Science.gov

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A two-year field trial was conducted to assess the impacts of two transgenic Bt rice lines, T1C-19 expressing Cry1C protein and T2A-1 expressing Cry2A protein, on the arthropod community sampled via vacuum. The arthropods were classified into five guilds, including herbivores, parasitoids, predators...

  2. 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

  3. Inheritance analysis of herbicide-resistant transgenic soybean lines.

    PubMed

    Zhang, Yong; Yang, Bao-Yu; Chen, Shi-Yun

    2006-12-01

    Four transgenic soybean lines generated via Agrobacterium-mediated transformation were used to analyze inheritance of the transgenes. Seed chip GUS assay and herbicide leaf painting and spraying assays were applied to test the gus reporter gene and the herbicide resistant bar selectable marker gene, respectively. Three of the four transgenic soybean lines were stably inherited in a Mendelian fashion with co-segregation of both transgenes in a 3:1 segregation ratio in the T(1) progeny, indicating that both transgenes were integrated into the same locus of the soybean genome. Homozygous transgenic progeny plants were obtained in the T(2) generation of these lines, and the transgenes were inherited in five successive generations. However, in one transgenic line, all the T(1) progeny plants showed GUS negative and herbicide sensitive. Southern blotting analysis confirmed that the transgenes were passed into the T(1) progeny, indicating that the transgenes were both silenced. To test if the transgene silencing was due to transcriptional or post-transcriptional level, Soybean mosaic virus (SMV) was inoculated on leaf tissues of the T(1) plants to test possible reverse effects on transgene silencing. Infection with SMV did not suppress transgene silencing, suggesting that transgene silencing in this transgenic line may not be due to post-transcriptional gene silencing. PMID:17185170

  4. 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

  5. Interhospital CT image communication: T-1 line versus courier service

    NASA Astrophysics Data System (ADS)

    Lou, Shyhliang A.; Huang, H. K.; Bazzill, Todd M.; Gould, Robert G.; Dillon, William P.; Schomer, Barbara G.

    1995-05-01

    The Mount Zion Hospital (MZH) in San Francisco, Calif. is associated with the University of California at San Francisco (UCSF) medical center. These hospitals are approximately two miles apart. The UCSF radiology department supports specialty image reading for MZH daily. The major issue involved with this service is the access of patient images. Currently, the patient image access is through two ways: (1) inter-hospital travel, and (2) image delivery. Both methods are neither efficient nor economic. If patient images can be transferred from MZH to UCSF to be viewed in digital form in a reasonable time period, the issue of patient image accession can be resolved. This study attempts to use an available digital communication technology, a T-1 line, to verify this hypothesis. The study is centered on the comparison between the T-1 line and courier service with respect to cost and image delivery performance. This comparison study focuses on CT images with an emphasis on neuroradiology application.

  6. 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...

  7. 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

  8. Germ-line transmission of transgenes in Xenopus laevis

    PubMed Central

    Marsh-Armstrong, Nicholas; Huang, Haochu; Berry, Deborah L.; Brown, Donald D.

    1999-01-01

    Adult Xenopus laevis frogs made transgenic by restriction enzyme-mediated integration were bred to test the feasibility of establishing lines of frogs that express transgenes. All of the 19 animals raised to sexual maturity generated progeny that expressed the transgene(s). The patterns and levels of expression of green fluorescent protein transgenes driven by a viral promoter, rat promoter, and four X. laevis promoters were all unaffected by passage through the germ line. These results demonstrate the ease of establishing transgenic lines in X. laevis. PMID:10588715

  9. Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S.

    PubMed

    Abe, Hirotake; Wada, Haruka; Baghdadi, Muhammad; Nakanishi, Sayaka; Usui, Yuu; Tsuchikawa, Takahiro; Shichinohe, Toshiaki; Hirano, Satoshi; Seino, Ken-Ichiro

    2016-04-01

    Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1-designated as 4T1-Sapporo (4T1-S)-which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines. PMID:26857856

  10. 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...

  11. [Analyses of virus resistance and transgenes for transgenic papaya].

    PubMed

    Ye, Chang-Ming; Wei, Xiang-Dong; Chen, Dong-Hong; Lan, Chong-Yu; Zhu, Li-Min

    2003-03-01

    Virus resistance in field and molecular biological characterizations of the transgenes were analyzed for two lines of T(1) generation of transgenic papaya with the replicase mutant gene from papaya ringspot virus (PRV). The transgenic plants showed highly resistant or immune against PRV. Results indicated that the transgenes inherited to and expressed at RNA level in the progenies. PMID:15639849

  12. 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.

