compared with the transgene-negative progeny and weedy parent in pure and mixed planting of transgenic and nontransgenic plants under environmental conditions with natural vs. low insect pressure. Results showed that under natural insect pressure the insect-resistant transgenes could effectively suppress target insects and bring significantly increased fitness to transgenic plants in pure planting, compared with nontransgenic plants (including weedy parent). In contrast, no significant differences in fitness were detected under low insect pressure. However, such increase in fitness was not detected in the mixed planting of transgenic and nontransgenic plants due to significantly reduced insect pressure. Conclusions/Significance Insect-resistance transgenes may have limited fitness advantages to hybrid progeny resulted from crop-weed transgene flow owning to the significantly reduced ambient target insect pressure when an insect-resistant GE crop is grown. Given that the extensive cultivation of an insect-resistant GE crop will ultimately reduce the target insect pressure, the rapid spread of insect-resistance transgenes in weedy populations in commercial GE crop fields may be not likely to happen. PMID:22815975

  6. Transgenic Farm Animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of recombinant DNA technology has enabled scientists to isolate single genes, analyze and modify their nucleotide structure(s), make copies of these isolated genes, and insert copies of these genes into the genome of plants and animals. The transgenic technology of adding genes to li...

  7. Viable transgenic goats derived from skin cells.

    PubMed

    Behboodi, Esmail; Memili, Erdogan; Melican, David T; Destrempes, Margaret M; Overton, Susan A; Williams, Jennifer L; Flanagan, Peter A; Butler, Robin E; Liem, Hetty; Chen, Li How; Meade, Harry M; Gavin, William G; Echelard, Yann

    2004-06-01

    The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line. PMID:15359599

  8. The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat.

    PubMed

    Han, Yonghua; Blechl, Ann; Wang, Daowen

    2015-12-01

    Although particle bombardment is the predominant method of foreign DNA direct transfer, whether transgene is integrated randomly into the genome has not been determined. In this study, we identified the distribution of transgene loci in 45 transgenic wheat (Triticum aestivum L.) lines containing co-transformed high molecular weight glutenin subunit genes and the selectable marker bar using fluorescence in situ hybridization. Transgene loci were shown to distribute unevenly throughout the genome and incorporate into different locations along individual chromosomes. There was only a slight tendency towards the localization of transgenes in distal chromosome regions. High proportions of transgenes in separate plasmids integrated at the same site and only 7 lines had 2 or 3 loci. Such loci may not segregate frequently in subsequent generations so it is difficult to remove selectable markers from transgenic lines after regeneration. We also found that three transgene lines were associated with rearranged chromosomes, suggesting a the close relationship between particle bombardment-mediated transgene integration and chromosomal rearrangements. PMID:26405007

  9. 2012 North Dakota Transgenic Barley FHB Nursery Report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2012 North Dakota transgenic field trials consisted of 23 barley lines, tested in three misted and three non-misted replicates. Plots were sown on May 9, 2012 in hill plots with 10 seed per hill spaced at 30 cm, and all plots were inoculated using the grain spawn method at heading. Lines include...

  10. A versatile transgenic allele for mouse overexpression studies.

    PubMed

    Dolatshad, Hamid; Biggs, Daniel; Diaz, Rebeca; Hortin, Nicole; Preece, Christopher; Davies, Benjamin

    2015-12-01

    For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies. PMID:26369329

  11. Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield

    PubMed Central

    Yadav, Dinesh; Shavrukov, Yuri; Bazanova, Natalia; Chirkova, Larissa; Borisjuk, Nikolai; Kovalchuk, Nataliya; Ismagul, Ainur; Parent, Boris; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2015-01-01

    Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1–T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20–30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance. PMID:26220082

  12. Constitutive overexpression of the TaNF-YB4 gene in transgenic wheat significantly improves grain yield.

    PubMed

    Yadav, Dinesh; Shavrukov, Yuri; Bazanova, Natalia; Chirkova, Larissa; Borisjuk, Nikolai; Kovalchuk, Nataliya; Ismagul, Ainur; Parent, Boris; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2015-11-01

    Heterotrimeric nuclear factors Y (NF-Ys) are involved in regulation of various vital functions in all eukaryotic organisms. Although a number of NF-Y subunits have been characterized in model plants, only a few have been functionally evaluated in crops. In this work, a number of genes encoding NF-YB and NF-YC subunits were isolated from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius was investigated. TaNF-YB4 was isolated as a result of two consecutive yeast two-hybrid (Y2H) screens, where ZmNF-YB2a was used as a starting bait. A new NF-YC subunit, designated TaNF-YC15, was isolated in the first Y2H screen and used as bait in a second screen, which identified two wheat NF-YB subunits, TaNF-YB2 and TaNF-YB4. Three-dimensional modelling of a TaNF-YB2/TaNF-YC15 dimer revealed structural determinants that may underlie interaction selectivity. The TaNF-YB4 gene was placed under the control of the strong constitutive polyubiquitin promoter from maize and introduced into wheat by biolistic bombardment. The growth and yield components of several independent transgenic lines with up-regulated levels of TaNF-YB4 were evaluated under well-watered conditions (T1-T3 generations) and under mild drought (T2 generation). Analysis of T2 plants was performed in large deep containers in conditions close to field trials. Under optimal watering conditions, transgenic wheat plants produced significantly more spikes but other yield components did not change. This resulted in a 20-30% increased grain yield compared with untransformed control plants. Under water-limited conditions transgenic lines maintained parity in yield performance. PMID:26220082

  13. [Effects of phytase transgenic corn planting on soil nematode community].

    PubMed

    Zhao, Zong-Chao; Su, Ying; Mou, Wen-Ya; Liu, Man-Qiang; Chen, Xiao-Yun; Chen, Fa-Jun

    2014-04-01

    A healthy soil ecosystem is essential for nutrient cycling and energy conversion, and the impact of exogenous genes from genetically modified crops had aroused wide concerns. Phytase transgenic corn (i. e., the inbred line BVLA430101) was issued a bio-safety certificate on 27 September 2009 in China, which could improve the efficiency of feed utilization, reduce environmental pollution caused by animal manure. In this study, the abundance of trophic groups, community structure and ecological indices of soil nematodes were studied over the growing cycle of phytase transgenic corn (ab. transgenic corn) and control conventional parental corn (ab. control corn) in the field. Totally 29 and 26 nematode genera were isolated from transgenic corn and control corn fields, respectively. The abundances of bacterivores and omnivores-predators, the total number of soil nematodes, and the Shannon index (H) were significantly greater under transgenic corn than under control corn, while the opposite trend was found for the relative abundance of herbivores and the maturity index (Sigma MI) of soil nematodes. Repeated-measures analysis of variance (ANOVA) did not detect any significant effects of transgenic corn on the composition and abundance of nematode trophic groups and ecological indices of soil nematodes. Furthermore, the Student-T test showed that the abundances of bacterivores and omnivores-predators and the total number of soil nematodes during the milk-ripe stage were significant higher in the transgenic corn field than in the control corn field. The effects of transgenic corn planting on soil nematodes might be related to the increase in the nitrogen content of field soil under transgenic corn compared to control corn. PMID:25011306

  14. Transgenic fish systems and their application in ecotoxicology.

    PubMed

    Lee, Okhyun; Green, Jon M; Tyler, Charles R

    2015-02-01

    The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4-UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities. PMID:25394772

  15. Generation of Transgenic C. elegans by Biolistic Transformation

    PubMed Central

    Hochbaum, Daniel; Ferguson, Annabel A.; Fisher, Alfred L.

    2010-01-01

    The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection. PMID:20811328

  16. Transgenics in crops

    NASA Technical Reports Server (NTRS)

    Li, Y.; Wu, Y. H.; McAvoy, R.; Duan, H.

    2001-01-01

    With rapid world population growth and declining availability of fresh water and arable land, a new technology is urgently needed to enhance agricultural productivity. Recent discoveries in the field of crop transgenics clearly demonstrate the great potential of this technology for increasing food production and improving food quality while preserving the environment for future generations. In this review, we briefly discuss some of the recent achievements in crop improvement that have been made using gene transfer technology.

  17. Comparative analysis of nutritional compositions of transgenic RNAi-mediated virus-resistant bean (event EMB-PV051-1) with its non-transgenic counterpart.

    PubMed

    Carvalho, José L V; de Oliveira Santos, Juliana; Conte, Carmine; Pacheco, Sidney; Nogueira, Elsa O P L; Souza, Thiago L P O; Faria, Josias C; Aragăo, Francisco J L

    2015-10-01

    Golden mosaic is among the most economically important diseases that severely reduce bean production in Latin America. In 2011, a transgenic bean event named Embrapa 5.1 (EMB-PV051-1), resistant to bean golden mosaic virus, was approved for commercial release in Brazil. The aim of this study was to measure and evaluate the nutritional components of the beans, as well as the anti-nutrient levels in the primary transgenic line and its derived near-isogenic lines after crosses and backcrosses with two commercial cultivars. Nutritional assessment of transgenic crops used for human consumption is an important aspect of safety evaluations. Results demonstrated that the transgenic bean event, cultivated under field conditions, was substantially equivalent to that of the non-transgenic bean plants. In addition, the amounts of the nutritional components are within the range of values observed for several bean commercial varieties grown across a range of environments and seasons. PMID:25894661

  18. Transgenic algae engineered for higher performance

    SciTech Connect

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

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  19. A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

    PubMed

    Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

    2001-02-01

    Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus. PMID:11302173

  20. Endoscopic ultrasonography for staging of T1a and T1b esophageal squamous cell carcinoma

    PubMed Central

    He, Long-Jun; Shan, Hong-Bo; Luo, Guang-Yu; Li, Yin; Zhang, Rong; Gao, Xiao-Yan; Wang, Guo-Bao; Lin, Shi-Yong; Xu, Guo-Liang; Li, Jian-Jun

    2014-01-01

    AIM: To investigate the accuracy of Endoscopic ultrasound (EUS) in staging and sub-staging T1a and T1b esophageal squamous cell carcinoma (ESCC). METHODS: A retrospective analysis involving 72 patients with pathologically confirmed T1a or T1b ESCC, was undertaken between January 2005 and December 2011 in Sun Yat-sen University Cancer Center. The accuracy and efficiency of EUS for detecting stages T1a and T1b ESCC were examined. RESULTS: The overall accuracy of EUS for detecting stage T1a or T1b ESCC was 70.8% (51/72), and the sensitivity was 74.3%. 77.8% (7/9) of lesions originated in the upper thoracic region, 73.1% (38/52) in the mid-thoracic region and 72.7% (8/11) in the lower thoracic region. Multivariate analysis revealed that the diagnostic accuracy of EUS was closely related to lesion length (F = 4.984, P = 0.029). CONCLUSION: EUS demonstrated median degree of accuracy for distinguishing between stages T1a and T1b ESCC. Therefore, it is necessary to improve EUS for staging early ESCC. PMID:24574809

  1. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

    PubMed

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV. PMID:25984767

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

    PubMed

    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. PMID:22101927

  3. Overexpression of host plant urease in transgenic silkworms.

    PubMed

    Jiang, Liang; Huang, Chunlin; Sun, Qiang; Guo, Huizhen; Peng, Zhengwen; Dang, Yinghui; Liu, Weiqiang; Xing, Dongxu; Xu, Guowen; Zhao, Ping; Xia, Qingyou

    2015-06-01

    Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms. PMID:25549597

  4. Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

    PubMed

    Leibman, Diana; Prakash, Shanmugam; Wolf, Dalia; Zelcer, Aaron; Anfoka, Ghandi; Haviv, Sabrina; Brumin, Marina; Gaba, Victor; Arazi, Tzahi; Lapidot, Moshe; Gal-On, Amit

    2015-11-01

    Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22 % of total small RNA) with dominant sizes of 21 nt (73 %) and 22 nt (22 %). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04 % of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved. PMID:26255053

  5. Purification and characterization of group A streptococcal T-1 antigen.

    PubMed Central

    Johnson, R H; Vosti, K L

    1977-01-01

    A method is described for the recovery of purified T-antigen from crude trypsin extracts of an avirulent strain of M-1 protein deficient, T-type 1 group A Streptococcus. The purified T-antigen was resistant to enzymatic degradation with trypsin and pepsin, formed a single precipitin line with standard T-1 antiserum, failed to react with antisera for teichoic acid, group A carbohydrate, and cross-reactive protein antigens, stimulated only a single precipitin system when rabbits were immunized, contained glycine, aspartic acid, glutamic acid, lysine, and serine as the five most predominant amino acids, and consisted of subunit size isomers. Images PMID:70408

  6. [Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

    PubMed

    Li, Jin-Di; Qin, Bai-Fu; Yang, Chun-Xian; Lan, Xiao-Zhong; Wu, Neng-Biao; Liao, Zhi-Hua

    2013-06-01

    Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed. PMID:24010284

  7. Epigenetic silencing in transgenic plants

    PubMed Central

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  8. Advances in transgenic rat production.

    PubMed

    Filipiak, Wanda E; Saunders, Thomas L

    2006-12-01

    Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models. PMID:17009096

  9. Distinct Human and Mouse Membrane Trafficking Systems for Sweet Taste Receptors T1r2 and T1r3

    PubMed Central

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system. PMID:25029362

  10. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    PubMed

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system. PMID:25029362

  11. Advanced Colloids Experiment (ACE-T1)

    NASA Technical Reports Server (NTRS)

    Meyer, William V.; Sicker, Ron; Brown, Dan; Eustace, John

    2015-01-01

    Increment 45 - 46 Science Symposium presentation of Advanced Colloids Experiment (ACE-T1) to RPO. The purpose of this event is for Principal Investigators to present their science objectives, testing approach, and measurement methods to agency scientists, managers, and other investigators.

  12. DISEASE RESISTANT TRANSGENIC COTTON TO PREVENT PREHARVEST AFLATOXIN CONTAMINATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing transgenic cottons that are resistant to the saprophytic fungus Aspergillus flavus, which produces carcinogenic aflatoxin on lipid-rich cottonseed. Several independently transformed lines of cotton expressing antifungal genes coding for either the chloroperoxidase (CPO-P) or the s...

  13. Next-generation transgenic mice for optogenetic analysis of neural circuits

    E-print Network

    Asrican, Brent

    Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of ...

  14. An inducible transgene reports activation of macrophages in live zebrafish larvae.

    PubMed

    Sanderson, Leslie E; Chien, An-Tzu; Astin, Jonathan W; Crosier, Kathryn E; Crosier, Philip S; Hall, Christopher J

    2015-11-01

    Macrophages are the most functionally heterogenous cells of the hematopoietic system. Given many diseases are underpinned by inappropriate macrophage activation, macrophages have emerged as a therapeutic target to treat disease. A thorough understanding of what controls macrophage activation will likely reveal new pathways that can be manipulated for therapeutic benefit. Live imaging fluorescent macrophages within transgenic zebrafish larvae has provided a valuable window to investigate macrophage behavior in vivo. Here we describe the first transgenic zebrafish line that reports macrophage activation, as evidenced by induced expression of an immunoresponsive gene 1(irg1):EGFP transgene. When combined with existing reporter lines that constitutively mark macrophages, we reveal this unique transgenic line can be used to live image macrophage activation in response to the bacterial endotoxin lipopolysaccharide and xenografted human cancer cells. We anticipate the Tg(irg1:EGFP) line will provide a valuable tool to explore macrophage activation and plasticity in the context of different disease models. PMID:26123890

  15. Post-mortem re-cloning of a transgenic red fluorescent protein dog.

    PubMed

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-12-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

  16. Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

    PubMed

    Chakraborti, Dipankar; Sarkar, Anindya; Mondal, Hossain A; Schuermann, David; Hohn, Barbara; Sarmah, Bidyut K; Das, Sampa

    2008-10-01

    A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view. PMID:18663453

  17. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

    PubMed Central

    2014-01-01

    Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G?+?C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

  18. Assessing Myocardial Disease Using T1? MRI

    PubMed Central

    Han, Yuchi; Liimatainen, Timo; Gorman, Robert C.

    2014-01-01

    There is great interest to use magnetic resonance imaging (MRI) for non-invasive assessment of myocardial disease in ischemic and non-ischemic cardiomyopathies. Recently, there has been a renewed interest to use a magnetic resonance imaging (MRI) technique utilizing spin locking radiofrequency (RF) pulses, called T1? MRI. The spin locking RF pulse creates sensitivity to some mechanisms of nuclear relaxation such as 1H exchange between water and amide, amine and hydroxyl functional groups in molecules; consequently, there is the potential to non-invasively, and without exogenous contrast agents, obtain important molecular information from diseased myocardial tissue. The purpose of this article is to review and critically examine the recent published literature in the field related to T1? MRI of myocardial disease. PMID:24688628

  19. Regeneration of transgenic tamarillo plants.

    PubMed

    Atkinson, R G; Gardner, R C

    1993-04-01

    Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the ? D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny. PMID:24197262

  20. Enhanced UV-induced skin carcinogenesis in transgenic mice overexpressing proprotein convertases.

    PubMed

    Fu, Jian; Bassi, Daniel E; Zhang, Jirong; Li, Tianyu; Cai, Kathy Q; Testa, Courtney Lyons; Nicolas, Emmanuelle; Klein-Szanto, Andres J

    2013-02-01

    The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice. PMID:23441131

  1. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

    PubMed

    Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

    2000-06-01

    Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed. PMID:10945344

  2. Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.).

    PubMed

    Zhang, S; Warkentin, D; Sun, B; Zhong, H; Sticklen, M

    1996-05-01

    Variation in the inheritance of expression among subclones for an unselected (uidA) and a selected (bar) transgene was analyzed in two individual transformation events in maize. The unselectable gene (uidA) and the selectable gene (bar), on two separate plasmids, were transferred to maize (Hi-II derivative) by particle bombardment of embryogenic calli or suspension cells. A total of 188 fertile T1 plants were obtained from one transformant (transformation event BG which integrated uidA and bar). A total of 98 fertile T1 plants were obtained from a second transformant (transformation event B which integrated bar). Through self-pollination and/or cross-pollination in the greenhouse, approximately 10 000 T2 progeny were obtained from event BG, and more than 1000 T2 progeny were obtained from event B. Segregation of transgene expression was analyzed statistically in a total of 2350 T2 progeny from 40 T1 subclones of event BG and in 217 T2 progeny from six T1 subclones from event B. Variation in the inheritance of expression among subclones for the two transgenes (uidA and bar) was observed in the two transformants. A significant difference was observed between the use of the female or male as the transgenic parent in the inheritance of expression for the two transgenes in event BG. No inheritance through the pollen was observed in two of four T1 subclones analyzed in event B. Co-expression analysis of event BG showed that both transgenes were co-expressed in 67.7% of the T2 plants which expressed at least one of the two transgenes. Of the T2 expressing plants, 30.4% expressed only bar, and 1.9% expressed only uidA. Inactivation of the unselected (uidA) and the selected (bar) transgenes was observed in individual T2 plants. PMID:24166400

  3. Suppression of intestinal immunity through silencing of TCTP by RNAi in transgenic silkworm, Bombyx mori.

    PubMed

    Hu, Cuimei; Wang, Fei; Ma, Sanyuan; Li, Xianyang; Song, Liang; Hua, Xiaoting; Xia, Qingyou

    2015-12-10

    Intestinal immune response is a front line of host defense. The host factors that participate in intestinal immunity response remain largely unknown. We recently reported that Translationally Controlled Tumor Protein (BmTCTP) was obtained by constructing a phage display cDNA library of the silkworm midgut and carrying out high throughput screening of pathogen binding molecules. To further address the function of BmTCTP in silkworm intestinal immunity, transgenic RNAi silkworms were constructed by microinjection piggBac plasmid to Dazao embryos. The antimicrobial capacity of transgenic silkworm decreased since the expression of gut antimicrobial peptide from transgenic silkworm was not sufficiently induced during oral microbial challenge. Moreover, dynamic ERK phosphorylation from transgenic silkworm midgut was disrupted. Taken together, the innate immunity of intestinal was suppressed through disruption of dynamic ERK phosphorylation after oral microbial infection as a result of RNAi-mediated knockdown of midgut TCTP in transgenic silkworm. PMID:26302749

  4. Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1

    PubMed Central

    Berquam-Vrieze, Katherine E.; Swing, Deborah A.; Tessarollo, Lino; J.Dupuy, Adam

    2013-01-01

    The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ~1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology. PMID:21898766

  5. Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations.

    PubMed

    Lonfat, Nicolas; Montavon, Thomas; Jebb, David; Tschopp, Patrick; Nguyen Huynh, Thi Hanh; Zakany, Jozsef; Duboule, Denis

    2013-07-16

    When positioned into the integrin ?-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting. PMID:23818637

  6. Pharming and transgenic plants.

    PubMed

    Liénard, David; Sourrouille, Christophe; Gomord, Véronique; Faye, Loďc

    2007-01-01

    Plant represented the essence of pharmacopoeia until the beginning of the 19th century when plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. In the last decades, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. More recently, molecular farming has rapidly pushed towards plants among the major players in recombinant protein production systems. Indeed, therapeutic protein production is safe and extremely cost-effective in plants. Unlike microbial fermentation, plants are capable of carrying out post-translational modifications and, unlike production systems based on mammalian cell cultures, plants are devoid of human infective viruses and prions. Furthermore, a large panel of strategies and new plant expression systems are currently developed to improve the plant-made pharmaceutical's yields and quality. Recent advances in the control of post-translational maturations in transgenic plants will allow them, in the near future, to perform human-like maturations on recombinant proteins and, hence, make plant expression systems suitable alternatives to animal cell factories. PMID:17875476

  7. Resistance of ?AI-1 transgenic chickpea (Cicer arietinum) and cowpea (Vigna unguiculata) dry grains to bruchid beetles (Coleoptera: Chrysomelidae).

    PubMed

    Lüthi, Christoph; Alvarez-Alfageme, Fernando; Ehlers, Jeffrey D; Higgins, Thomas J V; Romeis, Jörg

    2013-08-01

    Dry grain legume seeds possessing ?AI-1, an ?-amylase inhibitor from common bean (Phaseolus vulgaris), under the control of a cotyledon-specific promoter have been shown to be highly resistant to several important bruchid pest species. One transgenic chickpea and four cowpea lines expressing ?AI-1, their respective controls, as well as nine conventional chickpea cultivars were assessed for their resistance to the bruchids Acanthoscelides obtectus (Say), Callosobruchus chinensis L. and Callosobruchus maculatus F. All transgenic lines were highly resistant to both Callosobruchus species. A. obtectus, known to be tolerant to ?AI-1, was able to develop in all transgenic lines. While the cotyledons of all non-transgenic cultivars were highly susceptible to all bruchids, C. chinensis and C. maculatus larvae suffered from significantly increased mortality rates inside transgenic seeds. The main factor responsible for the partial resistance in the non-transgenic cultivars was deduced to reside in the seed coat. The ?AI-1 present in seeds of transgenic chickpea and cowpea lines significantly increases their resistance to two important bruchid pest species (C. chinensis and C. maculatus) essentially to immunity. To control ?AI-1 tolerant bruchid species such as A. obtectus and to avoid the development of resistance to ?AI-1, varieties carrying this transgene should be protected with additional control measures. PMID:23458831

  8. Transgenic maize plants expressing a fungal phytase gene.

    PubMed

    Chen, Rumei; Xue, Guangxing; Chen, Ping; Yao, Bin; Yang, Wenzhu; Ma, Qianli; Fan, Yunliu; Zhao, Zuoyu; Tarczynski, Mitchell C; Shi, Jinrui

    2008-08-01

    Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment. PMID:17932782

  9. Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.

    PubMed

    Graubert, T A; Hug, B A; Wesselschmidt, R; Hsieh, C L; Ryan, T M; Townes, T M; Ley, T J

    1998-06-15

    The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we designed a transgene that we expected to variegate, which consisted of a beta-globin locus control region 5' HS-2 linked in tandem to a tagged human beta-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of beta-galactosidase on red blood cell survival, by the half life of the beta-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription. PMID:9611227

  10. [Biofuels, food security and transgenic crops].

    PubMed

    Acosta, Orlando; Chaparro-Giraldo, Alejandro

    2009-01-01

    Soaring global food prices are threatening to push more poor people back below the poverty line; this will probably become aggravated by the serious challenge that increasing population and climate changes are posing for food security. There is growing evidence that human activities involving fossil fuel consumption and land use are contributing to greenhouse gas emissions and consequently changing the climate worldwide. The finite nature of fossil fuel reserves is causing concern about energy security and there is a growing interest in the use of renewable energy sources such as biofuels. There is growing concern regarding the fact that biofuels are currently produced from food crops, thereby leading to an undesirable competition for their use as food and feed. Nevertheless, biofuels can be produced from other feedstocks such as lingo-cellulose from perennial grasses, forestry and vegetable waste. Biofuel energy content should not be exceeded by that of the fossil fuel invested in its production to ensure that it is energetically sustainable; however, biofuels must also be economically competitive and environmentally acceptable. Climate change and biofuels are challenging FAO efforts aimed at eradicating hunger worldwide by the next decade. Given that current crops used in biofuel production have not been domesticated for this purpose, transgenic technology can offer an enormous contribution towards improving biofuel crops' environmental and economic performance. The present paper critically presents some relevant relationships between biofuels, food security and transgenic plant technology. PMID:19722000

  11. Impulsivity trait in the early symptomatic BACHD transgenic rat model of Huntington disease.

    PubMed

    Manfré, Giuseppe; Doyčre, Valérie; Bossi, Simon; Riess, Olaf; Nguyen, Huu Phuc; El Massioui, Nicole

    2016-02-15

    Impulsivity trait was characterized in 3-5 months old BACHD rats, a transgenic model of Huntington disease, using (1) the delay discounting task to assess cognitive/choice impulsivity, and (2) the Differential Reinforcement of Low Rate of Responding task to evaluate motor/action impulsivity. Transgenic animals showed a high level of choice impulsivity and, to a lesser extent, action impulsivity. Our results provide the first evidence that the transgenic BACHD rat (TG5 line) displays impulsivity disorder as early as 3 months old, as described in early symptomatic HD patients, thus adding to the face validity of the rat model. PMID:26592164

  12. Murine leukemia virus envelope protein in transgenic-mouse serum blocks infection in vitro.

    PubMed Central

    Nihrane, A; Fujita, K; Willey, R; Lyu, M S; Silver, J

    1996-01-01

    Transgenic mice bearing a murine retroviral envelope transgene (Fv4) have Fv4 gp70env (SU) in their serum in amounts sufficient to block infection by ecotropic virus in vitro. Fv4 Env in serum is derived largely but not exclusively from hematopoietic cells. Tail cells from Fv4 mice and cell lines transduced with the Fv4 env transgene synthesize both components of the envelope protein (gp70 SU and p15E TM) but secrete the gp70 moiety, in the absence of retroviral particles. Blocking of the ecotropic viral receptor by secreted gp70 SU may contribute to resistance to retroviral infection in these mice. PMID:8627713

  13. Comprehensive Assessment of Milk Composition in Transgenic Cloned Cattle

    PubMed Central

    Sui, Shunchao; Yu, Tian; Wang, Jianwu; Li, Ning

    2012-01-01

    The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human ?-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk. PMID:23185411

  14. Amino Acids Regulate Transgene Expression in MDCK Cells

    PubMed Central

    Torrente, Marta; Guetg, Adriano; Sass, Jörn Oliver; Arps, Lisa; Ruckstuhl, Lisa; Camargo, Simone M. R.; Verrey, François

    2014-01-01

    Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2? phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway. PMID:24797296

  15. High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

    PubMed

    Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

    2011-09-01

    To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding ?-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 ?M acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits. PMID:21516347

  16. Transgenic zebrafish using transposable elements.

    PubMed

    Clark, Karl J; Urban, Mark D; Skuster, Kimberly J; Ekker, Stephen C

    2011-01-01

    DNA transposons are effective chromosomal engineering vehicles for making transgenic zebrafish. We describe both autonomous and non-autonomous transposable elements, and we compare and contrast popular transposon systems. The Tol2 system is a robust gene transfer tool and has been selected as the primary transposon platform, facilitating the development of an array of reagents readily shared within the zebrafish community. We present common transposon and transposase vectors within the field based on the Tol2 system. We describe methods with a high success rate of generating transgenic zebrafish using Tol2 vectors, including key quality control steps during the transgenesis process. Together, these data should enable the ready generation of transgenic zebrafish for scientific inquiry. PMID:21924161

  17. How To Produce and Characterize Transgenic Plants.

    ERIC Educational Resources Information Center

    Savka, Michael A.; Wang, Shu-Yi; Wilson, Mark

    2002-01-01

    Explains the process of establishing transgenic plants which is a very important tool in plant biology and modern agriculture. Produces transgenic plants with the ability to synthesize opines. (Contains 17 references.) (YDS)

  18. Construction and long term preservation of clonal transgenic silkworms using a parthenogenetic strain.

    PubMed

    Zabelina, Valeriya; Uchino, Keiro; Mochida, Yuji; Yonemura, Naoyuki; Klymenko, Vyacheslav; Sezutsu, Hideki; Tamura, Toshiki; Sehnal, František

    2015-10-01

    For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace's medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene. PMID:26112978

  19. TRANSGENIC FISH: In Genomics and Genetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish into which foreign DNA is artificially introduced and integrated into their genome are called transgenic fish. Since the development of the first transgenic fish in 1985, techniques to produce transgenic fish have improved tremendously, resulting in the production of genetically modified (GM)...

  20. Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration

    PubMed Central

    DeLaurier, April; Eames, B. Frank; Blanco-Sánchez, Bernardo; Peng, Gang; He, Xinjun; Swartz, Mary E.; Ullmann, Bonnie; Westerfield, Monte; Kimmel, Charles B.

    2010-01-01

    Summary We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into 1 cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton. PMID:20506187

  1. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics.

    PubMed

    Howe, Douglas G; Bradford, Yvonne M; Conlin, Tom; Eagle, Anne E; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E; Westerfield, Monte

    2013-01-01

    ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos. PMID:23074187

  2. Full life-cycle assessment of gene flow consistent with fitness differences in transgenic and wild-type Japanese medaka fish (Oryzias latipes).

    PubMed

    Pennington, Kelly M; Kapuscinski, Anne R; Morton, Michael S; Cooper, Anne M; Miller, Loren M

    2010-01-01

    Transgenic fish in development for aquaculture could escape from farms and interbreed with wild relatives in the nearby environment. Predicting whether escapes would result in transgene introgression is a major challenge in assessing environmental risks of transgenic fish. Previous studies have simulated gene flow from transgenic fish using mathematical modeling of fitness traits to predict the relative selective value of transgenic genotypes. Here, we present the first study of gene flow over the full life cycle in openly-breeding populations of transgenic animals, along with measurement of fitness traits. We conducted two invasion experiments in which we released two lines of growth-enhanced transgenic fish (T67 and T400), Japanese medaka (Oryzias latipes), into populations of wild-type (W) medaka in structured mesocosms. After several generations, the frequency of transgenic fish varied across replicates in the first invasion experiment (6 months), but the frequency of transgenic fish decreased in the second experiment (19 months). We also measured selected fitness traits in transgenic and wild-type medaka because these traits could be used to predict the relative selective value of a genotype. We found that: T400 males were more fertile than W males; offspring of W females lived longer than those with transgenic mothers; and W and T67 females reached sexual maturity sooner than T400 females. In contrast with other research that reported larger transgenic males had a mating advantage, we found that W males obtained more matings with females than T males; genetic background effects may account for our differing results as we compared W and T fish derived from different strains. The decreasing frequency of transgenic fish in the second invasion experiment suggests that transgenic fish had a selective disadvantage in the experimental environment. Our finding of transgenic advantage of some fitness traits and wild-type advantage in others is consistent with our invasion experiment results. PMID:21122485

  3. Expression of human protamine P1 in sperm of transgenic mice

    SciTech Connect

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X.; Anderson, G.

    1994-12-31

    Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.

  4. Osmotin-expressing transgenic tea plants have improved stress tolerance and are of higher quality.

    PubMed

    Bhattacharya, Amita; Saini, Uksha; Joshi, Robin; Kaur, Devinder; Pal, Awadhesh Kumar; Kumar, Nitish; Gulati, Ashu; Mohanpuria, Prashant; Yadav, Sudesh Kumar; Kumar, Sanjay; Ahuja, Paramvir Singh

    2014-04-01

    Drought is a major stress that affects the yield and quality of tea, a widely consumed beverage crop grown in more than 20 countries of the world. Therefore, osmotin gene-expressing transgenic tea plants produced using earlier optimized conditions were evaluated for their tolerance of drought stress and their quality. Improved tolerance of polyethylene glycol-induced water stress and faster recovery from stress were evident in transgenic lines compared with the normal phenotype. Significant improvements in growth under in-vitro conditions were also observed. Besides enhanced reactive oxygen species-scavenging enzyme activity, the transgenic lines contained significantly higher levels of flavan-3-ols and caffeine, key compounds that govern quality and commercial yield of the beverage. The selected transgenic lines have the potential to meet the demands of the tea industry for stress-tolerant plants with higher yield and quality. These traits of the transgenic lines can be effectively maintained for generations because tea is commercially cultivated through vegetative propagation only. PMID:23982743

  5. Stable CoT-1 repeat RNA is abundant and associated with euchromatic interphase chromosomes

    PubMed Central

    Hall, Lisa L.; Carone, Dawn M.; Gomez, Alvin; Kolpa, Heather J.; Byron, Meg; Mehta, Nitish; Fackelmayer, Frank O.; Lawrence, Jeanne B.

    2014-01-01

    SUMMARY Recent studies recognize a vast diversity of non-coding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using CoT-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (“CoT-1 RNA”), including LINE-1. CoT-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis, and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The CoT-1 RNA territory resists mechanical disruption and fractionates with the non-chromatin scaffold, but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. CoT-1 RNA has several properties similar to XIST chromosomal RNA, but is excluded from chromatin condensed by XIST. These findings impact two “black boxes” of genome science: the poorly understood diversity of non-coding RNA and the unexplained abundance of repetitive elements. PMID:24581492

  6. Functional variability of the Lr34 durable resistance gene in transgenic wheat.

    PubMed

    Risk, Joanna M; Selter, Liselotte L; Krattinger, Simon G; Viccars, Libby A; Richardson, Terese M; Buesing, Gabriele; Herren, Gerhard; Lagudah, Evans S; Keller, Beat

    2012-05-01

    Breeding for durable disease resistance is challenging, yet essential to improve crops for sustainable agriculture. The wheat Lr34 gene is one of the few cloned, durable resistance genes in plants. It encodes an ATP binding cassette transporter and has been a source of resistance against biotrophic pathogens, such as leaf rust (Puccinina triticina), for over 100?years. As endogenous Lr34 confers quantitative resistance, we wanted to determine the effects of transgenic Lr34 with specific reference to how expression levels affect resistance. Transgenic Lr34 wheat lines were made in two different, susceptible genetic backgrounds. We found that the introduction of the Lr34 resistance allele was sufficient to provide comparable levels of leaf rust resistance as the endogenous Lr34 gene. As with the endogenous gene, we observed resistance in seedlings after cold treatment and in flag leaves of adult plants, as well as Lr34-associated leaf tip necrosis. The transgene-based Lr34 resistance did not involve a hypersensitive response, altered callose deposition or up-regulation of PR genes. Higher expression levels compared to endogenous Lr34 were observed in the transgenic lines both at seedling as well as adult stage and some improvement of resistance was seen in the flag leaf. Interestingly, in one genetic background the transgenic Lr34-based resistance resulted in improved seedling resistance without cold treatment. These data indicate that functional variability in Lr34-based resistance can be created using a transgenic approach. PMID:22321563

  7. Cu/Zn superoxide dismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum.

    PubMed

    Faize, Mohamed; Faize, Lydia; Petri, Cesar; Barba-Espin, Gregorio; Diaz-Vivancos, Pedro; Clemente-Moreno, María José; Koussa, Tayeb; Rifai, Lalla Aicha; Burgos, Lorenzo; Hernandez, José Antonio

    2013-05-01

    In this study we examined the role of antioxidant metabolism in in vitro shoot multiplication. We generated transgenic plum plantlets overexpressing the cytsod and cytapx genes in cytosol under the control of the constitutive promoter CaMV35S. Three transgenic lines with up-regulated sod at transcriptional levels that showed silenced cytapx expression displayed an elevated in vitro multiplication rate. By contrast, a transgenic line harboring several copies of cytapx and with elevated APX enzymatic activity did not show any improvement in plant vigor, measured as the number of axillary shoots and shoot length. All of the lines with elevated micropropagation ability exhibited intensive H2O2 accumulation, monitored by 3,3'-diaminobenzidine (DAB) staining as well as by colorimetric analysis, providing direct in vitro evidence of the role of H2O2 and antioxidant genes in in vitro shoot multiplication. PMID:23485261

  8. Functional screening of an asthma QTL in YAC transgenic mice

    SciTech Connect

    Symula, Derek J.; Frazer, Kelly A.; Ueda, Yukihiko; Denefle, Patrice; Stevens, Mary E.; Wang, Zhi-En; Locksley, Richard; Rubin, Edward M.

    1999-07-02

    While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q33, the authors characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a one megabase interva2048 chromosome 5q31 containing 23 genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180 kb region containing 5 genes, including human interleukin 4 (IL4) and interleukin 13 (IL13), which induce IgE class switching in B cells5. Further analysis of these mice and mice transgenic for only murine Il4 and Il13 demonstrated that moderate changes in murine Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled them to sift through multiple genes in the 5q3 asthma QTL without prior consideration of assumed individual gene function and identify genes that influence the QTL phenotype in vivo.

  9. Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment.

    PubMed

    Gao, Caixia; Long, Danfeng; Lenk, Ingo; Nielsen, Klaus Kristian

    2008-10-01

    Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants. PMID:18648817

  10. Native T1 Mapping of the Heart – A Pictorial Review

    PubMed Central

    Germain, Philippe; El Ghannudi, Soraya; Jeung, Mi-Young; Ohlmann, Patrick; Epailly, Eric; Roy, Catherine; Gangi, Afshin

    2014-01-01

    T1 mapping is now a clinically feasible method, providing pixel-wise quantification of the cardiac structure’s T1 values. Beyond focal lesions, well depicted by late gadolinium enhancement sequences, it has become possible to discriminate diffuse myocardial alterations, previously not assessable by noninvasive means. The strength of this method includes the high reproducibility and immediate clinical applicability, even without the use of contrast media injection (native or pre-contrast T1). The two most important determinants of native T1 augmentation are (1) edema related to tissue water increase (recent infarction or inflammation) and (2) interstitial space increase related to fibrosis (infarction scar, cardiomyopathy) or to amyloidosis. Conversely, lipid (Anderson–Fabry) or iron overload diseases are responsible for T1 reduction. In this pictorial review, the main features provided by native T1 mapping are discussed and illustrated, with a special focus on the awaited clinical purpose of this unique, promising new method. PMID:25525401

  11. Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange.

    PubMed

    Manivannan, Sathiya N; Jacobsen, Thomas L; Lyon, Peter; Selvaraj, Bhavani; Halpin, Peter; Simcox, Amanda

    2015-12-01

    Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the ?C31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ?6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied. PMID:26500255

  12. [Enhancement of tropane alkaloids production in transgenic hair roots of Atropa belladonna by overexpressing endogenous genes AbPMT and AbH6H].

    PubMed

    Long, Shi-Ping; Lu, Yan; Wang, Ya-Xiong; Yang, Chun-Xian; Lan, Xiao-Zhong; Liao, Zhi-Hua

    2013-02-01

    Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis. PMID:23672021

  13. T1R receptors mediate mammalian sweet and umami taste.

    PubMed

    Li, Xiaodong

    2009-09-01

    The T1R family of taste receptors mediates 2 taste qualities: T1R2/T1R3 for sweet taste and T1R1/T1R3 for umami taste. Functional expression in heterologous system and gene knockout studies has shown their functions as taste receptors. Structure-function relation studies on T1R2/T1R3 showed multiple ligand binding sites on both subunits. The umami taste of l-glutamate can be drastically enhanced by 5' ribonucleotides, and the synergy is a hallmark of this taste quality. On the basis of chimeric T1R receptors, site-directed mutagenesis, and molecular modeling data, we recently proposed a cooperative ligand binding model that involved the Venus flytrap domain of T1R1 in which l-glutamate binds close to the hinge region and 5' ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This novel mechanism may apply to other class C, G protein-coupled receptors. PMID:19656838

  14. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  15. Effects of Transgenic Cry1Ac + CpTI Cotton on Non-Target Mealybug Pest Ferrisia virgata and Its Predator Cryptolaemus montrouzieri

    PubMed Central

    Wu, Hongsheng; Zhang, Yuhong; Liu, Ping; Xie, Jiaqin; He, Yunyu; Deng, Congshuang; De Clercq, Patrick; Pang, Hong

    2014-01-01

    Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata. PMID:24751821

  16. Two Distinct Determinants of Ligand Specificity in T1R1/T1R3 (the Umami Taste Receptor)*

    PubMed Central

    Toda, Yasuka; Nakagita, Tomoya; Hayakawa, Takashi; Okada, Shinji; Narukawa, Masataka; Imai, Hiroo; Ishimaru, Yoshiro; Misaka, Takumi

    2013-01-01

    Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to l-Glu, whereas mouse T1R1/T1R3 responds more strongly to other l-amino acids than to l-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5?-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3. PMID:24214976

  17. Mouse neutrophils express functional umami taste receptor T1R1/T1R3

    PubMed Central

    Lee, NaHye; Jung, Young Su; Lee, Ha Young; Kang, NaNa; Park, Yoo Jung; Hwang, Jae Sam; Bahk, Young Yil; Koo, JaeHyung; Bae, Yoe-Sik

    2014-01-01

    Neutrophils play an important role in the initiation of innate immunity against infection and injury. Although many different types of G-protein coupled receptors are functionally expressed in neutrophils, no reports have demonstrated functional expression of umami taste receptor in these cells. We observed that mouse neutrophils express the umami taste receptor T1R1/T1R3 through RNA sequencing and quantitative RT-PCR analysis. Stimulation of mouse neutrophils with L-alanine or L-serine, which are ligands for the umami taste receptor, elicited not only ERK or p38 MAPK phosphorylation but also chemotactic migration. Moreover, addition of L-alanine or L-serine markedly reduced the production of several cytokines including TNF-? induced by lipopolysaccharide (LPS) through inhibition of NF-?B activity or STAT3 phosphorylation in neutrophils. Our findings demonstrate that neutrophils express the umami taste receptor, through which tastants stimulate neutrophils, resulting in chemotactic migration, and attenuation of LPS-induced inflammatory response. [BMB Reports 2014; 47(11): 649-654] PMID:25301019

  18. Targeted transgene integration overcomes variability of position effects in zebrafish

    PubMed Central

    Roberts, Jennifer Anne; Miguel-Escalada, Irene; Slovik, Katherine Joan; Walsh, Kathleen Theodora; Hadzhiev, Yavor; Sanges, Remo; Stupka, Elia; Marsh, Elizabeth Kate; Balciuniene, Jorune; Balciunas, Darius; Müller, Ferenc

    2014-01-01

    Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish. PMID:24449846

  19. TRANSGENE INTEGRATION INTO THE SAME CHROMOSOME LOCATION CAN PRODUCE ALLELES THAT EXPRESS AT A PREDICTABLE LEVEL, OR ALLELES THAT ARE DIFFERENTIALLY SILENCED

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single-copy of the transgene at the designated targ...

  20. Production of transgenic canine embryos using interspecies somatic cell nuclear transfer.

    PubMed

    Hong, So Gun; Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Kim, Geon A; Koo, Ok Jae; Jang, Goo; Lee, Byeong Chun

    2012-02-01

    Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models. PMID:21303585

  1. Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain

    PubMed Central

    2014-01-01

    Background Switchgrass is an abundant and dedicated bioenergy feedstock, however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The downregulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with Saccharomyces cerevisiae and wild-type Clostridium thermocellum (ATCC 27405) in comparison to the wild-type switchgrass. Results Here we examine the conversion and yield of the COMT transgenic and wild-type switchgrass lines with an engineered and evolved C. thermocellum (M1570) strain. The fermentation of the transgenic switchgrass by M1570 had superior conversion relative to the wild-type control switchgrass line with an increase in conversion of approximately 20% and ethanol being the primary product accounting for 90% of the total metabolites measured by HPLC analysis. Conclusions The engineered and evolved C. thermocellum M1570 was found to respond to the apparent reduced recalcitrance of the COMT switchgrass with no substrate inhibition, producing more ethanol on the transgenic feedstock than the wild-type substrate. Since ethanol was the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain, its ethanol yield on a transgenic switchgrass substrate (gram/gram (g/g) glucan liberated) is the highest produced thus far. This result indicates that the advantages of a modified feedstock can be combined with a modified consolidated bioprocessing microorganism as anticipated. PMID:24876889

  2. Herbicidal and antioxidant responses of transgenic rice overexpressing Myxococcus xanthus protoporphyrinogen oxidase.

    PubMed

    Jung, Sunyo; Back, Kyoungwhan

    2005-05-01

    We analyzed the herbicidal and antioxidant defense responses of transgenic rice plants that overexpressed the Myxococcus xanthus protoporphyrinogen oxidase gene. Leaf squares of the wild-type incubated with oxyfluorfen were characterized by necrotic leaf lesions and increases in conductivity and malonyldialdehyde levels, whereas transgenic lines M4 and M7 did not show any change with up to 100 microM oxyfluorfen. The wild-type had decreased F(v)/F(m) and produced a high level of H(2)O(2) at 18 h after foliar application of oxyfluorfen, whereas transgenic lines M4 and M7 were unaffected. In response to oxyfluorfen, violaxanthin, beta-carotene, and chlorophylls (Chls) decreased in wild-type plants, whereas antheraxanthin and zeaxanthin increased. Only a slight decline in Chls was observed in transgenic lines at 48 h after oxyfluorfen treatment. Noticeable increases of cytosolic Cu/Zn-superoxide dismutase, peroxidase isozymes 1 and 2, and catalase were observed after at 48 h of oxyfluorfen treatment in the wild-type. Non-enzymatic antioxidants appeared to respond faster to oxyfluorfen-induced photodynamic stress than did enzymatic antioxidants. Protective responses for the detoxification of active oxygen species were induced to counteract photodynamic stress in oxyfluorfen-treated, wild-type plants. However, oxyfluorfen-treated, transgenic plants suffered less oxidative stress, confirming increased herbicidal resistance resulted from dual expression of M. xanthus Protox in chloroplasts and mitochondria. PMID:15890521

  3. Myocardial T1 Mapping: Techniques and Potential Applications

    PubMed Central

    Burt, Jeremy R.; Zimmerman, Stefan L.; Kamel, Ihab R.; Halushka, Marc

    2014-01-01

    Myocardial fibrosis is a common endpoint in a variety of cardiac diseases and a major independent predictor of adverse cardiac outcomes. Short of histopathologic analysis, which is limited by sampling bias, most diagnostic modalities are limited in their depiction of myocardial fibrosis. Cardiac magnetic resonance (MR) imaging has the advantage of providing detailed soft-tissue characterization, and a variety of novel quantification methods have further improved its usefulness. Contrast material–enhanced cardiac MR imaging depends on differences in signal intensity between regions of scarring and adjacent normal myocardium. Diffuse myocardial fibrosis lacks these differences in signal intensity. Measurement of myocardial T1 times (T1 mapping) with gadolinium-enhanced inversion recovery–prepared sequences may depict diffuse myocardial fibrosis and has good correlation with ex vivo fibrosis content. T1 mapping calculates myocardial T1 relaxation times with image-based signal intensities and may be performed with standard cardiac MR imagers and radiologic workstations. Myocardium with diffuse fibrosis has greater retention of contrast material, resulting in T1 times that are shorter than those in normal myocardium. Early studies have suggested that diffuse myocardial fibrosis may be distinguished from normal myocardium with T1 mapping. Large multicenter studies are needed to define the role of T1 mapping in developing prognoses and therapeutic assessments. However, given its strengths as a noninvasive method for direct quantification of myocardial fibrosis, T1 mapping may eventually play an important role in the management of cardiac disease. © RSNA, 2014 PMID:24617686

  4. Next-generation sequencing is a robust strategy for the high-throughput detection of zygosity in transgenic maize.

    PubMed

    Fritsch, Leonie; Fischer, Rainer; Wambach, Christoph; Dudek, Max; Schillberg, Stefan; Schröper, Florian

    2015-08-01

    Simple and reliable, high-throughput techniques to detect the zygosity of transgenic events in plants are valuable for biotechnology and plant breeding companies seeking robust genotyping data for the assessment of new lines and the monitoring of breeding programs. We show that next-generation sequencing (NGS) applied to short PCR products spanning the transgene integration site provides accurate zygosity data that are more robust and reliable than those generated by PCR-based methods. The NGS reads covered the 5' border of the transgenic events (incorporating part of the transgene and the flanking genomic DNA), or the genomic sequences flanking the unfilled transgene integration site at the wild-type locus. We compared the NGS method to competitive real-time PCR with transgene-specific and wild-type-specific primer/probe pairs, one pair matching the 5' genomic flanking sequence and 5' part of the transgene and the other matching the unfilled transgene integration site. Although both NGS and real-time PCR provided useful zygosity data, the NGS technique was favorable because it needed fewer optimization steps. It also provided statistically more-reliable evidence for the presence of each allele because each product was often covered by more than 100 reads. The NGS method is also more suitable for the genotyping of large panels of plants because up to 80 million reads can be produced in one sequencing run. Our novel method is therefore ideal for the rapid and accurate genotyping of large numbers of samples. PMID:25648956

  5. Effect of the Citrus Lycopene ?-Cyclase Transgene on Carotenoid Metabolism in Transgenic Tomato Fruits

    PubMed Central

    Guo, Fei; Zhou, Wenjing; Zhang, Jiancheng; Xu, Qiang; Deng, Xiuxin

    2012-01-01

    Lycopene ?-cyclase (LYCB) is the key enzyme for the synthesis of ?-carotene, a valuable component of the human diet. In this study, tomato constitutively express Lycb-1 was engineered. The ?-carotene level of transformant increased 4.1 fold, and the total carotenoid content increased by 30% in the fruits. In the transgenic line, the downstream ?-branch metabolic fluxes were repressed during the three developmental stages while ?-carotene content increased in the ripe stage. Microarray analysis in the ripe stage revealed that the constitutive expression of Lycb-1 affected a number of pathways including the synthesis of fatty acids, flavonoids and phenylpropanoids, the degradation of limonene and pinene, starch and sucrose metabolism and photosynthesis. This study provided insight into the regulatory effect of Lycb-1 gene on plant carotenoid metabolism and fruit transcriptome. PMID:22384184

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

    PubMed

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

    2015-01-01

    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. PMID:26657016

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

    PubMed Central

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

    2015-01-01

    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. PMID:26657016

  8. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    PubMed

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peńa, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies. PMID:20204695

  9. Complementation of the pina (null) allele with the wild type Pina sequence restores a soft phenotype in transgenic wheat.

    PubMed

    Martin, J M; Meyer, F D; Smidansky, E D; Wanjugi, H; Blechl, A E; Giroux, M J

    2006-11-01

    The tightly linked puroindoline genes, Pina and Pinb, control grain texture in wheat, with wild type forms of both giving soft, and a sequence alteration affecting protein expression or function in either giving rise to hard wheat. Previous experiments have shown that addition of wild type Pina in the presence of mutated Pinb gave intermediate grain texture but addition of wild type Pinb gave soft grain. This raises questions as to whether Pina may be less functional than Pinb. Our goal here was to develop and characterize wheat lines expressing the wild type Pina-D1a sequence in hard wheat with the null mutation (Pina-D1b) for Pina. Three transgenic lines plus Bobwhite were evaluated in two environments. Grain texture, grain protein, and kernel weight were determined for the transgenic lines and Bobwhite. The three transgenic lines had soft phenotype, and none of the transgenic lines differed from Bobwhite for grain protein or kernel weight. The soft phenotype was accompanied by increases in Pina transcript accumulation. Total Triton X-114 extractable PINA and PINB increased from 2.5 to 5.5 times those from a soft wheat reference sample, and friabilin, PINA and PINB bound to starch, increased from 3.8 to 7.8 times those of the soft wheat reference. Bobwhite showed no starch bound PINA, but transgenic lines had levels from 5.3 to 13.7 times those of the soft wheat reference sample. Starch bound PINB in transgenic lines also increased from 0.9 to 2.5 times that for the soft wheat reference sample. The transgenic expression of wild type Pina sequence in the Pina null genotype gave soft grain with the characteristics of soft wheat including increased starch bound friabilin. The results support the hypothesis that both wild type Pin genes need to be present for friabilin formation and soft grain. PMID:16988815

  10. Transgene-host cell interactions mediate significant influences on the production, stability, and function of recombinant canine FVIII

    PubMed Central

    Crawford, Bredon; Ozelo, Margareth C; Ogiwara, Kenichi; Ahlin, James; Albanez, Silvia; Hegadorn, Carol; Harpell, Lori; Hough, Christine; Lillicrap, David

    2015-01-01

    Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not well understood and are of interest for next-generation protein and gene therapies. To investigate the effects of transgene design across different host cells, B-domain-deleted (BDD) and modified codon-optimized (CO-N6) transgenes were inserted via lentiviral delivery into cBOECs, HEK293T, and MDCK cells. The CO-N6 cFVIII transgene produced threefold more protein per transgene in HEK293T cells, and sixfold more protein in the two canine cell lines. However, pharmacokinetic analysis in hemophilia A dogs demonstrated that cFVIII produced from cBOECs transduced with the CO-N6 transgene had significantly reduced in vivo recovery. Furthermore, this product showed reduced in vitro stability and activity on thrombin activation versus the BDD product. This trend was reversed in HEK293T lines. Overall, our results demonstrate the need for an integrated approach that not only assesses protein expression levels but also considers the influence that host-cells have on preserving the molecular and biochemical properties of the naturally occurring FVIII. PMID:26636112

  11. Transgenic songbirds with suppressed or enhanced activity of CREB transcription factor.

    PubMed

    Abe, Kentaro; Matsui, Sumiko; Watanabe, Dai

    2015-06-16

    Songbirds postnatally develop their skill to utter and to perceive a vocal signal for communication. How genetic and environmental influences act in concert to regulate the development of such skill is not fully understood. Here, we report the phenotype of transgenic songbirds with altered intrinsic activity of cAMP response element-binding protein (CREB) transcription factor. By viral vector-mediated modification of genomic DNA, we established germ line-transmitted lines of zebra finches, which exhibited enhanced or suppressed activity of CREB. Although intrinsically acquired vocalizations or their hearing ability were not affected, the transgenic birds showed reduced vocal learning quality of their own songs and impaired audio-memory formation against conspecific songs. These results thus demonstrate that appropriate activity of CREB is necessary for the postnatal acquisition of learned behavior in songbirds, and the CREB transgenic birds offer a unique opportunity to separately manipulate both genetic and environmental factors that impinge on the postnatal song learning. PMID:26048905

  12. Targeted Expression of Cre Recombinase Provokes Placental-Specific DNA Recombination in Transgenic Mice

    PubMed Central

    Zhou, Cissy Chenyi; Chang, Jiang; Mi, Tiejuan; Abbasi, Shahrzad; Gu, Dongmin; Huang, Le; Zhang, WenZheng; Kellems, Rodney E.; Schwartz, Robert J.; Xia, Yang

    2012-01-01

    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 ? (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. PMID:22363401

  13. Iron homeostasis and fire blight susceptibility in transgenic pear plants overexpressing a pea ferritin gene.

    PubMed

    Djennane, Samia; Cesbron, Colette; Sourice, Sophie; Cournol, Raphael; Dupuis, Fabrice; Eychenne, Magali; Loridon, Karine; Chevreau, Elisabeth

    2011-05-01

    The bacterial pathogen Erwinia amylovora causes the devastating disease known as fire blight in some rosaceous plants including apple and pear. One of the pathogenicity factors affecting fire blight development is the production of a siderophore, desferrioxamine, which overcomes the limiting conditions in plant tissues and also protects bacteria against active oxygen species. In this paper we examine the effect of an iron chelator protein encoded by the pea ferritin gene on the fire blight susceptibility of pear (Pyrus communis). Transgenic pear clones expressing this gene controlled either by the constitutive promoter CaMV 35S or by the inducible promoter sgd24 promoter were produced. The transgenic clones produced were analysed by Q-RT-PCR to determine the level of expression of the pea transgene. A pathogen-inducible pattern of expression of the pea transgene was observed in sgd24-promoter transformants. Adaptation to iron deficiency in vitro was tested in some transgenic clones and different iron metabolism parameters were measured. No strong effect on iron and chlorophyll content, root reductase activity and fire blight susceptibility was detected in the transgenic lines tested. No transformants showed a significant reduction in susceptibility to fire blight in greenhouse conditions when inoculated with E. amylovora. PMID:21421420

  14. Impact of the ahas transgene for herbicides resistance on biological nitrogen fixation and yield of soybean.

    PubMed

    Hungria, Mariangela; Nakatani, André Shigueyoshi; Souza, Rosinei Aparecida; Sei, Fernando Bonafé; de Oliveira Chueire, Ligia Maria; Arias, Carlos Arrabal

    2015-02-01

    Studies on the effects of transgenes in soybean [Glycine max (L.) Merr.] and the associated use of specific herbicides on biological nitrogen fixation (BNF) are still few, although it is important to ensure minimal impacts on benefits provided by the root-nodule symbiosis. Cultivance CV127 transgenic soybean is a cultivar containing the ahas gene, which confers resistance to herbicides of the imidazolinone group. The aim of this study was to assess the effects of the ahas transgene and of imidazolinone herbicide on BNF parameters and soybean yield. A large-scale set of field experiments was conducted, for three cropping seasons, at nine sites in Brazil, with a total of 20 trials. The experiment was designed as a completely randomized block with four replicates and the following treatments: (T1) near isogenic transgenic soybean (Cultivance CV127) + herbicide of the imidazolinone group (imazapyr); (T2) near isogenic transgenic soybean + conventional herbicides; and (T3) parental conventional soybean (Conquista) + conventional herbicides; in addition, two commercial cultivars were included, Monsoy 8001 (M-SOY 8001) (T4), and Coodetec 217 (CD 217) (T5). At the R2 growth stage, plants were collected and BNF parameters evaluated. In general, there were no effects on BNF parameters due to the transgenic trait or associated with the specific herbicide. Similarly, at the final harvest, no grain-yield effects were detected related to the ahas gene or to the specific herbicide. However, clear effects on BNF and grain yield were attributed to location and cropping season. PMID:25201300

  15. Systematic T1 improvement for hyperpolarized 129xenon

    NASA Astrophysics Data System (ADS)

    Repetto, Maricel; Babcock, Earl; Blümler, Peter; Heil, Werner; Karpuk, Sergei; Tullney, Kathlynne

    2015-03-01

    The spin-lattice relaxation time T1 of hyperpolarized (HP)-129Xe was improved at typical storage conditions (i.e. low and homogeneous magnetic fields). Very long wall relaxation times T1wall of about 18 h were observed in uncoated, spherical GE180 glass cells of ? = 10 cm which were free of rubidium and not permanently sealed but attached to a standard glass stopcock. An "aging" process of the wall relaxation was identified by repeating measurements on the same cell. This effect could be easily removed by repeating the initial cleaning procedure. In this way, a constant wall relaxation was ensured. The Xe nuclear spin-relaxation rate 1 / T1Xe -Xe due to van der Waals molecules was investigated too, by admixing three different buffer gases (N2, SF6 and CO2). Especially CO2 exhibited an unexpected high efficiency (r) in shortening the lifetime of the Xe-Xe dimers and hence prolonging the total T1 relaxation even further. These measurements also yielded an improved accuracy for the van der Waals relaxation for pure Xe (with 85% 129Xe) of T1Xe -Xe = (4.6 ± 0.1)h . Repeating the measurements with HP 129Xe in natural abundance in mixtures with SF6, a strong dependence of T1Xe -Xe and r on the isotopic enrichment was observed, uncovering a shorter T1Xe -Xe relaxation for the 129Xe in natural composition as compared to the 85% isotopically enriched gas.

  16. Transgenic Papaya: Development, Release, Impact, and Challenges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the technology for developing virus-resistant transgenic plants through the use of the coat protein of a virus was unveiled twenty years ago, it is surprising to note that only a three virus-resistant plants (squash, potato, and papaya) have been commercialized in the U.S. The transgenic p...

  17. Transgenic Biofuel Feedstocks and Strategies for Biocontainment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are several reasons to believe that transgenic plant feedstocks will be required to realize the full economic and environmental benefits of cellulosic and other biofuels. Much of the commercialization potential for the use of transgenic plant cellulosic feedstocks may be impacted by regulatio...

  18. Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

    PubMed Central

    Lönnerberg, P; Lendahl, U; Funakoshi, H; Arhlund-Richter, L; Persson, H; Ibáńez, C F

    1995-01-01

    Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons. Images Fig. 1 Fig. 2 PMID:7732028

  19. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

    PubMed

    Ordovás, Laura; Boon, Ruben; Pistoni, Mariaelena; Chen, Yemiao; Wolfs, Esther; Guo, Wenting; Sambathkumar, Rangarajan; Bobis-Wozowicz, Sylwia; Helsen, Nicky; Vanhove, Jolien; Berckmans, Pieter; Cai, Qing; Vanuytsel, Kim; Eggermont, Kristel; Vanslembrouck, Veerle; Schmidt, Béla Z; Raitano, Susanna; Van Den Bosch, Ludo; Nahmias, Yaakov; Cathomen, Toni; Struys, Tom; Verfaillie, Catherine M

    2015-11-10

    Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes. PMID:26455413

  20. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance.

    PubMed

    Li, Li; Steffens, John C

    2002-06-01

    Polyphenol oxidases (PPOs; EC 1.10.3.2 or EC 1.14.18.1) catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms and assumed to be involved in plant defense against pests and pathogens. In order to investigate the role of PPO in plant disease resistance, we made transgenic tomato ( Lycopersicon esculentum Mill. cv. Money Maker) plants that overexpressed a potato ( Solanum tuberosum L.) PPO cDNA under control of the cauliflower mosaic virus 35S promoter. The transgenic plants expressed up to 30-fold increases in PPO transcripts and 5- to 10-fold increases in PPO activity and immunodetectable PPO. As expected, these PPO-overexpressing transgenic plants oxidized the endogenous phenolic substrate pool at a higher rate than control plants. Three independent transgenic lines were selected to assess their interaction with the bacterial pathogen Pseudomonas syringae pv. tomato. The PPO-overexpressing tomato plants exhibited a great increase in resistance to P. syringae. Compared with control plants, these transgenic lines showed less severity of disease symptoms, with over 15-fold fewer lesions, and strong inhibition of bacterial growth, with over 100-fold reduction of bacterial population in the infected leaves. These results demonstrate the importance of PPO-mediated phenolic oxidation in restricting plant disease development. PMID:12029473

  1. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis.

    PubMed

    Chen, Xiuping; Wang, Jiamei; Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 ?g L-1 or 10 ?g L-1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. PMID:26695426

  2. Effects of Transgenic cry1Ca Rice on the Development of Xenopus laevis

    PubMed Central

    Zhu, Haojun; Li, Yunhe; Ding, Jiatong; Peng, Yufa

    2015-01-01

    In fields of genetically modified, insect-resistant rice expressing Bacillus thuringiensis (Bt) proteins, frogs are exposed to Bt Cry proteins by consuming both target and non-target insects, and through their highly permeable skin. In the present study, we assessed the potential risk posed by transgenic cry1Ca rice (T1C-19) on the development of a frog species by adding purified Cry1Ca protein or T1C-19 rice straw into the rearing water of Xenopus laevis tadpoles, and by feeding X. laevis froglets diets containing rice grains of T1C-19 or its non-transformed counterpart MH63. Our results showed that there were no significant differences among groups receiving 100 ?g L–1 or 10 ?g L–1 Cry1Ca and the blank control in terms of time to completed metamorphosis, survival rate, body weight, body length, organ weight and liver enzyme activity after being exposed to the Cry1Ca (P > 0.05). Although some detection indices in the rice straw groups were significantly different from those of the blank control group (P < 0.05), there was no significant difference between the T1C-19 and MH63 rice straw groups. Moreover, there were no significant differences in the mortality rate, body weight, daily weight gain, liver and fat body weight of the froglets between the T1C-19 and MH63 dietary groups after 90 days, and there were no abnormal pathological changes in the stomach, intestines, livers, spleens and gonads. Thus, we conclude that the planting of transgenic cry1Ca rice will not adversely affect frog development. PMID:26695426

  3. Transgenic Field Trials for FHB Resistance and Related Research in Wheat and Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic wheat and barley lines expressing genes with the potential to reduce FHB and DON have been tested in field trials in Minnesota since 1997 and in North Dakota since 2001 (barley only). Replicated trials are planted, grown, and harvested to meet containment regulations of the Animal and Pla...

  4. Genomic evaluation of oxalate-degrading transgenic soybean in response to Sclerotinia sclerotiorum infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oxalate oxidases catalyze the degradation of oxalic acid (OA). Highly resistant transgenic soybean carrying an oxalate oxidase (OxO) gene and its susceptible parent soybean line, AC Colibri, were tested for genome-wide gene expression in response to the necrotrophic, OA producing pathogen Sclerotini...

  5. Evaluations of transgenic potatoes for resistance to potato tuberworm in the laboratory and field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potato variety ‘Spunta’ was previously transformed to constitutively express the cry1Ia1 gene from Bacillus thuringiensis from which three transgenic lines (Spunta G2, Spunta G3 and Spunta 6a3) were chosen to evaluate for resistance to potato tuberworm (Phthorimaea operculella Zeller). Because ...

  6. Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system

    E-print Network

    Amaya, Enrique

    Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system Katharine O phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show

  7. In vitro culture may be the major contributing factor for transgenic versus nontransgenic proteomic plant differences.

    PubMed

    Fonseca, Cátia; Planchon, Sébastien; Serra, Tânia; Chander, Subhash; Saibo, Nelson J M; Renaut, Jenny; Oliveira, M Margarida; Batista, Rita

    2015-01-01

    Identification of differences between genetically modified plants and their original counterparts plays a central role in risk assessment strategy. Our main goal was to better understand the relevance of transgene presence, genetic, and epigenetic changes induced by transgene insertion, and in vitro culture in putative unintended differences between a transgenic and its comparator. Thus, we have used multiplex fluorescence 2DE coupled with MS to characterize the proteome of three different rice lines (Oryza sativa L. ssp. japonica cv. Nipponbare): a control conventional line (C), an Agrobacterium-transformed transgenic line (Ta) and a negative segregant (NSb). We observed that Ta and NSb appeared identical (with only one spot differentially abundant--fold difference ? 1.5), contrasting with the control (49 spots with fold difference ? 1.5, in both Ta and NSb vs. control). Given that in vitro culture was the only event in common between Ta and NSb, we hypothesize that in vitro culture stress was the most relevant condition contributing for the observed proteomic differences. MS protein identification support our hypothesis, indicating that Ta and NSb lines adjusted their metabolic pathways and altered the abundance of several stress related proteins in order to cope with in vitro culture. PMID:25283639

  8. IMPACT OF TRANSGENIC BT-CORN ON MICROBIAL COMMUNITY COMPOSITION IN THREE SOIL TYPES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of Bt toxins expressed by transgenic corn on soil microbial communities were investigated in a growth chamber experiment. Two lines of Bt-corn, expressing different Cry proteins, and their non-Bt isolines, were grown in pots containing three different agricultural soils. The soils rang...

  9. MAIN-BELT COMET P/2012 T1 (PANSTARRS)

    E-print Network

    Hsieh, Henry H.

    We present initial results from observations and numerical analyses aimed at characterizing the main-belt comet P/2012 T1 (PANSTARRS). Optical monitoring observations were made between 2012 October and 2013 February using ...

  10. Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

    PubMed

    Park, Sung-Chul; Kim, Yun-Hee; Kim, Sun Ha; Jeong, Yu Jeong; Kim, Cha Young; Lee, Joon Seol; Bae, Ji-Yeong; Ahn, Mi-Jeong; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2015-04-01

    The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity. PMID:25220246

  11. Glyphostate-drift but not herbivory alters the rate of transgene flow from single and stacked trait transgenic canola (Brassica napus L.) to non-transgenic B. napus and B. rapa

    EPA Science Inventory

    While transgenic plants can offer agricultural benefits, the escape of transgenes out of crop fields is a major environmental concern. Escape of transgenic herbicide resistance has occurred between transgenic Brassica napus (canola) and weedy species in numerous locations. In t...