  13. 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

  14. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

    PubMed

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

    2015-12-01

    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 TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 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 TαT1 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

  15. Host status of three transgenic plum lines to Mesocriconema xenoplax

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gastrodianin anti-fungal protein (GAFP) increases tolerance against Phytophthora root rot and root knot nematode (Meloidogyne incognita) in transgenic plum lines. However, nothing is known about the potential of GAFP lectin to confer resistance to the ring nematode Mesocriconema xenoplax. Three t...

  16. Accumulation of nickel in transgenic tobacco

    NASA Astrophysics Data System (ADS)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TF<1) at all levels of metal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  17. Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus.

    PubMed

    Fan, Ming-Jen; Chen, Shu; Kung, Yi-Jung; Cheng, Ying-Huey; Bau, Huey-Jiunn; Su, Tien-Tsai; Yeh, Shyi-Dong

    2009-12-01

    The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program. PMID:19526355

  18. 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.

  19. Inheritance of transgenes in transgenic Bt lines resistance to Helicoerpa armigera in upland cotton.

    PubMed

    Zhang, Baolong; Guo, Wangzhen; Zhang, Tianzhen

    2013-01-01

    Six transgenic Bt cotton cultivars (lines) including GKsu12, GK19, MR1, GK5, 109B, and SGK1 are highly resistant to bollworm from the seedling to boll-setting stages in bioassays with detached cotton leaves, though there are differences in resistant level and Bt toxin content in these transgenic cottons. Genetics analysis reveals that the resistance to Helicoverpa armigera in these six transgenic Bt cotton cultivars (lines) are controlled by one pair of dominant genes. Allelic tests further demonstrate some populations are in Mendel segregation for two nonallelic genes, i.e., the inserted Bt gene in GKsu12 is nonallelic to that of SGK1, GK5, 109B, and GK19 and Bt genes in GK19 and SGK1 are likely inserted in the same or in close proximity (genetically closely linked), while some F(2) produce abnormal segregation patterns, with a segregation of resistance to Helicoerpa armigera vary between 15:1 and 3:1, though their Bt segregation fit into 15:1 by PCR analysis, suggesting Bt gene silence in these populations. Two genes silence may occur in these populations due to the homologous sequence by crossing since the silenced individuals accounted for 1/16 of the F(2) populations for allelic test. To those silenced populations, one of their parents all showed high resistance to bollworm. PMID:23143492

  20. Generation of Doubled Haploid Transgenic Wheat Lines by Microspore Transformation

    PubMed Central

    Liu, Weiguo; Konzak, Calvin F.; von Wettstein, Diter; Rustgi, Sachin

    2013-01-01

    Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1∶2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i) pretreatment of immature spikes with CuSO4 solution (500 mg/L) at 4°C for 10 days; (ii) electroporation of plasmid DNA in enlarged microspores by a single pulse of ∼375 V; (iii) induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv) co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v) elimination of AGL-1 cells after co-cultivation with timentin (200–400 mg/L). PMID:24260351

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

    PubMed Central

    Lovaglio, Jamie; Artwohl, James E; Ward, Christopher J; Diekwisch, Thomas GH; Ito, Yoshihiro; Fortman, Jeffrey D

    2014-01-01

    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 polycystic 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. PMID:24674586

  2. 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.

  3. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed

    Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

    1994-09-01

    Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium. PMID:7843139

  4. Detection of hepatocellular carcinoma in transgenic mice by Gd-DTPA- and rhodamine 123-conjugated human serum albumin nanoparticles in T1 magnetic resonance imaging.

    PubMed

    Watcharin, Waralee; Schmithals, Christian; Pleli, Thomas; Köberle, Verena; Korkusuz, Hüdayi; Hübner, Frank; Waidmann, Oliver; Zeuzem, Stefan; Korf, Horst-Werner; Terfort, Andreas; Gelperina, Svetlana; Vogl, Thomas J; Kreuter, Jörg; Piiper, Albrecht

    2015-02-10

    Nanoparticle (NP)-based contrast agents that enable high resolution anatomic T1-weighted magnetic resonance imaging (MRI) offer the prospect of improving differential diagnosis of liver tumors such as hepatocellular carcinoma (HCC). In the present study, we investigated the possibility of employing novel non-toxic human serum albumin nanoparticles conjugated with Gd-DTPA and rhodamine 123 (Gd-Rho-HSA-NPs) for the detection of HCC by T1-weighted MRI. In addition, the influence of surface coating of the NPs with poloxamine 908, which alters the absorptive behavior of NPs and changes their distribution between the liver and tumor was examined. MRI of transgenic mice with endogenously formed HCCs following intravenous injection of Gd-Rho-HSA-NPs revealed a strong negative contrast of the tumors. Contrasting of the HCCs by NP-enhanced MRI required less Gd as compared to gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid-enhanced MRI, which currently provides the most sensitive detection of HCC in patients. Immunohistochemical analyses revealed that the Gd-Rho-HSA-NPs were localized to macrophages, which were - similar to HCC in patients - fewer in number in HCC as compared to the liver tissue, which is in agreement with the negative contrasting of HCC in Gd-Rho-HSA-NP-enhanced MRI. Poloxamine-coated NPs showed lower accumulation in the tumor macrophages and caused a longer lasting enhancement of the MRI signal. These data indicate that Gd-Rho-HSA-NPs enable sensitive detection of HCC by T1-weighted MRI in mice with endogenous HCC through their uptake by macrophages. Poloxamine coating of the NPs delayed the tumor localization of the NPs. PMID:25499552

  5. 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

  6. 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

  7. Toxicity assessment of transgenic papaya ringspot virus of 823-2210 line papaya fruits.

    PubMed

    Lin, Hsin-Tang; Yen, Gow-Chin; Huang, Ting-Tzu; Chan, Lit-Fu; Cheng, Ying-Huey; Wu, Jhaol-Huei; Yeh, Shyi-Dong; Wang, Sheng-Yang; Liao, Jiunn-Wang

    2013-02-20

    The transgenic papaya is a valuable strategy for creating plants resistant to papaya ringspot virus (PRSV) infection and increasing production. This study was further performed to evaluate the comparative toxicity effects of the newly developed transgenic line of the fruits of two backcross transgenic papaya lines (2210 and 823) and one hybrid line (823-2210) and compare to their parent non-transgenic (TN-2) counterparts. The stability analysis of coat protein (CP) of PRSV was investigated using the digestion stability assays in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and bile salts to detect the CP fragments. Results revealed that the CP fragments were rapidly hydrolyzed in SGF and were undetectable in organs and gastrointestinal contents in rats. For the genotoxicity, three in vitro assays were conducted and exhibited that non-transgenic and backcross transgenic papaya fruits were negative. Moreover, a repeated animal feeding study was conducted by feeding 2 g/kg of body weight (bw) of non-transgenic and backcross transgenic papaya fruits for 28 days in rats. There were no biological or toxicological significances between non-transgenic and backcross transgenic papaya fruits in rats. The results demonstrated that the backcross transgenic papaya fruit can be recognized as an equivalent substitution for traditional papaya in food safety. PMID:23350793

  8. Safety assessment of transgenic Bacillus thuringiensis rice T1c-19 in Sprague-Dawley rats from metabonomics and bacterial profile perspectives.

    PubMed

    Cao, Sishuo; He, Xiaoyun; Xu, Wentao; Luo, YunBo; Yuan, Yanfang; Liu, Pengfei; Cao, Bo; Shi, Hui; Huang, Kunlun

    2012-03-01

    Bacillus thuringiensis rice is facing commercialization as the main food source in the near future. The unintended effects of genetically modified (GM) organisms are the most important barriers to their promotion. We aimed to establish a new in vivo evaluation model for genetically modified foods by using metabonomics and bacterial profile approaches. T1c-19 rice flour or its transgenic parent MH63 was used at 70% wt/wt to produce diets that were fed to rats for ∼ 90 days. Urine metabolite changes were detected using (1)H NMR. Denaturing gradient gel electrophoresis and real-time polymerase chain reaction (RT-PCR) were used to detect the bacterial profiles between the two groups. The metabonomics was analyzed for metabolite changes in rat urine, when compared with the non-GM rice group, where rats were fed a GM rice diet. Several metabolites correlated with rat age and sex but not with GM rice diet. Significant biological differences were not identified between the GM rice diet and the non-GM rice diet. The bacteria related to rat urine metabolites were also discussed. The results from metabonomics and bacterial profile analyses were comparable with the results attained using the traditional method. Because metabonomics and bacterial profiling offer noninvasive, dynamic approaches for monitoring food safety, they provide a novel process for assessing the safety of GM foods. PMID:22215564

  9. Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis (Lepidoptera: Crambidae).