  12. Efficacy and mechanism of action of Deguelin in suppressing metastasis of 4T1 cells.

    PubMed

    Mehta, Rajeshwari R; Katta, Harshadadevi; Kalra, Amit; Patel, Rutulkumar; Gupta, Akash; Alimirah, Fatouma; Murillo, Genoveva; Peng, Xinjian; Unni, Aditya; Muzzio, Miguel; Mehta, Rajendra G

    2013-10-01

    Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients. PMID:23645347

  13. Efficacy of Vip3A and Cry1Ab transgenic traits in cotton against various lepidopteran pests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    From 2004 through 2005, plots of experimental transgenic cotton lines containing the vegetative insecticidal protein, Vip3A; '-endotoxin, Cry1Ab; and both Vip3A and Cry1Ab were evaluated for efficacy against certain lepidopteran pests. Results showed that the cotton line containing Vip3A was more e...

  14. Markedly Increased Susceptibility to Natural Sheep Scrapie of Transgenic Mice Expressing Ovine PrP

    PubMed Central

    Vilotte, Jean-Luc; Soulier, Solange; Essalmani, Rachid; Stinnakre, Marie-George; Vaiman, Daniel; Lepourry, Laurence; Da Silva, Jose Costa; Besnard, Nathalie; Dawson, Mike; Buschmann, Anne; Groschup, Martin; Petit, Stephanie; Madelaine, Marie-Francoise; Rakatobe, Sabine; Le Dur, Annick; Vilette, Didier; Laude, Hubert

    2001-01-01

    The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrPVRQ-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrPVRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie. PMID:11390599

  15. In vivo quantification of hyperoxic arterial blood water T1.

    PubMed

    Siero, Jeroen C W; Strother, Megan K; Faraco, Carlos C; Hoogduin, Hans; Hendrikse, Jeroen; Donahue, Manus J

    2015-11-01

    Normocapnic hyperoxic and hypercapnic hyperoxic gas challenges are increasingly being used in cerebrovascular reactivity (CVR) and calibrated functional MRI experiments. The longitudinal arterial blood water relaxation time (T1a ) change with hyperoxia will influence signal quantification through mechanisms relating to elevated partial pressure of plasma-dissolved O2 (pO2 ) and increased oxygen bound to hemoglobin in arteries (Ya ) and veins (Yv ). The dependence of T1a on Ya and Yv has been elegantly characterized ex vivo; however, the combined influence of pO2 , Ya and Yv on T1a in vivo under normal ventilation has not been reported. Here, T1a is calculated during hyperoxia in vivo by a heuristic approach that evaluates T1 -dependent arterial spin labeling (ASL) signal changes to varying gas stimuli. Healthy volunteers (n?=?14; age, 31.5?±?7.2?years) were scanned using pseudo-continuous ASL in combination with room air (RA; 21% O2 /79% N2 ), hypercapnic normoxic (HN; 5% CO2 /21% O2 /74% N2 ) and hypercapnic hyperoxic (HH; 5% CO2 /95% O2 ) gas administration. HH T1a was calculated by requiring that the HN and HH cerebral blood flow (CBF) change be identical. The HH protocol was then repeated in patients (n?=?10; age, 61.4?±?13.3?years) with intracranial stenosis to assess whether an HH T1a decrease prohibited ASL from being performed in subjects with known delayed blood arrival times. Arterial blood T1a decreased from 1.65?s at baseline to 1.49?±?0.07?s during HH. In patients, CBF values in the affected flow territory for the HH condition were increased relative to baseline CBF values and were within the physiological range (RA CBF?=?36.6?±?8.2?mL/100?g/min; HH CBF?=?45.2?±?13.9?mL/100?g/min). It can be concluded that hyperoxic (95% O2 ) 3-T arterial blood T1aHH ?=?1.49?±?0.07?s relative to a normoxic T1a of 1.65?s. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26419505

  16. Broad-Spectrum Transgenic Resistance against Distinct Tospovirus Species at the Genus Level

    PubMed Central

    Raja, Joseph A. J.; Yang, Ching-Fu; Chien, Wan-Chu; Lin, Chen-Hsuan; Liu, Fang-Lin; Wu, Hui-Wen; Yeh, Shyi-Dong

    2014-01-01

    Thrips-borne tospoviruses cause severe damage to crops worldwide. In this investigation, tobacco lines transgenic for individual WLm constructs containing the conserved motifs of the L RNA-encoded RNA-dependent RNA polymerase (L) gene of Watermelon silver mottle virus (WSMoV) were generated by Agrobacterium-mediated transformation. The WLm constructs included: (i) translatable WLm in a sense orientation; (ii) untranslatable WLmt with two stop codons; (iii) untranslatable WLmts with stop codons and a frame-shift; (iv) untranslatable antisense WLmA; and (v) WLmhp with an untranslatable inverted repeat of WLm containing the tospoviral S RNA 3?-terminal consensus sequence (5?-ATTGCTCT-3?) and an NcoI site as a linker to generate a double-stranded hairpin transcript. A total of 46.7–70.0% transgenic tobacco lines derived from individual constructs showed resistance to the homologous WSMoV; 35.7–100% plants of these different WSMoV-resistant lines exhibited broad-spectrum resistance against four other serologically unrelated tospoviruses Tomato spotted wilt virus, Groundnut yellow spot virus, Impatiens necrotic spot virus and Groundnut chlorotic fan-spot virus. The selected transgenic tobacco lines also exhibited broad-spectrum resistance against five additional tospoviruses from WSMoV and Iris yellow spot virus clades, but not against RNA viruses from other genera. Northern analyses indicated that the broad-spectrum resistance is mediated by RNA silencing. To validate the L conserved region resistance in vegetable crops, the constructs were also used to generate transgenic tomato lines, which also showed effective resistance against WSMoV and other tospoviruses. Thus, our approach of using the conserved motifs of tospoviral L gene as a transgene generates broad-spectrum resistance against tospoviruses at the genus level. PMID:24811071

  17. [The zig-zagging legislative policies of the European Union in relation to transgenic products].

    PubMed

    Martín Uranga, Amelia

    2003-01-01

    The author analyses the E.U. legislative policies related to transgenic products from the 80s until nowadays, she stops in Directive 2001/18/Ce and its conversion into national law. She explains how the regulatory frame is after the recent approval of the newest rules about tracking and labelling genetically modified organisms (OMG). She also studies communitary jurisprudence about this subject according to the legislation, concretely sentence 9 September 2003 in the affair C-236/01 about commercialisation of two lines of transgenic corn. Finally she points out how legislative policies have influenced the creation and development of biotechnological enterprises. PMID:15032103

  18. Generation of red fluorescent protein transgenic dogs.

    PubMed

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. PMID:19358155

  19. [Transgenic animals generation. Regulation of gene expression].

    PubMed

    Cavagnari, Brian M

    2010-10-01

    Transgenic animals are organisms with a foreign gene incorporated into their genome, and their development was one of the most important advances in biotechnology. Although knockout animals have already been developed twenty years ago and being transgenic mice generation quite routine, many health care providers still do not understand their impact on biomedical research and their capability of improving human health. In this article, I present the most relevant approaches to generate transgenic animals and discuss how specific genes can be activated or inactivated in a living animal. PMID:21132233

  20. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D. (Madison, WI); Walker, Joseph M. (Madison, WI)

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  1. Systematic T1 improvement for hyperpolarized 129xenon.

    PubMed

    Repetto, Maricel; Babcock, Earl; Blümler, Peter; Heil, Werner; Karpuk, Sergei; Tullney, Kathlynne

    2015-03-01

    The spin-lattice relaxation time T1 of hyperpolarized (HP)-(129)Xe was improved at typical storage conditions (i.e. low and homogeneous magnetic fields). Very long wall relaxation times T(1)(wall) of about 18 h were observed in uncoated, spherical GE180 glass cells of ?=10 cm which were free of rubidium and not permanently sealed but attached to a standard glass stopcock. An "aging" process of the wall relaxation was identified by repeating measurements on the same cell. This effect could be easily removed by repeating the initial cleaning procedure. In this way, a constant wall relaxation was ensured. The Xe nuclear spin-relaxation rate 1/T1(Xe-Xe) due to van der Waals molecules was investigated too, by admixing three different buffer gases (N(2), SF(6) and CO(2)). Especially CO(2) exhibited an unexpected high efficiency (r) in shortening the lifetime of the Xe-Xe dimers and hence prolonging the total T1 relaxation even further. These measurements also yielded an improved accuracy for the van der Waals relaxation for pure Xe (with 85% (129)Xe) of T(1)(Xe-Xe)=(4.6±0.1)h. Repeating the measurements with HP (129)Xe in natural abundance in mixtures with SF6, a strong dependence of T(1)(Xe-Xe) and r on the isotopic enrichment was observed, uncovering a shorter T(1)(Xe-Xe) relaxation for the (129)Xe in natural composition as compared to the 85% isotopically enriched gas. PMID:25702572

  2. Hyperpolarized (129)Xe T (1) in oxygenated and deoxygenated blood

    NASA Technical Reports Server (NTRS)

    Albert, M. S.; Balamore, D.; Kacher, D. F.; Venkatesh, A. K.; Jolesz, F. A.

    2000-01-01

    The viability of the new technique of hyperpolarized (129)Xe MRI (HypX-MRI) for imaging organs other than the lungs depends on whether the spin-lattice relaxation time, T(1), of (129)Xe is sufficiently long in the blood. In previous experiments by the authors, the T(1) was found to be strongly dependent upon the oxygenation of the blood, with T(1) increasing from about 3 s in deoxygenated samples to about 10 s in oxygenated samples. Contrarily, Tseng et al. (J. Magn. Reson. 1997; 126: 79-86) reported extremely long T(1) values deduced from an indirect experiment in which hyperpolarized (129)Xe was used to create a 'blood-foam'. They found that oxygenation decreased T(1). Pivotal to their experiment is the continual and rapid exchange of hyperpolarized (129)Xe between the gas phase (within blood-foam bubbles) and the dissolved phase (in the skin of the bubbles); this necessitated a complicated analysis to extract the T(1) of (129)Xe in blood. In the present study, the experimental design minimizes gas exchange after the initial bolus of hyperpolarized (129)Xe has been bubbled through the sample. This study confirms that oxygenation increases the T(1) of (129)Xe in blood, from about 4 s in freshly drawn venous blood, to about 13 s in blood oxygenated to arterial levels, and also shifts the red blood cell resonance to higher frequency. Copyright 2000 John Wiley & Sons, Ltd. Abbreviations used BOLD blood oxygen level dependent NOE nuclear overhouses effect PO(2) oxygen partial pressure RBC red blood cells RF radio frequency SNR signal-to-noise ratio.

  3. Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties.

    PubMed

    Tamura, Ken-Ichi; Sanada, Yasuharu; Tase, Kazuhiro; Kawakami, Akira; Yoshida, Midori; Yamada, Toshihiko

    2014-04-01

    Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3-40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants. PMID:24385092

  4. Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain

    SciTech Connect

    Yee, Kelsey L; Rodriguez Jr, Miguel; Thompson, Olivia A; Fu, Chunxiang; Wang, Zeng-Yu; Davison, Brian H; Mielenz, Jonathan R

    2014-01-01

    Background: Switchgrass is an abundant and dedicated bioenergy feedstock however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The down-regulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with S. cerevisiae and C. thermocellum (ATCC 27405) in comparison to the wild-type switchgrass. Results: Here we examine the fermentation potential of the COMT transgenic switchgrass and its wild-type line, with an engineered and evolved Clostridium thermocellum (M1570) strain. The fermentation of the transgenic switchgrass had superior conversion relative to the control line with an increase of 20% and ethanol was the primary metabolite accounting for 90% of the total metabolites measured by HPLC. Conclusions: The down-regulation of the COMT gene in switchgrass reduced recalcitrance and improved microbial bioconversion yield. Moreover, these results showed ethanol as the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain grown on a transgenic switchgrass.

  5. Protocol: Whole Brain Mouse T1 Map, Page 1 of 1 Author: R.Bussell 8/9/2013 Protocol: Whole Brain Mouse T1 Map

    E-print Network

    California at San Diego, University of

    Protocol: Whole Brain Mouse T1 Map, Page 1 of 1 Author: R.Bussell 8/9/2013 Protocol: Whole Brain Mouse T1 Map Purpose Measurement of whole brain T1 maps in a mouse. Protocol Directory: "C_ MouseWholeBrainT1Map" Type Protocol Name In-plane resolution, slice thickness Slices Scan time Localizer 1_TripilotMouse

  6. Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1

    PubMed Central

    Anderson, Marilyn A.

    2014-01-01

    The plant defensin NaD1, from Nicotiana alata, has potent antifungal activity against a range of filamentous fungi including the two important cotton pathogens, Fusarium oxysporum f. sp. vasinfectum (Fov) and Verticillium dahliae. Transgenic cotton plants expressing NaD1 were produced and plants from three events were selected for further characterization. Homozygous plants were assessed in greenhouse bioassays for resistance to Fov. One line (D1) was selected for field trial testing over three growing seasons in soils naturally infested with Fov and over two seasons in soils naturally infested with V. dahliae. In the field trials with Fov-infested soil, line D1 had 2–3-times the survival rate, a higher tolerance to Fov (higher disease rank), and a 2–4-fold increase in lint yield compared to the non-transgenic Coker control. When transgenic line D1 was planted in V. dahliae-infested soil, plants had a higher tolerance to Verticillium wilt and up to a 2-fold increase in lint yield compared to the non-transgenic Coker control. Line D1 did not exhibit any detrimental agronomic features compared to the parent Coker control when plants were grown in non-diseased soil. This study demonstrated that the expression of NaD1 in transgenic cotton plants can provide substantial resistance to two economically important fungal pathogens. PMID:24502957

  7. GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat.

    PubMed

    Wang, Aiyun; Wei, Xuening; Rong, Wei; Dang, Liang; Du, Li-Pu; Qi, Lin; Xu, Hui-Jun; Shao, Yanjun; Zhang, Zengyan

    2015-05-01

    Take-all (caused by the fungal pathogen Gaeumannomyces graminis var. tritici, Ggt) and common root rot (caused by Bipolaris sorokiniana) are devastating root diseases of wheat (Triticum aestivum L.). Development of resistant wheat cultivars has been a challenge since no resistant wheat accession is available. GmPGIP3, one member of polygalacturonase-inhibiting protein (PGIP) family in soybean (Glycine max), exhibited inhibition activity against fungal endopolygalacturonases (PGs) in vitro. In this study, the GmPGIP3 transgenic wheat plants were generated and used to assess the effectiveness of GmPGIP3 in protecting wheat from the infection of Ggt and B. sorokiniana. Four independent transgenic lines were identified by genomic PCR, Southern blot, and reverse transcription PCR (RT-PCR). The introduced GmPGIP3 was integrated into the genomes of these transgenic lines and could be expressed. The expressing GmPGIP3 protein in these transgenic wheat lines could inhibit the PGs produced by Ggt and B. sorokiniana. The disease response assessments postinoculation showed that the GmPGIP3-expressing transgenic wheat lines displayed significantly enhanced resistance to both take-all and common root rot diseases caused by the infection of Ggt and B. sorokiniana. These data suggested that GmPGIP3 is an attractive gene resource in improving resistance to both take-all and common root rot diseases in wheat. PMID:25487419

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

    PubMed

    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. PMID:17947256

  9. The wheat aquaporin gene TaAQP7 confers tolerance to cold stress in transgenic tobacco.

    PubMed

    Huang, Chao; Zhou, Shiyi; Hu, Wei; Deng, Xiaomin; Wei, Shuya; Yang, Guangxiao; He, Guangyuan

    2014-01-01

    Aquaporin proteins (AQPs) have been shown to be involved in abiotic stress responses. However, the precise role of AQPs, especially in response to cold stress, is not understood in wheat (Triticum aestivum). In the present study, quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed that TaAQP7 expression increased in leaves, but decreased in roots after cold treatment. Expression of TaAQP7 in tobacco plants resulted in increased root elongation and better growth compared with wild-type (WT) plants under cold stress. Moreover, after cold treatment, the transgenic tobacco lines exhibited higher chlorophyll contents, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than WT plants. Thus, expression of TaAQP7 enhanced cold stress tolerance in transgenic tobacco. Taken together, our results suggest that TaAQP7 confers cold stress tolerance by relieving membrane damage in the transgenic plants. PMID:24873035

  10. Transgenic mouse model for neurocristopathy: Schwannomas and facial bone tumors.

    PubMed

    Jensen, N A; Rodriguez, M L; Garvey, J S; Miller, C A; Hood, L

    1993-04-15

    We have characterized a strain of double transgenic mice with simian virus 40 large tumor antigen and prokaryotic lacZ under the control of the myelin basic protein promoter that develops spindle-cell sarcomas and osteogenic sarcomas at 5-7 months of age. Although poorly differentiated, the spindle-cell sarcomas were characterized as malignant Schwannomas based on their neural association, the presence of basal lamina, and expression of Schwann cell-specific genes. The osteogenic sarcomas were often multiple and appeared predominantly in the facial bones, less frequently in the ribs and vertebral column, and only rarely in the appendicular skeleton. Benign osteoblastic lesions were often observed adjacent to these sarcomas. Both the osteoblastic cells in the facial skeleton and Schwann cells are regarded as neural crest derivatives. The biological properties and anatomical location of these tumors suggest that they may share a common origin from the neural crest or its derivatives. R.P. Bolande [Hum. Pathol. (1974) 5, 409-429] introduced the term neurocristopathy as a unifying concept to describe such lesions arising from the neural crest or its derivatives. Cell lines established from both bone and Schwann cell tumors arising in these transgenic mice express simian virus 40 large tumor antigen mRNA as well as functional large tumor antigen. Such cell lines are potentially valuable in the search for markers that identify mammalian neural crest derivatives. PMID:8386366

  11. Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene.

    PubMed

    Chen, Ming; Sun, Liying; Wu, Hongya; Chen, Jiong; Ma, Youzhi; Zhang, Xiaoxiang; Du, Lipu; Cheng, Shunhe; Zhang, Boqiao; Ye, Xingguo; Pang, Junlan; Zhang, Xinmei; Li, Liancheng; Andika, Ida B; Chen, Jianping; Xu, Huijun

    2014-05-01

    Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat-growing areas in China. Because it is vectored by the fungus-like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co-transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12-1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12-1 showed broad-spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild-type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus-derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad-spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV. PMID:24373454

  12. Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

    PubMed

    Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

    2013-12-01

    IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes. PMID:24378636

  13. AN APPROACH TO TRANSGENIC CROP MONITORING

    EPA Science Inventory

    Remote sensing by aerial or satellite images may provide a method of identifying transgenic pesticidal crop distribution in the landscape. Genetically engineered crops containing bacterial gene(s) that express an insecticidal protein from Bacillus thuringiensis (Bt) are regulated...

  14. Lens-specific expression of transforming growth factor beta1 in transgenic mice causes anterior subcapsular cataracts.

    PubMed Central

    Srinivasan, Y; Lovicu, F J; Overbeek, P A

    1998-01-01

    Transgenic mice were generated by microinjection of a construct containing a self-activating human TGF-beta1 cDNA driven by the lens-specific alphaA-crystallin promoter. Seven transgenic founder mice were generated, and four transgenic lines expressing TGF-beta1 were characterized. By postnatal day 21, mice from the four families exhibited anterior subcapsular cataracts. The lenses in these mice developed focal plaques of spindle-shaped cells that expressed alpha-smooth muscle actin, and that resembled the plaques seen in human anterior subcapsular cataracts. Transgenic mice showed additional ocular defects, including corneal opacification and structural changes in the iris and ciliary body. The corneal opacities were associated with increased exfoliation of the squamous layer of the corneal epithelium and increased DNA replication in the basal epithelium. PMID:9449696

  15. Transgenic Wheat, Barley and Oats: Future Prospects

    NASA Astrophysics Data System (ADS)

    Dunwell, Jim M.

    Following the success of transgenic maize and rice, methods have now been developed for the efficient introduction of genes into wheat, barley and oats. This review summarizes the present position in relation to these three species, and also uses information from field trial databases and the patent literature to assess the future trends in the exploitation of transgenic material. This analysis includes agronomic traits and also discusses opportunities in expanding areas such as biofuels and biopharming.

  16. [Detection of transgenic crop with gene chip].

    PubMed

    Huang, Ying-Chun; Sun, Chun-Yun; Feng, Hong; Hu, Xiao-Dong; Yin, Hai-Bin

    2003-05-01

    Some selected available sequences of reporter genes,resistant genes, promoters and terminators are amplified by PCR for the probes of transgenic crop detection gene chip. These probes are arrayed at definite density and printed on the surface of amino-slides by bioRobot MicroGrid II. Results showed that gene chip worked quickly and correctly, when transgenic rice, pawpaw,maize and soybean were applied. PMID:15639876

  17. Beyond the bolus: transgenic tools for investigating the neurophysiology of learning and memory.

    PubMed

    Lykken, Christine; Kentros, Clifford G

    2014-10-01

    Understanding the neural mechanisms underlying learning and memory in the entorhinal-hippocampal circuit is a central challenge of systems neuroscience. For more than 40 years, electrophysiological recordings in awake, behaving animals have been used to relate the receptive fields of neurons in this circuit to learning and memory. However, the vast majority of such studies are purely observational, as electrical, surgical, and pharmacological circuit manipulations are both challenging and relatively coarse, being unable to distinguish between specific classes of neurons. Recent advances in molecular genetic tools can overcome many of these limitations, enabling unprecedented control over neural activity in behaving animals. Expression of pharmaco- or optogenetic transgenes in cell-type-specific "driver" lines provides unparalleled anatomical and cell-type specificity, especially when delivered by viral complementation. Pharmacogenetic transgenes are specially designed neurotransmitter receptors exclusively activated by otherwise inactive synthetic ligands and have kinetics similar to traditional pharmacology. Optogenetic transgenes use light to control the membrane potential, and thereby operate at the millisecond timescale. Thus, activation of pharmacogenetic transgenes in specific neuronal cell types while recording from other parts of the circuit allows investigation of the role of those neurons in the steady state, whereas optogenetic transgenes allow one to determine the immediate network response. PMID:25225296

  18. Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease.

    PubMed

    Ignacimuthu, S; Ceasar, S Antony

    2012-03-01

    Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance. PMID:22357211

  19. Beyond the bolus: transgenic tools for investigating the neurophysiology of learning and memory

    PubMed Central

    Lykken, Christine

    2014-01-01

    Understanding the neural mechanisms underlying learning and memory in the entorhinal-hippocampal circuit is a central challenge of systems neuroscience. For more than 40 years, electrophysiological recordings in awake, behaving animals have been used to relate the receptive fields of neurons in this circuit to learning and memory. However, the vast majority of such studies are purely observational, as electrical, surgical, and pharmacological circuit manipulations are both challenging and relatively coarse, being unable to distinguish between specific classes of neurons. Recent advances in molecular genetic tools can overcome many of these limitations, enabling unprecedented control over neural activity in behaving animals. Expression of pharmaco- or optogenetic transgenes in cell-type-specific “driver” lines provides unparalleled anatomical and cell-type specificity, especially when delivered by viral complementation. Pharmacogenetic transgenes are specially designed neurotransmitter receptors exclusively activated by otherwise inactive synthetic ligands and have kinetics similar to traditional pharmacology. Optogenetic transgenes use light to control the membrane potential, and thereby operate at the millisecond timescale. Thus, activation of pharmacogenetic transgenes in specific neuronal cell types while recording from other parts of the circuit allows investigation of the role of those neurons in the steady state, whereas optogenetic transgenes allow one to determine the immediate network response. PMID:25225296

  20. Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D.

    PubMed

    Wang, Ping; Wei, ZhiGuo; Yan, BoWen; Huang, Tan; Gou, KeMian; Dai, YunPing; Zheng, Min; Wang, MeiLi; Cheng, XueQian; Wang, XiFeng; Xu, Chen; Sun, Yi

    2012-03-01

    Mutation of mevalonate kinase (MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome (HIDS) with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D (IgD) and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD (csIgD) specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS. PMID:22527518

  1. Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

    PubMed Central

    Chen, Xiang; Cui, Jing; Yan, Zhengjian; Zhang, Hongmei; Chen, Xian; Wang, Ning; Shah, Palak; Deng, Fang; Zhao, Chen; Geng, Nisha; Li, Melissa; Denduluri, Sahitya K.; Haydon, Rex C.; Luu, Hue H.; Reid, Russell R.; He, Tong-Chuan

    2015-01-01

    Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression. PMID:25815368

  2. Late Blight Resistance of RB Transgenic Potato Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Late blight of potato, caused by Phytophthora infestans, is a devastating disease effecting tuber yield and storage. Recent work has isolated a resistance gene, RB, from the wild species Solanum bulbocastanum. Field evaluations with a RB containing somatic hybrid have reported significant levels of ...

  3. Transgene flow: facts, speculations and possible countermeasures.

    PubMed

    Ryffel, Gerhart U

    2014-01-01

    Convincing evidence has accumulated that unintended transgene escape occurs in oilseed rape, maize, cotton and creeping bentgrass. The escaped transgenes are found in variant cultivars, in wild type plants as well as in hybrids of sexually compatible species. The fact that in some cases stacked events are present that have not been planted commercially, implies unintended recombination of transgenic traits. As the consequences of this continuous transgene escape for the ecosystem cannot be reliably predicted, I propose to use more sophisticated approaches of gene technology in future. If possible GM plants should be constructed using either site-directed mutagenesis or cisgenic strategies to avoid the problem of transgene escape. In cases where a transgenic trait is needed, efficient containment should be the standard approach. Various strategies available or in development are discussed. Such a cautious approach in developing novel types of GM crops will enhance the sustainable potential of GM crops and thus increase the public trust in green gene technology. PMID:25523171

  4. Viral and transgenic reporters and genetic analysis of adult neurogenesis.

    PubMed

    Enikolopov, Grigori; Overstreet-Wadiche, Linda; Ge, Shaoyu

    2015-08-01

    Stem and progenitor cells of the developing and adult brain can be effectively identified and manipulated using reporter genes, introduced into transgenic reporter mouse lines or recombinant viruses. Such reporters rely on an ever-increasing variety of fluorescent proteins and a continuously expanding list of regulatory elements and of mouse lines engineered for cell- or time-specific recombination. An important extension of stem-cell-based genetic strategies is an opportunity to explore the properties of newly generated neurons and their contribution to synaptic plasticity. Here, we review available strategies for marking and quantifying various classes of stem and progenitor cells in the adult brain, genetically tracing their progeny, and studying the properties of stem cells and new neurons. We compare various experimental approaches to labeling and investigating stem cells and their progeny and discuss caveats and limitations inherent to each approach. PMID:26238354

  5. Myocardial T1 mapping: modalities and clinical applications

    PubMed Central

    Jellis, Christine L.

    2014-01-01

    Myocardial fibrosis appears to be linked to myocardial dysfunction in a multitude of non-ischemic cardiomyopathies. Accurate non-invasive quantitation of this extra-cellular matrix has the potential for widespread clinical benefit in both diagnosis and guiding therapeutic intervention. T1 mapping is a cardiac magnetic resonance (CMR) imaging technique, which shows early clinical promise particularly in the setting of diffuse fibrosis. This review will outline the evolution of T1 mapping and the various techniques available with their inherent advantages and limitations. Histological validation of this technique remains somewhat limited, however clinical application in a range of pathologies suggests strong potential for future development. PMID:24834410

  6. Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene

    PubMed Central

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

    2014-01-01

    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. PMID:24595215

  7. Fitness Impact and Stability of a Transgene Conferring Resistance to Dengue-2 Virus following Introgression into a Genetically Diverse Aedes aegypti Strain

    PubMed Central

    Franz, Alexander W. E.; Sanchez-Vargas, Irma; Raban, Robyn R.; Black, William C.; James, Anthony A.; Olson, Ken E.

    2014-01-01

    In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement. PMID:24810399

  8. Fitness impact and stability of a transgene conferring resistance to dengue-2 virus following introgression into a genetically diverse Aedes aegypti strain.

    PubMed

    Franz, Alexander W E; Sanchez-Vargas, Irma; Raban, Robyn R; Black, William C; James, Anthony A; Olson, Ken E

    2014-05-01

    In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement. PMID:24810399

  9. Proline over-accumulation alleviates salt stress and protects photosynthetic and antioxidant enzyme activities in transgenic sorghum [Sorghum bicolor (L.) Moench].

    PubMed

    Surender Reddy, P; Jogeswar, Gadi; Rasineni, Girish K; Maheswari, M; Reddy, Attipalli R; Varshney, Rajeev K; Kavi Kishor, P B

    2015-09-01

    Shoot-tip derived callus cultures of Sorghum bicolor were transformed by Agrobacterium tumefaciens as well as by bombardment methods with the mutated pyrroline-5-carboxylate synthetase (P5CSF129A) gene encoding the key enzyme for proline biosynthesis from glutamate. The transgenics were selfed for three generations and T4 plants were examined for 100 mM NaCl stress tolerance in pot conditions. The effect of salt stress on chlorophyll and carotenoid contents, photosynthetic rate, stomatal conductance, internal carbon dioxide concentration, transpiration rates, intrinsic transpiration and water use efficiencies, proline content, MDA levels, and antioxidant enzyme activities were evaluated in 40-day-old transgenic lines and the results were compared with untransformed control plants. The results show that chlorophyll content declines by 65% in untransformed controls compared to 30-38% loss (significant at P < 0.05) in transgenics but not carotenoid levels. Photosynthetic rate (PSII activity) was reduced in untransformed controls almost completely, while it declined by 62-88% in different transgenic lines. Salinity induced ca 100% stomatal closure in untransformed plants, while stomatal conductance was decreased only by 64-81% in transgenics after 4 days. The intercellular CO2 decreased by ca 30% in individual transgenic lines. Malondialdehyde (MDA) content was lower in transgenics compared to untransformed controls. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and glutathione reductase (GR; EC1.8.1.7) were quantified in leaves exposed to 100 mM NaCl stress and found higher in transgenics. The results suggest that transgenic lines were able to cope better with salt stress than untransformed controls by protecting photosynthetic and antioxidant enzyme activities. PMID:26065619

  10. OBTAINING OF THE TRANSGENIC HELIANTHUS TUBEROSUS L. PLANTS, CALLUS AND "HAIRY" ROOT CULTURES ABLE TO EXPRESS THE RECOMBINANT HUMAN INTERFERON ALPHA-2b GENE.

    PubMed

    Maistrenko, O M; Luchakivska, Yu S; Zholobak, N M; Spivak, M Ya; Kuchuk, M V

    2015-01-01

    This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein. PMID:26638495

  11. A Primer for Using Transgenic Insecticidal Cotton in Developing Countries

    PubMed Central

    Showalter, Ann M.; Heuberger, Shannon; Tabashnik, Bruce E.; Carričre, Yves

    2009-01-01

    Many developing countries face the decision of whether to approve the testing and commercial use of insecticidal transgenic cotton and the task of developing adequate regulations for its use. In this review, we outline concepts and provide information to assist farmers, regulators and scientists in making decisions concerning this technology. We address seven critical topics: 1) molecular and breeding techniques used for the development of transgenic cotton cultivars, 2) properties of transgenic cotton cultivars and their efficacy against major insect pests, 3) agronomic performance of transgenic cotton in developing countries, 4) factors affecting transgene expression, 5) impact of gene flow between transgenic and non-transgenic cotton, 6) non-target effects of transgenic cotton, and 7) management of pest resistance to transgenic cotton. PMID:19613464

  12. Cardiac phenotype induced by a dysfunctional ? 1C transgene: a general problem for the transgenic approach.

    PubMed

    Asemu, Girma; Fishbein, Kenneth; Lao, Qi Zong; Ravindran, Arippa; Herbert, Ron; Canuto, Holly C; Spencer, Richard G; Soldatov, Nikolai M

    2011-01-01

    Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human CaV 1.2 ?(1C) cDNA deprived of 3'-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading "transgenic artifact" compatible with the expected function of the incorporated "correct" transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic ?-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of "incidental incorporation" leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains. PMID:21224729

  13. qPCR for quantification of transgene expression and determination of transgene copy number.

    PubMed

    Fletcher, Stephen J

    2014-01-01

    Quantitative real-time PCR (qPCR) is a mature technology that can be used to accurately quantify the number of copies of a target nucleic acid in a sample. Here, we describe a method for using this technology to determine the copy number of a transgene stably integrated into a plant's genome and to ascertain the level of transgene expression. PMID:24816671

  14. EUROGRAPHICS 2002 Tutorial T1: 3D Data Acquisition

    E-print Network

    Goesele, Michael

    Goesele, Hendrik P. A. Lensch, Roberto Scopigno Abstract 3D scanners and image acquisition systems (often called 3D scanners) are rapidly becom- ing more affordable and allow to build highly accurate modEUROGRAPHICS 2002 Tutorial T1: 3D Data Acquisition R. Scopigno, ISTI-CNR, PISA C. Andujar, UPC

  15. Be a Food Detective Grades 9-12: T-1

    E-print Network

    Miller, Scott

    Be a Food Detective Grades 9-12: T-1 Be a Food Detective Overview Students will explore the exciting area of food and nutrition and answer specific questions about prepared food products, such as, what a food is made from, the source of its ingredients, and how the ingredients were grown. Suggested

  16. Virus-induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation.

    PubMed

    Zhao, Mingmin; San León, David; Delgadillo, Ma Otilia; García, Juan Antonio; Simón-Mateo, Carmen

    2014-08-01

    We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so-called virus-induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5' end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3' end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3' end, and an overall increase in CG methylation in the 5' end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced. PMID:24916614

  17. Relationship between expression levels and atherogenesis in scavenger receptor Class B, Type I Transgenics

    SciTech Connect

    Ueda, Yukihiko; Gong, Elaine; Royer, Lori; Cooper, Philip N.; Francone, Omar L; Rubin, Edward M.