    PubMed

    Wang, Ya-Nan; Ke, Kai-Qie; Li, Yun-He; Han, Lan-Zhi; Liu, Yan-Min; Hua, Hong-Xia; Peng, Yu-Fa

    2016-02-01

    Two transgenic rice lines (T2A-1 and T1C-19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A-1 > Huahui 1 > T1C-19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C-19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests. PMID:25284137

  10. 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

  11. Changes in the protein expression profiles of the Hepa-T1 cell line when exposed to Cu2+.

    PubMed

    Chen, Dong-Shi; Chan, King Ming

    2009-09-14

    Copper is an essential element in a variety of biological processes, but it can be toxic when present in excessive amounts. The central regulators of cellular copper metabolism include copper-binding proteins, copper transporters, metal membrane active transporters and copper-dependent enzymes. However, the way in which cupric ions (Cu(2+)) cause cellular changes in proteins and lead to toxic effects is less well-known. The aim of this study is to identify the proteins related to Cu(2+) toxicity or detoxification mechanisms in tilapia (Oreochromis niloticus) using a proteomic approach. A cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa-T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2DE). It was found that Cu(2+) (120 and 300 microM) caused the differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. PMID:19616320

  12. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

    PubMed Central

    Praitis, V; Casey, E; Collar, D; Austin, J

    2001-01-01

    In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing. PMID:11238406

  13. Heat treatment results in a loss of transgene-encoded activities in several tobacco lines.

    PubMed Central

    Neumann, K; Dröge-Laser, W; Köhne, S; Broer, I

    1997-01-01

    Heat treatment (37 degrees C) of transgenic tobacco (Nicotiana tabacum) plants led to a reversible reduction or complete loss of transgene-encoded activities in about 40% of 10 independent transformants carrying the luciferase-coding region fused to the 355 cauliflower mosaic virus or the soybean small subunit promoter and the nopaline synthase promoter driving the neomycin phosphotransferase gene, whereas the other lines had temperature-tolerant activities. Temperature sensitivity or tolerance of transgene-encoded activities was heritable. In some of the lines, temperature sensitivity of the transgene-encoded activities depended on the stage of development, occurring in either seedlings (40% luciferase and 50% neomycin phosphotransferase) or adult plants (both 40%). The phenomenon did not correlate with copy numbers or the homo- or hemizygous state of the transgenes. In lines harboring a temperature-sensitive luciferase activity, reduction of bioluminescence was observed after 2 to 3 h at 37 degrees C. Activity was regained after 2 h of subsequent cultivation at 25 degrees C. Irrespective of the reaction to the heat treatment, the level of luciferase RNA was slightly increased at 37 degrees C. Only in lines showing temperature sensitivity of transgene-encoded activities was the amount of luciferase and neomycin phosphotransferase strongly reduced. In sterile culture, heat treatment for 15 d did not cause visible damage or changes in plant morphology. In all plants tested a slight induction of the heat-shock response was observed at 37 degrees C. PMID:9390430

  14. Generation of Transgenic Porcine Fibroblast Cell Lines Using Nanomagnetic Gene Delivery Vectors.

    PubMed

    Grześkowiak, Bartosz F; Hryhorowicz, Magdalena; Tuśnio, Karol; Grzeszkowiak, Mikołaj; Załęski, Karol; Lipiński, Daniel; Zeyland, Joanna; Mykhaylyk, Olga; Słomski, Ryszard; Jurga, Stefan; Woźniak, Anna

    2016-05-01

    The transgenic process allows for obtaining genetically modified animals for divers biomedical applications. A number of transgenic animals for xenotransplantation have been generated with the somatic cell nuclear transfer (SCNT) method. Thereby, efficient nucleic acid delivery to donor cells such as fibroblasts is of particular importance. The objective of this study was to establish stable transgene expressing porcine fetal fibroblast cell lines using magnetic nanoparticle-based gene delivery vectors under a gradient magnetic field. Magnetic transfection complexes prepared by self-assembly of suitable magnetic nanoparticles, plasmid DNA, and an enhancer under an inhomogeneous magnetic field enabled the rapid and efficient delivery of a gene construct (pCD59-GFPBsd) into porcine fetal fibroblasts. The applied vector dose was magnetically sedimented on the cell surface within 30 min as visualized by fluorescence microscopy. The PCR and RT-PCR analysis confirmed not only the presence but also the expression of transgene in all magnetofected transgenic fibroblast cell lines which survived antibiotic selection. The cells were characterized by high survival rates and proliferative activities as well as correct chromosome number. The developed nanomagnetic gene delivery formulation proved to be an effective tool for the production of genetically engineered fibroblasts and may be used in future in SCNT techniques for breeding new transgenic animals for the purpose of xenotransplantation. PMID:27048425

  15. Transposon-mediated Chromosomal Integration of Transgenes in the Parasitic Nematode Strongyloides ratti and Establishment of Stable Transgenic Lines

    PubMed Central

    Nolan, Thomas J.; Massey, Holman C.; Pearce, Edward J.; Lok, James B.