    2000-03-15

    Both in vitro and in vivo studies of SR-BI have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate SR-BI's effect on atherogenesis we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) in SR-BI expression in an inbred mouse background hemizygous for a human apo B transgene. Unlike non-HDL cholesterol levels which minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol while the high expression SR-BI transgene was associated with two-fold decreases in HDL as well as dramatic alterations in HDL composition and size including the near absence of a-migrating particles as determined by 2-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a two-fold decrease in the development of diet induced fatty streak lesions compared t o the apo B transgenics (4448{+-}1908 {mu}m2/aorta to 10133 {+-} 4035 {mu}m2/aorta; p<0.001), while the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692{+-}7238 {mu}m2/aorta) but three-fold greater than the low SR-BI/apo B mice (p<0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences illustrating the complexity of the relationship between SR-BI and atherogenesis.

  18. Presenilin transgenic mice as models of Alzheimer’s disease

    PubMed Central

    Elder, Gregory A.; Sosa, Miguel A. Gama; De Gasperi, Rita; Dickstein, Dara L.; Hof, Patrick R.

    2012-01-01

    Mutations in presenilin-1 (PS1) and presenilin-2 (PS2) cause familial Alzheimer’s disease (FAD). Presenilins influence multiple molecular pathways and are best known for their role in the ?-secretase cleavage of type I transmembrane proteins including the amyloid precursor protein (APP). PS1 and PS2 FAD mutant transgenic mice have been generated using a variety of promoters. PS1-associated FAD mutations have also been knocked into the endogenous mouse gene. PS FAD mutant mice consistently show elevations of A?42 with little if any effect on A?40. When crossed with plaque forming APP FAD mutant lines, the PS1 FAD mutants cause earlier and more extensive plaque deposition. Although single transgenic PS1 or PS2 mice do not form plaques, they exhibit a number of pathological features including age-related neuronal and synaptic loss as well as vascular pathology. They also exhibit increased susceptibility to excitotoxic injury most likely on the basis of exaggerated calcium release from the endoplasmic reticulum. Electrophysiologically long-term potentiation in the hippocampus is increased in young PS1 FAD mutant mice but this effect appears to be lost with aging. In most studies neurogenesis in the adult hippocampus is also impaired by PS1 FAD mutants. Mice in which PS1 has been conditionally knocked out in adult forebrain on a PS2 null background (PS1/2 cDKO) develop a striking neurodegeneration that mimics AD neuropathology in being associated with neuronal and synaptic loss, astrogliosis and hyperphosphorylation of tau, although it is not accompanied by plaque deposits. The relevance of PS transgenic mice as models of AD is discussed. PMID:19921519

  19. Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

    NASA Technical Reports Server (NTRS)

    Love, J.; Scott, A. C.; Thompson, W. F.; Brown, C. S. (Principal Investigator)

    2000-01-01

    We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

  20. The Ethics of Transgenic Art 

    E-print Network

    Hood, Jennifer L

    2011-11-23

    Is biotechnology an appropriate and ethical medium for contemporary art? In the past decade, the line between science and art has become increasingly blurred as artists have begun to harness the accelerating developments of the life...

  1. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    NASA Technical Reports Server (NTRS)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  2. Changes in oxidative stress in transgenic RNAi ACO1 tomato fruit during ripening

    NASA Astrophysics Data System (ADS)

    Eglous, Najat Mohamed; Ali, Zainon Mohd; Hassan, Maizom; Zainal, Zamri

    2013-11-01

    Tomato (Solanum Lycopersicum L.) is the second most cultivated vegetable in the world and widely used as a system for studying the role of ethylene during fruit ripening. Our objective was to study the oxidative stress and antioxidative metabolism during ripening of non transgenic tomato and transgenic line-21 tomato which reduced ethylene. The line-21 of transgenic tomato plants (RNAi ACO1) had lower ethylene production and longer shelf-life more than 32 days as compared to the wild-type fruits which have very short shelf-life. In this study, tomato fruit were divided into five different stages (MG: mature green 5%, B: breaker 25%, T: turning 50%, O: orange75%, RR: red ripe100%). The activity of lipoxygenase (LOX) and lipid peroxidation (MDA) were measured to assess changes in oxidative stress. The LOX activity and MDA content decreased significantly obtaining 2.6-fold and 1.2-fold, respectively, as compared to the wild type fruit. However, superoxide dismutase (SOD) and catalase (CAT) activities were increased to 1.9 and 1.2 folds from the mature green to the fully ripe stage in transgenic tomatoes. Furthermore, the wild type tomato increases 1.3 in SOD and 1.6 in CAT activities. The overall results indicate that the wild type tomato fruit showed a faster rate of ripening, parallel to decline in the rate of enzymatic antioxidative systems as compared to the transgenic line-21 tomato fruit. In addition, the results show that the antioxidant capacity is improved during the ripening process and is accompanied by an increase in the oxidative stress.

  3. Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation.

    PubMed

    Chattopadhyay, Tirthartha; Roy, Sheuli; Mitra, Adinpunya; Maiti, Mrinal K

    2011-04-01

    Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants. PMID:21153028

  4. Transgenic tobacco expressing Zephyranthes grandiflora agglutinin confers enhanced resistance to aphids.

    PubMed

    Ye, Song-hua; Chen, Sheng; Zhang, Fan; Wang, Wei; Tian, Qin; Liu, Jin-zhi; Chen, Fang; Bao, Jin-ku

    2009-09-01

    Plant lectins have been reported as transgenic resistance factors against a variety of insect pests. Herein, homologous analysis demonstrated that Zephyranthes grandiflora agglutinin (ZGA) exhibited high similarity with other monocot mannose-binding lectins (MBLs). Phylogenetic analysis revealed that it had taxonomical relationships with insecticidal MBLs. Subsequently, a plasmid expression vector pBI121 containing zga gene (pBIZGA) was constructed using the zga sequence, under the control of CaMV35S promoter and nos terminator. pBIZGA was then integrated into the genome of Nicotiana tabacum L. Polymerase chain reaction and Southern blot analysis demonstrated that this zga gene was integrated into the plant genome. Western blotting and agglutinating activity analysis also showed that transgenic tobacco plants expressed different levels of ZGA. Carbohydrate inhibition analysis indicated that recombinant ZGA and the native shared the same carbohydrate-binding specificity. Moreover, genetic analysis confirmed Mendelian segregation (3:1) of the transgenic in T1 progenies. In planta bioassays on T0 plants and their progenies indicated that expressed ZGA had an effect on reducing the survivability and fecundity of tobacco aphids (Myzus nicotianae). These findings demonstrate that the novel zga gene of ZGA can be expressed in crop plants susceptible to various sap-sucking insects. PMID:19067248

  5. Ouabain-resistant (Na+,K+)-ATPase enzyme activity in chemically induced ouabain-resistant C3H/10T1/2 cells.

    PubMed

    Shibuya, M L; Miura, T; Lillehaug, J R; Farley, R A; Landolph, J R

    1989-01-01

    To further understand the molecular nature of changes leading to chemically induced ouabain resistance in C3H/10T1/2 Cl 8 (10T1/2) cells, we isolated plasma membranes from wild-type and ouabain-resistant (Ouar) 10T1/2 cells and characterized (Na,K)-ATPase activity in the plasma membrane fraction. (Na+,K+)-ATPase enzyme activity in membrane fractions extracted from wild-type 10T1/2 cells was inhibited in a concentration-dependent manner by ouabain and was completely inhibited by 2.4 mM ouabain. Lineweaver-Burke and Eisenthal/Cornish-Bowden analysis indicated that the inhibition was uncompetitive. Ten to 45% of (Na+,K+)-ATPase enzyme activity extracted from three Ouar 10T1/2 cell lines cultured in 1 mM ouabain was resistant to 2.4 mM ouabain, depending on the cell line. Resistance of (Na+,K+)-ATPase activity in the plasma membrane fraction of Ouar cells to inhibition by ouabain and resistance of cultured Ouar cells to the cytotoxicity of ouabain occurred over similar concentrations of ouabain (0.1-3mM). Two ouabain-resistant cell lines, Ouar MNNG Cl 2 and Ouar MCA Cl 16-7, demonstrated the same total (Na+,K+)-ATPase specific activity as 10T1/2 cells, but the fraction of Ouar enzyme activity increased (from 18 to 40% in MNNG Cl 2 cells and from 10 to 25% in Sp Ouar Cl 16 cells) when the cells were cultured in ouabain. Thermal denaturation profiles and pH dependence profiles of (Na+,K+)-ATPase activity in plasma membranes from wild-type and Ouar 10T1/2 cells were identical. A 3.9-kb (Na,K)-ATPase alpha subunit mRNA transcript was found in 10T1/2 cells, and in the Ouar MNNG Cl 2 cell line cultured in the presence or absence of ouabain. There was no amplification of the gene coding for the alpha subunit of (Na+,K+)-ATPase in the chemically induced Ouar MNNG Cl 2 cell line, whether this cell line was cultured in the presence or absence of ouabain. These studies provide further evidence that the Ouar phenotype of chemically induced and spontaneous Ouar 10T1/2 cell lines derives from Ouar (Na+,K+)-ATPase activity, and that this Ouar (Na+,K+)-ATPase activity increases further in some cell lines cultured in the presence of ouabain. PMID:2562032

  6. Subchronic toxicity study of GH transgenic carp.

    PubMed

    Yong, Ling; Liu, Yu-Mei; Jia, Xu-Dong; Li, Ning; Zhang, Wen-Zhong

    2012-11-01

    A subchronic toxicity study of GH (growth hormone) transgenic carp was carried out with 60 SD rats aged 4 weeks, weight 115?125 g. Ten male and 10 female rats were allotted into each group. Animals of the three groups (transgenic carp group (GH-TC), parental carp group (PC) and control group) were fed soy- and alfalfa-free diet (SAFD) with 10% GH transgenic carp powder, 10% parental carp powder or 10% common carp powder for 90 consecutive days, respectively. In the end of study, animals were killed by exsanguination via the carotid artery under diethyl ether anesthesia, then weights of heart, liver, kidneys, spleen, thymus, brain, ovaries and uterus/testis were measured. Pathological examination of organs was determined. Endocrine hormones of triiodothyronine (T3), thyroid hormone (T4), follicle-stimulating hormone (FSH), 17?-estradiol (E2), progesterone (P) and testosterone (T) levels were detected by specific ELISA kit. Parameters of blood routine and blood biochemical were measured. The weights of the body and organs of the rats, food intake, blood routine, blood biochemical test and serum hormones showed no significant differences among the GH transgenic carp-treated, parental carp-treated and control groups (P>0.05). Thus, it was concluded that at the dose level of this study, GH transgenic carp showed no subchronic toxicity and endocrine disruption to SD rats. PMID:22889892

  7. BAC manipulations for making BAC transgene arrays.

    PubMed

    Khanna, Nimish; Bian, Qian; Plutz, Matt; Belmont, Andrew S

    2013-01-01

    Chromosome tagging using lac or tet operator repeats for in vivo visualization of chromosome dynamics has now become a standard methodology used in a range of organisms. One variation of this approach has been to build transgene arrays creating artificial chromosome blocks to study various aspects of chromatin structure, transcription, replication, or DNA repair. Previously, plasmid transgenes with or without subsequent gene amplification have been used to build these arrays. However, plasmid arrays typically show heterochromatic properties, while gene amplification typically results in chromosome instability of the amplified regions. To avoid these problems, we are now building transgene arrays from large genomic DNA inserts cloned in bacterial artificial chromosomes (BAC). These BAC transgenes show transcriptional levels within several fold of endogenous genes while also exhibiting targeting to specific nuclear compartments similar to the targeting of the endogenous genes. Here we describe Tn5 transposition and BAC recombineering methods used to retrofit BACs for their use in building BAC transgene arrays. This includes insertion of operator repeats and selectable markers into these BACs as well as targeted insertion or deletion of BAC sequences. PMID:23980009

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

    PubMed Central

    Dunn, David A.; Pinkert, Carl A.

    2012-01-01

    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; P < 0.05), no locomotor differences (gait analysis; P < 0.05) and enhanced endurance in Rota-Rod evaluations (P < 0.05). A6W mice exhibited inferior muscle strength (wire hang test; P < 0.05), no difference in balance beam footsteps, accelerating Rota-Rod, pole test and gait analyses; (P < 0.05) and superior performance in balance beam time-to-cross and constant velocity Rota-Rod analyses (P < 0.05) in comparison to non-transgenic control mice. Mice of both transgenic lines did not differ from non-transgenic controls in a number of bioenergetic and biochemical tests including measurements of serum lactate and mitochondrial MnSOD protein levels, ATP synthesis rate, and oxygen consumption (P > 0.05). 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. PMID:22778551

  9. Transgenic Resistance Confers Effective Field Level Control of Bacterial Spot Disease in Tomato

    PubMed Central

    Horvath, Diana M.; Stall, Robert E.; Jones, Jeffrey B.; Pauly, Michael H.; Vallad, Gary E.; Dahlbeck, Doug; Staskawicz, Brian J.; Scott, John W.

    2012-01-01

    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. PMID:22870280

  10. Transgenic mouse models of Alzheimer's disease: phenotype and application.

    PubMed

    Higgins, Guy A; Jacobsen, Heimut

    2003-09-01

    Alzheimer's disease (AD) is the most common cause of senile dementia, for which there is presently no disease-based treatment. The identification of genetic factors contributing to this disease, and the intense investigation into the cell biology of amyloid precursor protein (APP) and, to some extent, tau, has led to the development of several transgenic mouse models of this disease. These mice show some of the characteristic AD pathology, such as an age-dependent formation of amyloid plaques consisting of Abeta peptides. However, they usually lack both the tau pathology, i.e. neurofibrillary tangle formation, and the neurodegeneration also associated with AD. Importantly, many of these transgenic lines develop age-dependent deficits in some relevant behavioural tests and thus provide an animal model not only for amyloidosis but also for the cognitive deficits of AD patients. Incorporation of additional disease genes may lead to models that show a more complete disease phenotype. This review attempts to summarize much of this work, and describes how the availability of these models should assist in the understanding of AD aetiology and the identification of effective treatments for this disease. The review also considers the role behavioural testing may have in future AD drug discovery research. PMID:14501255

  11. Expression Analysis of CB2-GFP BAC Transgenic Mice

    PubMed Central

    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. PMID:26406232

  12. Expression of functional oat phytochrome A in transgenic rice.

    PubMed

    Clough, R C; Casal, J J; Jordan, E T; Christou, P; Vierstra, R D

    1995-11-01

    To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants. PMID:8552709

  13. Comparative Proteomics of Milk Fat Globule Membrane Proteins from Transgenic Cloned Cattle

    PubMed Central

    Wang, Jianwu; Zhang, Ran; Guo, Chengdong; Yu, Tian; Li, Ning

    2014-01-01

    The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human ?-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland. PMID:25133402

  14. Comparative proteomics of milk fat globule membrane proteins from transgenic cloned cattle.

    PubMed

    Sui, Shunchao; Zhao, Jie; Wang, Jianwu; Zhang, Ran; Guo, Chengdong; Yu, Tian; Li, Ning

    2014-01-01

    The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human ?-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland. PMID:25133402

  15. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    SciTech Connect

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup; Pedersen, Lene

    2014-08-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding. - Highlights: • Effects of elevated levels of the inorganic phosphate transporter PiT1 were studied. • The density-inhibited murine cell lines NIH3T3 and MC3T3-E1 showed faster adhesion. • NIH3T3 cells showed faster spreading. • We suggest that the faster adhesion/spreading contributes to faster proliferation.

  16. J=0 T=1 Pairing Interaction Selection Rules

    E-print Network

    Matthew Harper; Larry Zamick

    2015-05-10

    Wave functions arising form a pairing Hamiltonian E(0) i,e, one in which the interaction is only between J=0^{+}T=1 pairs, lead to magnetic dipole and Gamow-Teller transition rates that are much larger than those from an interaction E(J_{max}) in which a proton and a neutron couple to J=2j. With realistic interactions the results are in between the 2 extremes. In the course of this study we found that certain M1 and GT matrix elements vanish with E(0) . These are connected to seniority and reduced isospin isospin selection rules. We find the surprising result that The M1 strength to the single j scissors is larger for a J=0 T=1 pairing interaction than it is for Q.Q.

  17. Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

    PubMed Central

    Jhappan, C; Geiser, A G; Kordon, E C; Bagheri, D; Hennighausen, L; Roberts, A B; Smith, G H; Merlino, G

    1993-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland. Images PMID:8491177

  18. Toxins for transgenic resistance to hemipteran pests.

    PubMed

    Chougule, Nanasaheb P; Bonning, Bryony C

    2012-06-01

    The sap sucking insects (Hemiptera), which include aphids, whiteflies, plant bugs and stink bugs, have emerged as major agricultural pests. The Hemiptera cause direct damage by feeding on crops, and in some cases indirect damage by transmission of plant viruses. Current management relies almost exclusively on application of classical chemical insecticides. While the development of transgenic crops expressing toxins derived from the bacterium Bacillus thuringiensis (Bt) has provided effective plant protection against some insect pests, Bt toxins exhibit little toxicity against sap sucking insects. Indeed, the pest status of some Hemiptera on Bt-transgenic plants has increased in the absence of pesticide application. The increased pest status of numerous hemipteran species, combined with increased prevalence of resistance to chemical insecticides, provides impetus for the development of biologically based, alternative management strategies. Here, we provide an overview of approaches toward transgenic resistance to hemipteran pests. PMID:22822455

  19. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V. (Okemos, MI)

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  20. Production of homozygous transgenic rainbow trout with enhanced disease resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit antibacterial an...

  1. Vector Identities T 1. (f + g) = f + g

    E-print Network

    Fournier, John J.F.

    Vector Identities Gradient T 1. (f + g) = f + g T 2. (cf) = cf, for any constant c E 3. (fg) = fg + gf E 4. (f/g) = gf - fg /g2 at points x where g(x) = 0. Pcc 5. (F · G) = F � ( � G) - ( � F) � G + (G · )F + (F · )G Divergence T 6. · (F + G) = · F + · G T 7. · (cF) = c · F, for any constant c E , Pa

  2. Towards T1-limited magnetic resonance imaging using Rabi beats

    E-print Network

    H. Fedder; F. Dolde; F. Rempp; T. Wolf; P. Hemmer; F. Jelezko; J. Wrachtrup

    2010-09-03

    Two proof-of-principle experiments towards T1-limited magnetic resonance imaging with NV centers in diamond are demonstrated. First, a large number of Rabi oscillations is measured and it is demonstrated that the hyperfine interaction due to the NV's 14N can be extracted from the beating oscillations. Second, the Rabi beats under V-type microwave excitation of the three hyperfine manifolds is studied experimentally and described theoretically.

  3. GROWTH PATTERNS OF TRANSGENIC GUAYULE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study is being conducted to determine whether the insertion of genes that synthesize precursors in the rubber biosynthesis pathway can increase rubber production in guayule (Parthenium argentatum), thereby increasing overall rubber yield. Three lines, AZ101, N6-5 and G7-11, had either farnesyl ...

  4. Transgenically expressed Parascaris P-glycoprotein-11 can modulate ivermectin susceptibility in Caenorhabditis elegans

    PubMed Central

    Janssen, I. Jana I.; Krücken, Jürgen; Demeler, Janina; von Samson-Himmelstjerna, Georg

    2015-01-01

    P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility in a motility (thrashing) assay conducted in liquid medium. The EC50 values increased by 3.2- and 4.6-fold in the two lines relative to a transgenic control strain. This is the first report on the successful functional analysis of a parasitic nematode Pgp in the model organism C.?elegans. PMID:25905032

  5. Engineering the production of sugar alcohols in transgenic plants: Extending the limits of photosynthesis. Final technical report

    SciTech Connect

    1997-03-31

    In the different tobacco lines expressing different polyols, the authors have investigated how the presence of polyols affects ion uptake during short periods of stress. In addition, they began investigations on recovery from short periods of stress, e.g. eight days of drought and/or five days in 400 mM NaCl. The transgenic plants take up sodium more slowly. The next set of experiments, modeled after the experiments done with Mesembryanthemum will investigate ion transport and partitioning in control and transgenic tobacco. Photosynthetic activities of drought-stressed mannitol/ononitrol tobacco were investigated. Measurements of fluorescence, carbon fixation rates and electron transport indicated that the polyol-containing plants loose photosynthetic competence more slowly than controls. Transfer of the mtlD gene (mannitol production) into Arabidopsis has been accomplished. The transgenic plants are phenotypically normal. They survive 300 mM NaCl when the stress is started when the plants are mature--in contrast to wild type which is killed at 150 mM. Seeds from mannitol-containing plants germinate (100%) in 100 mM NaCl while germination rate of wild type is about 20%. In 200 mM NaCl n wild type germinates, while in some transgenic lines still 50% of the seeds germinated. At 250 mM NaCl during germination, the transgenic seeds are severely impaired, only 10 to 20% begin germination.

  6. Transgenic Quail as a Model for Research in the Avian Nervous System – A Comparative Study of the Auditory Brainstem

    PubMed Central

    Seidl, Armin H.; Sanchez, Jason Tait; Schecterson, Leslayann; Tabor, Kathryn M.; Wang, Yuan; Kashima, Daniel T.; Poynter, Greg; Huss, David; Fraser, Scott E.; Lansford, Rusty; Rubel, Edwin W

    2012-01-01

    Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proven problematic. As a result, experiments aimed at genetic manipulations on birds remained difficult for this popular research tool. Recently, lentiviral methods have enabled production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal-specificity and stable expression of eGFP across generations (termed here as GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of gene manipulation, we compared the development, organization, structure and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) to that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM) and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken. PMID:22806400

  7. Transgenic Mouse Models OHSU Research Cores and Shared Resources

    E-print Network

    Chapman, Michael S.

    Transgenic Mouse Models OHSU Research Cores and Shared Resources Mission Statement The TMM assists investigators with the development of genetically engineered mouse models of human diseases for studying.494.4705 Web www.ohsu/edu/transgenics To generate mouse models of human diseases the Transgenic Mouse Models

  8. IDENTIFICATION OF ESCAPED TRANSGENIC CREEPING BENTGRASS IN OREGON

    EPA Science Inventory

    When transgenic plants are cultivated near wild species that are sexually compatible with the crop, gene flow between the crop and wild plants is possible. A resultant concern is that transgene flow and transgene introgression within wild populations could have unintended ecologi...

  9. Maize transgenes containing zein promoters are regulated by opaque2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenes have great potential in crop improvement, but relatively little is known about the epistatic interaction of transgenes with the native genes in the genome. Understanding these interactions is critical for predicting the response of transgenes to different genetic backgrounds and environm...

  10. Drosophila transgenes Spectrum estimation from X-ray scattering

    E-print Network

    McCullagh, Peter

    Drosophila transgenes Spectrum estimation from X-ray scattering The big picture A miscellany University of Chicago Chicago May 2010 Peter McCullagh Consulting problems #12;Drosophila transgenes Spectrum estimation from X-ray scattering The big picture Outline 1 Drosophila transgenes 2 Spectrum estimation from X

  11. Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.

    PubMed

    Grisolano, J L; Sclar, G M; Ley, T J

    1994-09-13

    The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment. PMID:8090757

  12. Transgenic rats with green, red, and blue fluorescence: powerful tools for bioimaging, cell trafficking, and differentiation

    NASA Astrophysics Data System (ADS)

    Murakami, Takashi; Kobayashi, Eiji

    2005-04-01

    The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats" system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

  13. T1 and susceptibility contrast at high fields

    NASA Astrophysics Data System (ADS)

    Neelavalli, Jaladhar

    Clinical imaging at high magnetic field strengths (? 3Tesla) is sought after primarily due to the increased signal strength available at these fields. This increased SNR can be used to perform: (a) high resolution imaging in the same time as at lower field strengths; (b) the same resolution imaging with much faster acquisition; and (c) functional MR imaging (fMRI), dynamic perfusion and diffusion imaging with increased sensitivity. However they are also associated with increased power deposition (SAR) due to increase in imaging frequency and longer T1 relaxation times. Longer T1s mean longer imaging times for generating good T1 contrast images. On the other hand for faster imaging, at high fields fast spin echo or magnetization prepared sequences are conventionally proposed which are, however, associated with high SAR values. Imaging with low SAR is more and more important as we move towards high fields and particularly for patients with metallic implants like pacemakers or deep brain stimulator. The SAR limit acceptable for these patients is much less than the limit acceptable for normal subjects. A new method is proposed for imaging at high fields with good contrast with simultaneous reduction in power deposition. Further, T1 based contrast optimization problem in FLASH imaging is considered for tissues with different T1s but same spin densities. The solution providing optimal imaging parameters is simplified for quick and easy computation in a clinical setting. The efficacy of the simplification is evaluated and practical limits under which the simplification can be applied are worked out. The phase difference due to variation in magnetic susceptibility property among biological tissues is another unique source of contrast which is different from the conventional T1, T2 and T2* contrast. This susceptibility based phase contrast has become more and more important at high fields, partly due to contrast generation issues due to longer T 1s and shorter T2s and partly because of the invariance of most tissue susceptibilities with field strength. This essentially ensures a constant available phase contrast between tissues across field strengths. In fact, with the increased SNR at high fields, the phase CNR actually increases with field strength which is even better. Susceptibility weighted imaging, which uniquely combines this phase and magnitude information to generate enhanced susceptibility contrast magnitude images, has proven to be an important tool in the study of various neurological conditions like, Alzheimer's, Parkinson's, Huntington's disease and multiple sclerosis even at conventional field strength of 1.5T and should have more applicability at high fields. A major issue in using phase images for susceptibility contrast, directly or as processed SWI magnitude images, is the large scale background phase variations that obscure the local susceptibility based contrast. A novel method is proposed for removing such geometrically induced large scale phase variations using a Fourier Transform based field calculation method. It is shown that the new method is capable of successfully removing the background field effects. It is shown that the new method is not only capable of successfully removing the background field effects but also helps in preserving more local phase information.

  14. Genomic position effects lead to an inefficient reorganization of nucleosomes in the 5'-regulatory region of the chicken lysozyme locus in transgenic mice.

    PubMed Central

    Huber, M C; Krüger, G; Bonifer, C

    1996-01-01

    The chicken lysozyme locus is gradually activated during macrophage development exhibiting a specific chromatin structure with each differentiation state. Its small size and the extensive characterization of its cis-regulatory elements allows us to study even subtle changes in chromatin structure of the entire gene locus during transcriptional activation. Tissue-specific and position independent expression of the lysozyme locus in transgenic mice requires the cooperation of all cis-regulatory elements. In order to elucidate further the molecular basis of locus activation, we have determined nucleosome positions within the complete 5'-regulatory region of the chicken lysozyme locus in chicken myeloid cell lines and transgenic mice. Each cis-regulatory element develops its unique nucleosomal structure and each one remodels chromatin differently. The nucleosomal organization of the endogenous gene in chicken cell lines and the transgene in the mouse turned out to be identical, enabling us to study the influence of cis-regulatory deletions on the development of an active chromatin structure in transgenic mice. Transgenes with a deletion of an important cis-regulatory element show an impediment in nucleosome reorganization as compared with the complete lysozyme locus. We demonstrate that multicopy transgene-clusters in position dependently expressing mouse lines exhibit a heterogeneous chromatin organization. PMID:8628676

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

    SciTech Connect

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

    1995-06-01

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

  16. TRANSGENIC INDIAN MUSTARD OVEREXPRESSING SELENOCYSTEINE LYASE, SELENOCYSTEINE METHYLTRANSFERASE, OR METHIONINE METHYLTRANSFERASE EXHIBIT ENHANCED POTENTIAL FOR SELENIUM PHPYTOREMEDIATION UNDER FIELD CONDITIONS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effective use of phytoremediation of Se as a clean-up technology could be enhanced by improving the ability of plants to accumulate Se. In this regar, three transgenic Indican mustard (Brassica juncea (L.) Czern.) lines were tested under field conditions for their ability to accumulate selenium...

  17. Determining sex and screening for the adventitious presence of transgenic material in Carica papaya L. seed germplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Papaya Ringspot Virus (PRSV) is a devastating disease that has a detrimental impact on both commercial papaya production and Caricaceae germplasm conservation. The PRSV coat protein transgenic line 55-1 and derived progeny are resistant to PRSV and have saved the papaya industry in Hawaii. However,...

  18. Comparative genomic analysis of transgenic poplar dwarf mutant reveals numerous differentially expressed genes involved in energy flow.

    PubMed

    Chen, Su; Bai, Shuang; Liu, Guifeng; Li, Huiyu; Jiang, Jing

    2014-01-01

    In our previous research, the Tamarix androssowii LEA gene (Tamarix androssowii late embryogenesis abundant protein Mrna, GenBank ID: DQ663481) was transferred into Populus simonii × Populus nigra. Among the eleven transgenic lines, one exhibited a dwarf phenotype compared to the wild type and other transgenic lines, named dwf1. To uncover the mechanisms underlying this phenotype, digital gene expression libraries were produced from dwf1, wild-type, and other normal transgenic lines, XL-5 and XL-6. Gene expression profile analysis indicated that dwf1 had a unique gene expression pattern in comparison to the other two transgenic lines. Finally, a total of 1246 dwf1-unique differentially expressed genes were identified. These genes were further subjected to gene ontology and pathway analysis. Results indicated that photosynthesis and carbohydrate metabolism related genes were significantly affected. In addition, many transcription factors genes were also differentially expressed in dwf1. These various differentially expressed genes may be critical for dwarf mutant formation; thus, the findings presented here might provide insight for our understanding of the mechanisms of tree growth and development. PMID:25192286

  19. Expression Patterns in Transgenic Wheats with Elevated Levels of Dx5 and/or Dy10 Glutenin Subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to study the effects of independently increasing the levels of high-molecular-weight glutenin subunits Dx5 and Dy10, we added copies of their genes to wheat by genetic transformation. Among 30 lines produced, six exhibited transgene-mediated co-suppression and eight showed the presence of e...

  20. Diversity of arthropod community in transgenic poplar-cotton ecosystems.

    PubMed

    Zhang, D J; Lu, Z Y; Liu, J X; Li, C L; Yang, M S

    2015-01-01

    Poplar-cotton agro-ecosystems are the main agricultural planting modes of plain cotton fields in China. Here, we performed a systematic survey of the diversity and population of arthropod communities in four different combination of poplar-cotton eco-systems, including I) non-transgenic poplar and non-transgenic cotton fields; II) non-transgenic poplar and transgenic cotton fields [Bacillus thuringiensis (Bt) cotton]; III) Bt transgenic poplar (high insect resistant strain Pb29) and non-transgenic cotton; and IV) transgenic poplar and transgenic cotton fields, over a period of 3 years. Based on the statistical methods used to investigate community ecology, the effects of transgenic ecosystems on the whole structure of the arthropod community, on the structure of arthropods in the nutritive layer, and on the similarity of arthropod communities were evaluated. The main results were as follows: the transgenic poplar-cotton ecosystem has a stronger inhibitory effect on insect pests and has no impact on the structure of the arthropod community, and therefore, maintains the diversity of the arthropod community. The character index of the community indicated that the structure of the arthropod community of the transgenic poplar-cotton ecosystem was better than that of the poplar-cotton ecosystem, and that system IV had the best structure. As for the abundance of nutritional classes, the transgenic poplar-cotton ecosystem was also better than that of the non-transgenic poplar-cotton ecosystem. The cluster analysis and similarity of arthropod communities between the four different transgenic poplar-cotton ecosystems illustrated that the structure of the arthropod community excelled in the small sample of the transgenic poplar-cotton ecosystems. PMID:26634539

  1. Grouper tsh? Promoter-Driven Transgenic Zebrafish Marks Proximal Kidney Tubule Development

    PubMed Central

    Wang, Yang; Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Gui, Jian-Fang

    2014-01-01

    Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tsh? (gtsh?) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtsh? role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtsh?:GFP) with green fluorescent protein driven by the gtsh? promoter. Similar to the endogenous tsh? in zebrafish or in grouper, the gtsh? promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtsh? promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule. PMID:24905828

  2. Transgenic plants with increased calcium stores

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  3. Monitoring transgenic plants using in vivo markers

    SciTech Connect

    Stewart, C.N. Jr.

    1996-06-01

    The gene coding for green fluorecent protein (GFP), isolated and cloned from the jellyfish Aequorea victoria, is an ideal transgene for the monitoring of any plant species. It has the ability to fluoresce without added substrate, enzyme, or cofactor; it does not introduce morphological or sexual aberrations when expressed. 7 refs., 1 fig.

  4. Generation of Transgenic Rats Using Lentiviral Vectors.

    PubMed

    Reichardt, Holger M; Fischer, Henrike J

    2016-01-01

    Transgenesis is a valuable tool with which to study different aspects of gene function in the context of the intact organism. During the last two decades a tremendous number of transgenic animals have been generated, and the continuous improvement of technology and the development of new systems have fostered their widespread application in biomedical research. Generally, transgenic animals are generated by introducing foreign DNA into fertilized oocytes, which can be achieved either by injecting recombinant DNA into the pronucleus or by transferring lentiviral particles into the perivitelline space. While mice remain the favored species in many laboratories, there are a number of applications where the use of rats is advantageous. One such research area is multiple sclerosis. Here, several experimental models are available that are closely mimicking the human disease, and it is possible to induce neuroinflammation by transferring pathogenic T cells which can then be studied by flow cytometry and 2-photon live imaging. Unlike for mice, the development of transgenic rats has encountered some hurdles in the past, e.g., due to a complicated reproductive biology and the frailty of the fertilized oocytes in vitro. In this chapter we provide a protocol describing how we manipulate single cell embryos in our lab in order to efficiently generate transgenic rats in a variety of different strains using lentiviral gene transfer. PMID:25063498

  5. Improved calcium imaging in transgenic mice

    E-print Network

    Cai, Long

    Improved calcium imaging in transgenic mice expressing a troponin C­based biosensor Nicola Heim1C)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca2+ sensitivity both in vitro, and opens a new avenue for structure- function analysis of intact neuronal circuits. Genetically encoded

  6. Metal resistance sequences and transgenic plants

    DOEpatents

    Meagher, Richard Brian (Athens, GA); Summers, Anne O. (Athens, GA); Rugh, Clayton L. (Athens, GA)

    1999-10-12

    The present invention provides nucleic acid sequences encoding a metal ion resistance protein, which are expressible in plant cells. The metal resistance protein provides for the enzymatic reduction of metal ions including but not limited to divalent Cu, divalent mercury, trivalent gold, divalent cadmium, lead ions and monovalent silver ions. Transgenic plants which express these coding sequences exhibit increased resistance to metal ions in the environment as compared with plants which have not been so genetically modified. Transgenic plants with improved resistance to organometals including alkylmercury compounds, among others, are provided by the further inclusion of plant-expressible organometal lyase coding sequences, as specifically exemplified by the plant-expressible merB coding sequence. Furthermore, these transgenic plants which have been genetically modified to express the metal resistance coding sequences of the present invention can participate in the bioremediation of metal contamination via the enzymatic reduction of metal ions. Transgenic plants resistant to organometals can further mediate remediation of organic metal compounds, for example, alkylmetal compounds including but not limited to methyl mercury, methyl lead compounds, methyl cadmium and methyl arsenic compounds, in the environment by causing the freeing of mercuric or other metal ions and the reduction of the ionic mercury or other metal ions to the less toxic elemental mercury or other metals.

  7. Transgenic Mouse Model of Chronic Beryllium Disease

    SciTech Connect

    Gordon, Terry

    2009-05-26

    Animal models provide powerful tools for dissecting dose-response relationships and pathogenic mechanisms and for testing new treatment paradigms. Mechanistic research on beryllium exposure-disease relationships is severely limited by a general inability to develop a sufficient chronic beryllium disease animal model. Discovery of the Human Leukocyte Antigen (HLA) - DPB1Glu69 genetic susceptibility component of chronic beryllium disease permitted the addition of this human beryllium antigen presentation molecule to an animal genome which may permit development of a better animal model for chronic beryllium disease. Using FVB/N inbred mice, Drs. Rubin and Zhu, successfully produced three strains of HLA-DPB1 Glu 69 transgenic mice. Each mouse strain contains a haplotype of the HLA-DPB1 Glu 69 gene that confers a different magnitude of odds ratio (OR) of risk for chronic beryllium disease: HLA-DPB1*0401 (OR = 0.2), HLA-DPB1*0201 (OR = 15), HLA-DPB1*1701 (OR = 240). In addition, Drs. Rubin and Zhu developed transgenic mice with the human CD4 gene to permit better transmission of signals between T cells and antigen presenting cells. This project has maintained the colonies of these transgenic mice and tested the functionality of the human transgenes.

  8. Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

    PubMed Central

    Iglesias, V A; Moscone, E A; Papp, I; Neuhuber, F; Michalowski, S; Phelan, T; Spiker, S; Matzke, M; Matzke, A J

    1997-01-01

    To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome. PMID:9286104

  9. Field resistance of transgenic plantain to nematodes has potential for future African food security.

    PubMed

    Tripathi, Leena; Babirye, Annet; Roderick, Hugh; Tripathi, Jaindra N; Changa, Charles; Urwin, Peter E; Tushemereirwe, Wilberforce K; Coyne, Danny; Atkinson, Howard J

    2015-01-01

    Plant parasitic nematodes impose losses of up to 70% on plantains and cooking bananas in Africa. Application of nematicides is inappropriate and resistant cultivars are unavailable. Where grown, demand for plantain is more than for other staple crops. Confined field testing demonstrated that transgenic expression of a biosafe, anti-feedant cysteine proteinase inhibitor and an anti-root invasion, non-lethal synthetic peptide confers resistance to plantain against the key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line showed improved agronomic performance relative to non-transgenic controls and provided about 99% nematode resistance at harvest of the mother crop. Its yield was about 186% in comparison with the nematode challenged control non-transgenic plants based on larger bunches and diminished plant toppling in storms, due to less root damage. This is strong evidence for utilizing this resistance to support the future food security of 70 million, mainly poor Africans that depend upon plantain as a staple food. PMID:25634654

  10. Transgenic rice plants harboring the grain hardness-locus region of Aegilops tauschii.

    PubMed

    Suzuki, Go; Wada, Hideo; Goto, Hiromi; Nakano, Akiko; Oba, Haruna; Deno, Takuya; Rahman, Sadequr; Mukai, Yasuhiko

    2011-12-01

    Grain hardness of wheat is determined by the hardness (Ha)-locus region, which contains three friabilin-related genes: puroindoline-a (Pina), puroindoline-b (Pinb) and GSP-1. In our previous study, we produced the transgenic rice plants harboring the large genomic fragment of the Ha-locus region of Aegilops tauschii containing Pina and GSP-1 genes by Agrobacterium-mediated transformation. To examine the effects of the transgenes in the rice endosperms, we firstly confirmed the homozygosity of the T-DNAs in four independent T2 lines by using fluorescence in situ hybridization (FISH) and DNA gel blot analyses. The transgenes, Pina and GSP-1, were stably expressed in endosperms of the T3 and T4 seeds at RNA and protein levels, indicating that the promoters and other regulatory elements on the wheat Ha-locus region function in rice, and that multigene transformation using a large genomic fragment is a useful strategy. The functional contribution of the transgene-derived friabilins to the rice endosperm structure was considered as an increase of spaces between compound starch granules, resulting in a high proportion of white turbidity seeds. PMID:21850595

  11. A novel ?/?-hydrolase gene IbMas enhances salt tolerance in transgenic sweetpotato.

    PubMed

    Liu, Degao; Wang, Lianjun; Zhai, Hong; Song, Xuejin; He, Shaozhen; Liu, Qingchang

    2014-01-01

    Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to ?/?-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity. PMID:25501819

  12. Optimising ketocarotenoid production in potato tubers: effect of genetic background, transgene combinations and environment.

    PubMed

    Campbell, Raymond; Morris, Wayne L; Mortimer, Cara L; Misawa, Norihiko; Ducreux, Laurence J M; Morris, Jenny A; Hedley, Pete E; Fraser, Paul D; Taylor, Mark A

    2015-05-01

    Astaxanthin is a high value carotenoid produced by some bacteria, a few green algae, several fungi but only a limited number of plants from the genus Adonis. Astaxanthin has been industrially exploited as a feed supplement in poultry farming and aquaculture. Consumption of ketocarotenoids, most notably astaxanthin, is also increasingly associated with a wide range of health benefits, as demonstrated in numerous clinical studies. Currently astaxanthin is produced commercially by chemical synthesis or from algal production systems. Several studies have used a metabolic engineering approach to produce astaxanthin in transgenic plants. Previous attempts to produce transgenic potato tubers biofortified with astaxanthin have met with limited success. In this study we have investigated approaches to optimising tuber astaxanthin content. It is demonstrated that the selection of appropriate parental genotype for transgenic approaches and stacking carotenoid biosynthetic pathway genes with the cauliflower Or gene result in enhanced astaxanthin content, to give six-fold higher tuber astaxanthin content than has been achieved previously. Additionally we demonstrate the effects of growth environment on tuber carotenoid content in both wild type and astaxanthin-producing transgenic lines and describe the associated transcriptome and metabolome restructuring. PMID:25804807

  13. Production of recombinant human proinsulin in the milk of transgenic mice

    PubMed Central

    Qian, Xi; Kraft, Jana; Ni, Yingdong; Zhao, Feng-Qi

    2014-01-01

    There is a steady increasing demand for insulin worldwide. Current insulin manufacturing capacities can barely meet this increasing demand. The purpose of this study was to test the feasibility of producing human proinsulin in the milk of transgenic animals. Four lines of transgenic mice harboring a human insulin cDNA with expression driven by the goat ?-casein gene promoter were generated. The expression level of human proinsulin in milk was as high as 8.1?g/L. The expression of the transgene was only detected in the mammary gland during lactation, with higher levels at mid-lactation and lower levels at early and late lactation. The blood glucose and insulin levels and the major milk compositions were unchanged, and the transgenic animals had no apparent health defects. The mature insulin derived from the milk proinsulin retained its biological activity. In conclusion, our study provides supporting evidence to explore the production of high levels of human proinsulin in the milk of dairy animals. PMID:25267062

  14. Field resistance of transgenic plantain to nematodes has potential for future African food security

    PubMed Central

    Tripathi, Leena; Babirye, Annet; Roderick, Hugh; Tripathi, Jaindra N.; Changa, Charles; Urwin, Peter E.; Tushemereirwe, Wilberforce K.; Coyne, Danny; Atkinson, Howard J.

    2015-01-01

    Plant parasitic nematodes impose losses of up to 70% on plantains and cooking bananas in Africa. Application of nematicides is inappropriate and resistant cultivars are unavailable. Where grown, demand for plantain is more than for other staple crops. Confined field testing demonstrated that transgenic expression of a biosafe, anti-feedant cysteine proteinase inhibitor and an anti-root invasion, non-lethal synthetic peptide confers resistance to plantain against the key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line showed improved agronomic performance relative to non-transgenic controls and provided about 99% nematode resistance at harvest of the mother crop. Its yield was about 186% in comparison with the nematode challenged control non-transgenic plants based on larger bunches and diminished plant toppling in storms, due to less root damage. This is strong evidence for utilizing this resistance to support the future food security of 70 million, mainly poor Africans that depend upon plantain as a staple food. PMID:25634654

  15. A Novel ?/?-Hydrolase Gene IbMas Enhances Salt Tolerance in Transgenic Sweetpotato

    PubMed Central

    Song, Xuejin; He, Shaozhen; Liu, Qingchang

    2014-01-01

    Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to ?/?-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity. PMID:25501819

  16. Molecular Characterization of Transgene Integration by Next-Generation Sequencing in Transgenic Cattle

    PubMed Central

    Zhang, Ran; Yin, Yinliang; Zhang, Yujun; Li, Kexin; Zhu, Hongxia; Gong, Qin; Wang, Jianwu; Hu, Xiaoxiang; Li, Ning

    2012-01-01

    As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species. PMID:23185606

  17. Small subunit of a cold-resistant plant, timothy, does not significantly alter the catalytic properties of Rubisco in transgenic rice.

    PubMed

    Fukayama, Hiroshi; Koga, Atsushi; Hatanaka, Tomoko; Misoo, Shuji

    2015-04-01

    Effects of overexpression of high activity-type Rubisco small subunit (RbcS) from a cold-resistant plant, timothy (Phleum pratense), on kinetic properties of Rubisco were studied in rice (Oryza sativa). The full-length mRNA sequence of timothy RbcS (PpRbcS1) was determined by 5'RACE and 3'RACE. The coding sequence of PpRbcS1 was fused to the chlorophyll a/b-binding protein promoter and introduced into rice. PpRbcS was highly expressed in leaf blade and accounted for approximately 30 % of total RbcS in homozygous transgenic lines. However, the catalytic turnover rate and K m for CO2 of Rubisco did not significantly change in these transgenic lines compared to non-transgenic rice, suggesting that PpRbcS1 is not effective for improvement of catalytic efficiency of rice Rubisco. The photosynthetic rate and growth were essentially unchanged, whereas the photosynthetic rate at low CO2 condition was marginally increased in transgenic lines. Rubisco content was significantly increased, whereas soluble protein, nitrogen, and chlorophyll contents were unchanged in transgenic lines compared to non-transgenic rice. Because the kinetic properties were similar, observed slight increase in photosynthetic rate at low CO2 is considered to be large due to increase in Rubisco content in transgenic lines. Introduction of foreign RbcS is an effective approach for the improvement of Rubisco kinetics and photosynthesis. However, in this study, it was suggested that RbcS of high activity-type Rubisco, even showing higher amino acid identity with rice RbcS, did not always enhance the catalytic turnover rate of Rubisco in rice. Thus, we should carefully select RbcS to be overexpressed before introduction. PMID:25595546

  18. T-1 Test Program Ver. 6.0.1

    Energy Science and Technology Software Center (ESTSC)

    2004-05-21

    The software allows for easy setup and testing of a variety of RF Electronic Sensor Platforms (ESPs). The software interprets RF messages from the ESP and displays the information in a graphical user interface. This program is used primarily for testing of the T-1 Electronic Sensor Platform. The software imports Electronic Tag Data files which are created from the Electronic Sensor Platform Programmer (ESPP). The software will automatically add sensors to its database when amore »RF message s received that the program recognizes. Any data that is generated can be stored to a file for later analysis.« less

  19. The TOTEM T1 read out card motherboard

    NASA Astrophysics Data System (ADS)

    Minutoli, S.; Lo Vetere, M.; Robutti, E.

    2010-12-01

    This article describes the Read Out Card (ROC) motherboard, which is the main component of the T1 forward telescope front-end electronic system. The ROC main objectives are to acquire tracking data and trigger information from the detector. It performs data conversion from electrical to optical format and transfers the data streams to the next level of the system and it implements Slow Control modules which are able to receive, decode and distribute the LHC machine low jitter clock and fast command. The ROC also provides a spy mezzanine connection based on programmable FPGA and USB2.0 for laboratory and portable DAQ debugging system.

  20. SirT1 is required in the male germ cell for differentiation and fecundity in mice.

    PubMed

    Bell, Eric L; Nagamori, Ippei; Williams, Eric O; Del Rosario, Amanda M; Bryson, Bryan D; Watson, Nicki; White, Forest M; Sassone-Corsi, Paolo; Guarente, Leonard

    2014-09-01

    Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis. PMID:25142464

  1. Resveratrol inhibits insulin responses in a SirT1-independent pathway.

    PubMed

    Zhang, Jiandi

    2006-08-01

    Resveratrol mimics calorie restriction to extend lifespan of Caenorhabditis elegans, yeast and Drosophila, possibly through activation of Sir2 (silent information regulator 2), a NAD+-dependent histone deacetylase. In the present study, resveratrol is shown to inhibit the insulin signalling pathway in several cell lines and rat primary hepatocytes in addition to its broad-spectrum inhibition of several signalling pathways. Resveratrol effectively inhibits insulin-induced Akt and MAPK (mitogen-activated protein kinase) activation mainly through disruption of the interactions between insulin receptor substrates and its downstream binding proteins including p85 regulatory subunit of phosphoinositide 3-kinase and Grb2 (growth factor receptor-bound protein 2). The inhibitory effect of resveratrol on insulin signalling is also demonstrated at mRNA level, where resveratrol reverses insulin effects on phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, fatty acid synthase and glucokinase. In addition, RNA interference experiment shows that the inhibitory effect of resveratrol on insulin signalling pathway is not weakened in cells with reduced expression of SirT1, the mammalian counterpart of Sir2. These observations raise the possibility that resveratrol may additionally modulate lifespan through inhibition of insulin signalling pathway, independently of its activation of SirT1 histone deacetylase. Furthermore, the present study may help to explain a wide range of biological effects of resveratrol, and provides further insight into the molecular basis of calorie restriction. PMID:16626303

  2. Transgene-Free Disease-Specific iPSC Generation from Fibroblasts and Peripheral Blood Mononuclear Cells.

    PubMed

    Fidan, Kerem; Ebrahimi, Ayyub; Ça?layan, Özlem H; Özçimen, Burcu; Önder, Tamer T

    2016-01-01

    Induced pluripotent stem cells (iPSCs) offer great promise as tools for basic biomedical research, disease modeling, and drug screening. In this chapter, we describe the generation of patient-specific, transgene-free iPSCs from skin biopsies and peripheral blood mononuclear cells through electroporation of episomal vectors and growth under two different culture conditions. The resulting iPSC lines are characterized with respect to pluripotency marker expression through immunostaining, tested for transgene integration by PCR, and assayed for differentiation capacity via teratoma formation. PMID:26126451

  3. Heterologous Expression of the AtDREB1A Gene in Transgenic Peanut-Conferred Tolerance to Drought and Salinity Stresses

    PubMed Central

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P.; Dobaria, Jentilal Ramjibhai

    2014-01-01

    Research on genetic transformation in various crop plants using the DREB1A transcription factor has shown better abiotic stress tolerance in transgenic crops. The AtDREB1A transgenic peanut (Arachis hypogaea L. cv. GG 20), which was previously developed, was characterized in terms of its physio-biochemical, molecular and growth parameters. The tolerance of this transgenic peanut to drought and salinity stresses was evaluated at the seedling (18 days old) and maturity stages. Transgenic peanut lines showed improved tolerance to both stresses over wild-type, as observed by delayed and less severe wilting of leaves and by improved growth parameters that were correlated with physio-biochemical parameters such as proline content, total chlorophyll content, osmotic potential, electrolytic leakage and relative water content. The expression pattern of the AtDREB1A gene evaluated using qPCR at different time points demonstrated that transgene expression was induced within two hours of stress imposition. The better performance of transgenic AtDREB1A peanut at the seedling stage and the improved growth parameters were due to the expression of the transgene, which is a transcription factor, and the possible up-regulation of various stress-inducible, downstream genes in the signal transduction pathway under abiotic stress. PMID:25545786

  4. Effect of straw leachates from Cry1Ca-expressing transgenic rice on the growth of Chlorella pyrenoidosa.

    PubMed

    Wang, Jiamei; Chen, Xiuping; Li, Yunhe; Zhu, Haojun; Ding, Jiatong; Peng, Yufa

    2014-05-01

    Because of the prevalence of algae in rice paddy fields, they will be exposed to Bacillus thurigiensis (Bt) proteins released from Bt protein-expressing genetically engineered rice. To assess the effects of leachates extracted from Cry1Ca-expressing transgenic rice (T1C-19) straw on the microalga Chlorella pyrenoidosa, the authors added purified Cry1Ca (10?µg/L, 100?µg/L, and 1000?µg/L) and 5 concentrations of diluted extracts (5%, 10%, 20%, 40%, and 80%) from T1C-19 and the nontransformed control strain Minghui 63 (MH63) to the medium of C. pyrenoidosa. The authors found that the growth curves of C. pyrenoidosa treated with purified Cry1Ca overlapped with the medium control; that the order of C. pyrenoidosa growth rates for the T1C-19 leachate concentrations was 5%?>?10%?>?20%?>?control?>?40%?>?80%, and for the MH63 concentrations the order was 5%?>?10%?>?control?>?20%?>?40%?>?80%, but there were no statistical differences between the 20% T1C-19 or 20% MH63 leachate treatment and the medium control on day 8; and that after 7 d of culture, Cry1Ca could be detected in C. pyrenoidosa treated with different concentrations of T1C-19 leachate. The results demonstrated that Cry1Ca protein released from T1C-19 rice can be absorbed into C. pyrenoidosa but that purified Cry1Ca and leachates from T1C-19 rice have no obvious adverse effects on the growth of C. pyrenoidosa. PMID:24478192

  5. Role of nutrient-sensing taste 1 receptor (T1R) family members in gastrointestinal chemosensing.

    PubMed

    Shirazi-Beechey, Soraya P; Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew W; Bravo, David

    2014-06-01

    Luminal nutrient sensing by G-protein-coupled receptors (GPCR) expressed on the apical domain of enteroendocrine cells activates intracellular pathways leading to secretion of gut hormones that control vital physiological processes such as digestion, absorption, food intake and glucose homeostasis. The taste 1 receptor (T1R) family of GPCR consists of three members: T1R1; T1R2; T1R3. Expression of T1R1, T1R2 and T1R3 at mRNA and protein levels has been demonstrated in the intestinal tissue of various species. It has been shown that T1R2-T1R3, in association with G-protein gustducin, is expressed in intestinal K and L endocrine cells, where it acts as the intestinal glucose (sweet) sensor. A number of studies have demonstrated that activation of T1R2-T1R3 by natural sugars and artificial sweeteners leads to secretion of glucagon-like peptides 1&2 (GLP-1 and GLP-2) and glucose dependent insulinotropic peptide (GIP). GLP-1 and GIP enhance insulin secretion; GLP-2 increases intestinal growth and glucose absorption. T1R1-T1R3 combination co-expressed on the apical domain of cholecystokinin (CCK) expressing cells is a luminal sensor for a number of L-amino acids; with amino acid-activation of the receptor eliciting CCK secretion. This article focuses on the role of the gut-expressed T1R1, T1R2 and T1R3 in intestinal sweet and L-amino acid sensing. The impact of exploiting T1R2-T1R3 as a nutritional target for enhancing intestinal glucose absorption and gut structural maturity in young animals is also highlighted. PMID:24382171

  6. Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

    PubMed Central

    2012-01-01

    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 analysis of mRNA expression. Conclusions In conclusion, dHuEPO tg mice caused excessive erythrocytosis that led to abnormal blood composition, short lifespan, and abnormal splenomegaly. Further, we identified 2,672 genes associated with splenomegaly by microarray analysis. These results could be useful in the development of dHuEPO-producing tg animals. PMID:22284751

  7. Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene.

    PubMed

    Stewart, C N; Adang, M J; All, J N; Boerma, H R; Cardineau, G; Tucker, D; Parrott, W A

    1996-09-01

    Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene. PMID:8819322

  8. RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains

    PubMed Central

    Adkar-Purushothama, Charith Raj; Kasai, Atsushi; Sugawara, Kohei; Yamamoto, Hideki; Yamazaki, Yuto; He, Ying-Hong; Takada, Nobuyuki; Goto, Hideki; Shindo, Sahori; Harada, Takeo; Sano, Teruo

    2015-01-01

    Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection. PMID:26656294

  9. Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures.

    PubMed

    Kim, Sun Ha; Ahn, Young Ock; Ahn, Mi-Jeong; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2013-09-01

    The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of ?-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands. PMID:23835362

  10. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    SciTech Connect

    Tomioka, Yukiko; Morimatsu, Masami; Nishijima, Ken-ichi; Usui, Tatsufumi; Yamamoto, Sayo; Suyama, Haruka; Ozaki, Kinuyo; Ito, Toshihiro; 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.

  11. Transgenic expression of walleye dermal sarcoma virus rv-cyclin gene in zebrafish and its suppressive effect on liver tumor development after carcinogen treatment.

    PubMed

    Zhan, Huiqing; Spitsbergen, Jan M; Qing, Wei; Wu, Yi Lian; Paul, Thomas A; Casey, James W; Her, Guor Muor; Gong, Zhiyuan

    2010-11-01

    A retrovirus homologue gene of cellular cyclin D?, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors. PMID:20052603

  12. Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

    PubMed Central

    Wan, Shen; Johnson, Amanda M; Altosaar, Illimar

    2012-01-01

    The nitrous oxide (N2O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N2O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N2OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N2OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N2OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N2O reduced min?1 g?1 root protein. Another event, plant line 1.9, also demonstrated high specific activity of N2OR, 13.2 µmol N2O reduced min?1 g?1 root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N2O that has continued to increase linearly (about 0.26% year?1) over the past half-century. PMID:22423324

  13. Inhibition of Wnt16 in human acute lymphoblastoid leukemia cells containing the t(1;19) translocation induces apoptosis.

    PubMed

    Mazieres, Julien; You, Liang; He, Biao; Xu, Zhidong; Lee, Amie Y; Mikami, Iwao; McCormick, Frank; Jablons, David M

    2005-08-11

    The Wnt family of secreted glycoproteins is widely involved in cell proliferation, differentiation and oncogenesis. Many Wnt signaling genes are upregulated and activated in chronic lymphocytic leukemia. Less is known concerning acute leukemia. One subtype of acute lymphoblastoid leukemia (ALL) is characterized by a t(1;19) chromosomal translocation resulting in a fusion protein E2A-Pbx1 that promotes transformation and leukemogenesis. Wnt16 has been shown to be targeted by E2A-Pbx1. We performed a differential gene expression array in acute leukemia cell lines displaying or not displaying the t(1;19) translocation. We found that Wnt16 and many Wnt signaling-related genes were upregulated in the translocation-containing cells. As two isoforms of Wnt16, Wnt16a and Wnt16b, have been recently identified, we demonstrated by using RT-PCR and Western blot that Wnt16b (and not Wnt16a) is overexpressed in t(1;19)-containing cell lines. We then directly addressed the role played by both isoforms in this type of leukemia. Using specific short interfering RNA (siRNA) and an anti-Wnt16 antibody, we showed that targeted-Wnt16b inhibition leads to apoptotic cell death. We also demonstrated that Wnt16b mediates its effect through the canonical Wnt pathway involving dishevelled-2, beta-catenin and survivin. We thus propose that Wnt16 plays an important role in leukemogenesis, raising its therapeutic interest. PMID:16007226

  14. Composition of transgenic soybean seeds with higher ?-linolenic acid content is equivalent to that of conventional control.

    PubMed

    Qin, Fengyun; Kang, Linzhi; Guo, Liqiong; Lin, Junfang; Song, Jingshen; Zhao, Yinhua

    2012-03-01

    ?-Linolenic acid (GLA) has been used as a general nutraceutical for pharmacologic applications, particularly in the treatment of skin conditions such as eczema. Four transgenic soybean lines that produce GLA at high yields (4.21% of total fatty acids, up to 1002-fold) were generated through the stable insertion of the Delta-6-fatty acid desaturase gene isolated from Borago officinalis into the genome of a conventional soybean cultivar. As part of the safety assessment of genetically engineered crops, the transgenic soybean seeds were compared with their parental soybean seeds (nontransgenic) by applying the principle of substantial equivalence. Compositional analyses were conducted by measuring the fatty acids, proximate analysis (moisture, crude protein, crude fat, carbohydrates, TDF, and ash contents), amino acids, lectins, and trypsin inhibitor activity. The present results showed that the specific transgenic cultivar studied was similar to the conventional control. PMID:22324875

  15. Functional Lung MRI in Chronic Obstructive Pulmonary Disease: Comparison of T1 Mapping, Oxygen-Enhanced T1 Mapping and Dynamic Contrast Enhanced Perfusion

    PubMed Central

    Jobst, Bertram J.; Triphan, Simon M. F.; Sedlaczek, Oliver; Anjorin, Angela; Kauczor, Hans Ulrich; Biederer, Jürgen; Ley-Zaporozhan, Julia; Ley, Sebastian; Wielpütz, Mark O.

    2015-01-01

    Purpose Monitoring of regional lung function in interventional COPD trials requires alternative endpoints beyond global parameters such as FEV1. T1 relaxation times of the lung might allow to draw conclusions on tissue composition, blood volume and oxygen fraction. The aim of this study was to evaluate the potential value of lung Magnetic resonance imaging (MRI) with native and oxygen-enhanced T1 mapping for the assessment of COPD patients in comparison with contrast enhanced perfusion MRI. Materials and Methods 20 COPD patients (GOLD I-IV) underwent a coronal 2-dimensional inversion recovery snapshot flash sequence (8 slices/lung) at room air and during inhalation of pure oxygen, as well as dynamic contrast-enhanced first-pass perfusion imaging. Regional distribution of T1 at room air (T1), oxygen-induced T1 shortening (?T1) and peak enhancement were rated by 2 chest radiologists in consensus using a semi-quantitative 3-point scale in a zone-based approach. Results Abnormal T1 and ?T1 were highly prevalent in the patient cohort. T1 and ?T1 correlated positively with perfusion abnormalities (r = 0.81 and r = 0.80; p&0.001), and with each other (r = 0.80; p<0.001). In GOLD stages I and II ?T1 was normal in 16/29 lung zones with mildly abnormal perfusion (15/16 with abnormal T1). The extent of T1 (r = 0.45; p<0.05), ?T1 (r = 0.52; p<0.05) and perfusion abnormalities (r = 0.52; p<0.05) showed a moderate correlation with GOLD stage. Conclusion Native and oxygen-enhanced T1 mapping correlated with lung perfusion deficits and severity of COPD. Under the assumption that T1 at room air correlates with the regional pulmonary blood pool and that oxygen-enhanced T1 reflects lung ventilation, both techniques in combination are principally suitable to characterize ventilation-perfusion imbalance. This appears valuable for the assessment of regional lung characteristics in COPD trials without administration of i.v. contrast. PMID:25822195

  16. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits

    PubMed Central

    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; B?sze, Zsuzsanna; Rülicke, Thomas; Izsvák, Zsuzsanna

    2013-01-01

    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.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., B?sze, Z., Rülicke, T., Izsvák, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits. PMID:23195032

  17. A longitudinal FDG-PET study of transgenic mice overexpressing GSK- 3? in the brain.

    PubMed

    de Cristóbal, Javier; Garcia-Garcia, Luis; Delgado, Mercedes; Pozo, Miguel A; Medina, Miguel

    2014-02-01

    Increased Glycogen synthase kinase-3 (GSK-3) activity is believed to contribute to the etiology of chronic disorders such as Alzheimer's disease, one of the earliest diseases linked to GSK-3 dysfunction. Numerous mouse models with modified GSK-3 have been generated in order to study the physiology of GSK-3, its implication in diverse pathologies and the potential effect of GSK-3 inhibitors. In this study we have characterised and evaluated the brain metabolic changes induced by GSK-3? overexpression in transgenic mice throughout their lifespan. The conditional Tet/GSK-3? transgenic line used in this study has been previously extensively characterized at the pathological, biochemical and cognitive levels. Now we have investigated the effect GSK-3? overexpression on the (18)F-fluoro-deoxyglucose (FDG) uptake by positron emission tomography (PET), taking advantage from this non-invasive technique which has allowed us to track individually the same animals throughout their lives. The results obtained during the longitudinal analysis showed a reduction of metabolic activity in several brain regions, such as cortex, striatum and hippocampus, consistent with the areas where the transgene is being expressed. The reduction of the metabolic activity in these mice is observed from the first time point, performed at the age of 3 months, and maintained throughout the whole study, until the oldest age tested (19 months). This effect seems to be reverted in a satellite group of 3-month transgenic animals treated with the classical GSK-3 inhibitor lithium, as they show higher FDG uptake values compared with untreated age-matched transgenic animals. PMID:23905999

  18. Manipulation of the Rice L-Galactose Pathway: Evaluation of the Effects of Transgene Overexpression on Ascorbate Accumulation and Abiotic Stress Tolerance

    PubMed Central

    Zhang, Gui-Yun; Liu, Ru-Ru; Zhang, Chang-Quan; Tang, Ke-Xuan; Sun, Ming-Fa; Yan, Guo-Hong; Liu, Qiao-Quan

    2015-01-01

    Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress. PMID:25938231

  19. Transgenes for insect resistance reduce herbivory and enhance fecundity in advanced generations of crop–weed hybrids of rice

    PubMed Central

    Yang, Xiao; Xia, Hui; Wang, Wei; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2011-01-01

    Gene flow from transgenic crops allows novel traits to spread to sexually compatible weeds. Traits such as resistance to insects may enhance the fitness of weeds, but few studies have tested for these effects under natural field conditions. We created F2 and F3 crop–weed hybrid lineages of genetically engineered rice (Oryza sativa) using lines with two transgene constructs, cowpea trypsin inhibitor (CpTI) and a Bt transgene linked to CpTI (Bt/CpTI). Experiments conducted in Fuzhou, China, demonstrated that CpTI alone did not significantly affect fecundity, although it reduced herbivory. In contrast, under certain conditions, Bt/CpTI conferred up to 79% less insect damage and 47% greater fecundity relative to nontransgenic controls, and a 44% increase in fecundity relative to the weedy parent. A small fitness cost was detected in F3 progeny with Bt/CpTI when grown under low insect pressure and direct competition with transgene-negative controls. We conclude that Bt/CpTI transgenes may introgress into co-occurring weedy rice populations and contribute to greater seed production when target insects are abundant. However, the net fitness benefits that are associated with Bt/CpTI could be ephemeral if insect pressure is lacking, for example, because of widespread planting of Bt cultivars that suppress target insect populations. PMID:25568014

  20. Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons.

    PubMed

    Kim, Shin-Il; Oceguera-Yanez, Fabian; Sakurai, Chiho; Nakagawa, Masato; Yamanaka, Shinya; Woltjen, Knut

    2016-01-01

    Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs. PMID:26025620

  1. Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

    PubMed

    Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita

    2012-10-01

    Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ? 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes. PMID:22733208

  2. Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana.

    PubMed

    Yuan, Xiaowei; Li, Yaxiao; Liu, Shiyang; Xia, Fei; Li, Xinzheng; Qi, Baoxiu

    2014-04-01

    IgASE1, a C?? ?(9)-specific polyunsaturated fatty acid elongase from the marine microalga Isochrysis galbana, is able to convert linoleic acid and ?-linolenic acid to eicosadienoic acid and eicosatrienoic acid in Arabidopsis. Eicosadienoic acid and eicosatrienoic acid are precursors of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which are synthesized via the ?(8) desaturation biosynthetic pathways. This study shows that the IgASE1-expressing transgenic Arabidopsis exhibited altered morphology (decreased leaf area and biomass) and enhanced drought resistance compared to wild-type plants. The transgenic Arabidopsis were hypersensitive to abscisic acid (ABA) during seed germination, post-germination growth, and seedling development. They had elevated leaf ABA levels under well-watered and dehydrated conditions and their stomata were more sensitive to ABA. Exogenous application of eicosadienoic acid and eicosatrienoic acid can mimic ABA and drought responses in the wild type plants, similar to that found in the transgenic ones. The transcript levels of genes involved in the biosynthesis of ABA (NCED3, ABA1, AAO3) as well as other stress-related genes were upregulated in this transgenic line upon osmotic stress (300 mM mannitol). Taken together, these results indicate that these two eicosapolyenoic acids or their derived metabolites can mitigate the effects of drought in transgenic Arabidopsis, at least in part, through the action of ABA. PMID:24609499

  3. Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

    PubMed

    Melo, C H; Sousa, F C; Batista, R I P T; Sanchez, D J D; Souza-Fabjan, J M G; Freitas, V J F; Melo, L M; Teixeira, D I A

    2015-01-01

    The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia. PMID:26345799

  4. Transgenes for insect resistance reduce herbivory and enhance fecundity in advanced generations of crop-weed hybrids of rice.