    2012-01-01

    Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis. PMID:22912584

  16. A Transgenic Durum Wheat Line that is Free of Marker Genes and Expresses 1dy10

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We used a combination of “clean gene” technology and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments exc...

  17. Assessment of Fecundity and Germ Line Transmission in Two Transgenic Pig Lines Produced by Sleeping Beauty Transposition

    PubMed Central

    Garrels, Wiebke; Holler, Stephanie; Cleve, Nicole; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A.

    2012-01-01

    Recently, we described a simplified injection method for producing transgenic pigs using a non-autonomous Sleeping Beauty transposon system. The founder animals showed ubiquitous expression of the Venus fluorophore in almost all cell types. To assess, whether expression of the reporter fluorophore affects animal welfare or fecundity, we analyzed reproductive parameters of two founder boars, germ line transmission, and organ and cell specific transgene expression in animals of the F1 and F2 generation. Molecular analysis of ejaculated sperm cells suggested three monomeric integrations of the Venus transposon in both founders. To test germ line transmission of the three monomeric transposon integrations, wild-type sows were artificially inseminated. The offspring were nursed to sexual maturity and hemizygous lines were established. A clear segregation of the monomeric transposons following the Mendelian rules was observed in the F1 and F2 offspring. Apparently, almost all somatic cells, as well as oocytes and spermatozoa, expressed the Venus fluorophore at cell-type specific levels. No detrimental effects of Venus expression on animal health or fecundity were found. Importantly, all hemizygous lines expressed the fluorophore in comparable levels, and no case of transgene silencing or variegated expression was found after germ line transmission, suggesting that the insertions occurred at transcriptionally permissive loci. The results show that Sleeping Beauty transposase-catalyzed transposition is a promising approach for stable genetic modification of the pig genome. PMID:24705079

  18. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

    PubMed

    Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

    2015-04-01

    Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. PMID:25650551

  19. Production of fat-1 transgenic rats using a post-natal female germline stem cell line.

    PubMed

    Zhou, Li; Wang, Lei; Kang, Jing X; Xie, Wenhai; Li, Xiaoyong; Wu, Changqing; Xu, Bo; Wu, Ji

    2014-03-01

    Germline stem cell lines possess the abilities of self-renewal and differentiation, and have been established from both mouse and human ovaries. Here, we established a new female germline stem cell (FGSC) line from post-natal rats by immunomagnetic sorting for Fragilis, which showed a normal karyotype, high telomerase activity, and a consistent gene expression pattern of primordial germ cells after 1 year of culture. Using an in vitro differentiation system, the FGSC line could differentiate into oocytes. After liposome-based transfection with green fluorescent protein (GFP) or fat-1 vectors, the FGSCs were transplanted into the ovaries of infertile rats. The transplanted FGSCs underwent oogenesis, and the rats produced offspring carrying the GFP or fat-1 transgene after mating with wild-type male rats. The efficiency of gene transfer was 27.86-28.00%, and 2 months was needed to produce transgenic rats. These findings have implications in biomedical research and potential applications in biotechnology. PMID:24258451

  20. A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

    PubMed

    Norum, Jens Henrik; Bergström, Åsa; Andersson, Agneta Birgitta; Kuiper, Raoul V; Hoelzl, Maria A; Sørlie, Therese; Toftgård, Rune

    2015-08-15

    LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis. PMID:26003047

  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. Retinal Degeneration in Two Lines of Transgenic S334ter Rats

    PubMed Central

    Martinez-Navarrete, G.; Seiler, M.J.; Aramant, R.B.; Fernandez-Sanchez, L.; Pinilla, I.; Cuenca, N.

    2011-01-01

    Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1–2 layers remaining at P30, and only cones left at P60. Line-5 had 4–5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those more exposed to light close to the OPL. PMID:21147100

  3. Transgenic retinoic acid sensor lines in zebrafish indicate regions of available embryonic retinoic acid

    PubMed Central

    Mandal, Amrita; Rydeen, Ariel; Anderson, Jane; Sorrell, Mollie R.J.; Zygmunt, Tomas; Torres-Vzquez, Jess; Waxman, Joshua S.