    PubMed

    Yang, Xiao; Xia, Hui; Wang, Wei; Wang, Feng; Su, Jun; Snow, Allison A; Lu, Bao-Rong

    2011-09-01

    Gene flow from transgenic crops allows novel traits to spread to sexually compatible weeds. Traits such as resistance to insects may enhance the fitness of weeds, but few studies have tested for these effects under natural field conditions. We created F 2 and F 3 crop-weed hybrid lineages of genetically engineered rice (Oryza sativa) using lines with two transgene constructs, cowpea trypsin inhibitor (CpTI) and a Bt transgene linked to CpTI (Bt/CpTI). Experiments conducted in Fuzhou, China, demonstrated that CpTI alone did not significantly affect fecundity, although it reduced herbivory. In contrast, under certain conditions, Bt/CpTI conferred up to 79% less insect damage and 47% greater fecundity relative to nontransgenic controls, and a 44% increase in fecundity relative to the weedy parent. A small fitness cost was detected in F 3 progeny with Bt/CpTI when grown under low insect pressure and direct competition with transgene-negative controls. We conclude that Bt/CpTI transgenes may introgress into co-occurring weedy rice populations and contribute to greater seed production when target insects are abundant. However, the net fitness benefits that are associated with Bt/CpTI could be ephemeral if insect pressure is lacking, for example, because of widespread planting of Bt cultivars that suppress target insect populations. PMID:25568014

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

    PubMed Central

    2010-01-01

    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. PMID:20525262

  6. The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin.

    PubMed

    Ohta, Keisuke; Masuda, Tetsuya; Tani, Fumito; Kitabatake, Naofumi

    2011-03-18

    Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2-T1R3. Chimeric human- mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2- mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners. PMID:21329673

  7. TRANSFECTION OF A RAT CYTOCHROME P-450B CDNA INTO C3H10T1/2CL8 MOUSE EMBRYO FIBROBLASTS

    EPA Science Inventory

    A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. his bifunctional plasmid (pJRSL100) was transfected into the C3H10T1/2CL8 mouse embryo fibroblast cell line. 418-r...

  8. The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development

    PubMed Central

    Beck, Laurent; Leroy, Christine; Beck-Cormier, Sarah; Forand, Anne; Salaün, Christine; Paris, Nadine; Bernier, Adeline; Ureńa-Torres, Pablo; Prié, Dominique; Ollero, Mario; Coulombel, Laure; Friedlander, Gérard

    2010-01-01

    Background PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na+-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing. Methodology/Principal Findings To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5–E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation. Conclusion/Significance This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth. PMID:20161774

  9. Selection of Thymocytes Expressing Transgenic TCR Specific for a Minor Histocompatibility Antigen, H60

    PubMed Central

    Ju, Ji-Min; Kim, Min Bum; Ryu, Su Jeong; Kim, Joo Young; Chang, Jun

    2015-01-01

    Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2Kb, and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8+ single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR. PMID:26557806

  10. Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress.

    PubMed

    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. PMID:26160315

  11. MAIN-BELT COMET P/2012 T1 (PANSTARRS)

    SciTech Connect

    Hsieh, Henry H.; Kaluna, Heather M.; Yang Bin; Haghighipour, Nader; Micheli, Marco; Denneau, Larry; Jedicke, Robert; Kleyna, Jan; Veres, Peter; Wainscoat, Richard J.; Ansdell, Megan; Elliott, Garrett T.; Keane, Jacqueline V.; Meech, Karen J.; Riesen, Timm E.; Sonnett, Sarah; Novakovic, Bojan; Fitzsimmons, Alan; Moskovitz, Nicholas A.; Sheppard, Scott S.; and others

    2013-07-01

    We present initial results from observations and numerical analyses aimed at characterizing the main-belt comet P/2012 T1 (PANSTARRS). Optical monitoring observations were made between 2012 October and 2013 February using the University of Hawaii 2.2 m telescope, the Keck I telescope, the Baade and Clay Magellan telescopes, Faulkes Telescope South, the Perkins Telescope at Lowell Observatory, and the Southern Astrophysical Research Telescope. The object's intrinsic brightness approximately doubles from the time of its discovery in early October until mid-November and then decreases by {approx}60% between late December and early February, similar to photometric behavior exhibited by several other main-belt comets and unlike that exhibited by disrupted asteroid (596) Scheila. We also used Keck to conduct spectroscopic searches for CN emission as well as absorption at 0.7 {mu}m that could indicate the presence of hydrated minerals, finding an upper limit CN production rate of Q{sub CN} < 1.5 Multiplication-Sign 10{sup 23} mol s{sup -1}, from which we infer a water production rate of Q{sub H{sub 2O}}<5 Multiplication-Sign 10{sup 25} mol s{sup -1}, and no evidence of the presence of hydrated minerals. Numerical simulations indicate that P/2012 T1 is largely dynamically stable for >100 Myr and is unlikely to be a recently implanted interloper from the outer solar system, while a search for potential asteroid family associations reveals that it is dynamically linked to the {approx}155 Myr old Lixiaohua asteroid family.

  12. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    PubMed Central

    Guo, Jinlong; Gao, Shiwu; Lin, Qinliang; Wang, Hengbo; Que, Youxiong; Xu, Liping

    2015-01-01

    As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423?bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms. PMID:25685813

  13. Competitive Expression of Endogenous Wheat CENH3 May Lead to Suppression of Alien ZmCENH3 in Transgenic Wheat × Maize Hybrids.

    PubMed

    Chen, Wei; Zhu, Qilin; Wang, Haiyan; Xiao, Jin; Xing, Liping; Chen, Peidu; Jin, Weiwei; Wang, Xiu-E

    2015-11-20

    Uniparental chromosome elimination in wheat × maize hybrid embryos is widely used in double haploid production of wheat. Several explanations have been proposed for this phenomenon, one of which is that the lack of cross-species CENH3 incorporation may act as a barrier to interspecies hybridization. However, it is unknown if this mechanism applies universally. To study the role of CENH3 in maize chromosome elimination of wheat × maize hybrid embryos, maize ZmCENH3 and wheat ?TaCENH3-B driven by the constitutive CaMV35S promoter were transformed into wheat variety Yangmai 158. Five transgenic lines for ZmCENH3 and six transgenic lines for ?TaCENH3-B were identified. RT-PCR analysis showed that the transgene could be transcribed at a low level in all ZmCENH3 transgenic lines, whereas transcription of endogenous wheat CENH3 was significantly up-regulated. Interestingly, the expression levels of both wheat CENH3 and ZmCENH3 in the ZmCENH3 transgenic wheat × maize hybrid embryos were higher than those in the non-transformed Yangmai 158 × maize hybrid embryos. This indicates that the alien ZmCENH3 in wheat may induce competitive expression of endogenous wheat CENH3, leading to suppression of ZmCENH3 over-expression. Eliminations of maize chromosomes in hybrid embryos of ZmCENH3 transgenic wheat × maize and Yangmai 158 × maize were compared by observations on micronuclei presence, by marker analysis using maize SSRs (simple sequence repeats), and by FISH (fluorescence in situ hybridization) using 45S rDNA as a probe. The results indicate that maize chromosome elimination events in the two crosses are not significantly different. Fusion protein ZmCENH3-YFP could not be detected in ZmCENH3 transgenic wheat by either Western blotting or immnunostaining, whereas accumulation and loading of the ?TaCENH3-B-GFP fusion protein was normal in ?TaCENH3-B transgenic lines. As ZmCENH3-YFP did not accumulate after AM114 treatment, we speculate that low levels of ZmCENH3 protein in transgenic wheat may be one of the factors that lead to failure of suppression of maize chromatin elimination in ZmCENH3 transgenic wheat × maize hybrids. PMID:26674381

  14. Transgenic oil palm: production and projection.

    PubMed

    Parveez, G K; Masri, M M; Zainal, A; Majid, N A; Yunus, A M; Fadilah, H H; Rasid, O; Cheah, S C

    2000-12-01

    Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020. PMID:11171275

  15. An Apo-14 Promoter-Driven Transgenic Zebrafish That Marks Liver Organogenesis

    PubMed Central

    Wang, Rui; Li, Zhi; Wang, Yang; Gui, Jian-Fang

    2011-01-01

    Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5?-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16–17 hpf, a typical “salt-and-pepper” expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis. PMID:21799896

  16. Using metabolomics to estimate unintended effects in transgenic crop plants: problems, promises and opportunities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic crops are widespread in some countries and sectors of the agro-economy, but are also highly contentious. Proponents of transgenic crop improvement often cite the “substantial equivalence” of transgenic crops to their non-transgenic parents and sibling varieties. Opponents of transgenic cr...

  17. Transporter mRNA expression in a conditionally immortalized rat small intestine epithelial cell line (TR-SIE).

    PubMed

    Hosoya, Ken-ichi; Tomi, Masatoshi; Takayama, Megumi; Komokata, Yuko; Nakai, Daisuke; Tokui, Taro; Nishimura, Kenji; Ueda, Masatsugu; Obinata, Masuo; Hori, Satoko; Ohtsuki, Sumio; Amidon, Gordon L; Terasaki, Tetsuya

    2004-08-01

    Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters. PMID:15499194

  18. Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol.

    PubMed

    Oraby, Hesham; Venkatesh, Balan; Dale, Bruce; Ahmad, Rashid; Ransom, Callista; Oehmke, James; Sticklen, Mariam

    2007-12-01

    The catalytic domain of Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1,4-beta-glucanase enzyme or E1) was constitutively expressed in rice. Molecular analyses of T1 plants confirmed presence and expression of the transgene. The amount of E1 enzyme accounted for up to 4.9% of the plant total soluble proteins, and its accumulation had no apparent deleterious effects on plant growth and development. Approximately 22 and 30% of the cellulose of the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize biomass respectively was converted into glucose using rice E1 heterologous enzyme. As rice is the major food crop of the world with minimal use for its straw, our results suggest a successful strategy for producing biologically active hydrolysis enzymes in rice to help generate alcohol fuel, by substituting the wasteful and polluting practice of rice straw burning with an environmentally friendly technology. PMID:17237981

  19. Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon.

    PubMed

    Kendig, Derek M; Hurst, Norman R; Bradley, Zachary L; Mahavadi, Sunila; Kuemmerle, John F; Lyall, Vijay; DeSimone, John; Murthy, Karnam S; Grider, John R

    2014-12-01

    Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5'-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1(-/-) mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. PMID:25324508

  20. Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon

    PubMed Central

    Kendig, Derek M.; Hurst, Norman R.; Bradley, Zachary L.; Mahavadi, Sunila; Kuemmerle, John F.; Lyall, Vijay; DeSimone, John; Murthy, Karnam S.

    2014-01-01

    Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5?-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1?/? mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion. PMID:25324508

  1. Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion

    PubMed Central

    Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew; Marcello, Marco; Ninomiya, Yuzo

    2013-01-01

    CCK is secreted by endocrine cells of the proximal intestine in response to dietary components, including amino acids. CCK plays a variety of roles in digestive processes, including inhibition of food intake, consistent with a role in satiety. In the lingual epithelium, the sensing of a broad spectrum of l-amino acids is accomplished by the heteromeric amino acid (umami) taste receptor (T1R1-T1R3). T1R1 and T1R3 subunits are also expressed in the intestine. A defining characteristic of umami sensing by T1R1-T1R3 is its potentiation by IMP or GMP. Furthermore, T1R1-T1R3 is not activated by Trp. We show here that, in response to l-amino acids (Phe, Leu, Glu, and Trp), but not d-amino acids, STC-1 enteroendocrine cells and mouse proximal small intestinal tissue explants secrete CCK and that IMP enhances Phe-, Leu-, and Glu-induced, but not Trp-induced, CCK secretion. Furthermore, small interfering RNA inhibition of T1R1 expression in STC-1 cells results in significant diminution of Phe-, Leu-, and Glu-stimulated, but not Trp-stimulated, CCK release. In STC-1 cells and mouse intestine, gurmarin inhibits Phe-, Leu-, and Glu-induced, but not Trp-stimulated, CCK secretion. In contrast, the Ca2+-sensing receptor antagonist NPS2143 inhibits Phe-stimulated CCK release partially and Trp-induced CCK secretion totally in mouse intestine. However, NPS2143 has no effect on Leu- or Glu-induced CCK secretion. Collectively, our data demonstrate that functional characteristics and cellular location of the gut-expressed T1R1-T1R3 support its role as a luminal sensor for Phe-, Leu-, and Glu-induced CCK secretion. PMID:23203156

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

    PubMed Central

    2010-01-01

    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. PMID:21040591

  3. Control of Pollen-Mediated Gene Flow in Transgenic Trees[W][OA

    PubMed Central

    Zhang, Chunsheng; Norris-Caneda, Kim H.; Rottmann, William H.; Gulledge, Jon E.; Chang, Shujun; Kwan, Brian Yow-Hui; Thomas, Anita M.; Mandel, Lydia C.; Kothera, Ronald T.; Victor, Aditi D.; Pearson, Leslie; Hinchee, Maud A.W.

    2012-01-01

    Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees. PMID:22723085

  4. The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model.

    PubMed

    Ravelonandro, M; Scorza, R; Callahan, A; Levy, L; Jacquet, C; Monsion, M; Damsteegt, V

    2000-11-01

    Sharka or plum pox, caused by Plum pox virus (PPV: genus Potyvirus; Family Potyviridae), is the most serious disease of Prunus. Most cultivated Prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties. Success in developing virus-resistant herbaceous crops through genetic engineering led us to investigate this approach for resistance to PPV. Our programme aims to develop a biotechnological approach to PPV control that is effective and shown to be environmentally safe. The programme began with the cloning of the PPV coat protein (CP) gene and the development of a transformation system for plum (Prunus domestica). The CP construct was first tested in Nicotiana benthamiana in which it proved effective in producing transgenic plants with varying levels of CP expression. Some of these plants, particularly low PPV CP expressers, were resistant to PPV, or recovered from initial infection. Based on these results plum was transformed using the Agrobacterium tumefaciens system and both low and high PPV CP-expressing transgenic plum lines were obtained. These were inoculated with PPV by bud grafts in the greenhouse. Line C-5 proved to be highly resistant. It contained multiple copies of the insert, produced low levels of PPV CP mRNA, no detectable CP and the insert appeared to be methylated. These characteristics all suggest that the resistance of the C-5 clone is based on post-transcriptional gene silencing (PTGS). Field tests of C-5 and other transgenic lines in Poland, Romania and Spain have demonstrated that such trees when inoculated by bud-grafts allow a low level of PPV multiplication, from which they rapidly recover. C-5 plants exposed to natural infection for 3 years did not become infected, whereas control trees were infected in the first year. Hybrid plums having the C-5 PPV CP insert inherited from C-5 are virus-resistant, demonstrating the usefulness of C-5 as a parent in developing new PPV-resistant plum varieties. Research is in progress on the biorisks of PPV CP transgenic plants. Gene constructs that either produce no CP or CP that cannot be transmitted by aphids have been developed, tested in N. benthamiana and transferred to plum. Studies have begun on the potential for synergistic interactions between the PPV CP gene and the other common viruses of Prunus spp. In the future we will be participating in investigating the toxicity or/and the allergenicity of transgenic fruit products and, more importantly, transgenic lines will be developed that express transgenes only in vegetative parts of the plant and not in the fruit. PMID:11137162

  5. Characterization of novel nitrate reductase-deficient mutants for transgenic Dunaliella salina systems.

    PubMed

    Gao, L J; Jia, Y L; Li, S K; Qiu, L L

    2015-01-01

    The aim of the present study was to isolate and characterize novel nitrate reductase (NR)-deficient mutants, which may be useful for the transgenic manipulation of Dunaliella salina. Three NR-deficient mutants of D. salina, J-1, J-2, and J-3, were successfully isolated by screening for chlorate resistance after chemical mutagenesis with ethylnitrosourea. NR activity was not detected in the mutants and the expression of NR mRNA was significantly decreased. Growth analysis of D. salina strains grown in media containing different nitrogen sources revealed that these mutants were capable of utilizing nitrite and urea, but not nitrate as a nitrogen source, indicating that these mutants are indeed NR-deficient. Mutation analysis of NR cDNA sequences revealed that there were 11 point mutations shared by the J-1, J-2, and J-3 mutants. Furthermore, the results of the functional complementation experiment showed that NR activity of transformant T-1 derived from J-1 was recovered to 48.1 % of that of the wild-type D. salina. The findings of the present study indicate that nitrate may be used as a selective agent rather than antibiotics or herbicides for the isolated NR-deficient mutants in future transgenic D. salina systems. PMID:26535642

  6. Potential of MuS1 Transgenic Tobacco for Phytoremediation of the Urban Soils Contaminated with Cadmium

    NASA Astrophysics Data System (ADS)

    Kim, K. H.; Kim, Y. N.; Kim, S. H.

    2010-05-01

    Urban soils are prone to contamination by trace elements such as Cd, Cu, Pb and Zn. Phytoremediation is one of the attractive remediation methods for soils contaminated with trace elements due to its non-destructive and environmentally-friendly characteristic. Scientists have tried to find hyper-accumulator plants in nature or to develop transgenic plant through genetic engineering. This study was carried out to identify a potential of MuS1 transgenic tobacco for phytoremediation of the urban soils contaminated with Cd. MuS1 is known as a multiple stress related gene with several lines. The previous study using RT-PCR showed that the expression of MuS1 gene in tobacco plant induced tolerance to Cd stress. For this study, MuS1 transgenic tobacco and wild-type tobacco (control) were cultivated in a hydroponic system treated with Cd (0, 50, 100 and 200?M Cd) for 3 weeks. At harvest, both tobacco and nutrient solution were collected and were analyzed for Cd. Effect of Cd treatment on morphological change of the tobacco leaves was also observed by variable-pressure scanning electron microscopy (VP-SEM). The tolerance of MuS1 transgenic tobacco to Cd stress was better than that of wild-type tobacco at all Cd levels. Especially, wild-type tobacco showed chlorosis and withering with 200?M Cd treatment, whereas MuS1 transgenic tobacco gradually recovered from Cd damage. Wild-type tobacco accumulated more Cd (4.65mg per plant) than MuS1 transgenic tobacco (2.37mg per plant) with 200?M Cd treatment. Cd translocation rate from root to leaves was 81.8 % for wild-type tobacco compared to 37.1 % for MuS1 transgenic tobacco. Result of VP-SEM showed that the number of trichome in the leaves for wild-type tobacco increased in comparison with that for untreated samples after 3 weeks, while that for MuS1 transgenic tobacco was not changed by Cd treatment. Results showed that the mechanism of the recovery of the MuS1 tobacco plant was not by high level of Cd uptake and accumulation in the plant but by revealing resistance to Cd through inducing less Cd uptake and/or more Cd immobilization around roots, resulting in less translocation to shoot. In conclusion, this study showed a potential to use MuS1 transgenic tobacco for phytoremediation of the urban soils contaminated with Cd.

  7. Increased Apopototic Chondrocytes in Articular Cartilage of Adults Heterozygous SirT1 Mice

    PubMed Central

    Gabay, Odile; Oppenhiemer, Hanna; Meir, Hadar; Zaal, Kristien; Sanchez, Christelle; Dvir-Ginzberg, Mona

    2012-01-01

    Objective A growing body of evidence indicates that the protein deacetylase SirT1 affects chondrocyte biology and survival. This report aims to evaluate in-vivo effects of SirT1 on cartilage biology in 9 month-old mice 129/J murine strains. Methods Heterozygous (SirT1+/?) and wild type (WT; SirT1+/+) mice were systematically compared for musculoskeletal features, scored for OA severity and chondrocyte viability in articular cartilage at 1 month and 9 months post-natally. Sections of femoro-tibial joints were stained for type II collagen, aggrecan and active caspase 3. Articular murine chondrocytes were isolated and immunoblotted for SirT1 and active caspase 3. Results Phenotypic observations show that at the age of 1 month SirT1+/? mice were smaller than the wild type and presented a significant reduction in full-length SirT1 (FLSirT1; 110kDa) protein levels. Nine month-old SirT1+/? and WT mice did not express FLSirT1, however immunoblot assays of 9M articular cartilage-derived protein extracts revealed the inactive cleaved variant of SirT1 (75SirT1; 75kDa) was decreased in the SirT1+/? compared to WT mice. Nine month-old SirT1+/? mice possessed enhanced OA and enhanced levels of active caspase 3, compared to age-matched WT mice. Conclusion The data suggest that the presence of 75SirT1 may prolong viability of articular chondrocytes in adult 9 month-old mice. PMID:22258484

  8. Muscle regulatory factors regulate T1R3 taste receptor expression.

    PubMed

    Kokabu, Shoichiro; Lowery, Jonathan W; Toyono, Takashi; Seta, Yuji; Hitomi, Suzuro; Sato, Tsuyoshi; Enoki, Yuichiro; Okubo, Masahiko; Fukushima, Yosuke; Yoda, Tetsuya

    2015-12-25

    T1R3 is a T1R class of G protein-coupled receptors, composing subunit of the umami taste receptor when complexed with T1R1. T1R3 was originally discovered in gustatory tissue but is now known to be expressed in a wide variety of tissues and cell types such the intestine, pancreatic ?-cells, skeletal muscle, and heart. In addition to taste recognition, the T1R1/T1R3 complex functions as an amino acid sensor and has been proposed to be a control mechanism for the secretion of hormones, such as cholecystokinin, insulin, and duodenal HCO3(-) and activates the mammalian rapamycin complex 1 (MTORC1) to inhibit autophagy. T1R3 knockout mice have increased rate of autophagy in the heart, skeletal muscle and liver. Thus, T1R3 has multiple physiological functions and is widely expressed in vivo. However, the exact mechanisms regulating T1R3 expression are largely unknown. Here, we used comparative genomics and functional analyses to characterize the genomic region upstream of the annotated transcriptional start of human T1R3. This revealed that the T1R3 promoter in human and mouse resides in an evolutionary conserved region (ECR). We also identified a repressive element located upstream of the human T1R3 promoter that has relatively high degree of conservation with rhesus macaque. Additionally, the muscle regulatory factors MyoD and Myogenin regulate T1R3 expression and T1R3 expression increases with skeletal muscle differentiation of murine myoblast C2C12 cells. Taken together, our study raises the possibility that MyoD and Myogenin might control skeletal muscle metabolism and homeostasis through the regulation of T1R3 promoter activity. PMID:26545778

  9. Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

    PubMed Central

    Khan, Kasim; Agarwal, Pallavi; Shanware, Arti; Sane, Vidhu Aniruddha

    2015-01-01

    Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils. PMID:26067295

  10. T1R2 and T1R3 subunits are individually unnecessary for normal affective licking responses to Polycose: implications for saccharide taste receptors in mice.

    PubMed

    Treesukosol, Yada; Blonde, Ginger D; Spector, Alan C

    2009-04-01

    The T1R2 and T1R3 proteins are expressed in taste receptor cells and form a heterodimer binding with compounds described as sweet by humans. We examined whether Polycose taste might be mediated through this heterodimer by testing T1R2 knockout (KO) and T1R3 KO mice and their wild-type (WT) littermate controls in a series of brief-access taste tests (25-min sessions with 5-s trials). Sucrose, Na-saccharin, and Polycose were each tested for three consecutive sessions with order of presentation varied among subgroups in a Latin-Square manner. Both KO groups displayed blunted licking responses and initiated significantly fewer trials of sucrose and Na-saccharin across a range of concentrations. KO mice tested after Polycose exposure demonstrated some degree of concentration-dependent licking of sucrose, likely attributable to learning related to prior postingestive experience. These results are consistent with prior findings in the literature, implicating the T1R2+3 heterodimer as the principal taste receptor for sweet-tasting ligands, and also provide support for the potential of postingestive experience to influence responding in the KO mice. In contrast, T1R2 KO and T1R3 KO mice displayed concentration-dependent licking responses to Polycose that tracked those of their WT controls and in some cases licked midrange concentrations more; the number of Polycose trials initiated overall did not differ between KO and WT mice. Thus, the T1R2 and T1R3 proteins are individually unnecessary for normal concentration-dependent licking of Polycose to be expressed in a brief-access test. Whether at least one of these T1R protein subunits is necessary for normal Polycose responsiveness remains untested. Alternatively, there may be a novel taste receptor(s) that mediates polysaccharide taste. PMID:19158407

  11. Main-Belt Comet P/2012 T1 (PANSTARRS)

    E-print Network

    Hsieh, Henry H; Novakovic, Bojan; Yang, Bin; Haghighipour, Nader; Micheli, Marco; Denneau, Larry; Fitzsimmons, Alan; Jedicke, Robert; Kleyna, Jan; Veres, Peter; Wainscoat, Richard J; Ansdell, Megan; Elliott, Garrett T; Keane, Jacqueline V; Meech, Karen J; Moskovitz, Nicholas A; Riesen, Timm E; Sheppard, Scott S; Sonnett, Sarah; Tholen, David J; Urban, Laurie; Kaiser, Nick; Chambers, K C; Burgett, William S; Magnier, Eugene A; Morgan, Jeffrey S; Price, Paul A

    2013-01-01

    We present initial results from observations and numerical analyses aimed at characterizing main-belt comet P/2012 T1 (PANSTARRS). Optical monitoring observations were made between October 2012 and February 2013 using the University of Hawaii 2.2 m telescope, the Keck I telescope, the Baade and Clay Magellan telescopes, Faulkes Telescope South, the Perkins Telescope at Lowell Observatory, and the Southern Astrophysical Research (SOAR) telescope. The object's intrinsic brightness approximately doubles from the time of its discovery in early October until mid-November and then decreases by ~60% between late December and early February, similar to photometric behavior exhibited by several other main-belt comets and unlike that exhibited by disrupted asteroid (596) Scheila. We also used Keck to conduct spectroscopic searches for CN emission as well as absorption at 0.7 microns that could indicate the presence of hydrated minerals, finding an upper limit CN production rate of QCN100 Myr and is unlikely to be a rec...

  12. Clinical implications of proliferation activity in T1 or T2 male gastric cancer patients

    PubMed Central

    Kim, Young-Woo; Eom, Bang Wool; Kook, Myeong-Cherl; Kim, Han-Seong; Kim, Mi-Kyung; Hwang, Hai-Li; Chandra, Vishal; Poojan, Shiv; Song, Yura; Koh, Jae-Soo; Bae, Chang-Dae; Ro, Jungsil; Hong, Kyeong-Man

    2015-01-01

    Proliferation activity has already been established as a prognostic marker or as a marker for anticancer drug sensitivity. In gastric cancer, however, the prognostic significance of proliferation activity is still being debated. Several studies evaluating proliferation activity using Ki-67 have shown controversial results in terms of the relationship between proliferation activity and overall survival (OS) or drug sensitivity in gastric cancer patients. Because cytoskeleton-associated protein 2 (CKAP2) staining has recently been introduced as a marker of proliferation activity, we analyzed 437 gastric cancer tissues through CKAP2 immunohistochemistry, and we evaluated the chromatin CKAP2-positive cell count (CPCC) for proliferation activity. Although the CPCC did not show any significant correlation with OS in the male, female or total number of cases, it did show a significant correlation in the T1 or T2 male patient subgroup, according to log-rank tests (P=0.001) and univariate analysis (P=0.045). Additionally, multivariate analysis with the Cox proportional hazard regression model showed a significant correlation between the CPCC and OS (P=0.039) for the co-variables of age, gender, T stage, N stage, histology, tumor location, tumor size and adjuvant chemotherapy. In male gastric cancer cell lines, faster-growing cancer cells showed higher sensitivity to cisplatin than slow-growing cells. Thus our study indicates that CPCC-measured proliferation activity demonstrates a significantly worse prognosis in T1 or T2 male gastric cancer patients. The CPCC will help to more precisely classify gastric cancer patients and to select excellent candidates for adjuvant chemotherapy, which in turn will facilitate further clinical chemotherapeutic trials. PMID:26542785

  13. Spermidine levels are implicated in heavy metal tolerance in a spermidine synthase overexpressing transgenic European pear by exerting antioxidant activities.

    PubMed

    Wen, Xiao-Peng; Ban, Yusuke; Inoue, Hiromichi; Matsuda, Narumi; Moriguchi, Takaya

    2010-02-01

    To verify whether spermidine synthase (SPDS) can confer long-term multi-heavy metal tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. 'Ballad') line #32 overexpressing apple SPDS (MdSPDS1), as well as a wild type (WT) line, were subjected to stress using either CdCl(2), PbCl(2), ZnCl(2), or a combination thereof. Based on either shoot height increment or fresh weight and morphological changes upon heavy metal stress, the performance of the transgenic line #32 was better than that of WT. Although SPDS expression levels and spermidine (Spd) contents in line #32 were higher than those in WT, possibly due to transgene (MdSPDS1) expression, no obvious inductions of SPDS expression and increases in Spd-content were observed by long-term stress treatments in both lines. When the glutathione (GSH) content was compared with or without stress in each line, GSH was significantly depleted in line #32 with stress, but not as much as in WT. The activities of glutathione reductase and superoxide dismutase and the content of malondialdehyde, an indicator for lipid peroxidation, changed upon stress toward a more favorable status for survival in line #32 than in WT. These antioxidant parameters were positively related to Spd-content. The accumulation of heavy metals tended to be less in line #32 than in WT except for Zn stress, and the Ca content showed an opposite trend. These results suggest that Spd-levels are implicated in enhanced heavy metal tolerance, possibly by exerting an antioxidant activity as well as by the properties of Spd per se including metal chelator. PMID:19544002

  14. Comparisons of EPR imaging and T 1-weighted MRI for efficient imaging of nitroxyl contrast agents

    NASA Astrophysics Data System (ADS)

    Matsumoto, Ken-ichiro; Narazaki, Michiko; Ikehira, Hiroo; Anzai, Kazunori; Ikota, Nobuo

    2007-07-01

    The resolution and signal to noise ratio of EPR imaging and T 1-weighted MRI were compared using an identical phantom. Several solutions of nitroxyl contrast agents with different EPR spectral shapes were tested. The feasibility of T 1-weighted MRI to detect nitroxyl contrast agents was described. T 1-weighted MRI can detect nitroxyl contrast agents with a complicated EPR spectrum easier and quicker; however, T 1-weighted MRI has less quantitative ability especially for lipophilic nitroxyl contrast agents, because T 1-relaxivity, i.e. accessibility to water, is affected by the hydrophilic/hydrophobic micro-environment of a nitroxyl contrast agent. The less quantitative ability of T 1-weighted MRI may not be a disadvantage of redox imaging, which obtains reduction rate of a nitroxyl contrast. Therefore, T 1-weighted MRI has a great advantage to check the pharmacokinetics of newly modified and/or designed nitroxyl contrast agents.

  15. Expression of T1Rs and gustducin in palatal taste buds of mice.

    PubMed

    Stone, Leslie M; Barrows, Jennell; Finger, Thomas E; Kinnamon, Sue C

    2007-03-01

    The palatal region of the oral cavity in rodents houses 100-300 taste buds and is particularly sensitive to sweet and umami compounds; yet, few studies have examined the expression patterns of transduction-related molecules in this taste field. We investigated the interrelationships between members of the T1R family and between each T1R and gustducin in palatal taste buds. Similar to lingual taste buds, T1R1 and T1R2 are generally expressed in separate palatal taste cells. In contrast to lingual taste buds, however, T1R2 and T1R3-positive palatal taste cells almost always coexpress gustducin, suggesting that sweet taste transduction in the palate is almost entirely dependent on gustducin. T1R1-positive palate taste cells coexpress gustducin about half the time, suggesting that other G proteins may contribute to the transduction of umami stimuli in this taste field. PMID:17229761

  16. The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-Waxy gene

    PubMed Central

    Yang, Rui; Zhou, Yongguo; Cao, Yueqin; Yin, Zhongming; Yang, Lijun; Li, Jianyue

    2013-01-01

    Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. “261S” plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F2 hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F2 hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F2 hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected. PMID:23853508

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

    PubMed

    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. PMID:25416172

  18. Butyrate Transcriptionally Enhances Peptide Transporter PepT1 Expression and Activity

    PubMed Central

    Dalmasso, Guillaume; Nguyen, Hang Thi Thu; Yan, Yutao; Charrier-Hisamuddin, Laetitia; Sitaraman, Shanthi V.; Merlin, Didier

    2008-01-01

    Background PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. Results We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ?2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. Conclusions Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation. PMID:18575574

  19. The functional role of the T1R family of receptors in sweet taste and feeding.

    PubMed

    Treesukosol, Yada; Smith, Kimberly R; Spector, Alan C

    2011-11-30

    The discovery of the T1R family of Class C G protein-coupled receptors in the peripheral gustatory system a decade ago has been a tremendous advance for taste research, and its conceptual reach has extended to other organ systems. There are three proteins in the family, T1R1, T1R2, and T1R3, encoded by their respective genes, Tas1r1, Tas1r2, and Tas1r3. T1R2 combines with T1R3 to form a heterodimer that binds with sugars and other sweeteners. T1R3 also combines with T1R1 to form a heterodimer that binds with l-amino acids. These proteins are expressed not only in taste bud cells, but one or more of these T1Rs have also been identified in the nasal epithelium, gut, pancreas, liver, kidney, testes and brain in various mammalian species. Here we review current perspectives regarding the functional role of these receptors, concentrating on sweet taste and feeding. We also discuss behavioral findings suggesting that a glucose polymer mixture, Polycose, which rodents avidly prefer, appears to activate a receptor that does not depend on the combined expression of T1R2 and T1R3. In addition, although the T1Rs have been implicated as playing a role in glucose sensing, T1R2 knock-out (KO) and T1R3 KO mice display normal chow and fluid intake as well as normal body weight compared with same-sex littermate wild type (WT) controls. Moreover, regardless of whether they are fasted or not, these KO mice do not differ from their WT counterparts in their Polycose intake across a broad range of concentrations in 30-minute intake tests. The functional implications of these results and those in the literature are considered. PMID:21376068

  20. The Functional Role of the T1R Family of Receptors in Sweet Taste and Feeding

    PubMed Central

    Treesukosol, Yada; Smith, Kimberly R.; Spector, Alan C.

    2011-01-01

    The discovery of the T1R family of Class C G protein-coupled receptors in the peripheral gustatory system a decade ago has been a tremendous advance for taste research, and its conceptual reach has extended to other organ systems. There are three proteins in the family, T1R1, T1R2, and T1R3, encoded by their respective genes, Tas1r1, Tas1r2, and Tas1r3. T1R2 combines with T1R3 to form a heterodimer that binds with sugars and other sweeteners. T1R3 also combines with T1R1 to form a heterodimer that binds with L-amino acids. These proteins are expressed not only in taste bud cells, but one or more of these T1Rs have also been identified in the nasal epithelium, gut, pancreas, liver, kidney, testes and brain in various mammalian species. Here we review current perspectives regarding the functional role of these receptors, concentrating on sweet taste and feeding. We also discuss behavioral findings suggesting that a glucose polymer mixture, Polycose, which rodents avidly prefer, appears to activate a receptor that does not depend on the combined expression of T1R2 and T1R3. In addition, although the T1Rs have been implicated as playing a role in glucose sensing, T1R2 knock-out (KO) and T1R3 KO mice display normal chow and fluid intake as well as normal body weight compared with same-sex littermate wild type (WT) controls. Moreover, regardless of whether they are fasted or not, these KO mice do not differ from their WT counterparts in their Polycose intake across a broad range of concentrations in 30-min intake tests. The functional implications of these results and those in the literature are considered. PMID:21376068

  1. Analysis of Quality-Related Parameters in Mature Kernels of Polygalacturonase Inhibiting Protein (PGIP) Transgenic Bread Wheat Infected with Fusarium graminearum.

    PubMed

    Masci, Stefania; Laino, Paolo; Janni, Michela; Botticella, Ermelinda; Di Carli, Mariasole; Benvenuto, Eugenio; Danieli, Pier Paolo; Lilley, Kathryn S; Lafiandra, Domenico; D'Ovidio, Renato

    2015-04-22

    Fusarium head blight, caused by the fungus Fusarium graminearum, has a detrimental effect on both productivity and qualitative properties of wheat. To evaluate its impact on wheat flour, we compared its effect on quality-related parameters between a transgenic bread wheat line expressing a bean polygalacturonase inhibiting protein (PGIP) and its control line. We have compared metabolic proteins, the amounts of gluten proteins and their relative ratios, starch content, yield, extent of pathogen contamination, and deoxynivalenol (DON) accumulation. These comparisons showed that Fusarium significantly decreases the amount of starch in infected control plants, but not in infected PGIP plants. The flour of PGIP plants contained also a lower amount of pathogen biomass and DON accumulation. Conversely, both gluten and metabolic proteins were not significantly influenced either by the transgene or by fungal infection. These results indicate that the transgenic PGIP expression reduces the level of infection, without changing significantly the wheat seed proteome and other quality-related parameters. PMID:25823882

  2. Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, we isolated the Or gene from an orange cauliflower mutant and showed that the Or gene could serve as a novel genetic tool to enrich carotenoid content in tr...

  3. Synthesis of minus-strand copies of a viral transgene during viral infections of transgenic plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants can be genetically engineered to express viral sequences, often resulting in resistance to the virus from which the sequence was derived. The generally accepted mechanism for this pathogen induced resistance is gene silencing. Previous work has demonstrated that viral transgenes can be incorp...

  4. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms

    PubMed Central

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control. PMID:26435695

  5. Anticancer Activity of Saponins from Allium chinense against the B16 Melanoma and 4T1 Breast Carcinoma Cell

    PubMed Central

    Yu, Zhihui; Zhang, Tong; Zhou, Fengjuan; Xiao, Xiuqing; Ding, Xuezhi; He, Hao; Rang, Jie; Quan, Meifang; Wang, Ting; Zuo, Mingxing; Xia, Liqiu

    2015-01-01

    The cytotoxic substance of A. chinense saponins (ACSs) was isolated using ethanol extraction and purified with the D101 macroporous adsorption resin approach. We investigated the anticancer activity of ACSs in the B16 melanoma and 4T1 breast carcinoma cell lines. Methylthioninium chloride and hematoxylin-eosin staining with Giemsa dyestuff were used when the cells were treated with ACSs. The results showed that the cells morphologies changed significantly; ACSs induced cell death in B16 and 4T1 cells based on acridine orange/ethidium bromide double fluorescence staining, with the number and degree of apoptotic tumor cells increasing as ACS concentration increased. ACSs inhibited the proliferation of B16 and 4T1 cells in a dose-dependent manner. They also inhibited cell migration and colony formation and exhibited a concentration-dependent effect. In addition, ACSs apparently inhibited the growth of melanoma in vivo. The preliminary antitumor in vivo assay revealed that early medication positively affected tumor inhibition action and effectively protected the liver and spleen of C57 BL/6 mice from injury. This study provides evidence for the cytotoxicity of ACSs and a strong foundation for further research to establish the theoretical basis for cell death and help in the design and development of new anticancer drugs. PMID:26146506

  6. Osteogenic capacity of transgenic flax scaffolds.

    PubMed

    Gredes, Tomasz; Wróbel-Kwiatkowska, Magdalena; Dominiak, Marzena; Gedrange, Tomasz; Kunert-Keil, Christiane

    2012-02-01

    The modification of flax fibers to create biologically active dressings is of undoubted scientific and practical interest. Flax fibers, derived from transgenic flax expressing three bacterial genes for the synthesis of poly-3-hydroxybutyric acid (PHB), have better mechanical properties than unmodified flax fibers; do not show any inflammation response after subcutaneous insertion; and have a good in vitro and in vivo biocompatibility. The aim of this study was to examine the applicability of composites containing flax fibers of genetically modified (M50) or non-modified (wt-Nike) flax within a polylactide (PLA) matrix for bone regeneration. For this, the mRNA expression of genes coding for growth factors (insulin-like growth factor IGF1, IGF2, vascular endothelial growth factor), for osteogenic differentiation (alkaline phosphatase, osteocalcin, Runx2, Phex, type 1 and type 2 collagen), and for bone resorption markers [matrix metalloproteinase 8 (MMP8), acid phosphatase type 5] were analyzed using quantitative real-time polymerase chain reaction. We found a significant elevated mRNA expression of IGF1 with PLA and PLA-wt-Nike composites. The mRNA amount of MMP8 and osteocalcin was significantly decreased in all biocomposite-treated cranial tissue samples compared to controls, whereas the expression of all other tested transcripts did not show any differences. It is assumed that both flax composites are able to stimulate bone regeneration, but composites from transgenic flax plants producing PHB showed faster bone regeneration than composites of non-transgenic flax plants. The application of these linen membranes for bone tissue engineering should be proved in further studies. PMID:22718592

  7. Transgene detection by digital droplet PCR.

    PubMed

    Moser, Dirk A; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future. PMID:25375130

  8. A transgenic rat that develops Alzheimer's disease-like amyloid pathology, deficits in synaptic plasticity and cognitive impairment.

    PubMed

    Liu, Li; Orozco, Ian J; Planel, Emmanuel; Wen, Yi; Bretteville, Alexis; Krishnamurthy, Pavan; Wang, Lili; Herman, Mathieu; Figueroa, Helen; Yu, W Haung; Arancio, Ottavio; Duff, Karen

    2008-07-01

    In the last decade, multiple lines of transgenic APP overexpressing mice have been created that recapitulate certain aspects of Alzheimer's disease (AD). However, none of the previously reported transgenic APP overexpressing rat models developed AD-like beta-amyloid (Abeta) deposits, or age-related learning and memory deficits. In the present study, we have characterized a transgenic rat model overexpressing transgenes with three, familial AD mutations (two in APP and one in PS1) that were developed by Flood et al. [Flood, D.G., et al., Abeta deposition in a transgenic rat model of Alzheimer's disease. Society for Neuroscience 2003, Washington, DC, 2003]. From the age of 9 months, these rats develop Abeta deposits in both diffuse and compact forms, with the latter being closely associated with activated microglia and reactive astrocytes. Impaired long-term potentiation (LTP) was revealed by electrophysiological recordings performed on hippocampal slices from rats at 7 months of age, which is 2 months before the appearance of amyloid plaques. The deficit in LTP was accompanied by impaired spatial learning and memory in the Morris water maze, which became more pronounced in transgenic rats of 13 months of age. For Tg rats of both ages, there was a trend for cognitive impairment to correlate with total Abeta42 levels in the hippocampus. The rat model therefore recapitulates AD-like amyloid pathology and cognitive impairment. The advantage of the rat model over the available mouse models is that rats provide better opportunities for advanced studies, such as serial CSF sampling, electrophysiology, neuroimaging, cell-based transplant manipulations, and complex behavioral testing. PMID:18504134

  9. Transgenic Phytoremediation Blasts onto the Scene

    SciTech Connect

    Hooker, Brian S.; Skeen, R S.

    1999-05-01

    The EPA National Priority List contains 22 ammunition production and processing sites that are laden with explosive and propellant wastes. With levels of 2,4,6-trinitrotoluene (TNT) contamination as high as 200 g/kg of solids, some of these sites are literally on the verge of exploding. They also present serious exposure risks to humans and wildlife, as many of these contaminants are also strong toxins and mutagens. In this issue, French et al. describe a new option for cleaning up this dangerous mixture: the use of transgenic plants. They engineered plants to express a bacterial enzyme that can completely denitrify TNT and trinitroglycerin (GTN) into harmless compounds.

  10. Ultra-low field T1 vs. T1rho at 3T and 7T: study of rotationally immobilized protein gels and animal brain tissues

    NASA Astrophysics Data System (ADS)

    Dong, Hui; Inglis, Ben; Barr, Ian; Clarke, John

    2015-03-01

    Clinical magnetic resonance imaging (MRI) machines operating in static fields of typically 1.5 T or 3 T can capture information on slow molecular dynamics utilizing the so-called T1rho technique. This technique, in which a radiofrequency (RF) spin-lock field is applied with microtesla amplitude, has been used, for example, to determine the onset time of stroke in studies on rats. The long RF pulse, however, may exceed the specific absorption rate (SAR) limit, putting subjects at risk. Ultra-low-field (ULF) MRI, based on Superconducting Quantum Interference Devices (SQUIDs), directly detects proton signals at a static magnetic field of typically 50-250 ?T. Using our ULF MRI system with adjustable static field of typically 55 to 240 ?T, we systematically measured the T1 and T2 dispersion profiles of rotationally immobilized protein gels (bovine serum albumin), ex vivo pig brains, and ex vivo rat brains with induced stroke. Comparing the ULF results with T1rho dispersion obtained at 3 T and 7 T, we find that the degree of protein immobilization determines the frequency-dependence of both T1 and T1rho. Furthermore, T1rho and ULF T1 show similar results for stroke, suggesting that ULF MRI may be used to image traumatic brain injury with negligible SAR. This research was supported by the Henry H. Wheeler, Jr. Brain Imaging Center and the Donaldson Trust.

  11. A 700-bp fragment of the human antithrombin III promoter is sufficient to confer high, tissue-specific expression on human apolipoprotein A-II in transgenic mice.

    PubMed

    Tremp, G L; Duchange, N; Branellec, D; Cereghini, S; Tailleux, A; Berthou, L; Fievet, C; Touchet, N; Schombert, B; Fruchart, J C

    1995-04-24

    The human antithrombin III-encoding gene (hAT-III) promoter (phAT-III) was used to generate transgenic mice producing a human hepatic apolipoprotein, apolipoprotein A-II (hApoA-II). Integration of the transgene into the mouse genome resulted in the efficient production of hApoA-II in plasma, reaching up to 0.40 g/l in two transgenic lines. The human ApoA-II mRNA was detected at high levels, both in the liver and in the kidney of transgenic mice. The rat AT-III gene shows the same expression pattern. In contrast, as previously described, the same promoter permitted the expression of the SV40 large T antigen only in the liver of transgenic mice. In view of the extra-hepatic distribution of the ApoA-II mRNA, a preliminary characterization of the hAT-III proximal promoter (phAT-III), driving the expression of the transgene, was realized. Using DNase I footprinting analysis with liver nuclear extracts, four protected regions (I-IV) were identified in the first 175 bp of the 5' region of hAT-III. Electrophoretic mobility shift assays performed with liver and kidney nuclear extracts indicate that region III (nucleotides (nt) -67 to -90) interacts with the liver-enriched HNF4 nuclear factor. Furthermore, our data suggest that region I (nt -123 to -138) interacts with the liver-enriched HNF3 transcription factor family, both in liver and kidney. Taken together, these results demonstrate that phAT-III is a useful tool to create transgenic mice producing high plasma levels of a human apolipoprotein due to expression of the transgene in liver and kidney.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7758957

  12. Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease

    PubMed Central

    Liu, Xin; Yang, Lihua; Zhou, Xianyao; Zhou, Miaoping; Lu, Yan; Ma, Lingjian; Ma, Hongxiang; Zhang, Zengyan

    2013-01-01

    The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription–PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops. PMID:23547108

  13. Orosensory detection of sucrose, maltose, and glucose is severely impaired in mice lacking T1R2 or T1R3, but Polycose sensitivity remains relatively normal.

    PubMed

    Treesukosol, Yada; Spector, Alan C

    2012-07-15

    Evidence in the literature supports the hypothesis that the T1R2+3 heterodimer binds to compounds that humans describe as sweet. Here, we assessed the necessity of the T1R2 and T1R3 subunits in the maintenance of normal taste sensitivity to carbohydrate stimuli. We trained and tested water-restricted T1R2 knockout (KO), T1R3 KO and their wild-type (WT) same-sex littermate controls in a two-response operant procedure to sample a fluid and differentially respond on the basis of whether the stimulus was water or a tastant. Correct responses were reinforced with water and incorrect responses were punished with a time-out. Testing was conducted with a modified descending method of limits procedure across daily 25-min sessions. Both KO groups displayed severely impaired performance and markedly decreased sensitivity when required to discriminate water from sucrose, glucose, or maltose. In contrast, when Polycose was tested, KO mice had normal EC(50) values for their psychometric functions, with some slight, but significant, impairment in performance. Sensitivity to NaCl did not differ between these mice and their WT controls. Our findings support the view that the T1R2+3 heterodimer is the principal receptor that mediates taste detection of natural sweeteners, but not of all carbohydrate stimuli. The combined presence of T1R2 and T1R3 appears unnecessary for the maintenance of relatively normal sensitivity to Polycose, at least in this task. Some detectability of sugars at high concentrations might be mediated by the putative polysaccharide taste receptor, the remaining T1R subunit forming either a homodimer or heteromer with another protein(s), or nontaste orosensory cues. PMID:22621968

  14. Ethylene insensitivity conferred by a mutated Arabidopsis ethylene receptor gene alters nodulation in transgenic Lotus japonicus

    PubMed Central

    Lohar, Dasharath; Stiller, Jiri; Kam, Jason; Stacey, Gary; Gresshoff, Peter M.

    2009-01-01

    Background and Aims Transgenics are used to demonstrate a causal relationship between ethylene insensitivity of a seedling legume plant, the level of ethylene receptor gene expression, lateral root growth and Mesorhizobium loti-induced nodule initiation. Methods Lotus japonicus plants expressing the dominant etr1-1 allele of the Arabidopsis thaliana gene encoding a well-characterized mutated ethylene receptor were created by stable Agrobacterium tumefaciens transformation. Single insertion, homozygous lines were characterized for symbiotic properties. Key Results Transgenic plants were ethylene insensitive as judged by the lack of the ‘Triple Response’, and their continued ability to grow and nodulate in the presence of inhibitory concentrations of ACC (1-aminocyclopropane-1-carboxylic acid; an ethylene precursor). Transgenic plants with high insensitivity to ACC had significantly fewer lateral roots and exhibited increased nodulation while showing no altered nitrate sensitivity or lack of systemic autoregulation. Whereas ACC-insensitive shoot growth and nodulation were observed in transformants, root growth was inhibited similarly to the wild type. Increased nodulation was caused by increased infection and a seven-fold increase in nodules developing between xylem poles. Bacteroid numbers per symbiosome increased about 1·7-fold in ethylene-insensitive plants. Conclusions The study further demonstrates multiple roles for ethylene in nodule initiation by influencing root cell infections and radial positioning, independent of autoregulation and nitrate inhibition of nodulation. PMID:19505874

  15. Postnatal lineage mapping of follicular melanocytes with the Tyr::CreER(T) (2) transgene.

    PubMed

    Harris, Melissa L; Pavan, William J

    2013-03-01

    One of the main advantages of using inducible and conditional transgenes to study pigment cell biology is that they allow for genetic manipulation within melanocytes after roles in general neural crest or melanoblast development have been fulfilled. Specifically, we focus here on the ability of the Tyr::CreER(T) (2) transgenic line to alter genes within follicular melanocytes postnatally. Using the Gt(ROSA)26Sor(tm1sor) reporter allele, we present in detail the expression and activity of Tyr::CreER(T) (2) when induced during hair morphogenesis and adult hair cycling. We find that despite similarities in expression pattern to endogenous TYR, Tyr::CreER(T) (2) is effective at targeting both undifferentiated and differentiated melanocytes within the hair follicle. We also find that Tyr::CreER(T) (2) provides the highest levels of recombination when induced during the early phases of hair growth. In conclusion, the descriptions provided here will guide future analyses of gene function within the melanocyte system of the hair follicle when using this Tyr::CreER(T) (2) transgene. PMID:23176440

  16. Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana.

    PubMed

    Ma, Xiaoli; Cui, Weina; Liang, Wenji; Huang, Zhanjing

    2015-12-01

    A novel salt-induced gene with unknown functions was cloned through analysis of gene expression profile of a salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt-related protein (TaSP) and deposited in GenBank (Accession No. KF307326). Quantitative polymerase chain reaction (qPCR) results showed that TaSP expression was induced under salt, abscisic acid (ABA), and polyethylene glycol (PEG) stresses. Subcellular localization revealed that TaSP was mainly localized in cell membrane. Overexpression of TaSP in Arabidopsis could improve salt tolerance of 35S::TaSP transgenic Arabidopsis. 35S::TaSP transgenic Arabidopsis lines after salt stress presented better physiological indexes than the control group. In the non-invasive micro-test (NMT), an evident Na(+) excretion was observed at the root tip of salt-stressed 35S::TaSP transgenic Arabidopsis. TaSP promoter was cloned, and its beta-glucuronidase (GUS) activities before and after ABA, salt, cold, heat, and salicylic acid (SA) stresses were determined. Full-length TaSP promoter contained ABA and salt response elements. PMID:26476792

  17. Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins.

    PubMed

    Soria, Cristina; Coccini, Teresa; De Simone, Uliana; Marchese, Loredana; Zorzoli, Irene; Giorgetti, Sofia; Raimondi, Sara; Mangione, P Patrizia; Ramat, Stefano; Bellotti, Vittorio; Manzo, Luigi; Stoppini, Monica

    2015-12-01

    The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: ?2-microglobulin and A? peptide3-42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm's life cycle (LC50 survival, 0.9?µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer's disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a "fragile population", where an underlying pathologic state may amplify their toxicity. PMID:26466638

  18. Postharvest Analysis of Lowland Transgenic Tomato Fruits Harboring hpRNAi-ACO1 Construct

    PubMed Central

    Behboodian, Bita; Mohd Ali, Zainon; Ismail, Ismanizan; Zainal, Zamri

    2012-01-01

    The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of ?-galactosidase (?-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid. PMID:22919320

  19. Overview of the investigation of transgenic plums in Romania

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment, PPV-CP transgenic clones C2, C3, C4, C5, C6 and PT3 were evaluated for Sharka resistance under high natural i...

  20. Overview on the investigations of transgenic plums in Romania

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic plums of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were the subjects of three experiments undertaken in Romania. In the first experiment, PPV-CP transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 were evaluated for Sharka resistance under high natu...

  1. Principles and application of transgenic technology in marine organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marine organisms into which a foreign gene or noncoding DNA fragment is artificially introduced and stably integrated in their genomes are termed transgenic marine organisms. Since the first report in 1985, a wide range of transgenic fish and marine bivalve mollusks have been produced by microinjec...

  2. Gene flow in genetically altered crops helps progress transgenic turfgrass.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous useful traits are being imparted into transgenic and non-transgenic plants. Gene flow as indicated in a recent publication from the Council for Agricultural Science and Technology (CAST 2007) is the successful transfer of genetic information between different individuals, populations, and g...

  3. Transgenes sustain epigeal insect biodiversity in diversified vegetable farm systems.

    PubMed

    Leslie, T W; Hoheisel, G A; Biddinger, D J; Rohr, J R; Fleischer, S J

    2007-02-01

    Many ecological studies have focused on the effects of transgenes in field crops, but few have considered multiple transgenes in diversified vegetable systems. We compared the epigeal, or soil surface-dwelling, communities of Coleoptera and Formicidae between transgenic and isoline vegetable systems consisting of sweet corn, potato, and acorn squash, with transgenic cultivars expressing Cry1(A)b, Cry3, or viral coat proteins. Vegetables were grown in replicated split plots over 2 yr with integrated pest management (IPM) standards defining insecticide use patterns. More than 77.6% of 11,925 insects from 1,512 pitfall traps were identified to species, and activity density was used to compare dominance distribution, species richness, and community composition. Measures of epigeal biodiversity were always equal in transgenic vegetables, which required fewer insecticide applications than their near isolines. There were no differences in species richness between transgenic and isoline treatments at the farm system and individual crop level. Dominance distributions were also similar between transgenic and isoline farming systems. Crop type, and not genotype, had a significant influence on Carabidae and Staphylinidae community composition in the first year, but there were no treatment effects in the second year, possibly because of homogenizing effects of crop rotations. Communities were more influenced by crop type, and possibly crop rotation, than by genotype. The heterogeneity of crops and rotations in diversified vegetable farms seems to aid in preserving epigeal biodiversity, which may be supplemented by reductions in insecticide use associated with transgenic cultivars. PMID:17349138

  4. Enhanced phytoremediation of volatile environmental pollutants with transgenic trees

    E-print Network

    Brown, Sally

    pollutants that pose serious health effects. We have developed transgenic poplar (Populus tremula Populus alba) plants with greatly increased rates of metabolism and re- moval of these pollutants through. The transgenic poplar plants exhibited increased removal rates of these pollutants from hydroponic so- lution

  5. Expression Systems and Species Used for Transgenic Animal Bioreactors

    PubMed Central

    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. PMID:23586046

  6. Transgenic phenolic production in corn silks moderately enhances insect resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some phenolic compounds produced in corn silks, such as maysin, can promote resistance to caterpillar pests. We evaluated transgenic maize engineered to express a maize cDNA controlled by a putative silk specific promoter for secondary metabolite production and corn earworm resistance. Transgene e...

  7. Transgenes and their contributions to epigenetic research PETER MEYER*

    E-print Network

    Meyer, Peter

    Transgenes and their contributions to epigenetic research PETER MEYER* Center for Plant Sciences to un- stable expression.Initially,the erratic expression behavior of transgenes was considered and that led to the discovery of a new molecular world of small RNAs and epigenetic marks that regulate plant

  8. Fitness Effects of Transgenic Disease Resistance in Sunflowers

    E-print Network

    Burke, John M.

    Fitness Effects of Transgenic Disease Resistance in Sunflowers John M. Burke1 * and Loren H resistance to white mold (Sclerotinia sclerotiorum) in sunflower (He- lianthus annuus). Unfortunately (OxOx) transgene has now been used to enhance white mold resis- tance in cultivated sunflower

  9. Cardiac phenotype induced by a dysfunctional ?1C transgene

    PubMed Central

    Lao, Qi Zong; Ravindran, Arippa; Herbert, Ron; Canuto, Holly C

    2011-01-01

    Based on stable integration of recombinant DNA into a host genome, transgenic technology has become an important genetic engineering methodology. An organism whose genetic characteristics have been altered by the insertion of foreign DNA is supposed to exhibit a new phenotype associated with the function of the transgene. However, successful insertion may not be sufficient to achieve specific modification of function. In this study we describe a strain of transgenic mouse, G7-882, generated by incorporation into the mouse genome of human Cav1.2 ?1C cDNA deprived of 3?-UTR to exclude transcription. We found that, in response to chronic infusion of isoproterenol, G7-882 develops dilated cardiomyopathy, a misleading “transgenic artifact” compatible with the expected function of the incorporated “correct” transgene. Specifically, using magnetic resonance imaging (MRI), we found that chronic ?-adrenergic stimulation of G7-882 mice caused left ventricular hypertrophy and aggravated development of dilated cardiomyopathy, although no significant changes in the kinetics, density and voltage dependence of the calcium current were observed in G7-882 cardiomyocytes as compared to cells from wild type mice. This result illustrates the possibility that even when a functional transgene is expressed, an observed change in phenotype may be due to the artifact of “incidental incorporation” leading to misleading conclusions. To exclude this possibility and thus provide a robust tool for exploring biological function, the new transgenic phenotype must be replicated in several independently generated transgenic strains. PMID:21224729

  10. Transgenic Crops and Sustainable Agriculture in the European Context

    ERIC Educational Resources Information Center

    Ponti, Luigi

    2005-01-01

    The rapid adoption of transgenic crops in the United States, Argentina, and Canada stands in strong contrast to the situation in the European Union (EU), where a de facto moratorium has been in place since 1998. This article reviews recent scientific literature relevant to the problematic introduction of transgenic crops in the EU to assess if…

  11. Fully automatic detection of deep white matter T1 hypointense lesions in multiple sclerosis.

    PubMed

    Spies, Lothar; Tewes, Anja; Suppa, Per; Opfer, Roland; Buchert, Ralph; Winkler, Gerhard; Raji, Alaleh

    2013-12-01

    A novel method is presented for fully automatic detection of candidate white matter (WM) T1 hypointense lesions in three-dimensional high-resolution T1-weighted magnetic resonance (MR) images. By definition, T1 hypointense lesions have similar intensity as gray matter (GM) and thus appear darker than surrounding normal WM in T1-weighted images. The novel method uses a standard classification algorithm to partition T1-weighted images into GM, WM and cerebrospinal fluid (CSF). As a consequence, T1 hypointense lesions are assigned an increased GM probability by the standard classification algorithm. The GM component image of a patient is then tested voxel-by-voxel against GM component images of a normative database of healthy individuals. Clusters (?0.1 ml) of significantly increased GM density within a predefined mask of deep WM are defined as lesions. The performance of the algorithm was assessed on voxel level by a simulation study. A maximum dice similarity coefficient of 60% was found for a typical T1 lesion pattern with contrasts ranging from WM to cortical GM, indicating substantial agreement between ground truth and automatic detection. Retrospective application to 10 patients with multiple sclerosis demonstrated that 93 out of 96 T1 hypointense lesions were detected. On average 3.6 false positive T1 hypointense lesions per patient were found. The novel method is promising to support the detection of hypointense lesions in T1-weighted images which warrants further evaluation in larger patient samples. PMID:24216694

  12. Fully automatic detection of deep white matter T1 hypointense lesions in multiple sclerosis

    NASA Astrophysics Data System (ADS)

    Spies, Lothar; Tewes, Anja; Suppa, Per; Opfer, Roland; Buchert, Ralph; Winkler, Gerhard; Raji, Alaleh

    2013-12-01

    A novel method is presented for fully automatic detection of candidate white matter (WM) T1 hypointense lesions in three-dimensional high-resolution T1-weighted magnetic resonance (MR) images. By definition, T1 hypointense lesions have similar intensity as gray matter (GM) and thus appear darker than surrounding normal WM in T1-weighted images. The novel method uses a standard classification algorithm to partition T1-weighted images into GM, WM and cerebrospinal fluid (CSF). As a consequence, T1 hypointense lesions are assigned an increased GM probability by the standard classification algorithm. The GM component image of a patient is then tested voxel-by-voxel against GM component images of a normative database of healthy individuals. Clusters (?0.1 ml) of significantly increased GM density within a predefined mask of deep WM are defined as lesions. The performance of the algorithm was assessed on voxel level by a simulation study. A maximum dice similarity coefficient of 60% was found for a typical T1 lesion pattern with contrasts ranging from WM to cortical GM, indicating substantial agreement between ground truth and automatic detection. Retrospective application to 10 patients with multiple sclerosis demonstrated that 93 out of 96 T1 hypointense lesions were detected. On average 3.6 false positive T1 hypointense lesions per patient were found. The novel method is promising to support the detection of hypointense lesions in T1-weighted images which warrants further evaluation in larger patient samples.

  13. [Transgene complete silencing may associate with rearrangement of retroviral vector].

    PubMed

    Wang, Dan; Xiao, Lejia; Ma, Qingxin; Zhai, Fen; Ren, Sichong; Li, Changlong

    2011-04-01

    Transgene silencing is one of two major obstacles in both basic biomedical research for transgene and clinical practice of gene therapy. Based on the model of HT1080 cell clones, which transduced single copy of retroviral vector MGPN2, the mechanism of transgene silencing was explored in this investigation by a serial molecular techniques. In the HT1080 cell clone that absence of GFP protein synthesized, no significant aberration of epigenetic modification was detected, but the transcript size and the sequence changed that resulted in the reading frame shift. In addition to chromosomal position effect leading to transgene silencing, the transcript reading frame shift associated with retroviral vector rearrangements could induce complete silencing of transgene. PMID:21604499

  14. Investigations into the hypothesis of transgenic cannabis.

    PubMed

    Cascini, Fidelia

    2012-05-01

    The unusual concentration of cannabinoids recently found in marijuana samples submitted to the forensic laboratory for chemical analysis prompted an investigation into whether genetic modifications have been made to the DNA of Cannabis sativa L. to increase its potency. Traditional methods for the detection of genetically modified organisms (GMO) were used to analyze herbal cannabis preparations. Our analyses support the hypothesis that marijuana samples submitted to forensic laboratories and characterized by an abnormal level of ?(9)-THC are the product of breeding selection rather than of transgenic modifications. Further, this research has shown a risk of false positive results associated with the poor quality of the seized samples and probably due to the contamination by other transgenic vegetable products. On the other hand, based on these data, a conclusive distinction between the hypothesis of GMO plant contamination and the other of genetic modification of cannabis cannot be made requiring further studies on comparative chemical and genetic analyses to find out an explanation for the recently detected increased potency of cannabis. PMID:22211569

  15. Increased adenovirus Type 5 mediated transgene expression due to RhoB down-regulation.

    PubMed

    Majhen, Dragomira; Stojanovi?, Nikolina; Vuki?, Dunja; Pichon, Chantal; Leduc, Chloé; Osmak, Maja; Ambriovi?-Ristov, Andreja

    2014-01-01

    Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking. PMID:24466204

  16. Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy.

    PubMed Central

    Kohno, K.; Palha, J. A.; Miyakawa, K.; Saraiva, M. J.; Ito, S.; Mabuchi, T.; Blaner, W. S.; Iijima, H.; Tsukahara, S.; Episkopou, V.; Gottesman, M. E.; Shimada, K.; Takahashi, K.; Yamamura, K.; Maeda, S.

    1997-01-01

    Amyloid fibrils derived from the Japanese, Portuguese, and Swedish types of familial amyloidotic polyneuropathy all consist of a variant transthyretin (TTR) with a substitution of methionine for valine at position 30 (TTR Met 30). In an attempt to establish an animal model of TTR Met-30-associated homozygous familial amyloidotic polyneuropathy and to study the structural and functional properties of human TTR Met 30, we generated a mouse line carrying a null mutation at the endogenous ttr locus (ttr-/-) and the human mutant ttr gene (6.0-hMet 30) as a transgene. In these mice, human TTR Met-30-derived amyloid deposits were first observed in the esophagus and stomach when the mice were 11 months of age. With advancing age, amyloid deposits extended to various other tissues. Because no significant difference was detected in the onset, progression, and tissue distribution of amyloid deposition between the ttr-/- and ttr+/+ transgenic mice expressing 6.0-hMet 30, endogenous normal mouse TTR probably does not affect the deposition of human TTR Met-30-derived amyloid in mice. TTR is a tetramer composed of four identical subunits that binds thyroxine (T4) and plasma retinol-binding protein. The introduction of 6.0-hMet 30 into the ttr-/- mice significantly increased their depressed serum levels of T4 and retinol-binding protein, suggesting that human TTR Met 30 binds T4 and retinol-binding protein in vivo. The T4-binding ability of human TTR Met 30 was confirmed by the analysis of T4-binding proteins in the sera of ttr-/- transgenic mice expressing 6.0-hMet 30. The T4-binding studies also demonstrated the presence of hybrid tetramers between mouse and human TTR subunits in the ttr+/+ transgenic mice expressing 6.0-hMet 30. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9095004

  17. Transgenic rabbits as therapeutic protein bioreactors and human disease models.

    PubMed

    Fan, Jianglin; Watanabe, Teruo

    2003-09-01

    Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to directly examine structure-function and cause-and-effect relationships in pathophysiological processes. Today, transgenic mice are available as a research tool in almost every research institution. On the other hand, the development of a relatively large mammalian transgenic model, transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also provided an alternative way to produce therapeutic proteins to treat human diseases. Transgenic rabbits expressing human genes have been used as a model for cardiovascular disease, AIDS, and cancer research. The recombinant proteins can be produced from the milk of transgenic rabbits not only at lower cost but also on a relatively large scale. One of the most promising and attractive recombinant proteins derived from transgenic rabbit milk, human alpha-glucosidase, has been successfully used to treat the patients who are genetically deficient in this enzyme. Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic cell nuclear transfer in the future. Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbits. PMID:12951161

  18. Taste responses in mice lacking taste receptor subunit T1R1

    PubMed Central

    Kusuhara, Yoko; Yoshida, Ryusuke; Ohkuri, Tadahiro; Yasumatsu, Keiko; Voigt, Anja; Hübner, Sandra; Maeda, Katsumasa; Boehm, Ulrich; Meyerhof, Wolfgang; Ninomiya, Yuzo

    2013-01-01

    The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1?/?) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1?/? mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1?/? mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/? mice responded to sweet and umami compounds, whereas those in T1R1?/? mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1?/? than in T1R1+/? mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1?/? and T1R1+/? mice. Conditioned taste aversion tests demonstrated that both T1R1?/? and T1R1+/? mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds. PMID:23339178

  19. Transgene Expression and Bt Protein Content in Transgenic Bt Maize (MON810) under Optimal and Stressful Environmental Conditions

    PubMed Central

    Trtikova, Miluse; Wikmark, Odd Gunnar; Zemp, Niklaus; Widmer, Alex; Hilbeck, Angelika

    2015-01-01

    Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict. PMID:25853814

  20. Minute Pirate Bug (Orius Insidiosus Say) populations on transgenic and non-transgenic maize using different sampling techniques

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field experiments were conducted to evaluate the populations of minute pirate bug [Orius insidiosus (Say)] using visual, sticky cards, and destructive sampling techniques in transgenic and non-transgenic maize in three locations in Nebraska (Mead, Clay Center, and Concord), United States of America,...

  1. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing

    PubMed Central

    Madisen, Linda; Mao, Tianyi; Koch, Henner; Zhuo, Jia-min; Berenyi, Antal; Fujisawa, Shigeyoshi; Hsu, Yun-Wei A.; Garcia, Alfredo J.; Gu, Xuan; Zanella, Sebastien; Kidney, Jolene; Gu, Hong; Mao, Yimei; Hooks, Bryan M.; Boyden, Edward S.; Buzsáki, György; Ramirez, Jan Marino; Jones, Allan R.; Svoboda, Karel; Han, Xue; Turner, Eric E.; Zeng, Hongkui

    2012-01-01

    Cell-type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of 4 knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0, and archaerhodopsin Arch-ER2. All 4 transgenes mediate Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent, and inducible nature of our ChR2 mice represents a significant advancement over previous lines, whereas the Arch-ER2 and eNpHR3.0 mice are the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre-driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy. PMID:22446880

  2. Generation of transgenic plantain (Musa spp.) with resistance to plant pathogenic nematodes.

    PubMed

    Roderick, Hugh; Tripathi, Leena; Babirye, Annet; Wang, Dong; Tripathi, Jaindra; Urwin, Peter E; Atkinson, Howard J

    2012-10-01

    Plant parasitic nematodes impose a severe constraint on plantain and banana productivity; however, the sterile nature of many cultivars precludes conventional breeding for resistance. Transgenic plantain cv. Gonja manjaya (Musa AAB) plants, expressing a maize cystatin that inhibits nematode digestive cysteine proteinases and a synthetic peptide that disrupts nematode chemoreception, were assessed for their ability to resist nematode infection. Lines were generated that expressed each gene singly or both together in a stacked defence. Nematode challenge with a single species or a mixed population identified 10 lines with significant resistance. The best level of resistance achieved against the major pest species Radopholus similis was 84% ± 8% for the cystatin, 66% ± 14% for the peptide and 70% ± 6% for the dual defence. In the mixed population, trial resistance was also demonstrated to Helicotylenchus multicinctus. A fluorescently labelled form of the chemodisruptive peptide underwent retrograde transport along certain sensory dendrites of R. similis as required to disrupt chemoreception. The peptide was degraded after 30 min in simulated intestinal fluid or boiling water and after 1 h in nonsterile soil. In silico sequence analysis suggests that the peptide is not a mammalian antigen. This work establishes the mode of action of a novel nematode defence, develops the evidence for its safe and effective deployment against multiple nematode species and identifies transgenic plantain lines with a high level of resistance for a proposed field trial. PMID:22435592

  3. Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis

    PubMed Central

    Liu, Xin; Zhang, Jiwen; Zhang, Cuicui; Wang, Liangchao; Chen, Hao; Zhu, Zengrong; Tu, Jumin

    2015-01-01

    Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding. PMID:26366116

  4. Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis.

    PubMed

    Liu, Xin; Zhang, Jiwen; Zhang, Cuicui; Wang, Liangchao; Chen, Hao; Zhu, Zengrong; Tu, Jumin

    2015-09-01

    Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding. PMID:26366116

  5. Generation of a transgenic ORFeome library in Drosophila

    PubMed Central

    Bischof, Johannes; Sheils, Emma M.; Björklund, Mikael; Basler, Konrad

    2014-01-01

    Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open reading frames (ORFs) regulated by Upstream Activation Sequences (UAS sites); the resulting Gal4-inducible UAS-ORF plasmid library is then used to generate Drosophila strains by ?C31 integrase-mediated site-specific integration. We also provide details for FLP/FRT-mediated in vivo exchange of epitope tags (or regulatory regions) in the ORF library strains, which further extends their potential applications. These transgenic UAS-ORF strains are a useful resource to complement and validate genetic experiments performed with loss-of-function mutants and RNAi lines. The duration of the complete protocol strongly depends on the number of ORFs required, but the procedure of injection and establishing balanced fly stocks can be completed within approx. 6-7 weeks for a few genes. PMID:24922270

  6. Transmissibility of caprine scrapie in ovine transgenic mice

    PubMed Central

    2012-01-01

    Background The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. Results First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. Conclusions These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program. PMID:22472560

  7. Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

    PubMed

    Vickery, Christopher M; Lockey, Richard; Holder, Thomas M; Thorne, Leigh; Beck, Katy E; Wilson, Christina; Denyer, Margaret; Sheehan, John; Marsh, Sarah; Webb, Paul R; Dexter, Ian; Norman, Angela; Popescu, Emma; Schneider, Amanda; Holden, Paul; Griffiths, Peter C; Plater, Jane M; Dagleish, Mark P; Martin, Stuart; Telling, Glenn C; Simmons, Marion M; Spiropoulos, John

    2014-02-01

    Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer. PMID:24257620

  8. The SbMT-2 Gene from a Halophyte Confers Abiotic Stress Tolerance and Modulates ROS Scavenging in Transgenic Tobacco

    PubMed Central

    Chaturvedi, Amit Kumar; Patel, Manish Kumar; Mishra, Avinash; Tiwari, Vivekanand; Jha, Bhavanath

    2014-01-01

    Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13) showed significantly enhanced salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA) analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2?; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed) condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance. PMID:25340650

  9. Effects of ambient and elevated carbon dioxide on the responses of leaf gas exchange, multispectral imaging and primary metabolism of water-stress resistant transgenic maize to drought

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ¬ Both traditional breeding methods and recombinant DNA techniques have been employed to develop commercial maize germplasm (Zea mays L.) with improved yields under moisture stressed conditions. In the current study, we compared responses to water stress of two experimental, transgenic maize lines ...

  10. Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

    SciTech Connect

    Strauss, W.M.; Dausman, J.; Beard, C.; Jaenisch, R. ); Johnson, C.; Lawrence, J.B. )

    1993-03-26

    Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protection analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.

  11. Predictors of associated autoimmune diseases (AAID) in families with type 1 diabetes (T1D). Results from the Type 1 Diabetes Genetics Consortium (T1DGC)

    PubMed Central

    Wägner, Ana M; Santana, Ángelo; Hernández, Marta; Wiebe, Julia C; Nóvoa, Javier; Mauricio, Didac

    2011-01-01

    Background Type 1 diabetes (T1D) is a clinically heterogeneous disease. The presence of associated autoimmune diseases (AAID) may represent a distinct form of autoimmune diabetes, with involvement of specific mechanisms. The aim of this study was to find predictors of AAID in the Type 1 Diabetes Genetics Consortium (T1DGC) data set. Methods 3263 families with at least 2 siblings with T1D were included. Clinical information was obtained using questionnaires, anti-GAD and anti-IA-2 were measured and HLA-genotyping was performed. Siblings with T1D with and without AAID were compared and a multivariate regression analysis was performed to find predictors of AAID. T1D-associated HLA haplotypes were defined as the 4 most susceptible and protective, respectively. Results AAID was present in 14.4% of the T1D affected siblings. Age of diabetes onset, current age and time since diagnosis were higher, and there was a female predominance and more family history of AAID in the group with AAID, as well as more frequent anti-GAD and less frequent anti-IA2 positivity. Risk and protective HLA haplotype distributions were similar, though DRB1*0301-DQA1*0501-DQB1*0201 was more frequent in the group with AAID. In the multivariate analysis, female gender, age of onset, family history of AAID, time since diagnosis and anti-GAD positivity were significantly associated with AAID. Conclusions In patients with T1D, the presence of AAID is associated with female predominance, more frequent family history of AAID, later onset of T1D and more anti-GAD antibodies, despite longer duration of the disease. The predominance of certain HLA haplotypes suggests that specific mechanisms of disease may be involved. PMID:21744463

  12. Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice.

    PubMed

    Glendinning, John I; Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F; Vasselli, Joseph R; Sclafani, Anthony

    2015-09-01

    Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. PMID:26157055

  13. Sugar-induced cephalic-phase insulin release is mediated by a T1r2+T1r3-independent taste transduction pathway in mice

    PubMed Central

    Stano, Sarah; Holter, Marlena; Azenkot, Tali; Goldman, Olivia; Margolskee, Robert F.; Vasselli, Joseph R.; Sclafani, Anthony

    2015-01-01

    Sensory stimulation from foods elicits cephalic phase responses, which facilitate digestion and nutrient assimilation. One such response, cephalic-phase insulin release (CPIR), enhances glucose tolerance. Little is known about the chemosensory mechanisms that activate CPIR. We studied the contribution of the sweet taste receptor (T1r2+T1r3) to sugar-induced CPIR in C57BL/6 (B6) and T1r3 knockout (KO) mice. First, we measured insulin release and glucose tolerance following oral (i.e., normal ingestion) or intragastric (IG) administration of 2.8 M glucose. Both groups of mice exhibited a CPIR following oral but not IG administration, and this CPIR improved glucose tolerance. Second, we examined the specificity of CPIR. Both mouse groups exhibited a CPIR following oral administration of 1 M glucose and 1 M sucrose but not 1 M fructose or water alone. Third, we studied behavioral attraction to the same three sugar solutions in short-term acceptability tests. B6 mice licked more avidly for the sugar solutions than for water, whereas T1r3 KO mice licked no more for the sugar solutions than for water. Finally, we examined chorda tympani (CT) nerve responses to each of the sugars. Both mouse groups exhibited CT nerve responses to the sugars, although those of B6 mice were stronger. We propose that mice possess two taste transduction pathways for sugars. One mediates behavioral attraction to sugars and requires an intact T1r2+T1r3. The other mediates CPIR but does not require an intact T1r2+T1r3. If the latter taste transduction pathway exists in humans, it should provide opportunities for the development of new treatments for controlling blood sugar. PMID:26157055

  14. Generation and characterization of T1R2-LacZ knock-in mouse.

    PubMed

    Iwatsuki, Ken; Nomura, Masatoshi; Shibata, Atsushi; Ichikawa, Reiko; Enciso, Patricio L M; Wang, Lixiang; Takayanagi, Ryoichi; Torii, Kunio; Uneyama, Hisayuki

    2010-11-19

    Taste cells are chemosensory epithelial cells that sense distinct taste quality such as umami, sweet, bitter, sour and salty. Taste cells utilize G protein-coupled receptors to detect umami, sweet and bitter taste whereas ion channels are responsible for detecting salty and sour taste. Among these taste receptors, taste receptor type 2, T1R2 (or Tas1r2), has been identified as a sole sweet taste receptor in mammals that mediates sweet signals upon dimerization with T1R3. However, because of limited availability of reliable antibodies and low expression level of G protein-coupled receptors, it is uneasy to identify the cell-types that express these receptors in non-taste tissues. In this study, we have generated a T1R2-LacZ reporter knock-in mouse to investigate tissue distribution of T1R2 at a single-cell level. We found that the LacZ gene expression in these mice was faithful to the expression of T1R2 in the taste tissue and in the gastrointestinal tract where T1R3 expression has been reported. Surprisingly, T1R2 expression was also found in the testis. Mice homozygous for T1R2 deletion lacked T1R2 protein analyzed by the antibody raised against T1R2 peptide sequences. In summary, the T1R2 knock-in mouse is a powerful tool to analyze the putative targets for sweeteners as well as to study the physiological roles of T1R2 in detecting sugars. PMID:20965149

  15. DETERMINATION OF AN UPPER LIMIT FOR THE WATER OUTGASSING RATE OF MAIN-BELT COMET P/2012 T1 (PANSTARRS)

    SciTech Connect

    O'Rourke, L.; Teyssier, D.; Kueppers, M.; Snodgrass, C.; De Val-Borro, M.; Hartogh, P.; Biver, N.; Bockelee-Morvan, D.; Hsieh, H.; Micheli, M.; Fernandez, Y.

    2013-09-01

    A new Main-Belt Comet (MBC) P/2012 T1 (PANSTARRS) was discovered on 2012 October 6, approximately one month after its perihelion, by the Pan-STARRS1 survey based in Hawaii. It displayed cometary activity upon its discovery with one hypothesis being that the activity was driven by sublimation of ices; as a result, we searched for emission assumed to be driven by the sublimation of subsurface ices. Our search was of the H{sub 2}O 1{sub 10}-1{sub 01} ground state rotational line at 557 GHz from P/2012 T1 (PANSTARRS) with the Heterodyne Instrument for the Far Infrared on board the Herschel Space Observatory on 2013 January 16, when the object was at a heliocentric distance of 2.504 AU and a geocentric distance of 2.064 AU. Perihelion was in early 2012 September at a distance of 2.411 AU. While no H{sub 2}O line emission was detected in our observations, we were able to derive sensitive 3{sigma} upper limits for the water production rate and column density of <7.63 Multiplication-Sign 10{sup 25} molecules s{sup -1} and of <1.61 Multiplication-Sign 10{sup 11} cm{sup -2}, respectively. An observation taken on 2013 January 15 using the Very Large Telescope found the MBC to be active during the Herschel observation, suggesting that any ongoing sublimation due to subsurface ice was lower than our upper limit.

  16. The T1R2/T1R3 sweet receptor and TRPM5 ion channel taste targets with therapeutic potential.

    PubMed

    Sprous, Dennis; Palmer, Kyle R

    2010-01-01

    Taste signaling is a critical determinant of ingestive behaviors and thereby linked to obesity and related metabolic dysfunctions. Recent evidence of taste signaling pathways in the gut suggests the link to be more direct, raising the possibility that taste receptor systems could be regarded as therapeutic targets. T1R2/T1R3, the G protein coupled receptor that mediates sweet taste, and the TRPM5 ion channel have been the focus of discovery programs seeking novel compounds that could be useful in modifying taste. We review in this chapter the hypothesis of gastrointestinal taste signaling and discuss the potential for T1R2/T1R3 and TRPM5 as targets of therapeutic intervention in obesity and diabetes. Critical to the development of a drug discovery program is the creation of libraries that enhance the likelihood of identifying novel compounds that modulate the target of interest. We advocate a computer-based chemoinformatic approach for assembling natural and synthetic compound libraries as well as for supporting optimization of structure activity relationships. Strategies for discovering modulators of T1R2/T1R3 and TRPM5 using methods of chemoinformatics are presented herein. PMID:20691962

  17. T1rho MRI and CSF biomarkers in diagnosis of Alzheimer's disease.

    PubMed

    Haris, Mohammad; Yadav, Santosh K; Rizwan, Arshi; Singh, Anup; Cai, Kejia; Kaura, Deepak; Wang, Ena; Davatzikos, Christos; Trojanowski, John Q; Melhem, Elias R; Marincola, Francesco M; Borthakur, Arijitt

    2015-01-01

    In the current study, we have evaluated the performance of magnetic resonance (MR) T1rho (T1?) imaging and CSF biomarkers (T-tau, P-tau and A?-42) in characterization of Alzheimer's disease (AD) patients from mild cognitive impairment (MCI) and control subjects. With informed consent, AD (n = 27), MCI (n = 17) and control (n = 17) subjects underwent a standardized clinical assessment and brain MRI on a 1.5-T clinical-scanner. T1? images were obtained at four different spin-lock pulse duration (10, 20, 30 and 40 ms). T1? maps were generated by pixel-wise fitting of signal intensity as a function of the spin-lock pulse duration. T1? values from gray matter (GM) and white matter (WM) of medial temporal lobe were calculated. The binary logistic regression using T1? and CSF biomarkers as variables was performed to classify each group. T1? was able to predict 77.3% controls and 40.0% MCI while CSF biomarkers predicted 81.8% controls and 46.7% MCI. T1? and CSF biomarkers in combination predicted 86.4% controls and 66.7% MCI. When comparing controls with AD, T1? predicted 68.2% controls and 73.9% AD, while CSF biomarkers predicted 77.3% controls and 78.3% for AD. Combination of T1? and CSF biomarkers improved the prediction rate to 81.8% for controls and 82.6% for AD. Similarly, on comparing MCI with AD, T1? predicted 35.3% MCI and 81.9% AD, whereas CSF biomarkers predicted 53.3% MCI and 83.0% AD. Collectively CSF biomarkers and T1? were able to predict 59.3% MCI and 84.6% AD. On receiver operating characteristic analysis T1? showed higher sensitivity while CSF biomarkers showed greater specificity in delineating MCI and AD from controls. No significant correlation between T1? and CSF biomarkers, between T1? and age, and between CSF biomarkers and age was observed. The combined use of T1? and CSF biomarkers have promise to improve the early and specific diagnosis of AD. Furthermore, disease progression form MCI to AD might be easily tracked using these two parameters in combination. PMID:25844314

  18. Phenoxy herbicides and fibrates potently inhibit the human chemosensory receptor subunit T1R3

    PubMed Central

    Maillet, Emeline L.; Margolskee, Robert F.; Mosinger, Bedrich

    2009-01-01

    We show that phenoxy-auxin herbicides and lipid-lowering fibrates inhibit human but not rodent T1R3. T1R3 as a co-receptor in taste cells responds to sweet compounds and amino-acids; in endocrine cells of gut and pancreas T1R3 contributes to glucose sensing. Thus, certain effects of fibrates in treating hyperlipidemia and type II diabetes may be via actions on T1R3. Likewise, phenoxy-herbicides may have adverse metabolic effects in humans that would have gone undetected in studies on rodents. PMID:19817384

  19. Myocardial T1 and T2 mapping at 3 T: reference values, influencing factors and implications

    PubMed Central

    2013-01-01

    Background Myocardial T1 and T2 mapping using cardiovascular magnetic resonance (CMR) are promising to improve tissue characterization and early disease detection. This study aimed at analyzing the feasibility of T1 and T2 mapping at 3 T and providing reference values. Methods Sixty healthy volunteers (30 males/females, each 20 from 20–39 years, 40–59 years, 60–80 years) underwent left-ventricular T1 and T2 mapping in 3 short-axis slices at 3 T. For T2 mapping, 3 single-shot steady-state free precession (SSFP) images with different T2 preparation times were acquired. For T1 mapping, modified Look-Locker inversion recovery technique with 11 single shot SSFP images was used before and after injection of gadolinium contrast. T1 and T2 relaxation times were quantified for each slice and each myocardial segment. Results Mean T2 and T1 (pre-/post-contrast) times were: 44.1 ms/1157.1 ms/427.3 ms (base), 45.1 ms/1158.7 ms/411.2 ms (middle), 46.9 ms/1180.6 ms/399.7 ms (apex). T2 and pre-contrast T1 increased from base to apex, post-contrast T1 decreased. Relevant inter-subject variability was apparent (scatter factor 1.08/1.05/1.11 for T2/pre-contrast T1/post-contrast T1). T2 and post-contrast T1 were influenced by heart rate (p?T1 by age (p?T1 (r?=?0.91; r?=?0.93) were high. T2 maps: 97.7% of all segments were diagnostic and 2.3% were excluded (susceptibility artifact). T1 maps (pre-/post-contrast): 91.6%/93.9% were diagnostic, 8.4%/6.1% were excluded (predominantly susceptibility artifact 7.7%/3.2%). Conclusions Myocardial T2 and T1 reference values for the specific CMR setting are provided. The diagnostic impact of the high inter-subject variability of T2 and T1 relaxation times requires further investigation. PMID:23777327

  20. Measurement of T1 of human arterial and venous blood at 7T

    PubMed Central

    Rane, S.; Gore, J.C.

    2012-01-01

    Techniques for measuring cerebral perfusion require accurate longitudinal relaxation (T1) of blood, a MRI parameter that is field dependent. T1 of arterial and venous human blood was measured at 7T using three different sources – pathology laboratory, blood bank and in vivo. The T1 of venous blood was measured from sealed samples from a pathology lab and in vivo. Samples from a blood bank were oxygenated and mixed to obtain different physiological concentrations of hematocrit and oxygenation. T1 relaxation times were estimated using a three-point fit to a simple inversion recovery equation. At 37° C, the T1 of blood at arterial pO2was 2.29 ± 0.1 s and 2.07 ± 0.12 at venous pO2. The in vivo T1 of venous blood, in three subjects, was slightly longer at 2.45 ± 0.11s. T1 of arterial and venous blood at 7T was measured and found to be significantly different. The T1 values were longer in vivo than in vitro. While the exact cause for the discrepancy is unknown, the additives in the blood samples, degradation during experiment, oxygenation differences, and the non-stagnant nature of blood in vivo could be potential contributors to the lower values of T1 in the venous samples. PMID:23102945

  1. The Use of T1 Sagittal Angle in Predicting Cervical Disc Degeneration

    PubMed Central

    Yang, Bong-Seok; Lee, Su-Keon; Song, Kyung-Sub; Yoon, Sang-Pil; Jang, Geun; Lee, Chae-Chul; Moon, Seong-Hwan; Lee, Hwan-Mo

    2015-01-01

    Study Design Retrospective evaluation. Purpose To analyze the effect of T1 slope on degree of degeneration in patients with cervical disc degeneration. Overview of Literature The T1 slope is well known parameter that may be very useful in evaluating sagittal balance. There are no reports on the analysis of the relationship between T1 slope and cervical disc degeneration. We hypothesized that T1 slope has an effect on the degree of cervical degeneration. Methods Sixty patients who had cervical spine magnetic resonance imaging (MRI) in our orthopedic clinic were enrolled. Patients were divided into two groups according to T1 slope. Radiologic parameters obtained from radiography and cervical spine MRI were compared between low T1 slope group (?25) and high T1 slope group (>25). Results Among low T1 slope group, average degeneration grade of each cervical segment was 2.65 in C2-3, 2.50 in C3-4, 2.62 in C4-5, 3.23 in C5-6, and 2.81 in C6-7. And that of high T1 group was 2.35 in C2-3, 2.32 in C3-4, 2.59 in C4-5, 2.79 in C5-6, and 2.32 in C6-7. Grade of degeneration of low T1 group was significantly higher, as compared with high T1 group in C5-6 (p=0.028) and C6-7 (p=0.009). Percentage of high grade degeneration of more than grand III was 65.4% in low T1 group and 32.4% in high T1 group (p=0.018). Risk of high grade degeneration of C6-7 was significantly higher in low T1 group (odds ratio, 5.63; 95% confidence interval, 1.665-19.057; p=0.005). Conclusions Patients with low T1 slope had higher grade of degeneration regardless of age and gender. Low T1 slope is a potential risk factor of cervical spondylosis especially in the C6-7 cervical segment. PMID:26435795

  2. Fully functional HLA B27-restricted CD4+ as well as CD8+ T cell responses in TCR transgenic mice.

    PubMed

    Roddis, Matthew; Carter, Robert W; Sun, Mei-Yi; Weissensteiner, Thomas; McMichael, Andrew J; Bowness, Paul; Bodmer, Helen C

    2004-01-01

    The strong association of HLA B27 with spondyloarthropathies contrasts strikingly with most autoimmune diseases, which are HLA class II associated and thought to be mediated by CD4+ T lymphocytes. By introducing a human-derived HLA B27-restricted TCR into HLA B27 transgenic mice, we have obtained a functional TCR transgenic model, GRb, dependent on HLA B27 for response. Surprisingly, HLA B27 supported CD4+ as well as CD8+ T cell responses in vivo and in vitro. Further, HLA B27-restricted CD4+ T cells were capable of differentiation into a range of Th1 and Th2 T cell subsets with normal patterns of cytokine expression. The transgenic T cells were also able to enhance clearance of recombinant vaccinia virus containing influenza nucleoprotein in vivo. This is the first description of a human HLA class I-restricted TCR transgenic line. The existence of CD4+ MHC class I-restricted T cells has significant implications for immune regulation in autoimmunity and, in particular, in HLA B27-associated arthritis. We believe that this model provides a novel system for the study of unusual T cell behavior in vivo. PMID:14688321

  3. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human ?-Globin Gene with the IVSI-6 Thalassemia Mutation

    PubMed Central

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the ?-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-?-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human ?-globin gene. In the TG-?-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human ?-globin mRNA is produced, giving rise to ?-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu?-globin2/hu?-globin2 and, more importantly, (d) the aberrant ?-globin-IVSI-6 RNAs are present in blood cells. The TG-?-IVSI-6 mouse reproduces the molecular features of IVSI-6 ?-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced ?-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for ?-thalassemia. PMID:26097845

  4. The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

    PubMed

    Danielsson, Angelika; Dzojic, Helena; Rashkova, Victoria; Cheng, Wing-Shing; Essand, Magnus

    2011-01-01

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected. PMID:21379379

  5. Ectomycorrhizal Colonization and Diversity in Relation to Tree Biomass and Nutrition in a Plantation of Transgenic Poplars with Modified Lignin Biosynthesis

    PubMed Central

    Danielsen, Lara; Lohaus, Gertrud; Sirrenberg, Anke; Karlovsky, Petr; Bastien, Catherine; Pilate, Gilles; Polle, Andrea

    2013-01-01

    Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R2?=?97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations. PMID:23516610

  6. Ectomycorrhizal colonization and diversity in relation to tree biomass and nutrition in a plantation of transgenic poplars with modified lignin biosynthesis.

    PubMed

    Danielsen, Lara; Lohaus, Gertrud; Sirrenberg, Anke; Karlovsky, Petr; Bastien, Catherine; Pilate, Gilles; Polle, Andrea

    2013-01-01

    Wood from biomass plantations with fast growing tree species such as poplars can be used as an alternative feedstock for production of biofuels. To facilitate utilization of lignocellulose for saccharification, transgenic poplars with modified or reduced lignin contents may be useful. However, the potential impact of poplars modified in the lignification pathway on ectomycorrhizal (EM) fungi, which play important roles for plant nutrition, is not known. The goal of this study was to investigate EM colonization and community composition in relation to biomass and nutrient status in wildtype (WT, Populus tremula × Populus alba) and transgenic poplar lines with suppressed activities of cinnamyl alcohol dehydrogenase, caffeate/5-hydroxyferulate O-methyltransferase, and cinnamoyl-CoA reductase in a biomass plantation. In different one-year-old poplar lines EM colonization varied from 58% to 86%, but the EM community composition of WT and transgenic poplars were indistinguishable. After two years, the colonization rate of all lines was increased to about 100%, but separation of EM communities between distinct transgenic poplar genotypes was observed. The differentiation of the EM assemblages was similar to that found between different genotypes of commercial clones of Populus × euramericana. The transgenic poplars exhibited significant growth and nutrient element differences in wood, with generally higher nutrient accumulation in stems of genotypes with lower than in those with higher biomass. A general linear mixed model simulated biomass of one-year-old poplar stems with high accuracy (adjusted R(2)?=?97%) by two factors: EM colonization and inverse wood N concentration. These results imply a link between N allocation and EM colonization, which may be crucial for wood production in the establishment phase of poplar biomass plantations. Our data further support that multiple poplar genotypes regardless whether generated by transgenic approaches or conventional breeding increase the variation in EM community composition in biomass plantations. PMID:23516610

  7. Two P5CS genes from common bean exhibiting different tolerance to salt stress in transgenic Arabidopsis.

    PubMed

    Chen, Ji Bao; Yang, Jian Wei; Zhang, Zhao Yuan; Feng, Xiao Fan; Wang, Shu Min

    2013-12-01

    Many plants accumulate proline in response to salt stress. ?-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme in proline biosynthesis in plants. Plasmid DNA (pCHF3-PvP5CS1 and pCHF3-PvP5CS2) containing the selectable neomycin phosphotransferase gene for kanamycin resistance and Phaseolus vulgaris P5CS (PvP5CS1 and PvP5CS2) cDNA was introduced into Arabidopsis plants using Agrobacterium-mediated gene transfer. Southern blot, northern blot and RT-PCR analyses demonstrated that the foreign genes were integrated into Arabidopsis chromosomal DNA and expressed. Single-gene transformants were analysed in this study. Transgenic plants expressed higher levels of PvP5CS1 and PvP5CS2 transcripts under salt stress conditions than under normal conditions. When treated with 0, 100 and 200 mM NaCl, the average proline content in leaves of transgenic plants was significantly higher (P < 0.01) than control plants. The average relative electrical conductivity (REC) of transgenic lines was significantly lower (P < 0.01) than control plants under salt stress condition. Biomass production of transgenic lines was significantly higher (P < 0.05) than control plants under 200 mM NaCl stress treatment. These results indicated that introducing PvP5CS1 and PvP5CS2 cDNA into transgenic Arabidopsis caused proline overproduction, increasing salt tolerance. Although the expression of PvP5CS1 in L4 lines and PvP5CS2 in S4 lines was the same under salt stress condition, the S4 lines accumulated 1.6 and 1.9 times more proline than the L4 lines under 100 and 200 mM NaCl treatments, respectively. The REC of S4 plants was 0.5 (100 mM NaCl) and 0.6 times (200 mM NaCl) that of L4 plants. The biomass production of S4 plants was 1.6 times (200 mM NaCl) more than in L4 plants. Total P5CS enzyme activity of S4 was significantly higher than that of L4. These results implied that the PvP5CS2 protein had stronger capacity to catalyze proline synthesis than PvP5CS1 under salt stress condition. PMID:24371167

  8. Comparative carcass and tissue nutrient composition of transgenic Yorkshire pigs expressing phytase in the saliva and conventional Yorkshire pigs.

    PubMed

    Forsberg, C W; Meidinger, R G; Ajakaiye, A; Murray, D; Fan, M Z; Mandell, I B; Phillips, J P

    2014-10-01

    A transgenic line of Yorkshire (YK) pigs named the Cassie (CA) line was produced with a low copy number phytase transgene inserted in the genome. The transgenic line efficiently digests P, Ca, and other major minerals of plant dietary origin. The objectives of this study were to 1) compare carcass and tissue nutrient composition and meat quality traits for third generation hemizygous CA line market BW finisher pigs (n = 24) with age-matched conventional YK finisher pigs (n = 24) and 2) examine effects of outbreeding with high-index conventional YK boars on modifying carcass leanness from the third to sixth generations in CA line finisher boars (n = 73) and gilts (n = 103). Cassie boars (n = 12) and CA gilts (n = 12) were fed diets without supplemental P and comparable numbers of age-matched YK boars and gilts fed diets containing supplement P were raised throughout the finisher phase. The pigs were slaughtered and then fabricated into commercial pork primals before meat composition and quality evaluation. Proximate and major micronutrient composition was determined on tissues including fat, kidney, lean, liver, and skin. The main difference observed was greater (P = 0.033) crude fat content in CA boar carcasses and increased (P < 0.04) leaf lard in both CA boars and gilts but no differences were observed (P = 0.895 and P = 0.223, respectively) in carcass backfat thickness as compared with YK pigs. There were no substantive differences in tissue composition, except for CA boar kidneys. Numerous changes in the mineral, fatty acid, and indispensable AA composition for CA boar kidneys were not apparent in CA gilts. These changes may point to adaptive physiological changes in the boar kidney necessary for homeostatic regulation of mineral retention related to phytase action rather than to insertion of the transgene. However, from a meat composition perspective, transgenic expression of phytase in the CA line of YK pigs had little overall effect on meat composition. Outbreeding of high-index CA gilts with high-index commercial YK boars linearly reduced (P = 0.002) back fat thickness with a corresponding linear increase (P = 0.001) in lean yield in finisher CA gilts, although no change in these parameters was observed in CA finisher boars. The increase in lean yield in CA gilts by selective breeding without affecting the level of salivary phytase activity documents the value of conventional genetic selection in conjunction with genetic modification. PMID:25085399

  9. Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice

    PubMed Central

    Sakaguchi, Masanori; Kim, Karam; Yu, Lily Mae Yee; Hashikawa, Yoshiko; Sekine, Yukiko; Okumura, Yuki; Kawano, Masako; Hayashi, Masanobu; Kumar, Deependra; Boyden, Edward S.; McHugh, Thomas J.; Hayashi, Yasunori

    2015-01-01

    The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKII?-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior. PMID:26075894

  10. Low MHD activity using resonant helical field and limiter biasing in IR-T1 tokamak

    NASA Astrophysics Data System (ADS)

    Lafouti, M.; Ghoranneviss, M.; Meshkani, S.; Salar Elahi, A.; Salar Elahi

    2013-10-01

    In this paper, the effect of resonant helical magnetic field (RHF) and cold biased local limiter on plasma current, loop voltage, confinement time energy, poloidal beta, line emission intensity H ?, and magnetohydrodynamic (MHD) behavior based on Mirnov oscillations in the IR-T1 tokamak has been investigated. The experiments have been done in different regimes as cold biased local limiter, magnetic perturbation application, and both of these. At first the effect of the positive and negative bias voltage on plasma parameters and magnetic fluctuations detected by Mirnov coils has been investigated. Also, the effects of RHF on plasma parameters have been investigated in the edge region. Then the effects of applied biasing and RHF at the same time are analyzed. The bias voltage has been restricted to (-320 <= V bias <= +320) and it has been applied with the limiter biasing that is fixed at r = 11.25 cm. These parameters have been measured using a diamagnetic loop, spectrometer, which is a visible light spectrometer for H-alpha, and Mirnov coils. The time-resolved frequency component analysis has been performed using short-time Fourier transforms. Fourier-based techniques and auto-correlation have been employed to analyze the frequency of MHD fluctuations. The results show that applied radial electric field at the plasma edge could control electromagnetic instabilities in the tokamak. In the positive biased regime, the amplitude of low-frequency Mirnov oscillations (<30 kHz) decreases after the application of biasing. Confinement time increases and line emission intensity decreases in all situations. The results are reversed when negative biasing has been applied. When RHF and biasing are applied to the plasma at the same time, the plasma parameters do not change any more compared to corresponding discharges with only RHF (L = 3). In other words, the amplitude of MHD activity can be totally controlled when a convenient biasing is applied to the limiter in the phases of current flat-top.

  11. Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome.

    PubMed

    Kaufman, R M; Pham, C T; Ley, T J

    1999-11-01

    To date, the normal transcriptional regulation of the human beta-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human beta-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human beta-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F(1) mice showed that 2 lines express human beta globin at levels approximately equivalent to the endogenous mouse beta-major genes. One line expresses no human beta globin, while the remaining 6 lines show intermediate expression levels. Complete gamma-->beta-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic-->adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size. PMID:10556205

  12. The effect of Bt-transgene introgression on plant growth and reproduction in wild Brassica juncea.

    PubMed

    Liu, Yong-Bo; Darmency, Henry; Stewart, C Neal; Wei, Wei; Tang, Zhi-Xi; Ma, Ke-Ping

    2015-06-01

    This study aims to investigate the relative plant growth and reproduction of insect-resistant and susceptible plants following the introgression of an insect-resistance Bt-transgene from Brassica napus, oilseed rape, to wild Brassica juncea. The second backcrossed generation (BC2) from a single backcross family was grown in pure and mixed stands of Bt-transgenic and non-transgenic siblings under two insect treatments. Various proportions of Bt-transgenic plants were employed in mixed stands to study the interaction between resistant and susceptible plants. In the pure stands, Bt-transgenic BC2 plants performed better than non-transgenic plants with or without insect treatments. In mixed stands, Bt-transgenic BC2 plants produced fewer seeds than their non-Bt counterparts at low