    2013-01-01

    Background Retinoic acid (RA) signaling plays a critical role in vertebrate development. Transcriptional reporters of RA signaling in zebrafish, thus far, have not reflected the broader availability of embryonic RA, necessitating additional tools to enhance our understanding of the spatial and temporal activity of RA signaling in vivo. Results We have generated novel transgenic RA sensors in which a RA receptor (RAR) ligand-binding domain (RLBD) is fused to the Gal4 DNA binding domain (GDBD) or a VP16-GDBD (VPBD) construct. Stable transgenic lines expressing these proteins when crossed with UAS reporter lines are responsive to RA. Interestingly, the VPBD RA sensor is significantly more sensitive than the GDBD sensor and demonstrates there may be almost ubiquitous availability of RA within the early embryo. Using confocal microscopy to compare the expression of the GDBD RA sensor to our previously established RA signaling transcriptional reporter line, Tg(12XRARE:EGFP), illustrates these reporters have significant overlap, but that expression from the RA sensor is much broader. We also identify previously unreported domains of expression for the Tg(12XRARE:EGFP) line. Conclusions Our novel RA sensor lines will be useful and complementary tools for studying RA signaling during development and anatomical structures independent of RA signaling. PMID:23703807

  4. (E)-β-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines

    PubMed Central

    Verma, Shiv Shankar; Sinha, Rakesh Kumar; Jajoo, Anajna

    2015-01-01

    Aphids are the major concern that significantly reduces the yield of crops. (E)-β-farnesene (Eβf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-β-farnesene (Eβf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eβf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-β-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using Eβf, a non-toxic mode of action. PMID:26251882

  5. A Tie2-driven BAC-TRAP transgenic line for in vivo endothelial gene profiling.

    PubMed

    Santhosh, Devi; Huang, Zhen

    2016-03-01

    Recent technological innovations including bacterial artificial chromosome-based translating ribosome affinity purification (BAC-TRAP) have greatly facilitated analysis of cell type-specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC-TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium-specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre- and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium-specific mRNAs from brain tissues at different stages. This BAC-TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136-145, 2016. © 2016 Wiley Periodicals, Inc. PMID:26817747

  6. Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters

    PubMed Central

    Burket, Christopher T.; Montgomery, Jacob E.; Thummel, Ryan; Kassen, Sean C.; LaFave, Matthew C.; Langenau, David M.; Zon, Leonard I.

    2013-01-01

    Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 α promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish. PMID:17968670

  7. 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

  8. 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

  9. 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

  10. 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

  11. A human beta cell line with drug inducible excision of immortalizing transgenes

    PubMed Central

    Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

    2015-01-01

    Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

  12. The histone deacetylase inhibitor Entinostat enhances polymer-mediated transgene expression in cancer cell lines.

    PubMed

    Elmer, Jacob J; Christensen, Matthew D; Barua, Sutapa; Lehrman, Jennifer; Haynes, Karmella A; Rege, Kaushal

    2016-06-01

    Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer- mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345-1356. © 2015 Wiley Periodicals, Inc. PMID:26614912

  13. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  14. 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 ...

  15. Molecular biology of deregulated gene expression in transformed C3H/10T1/2 mouse embryo cell lines induced by specific insoluble carcinogenic nickel compounds.

    PubMed Central

    Landolph, Joseph R; Verma, Anuradha; Ramnath, Jamuna; Clemens, Farrah

    2002-01-01

    In the past, exposure of workers to mixtures of soluble and insoluble nickel compounds by inhalation during nickel refining correlated with increased incidences of lung and nasal cancers. Insoluble nickel subsulfide and nickel oxide (NiO) are carcinogenic in animals by inhalation; soluble nickel sulfate is not. Particles of insoluble nickel compounds were phagocytized by C3H/10T1/2 mouse embryo cells and induced morphological transformation in these cells with the following order of potency: NiO (black) > NiO (green) > nickel subsulfide. Foci induced by black/green NiO and nickel monosulfide developed into anchorage-independent transformed cell lines. Random arbitrarily primed-polymerase chain reaction mRNA differential display showed that nine c-DNA fragments are differentially expressed between nontransformed and nickel compound-transformed 10T1/2 cell lines in 6% of total mRNA; 130 genes would be differentially expressed in 100% of the mRNA. Fragment R3-2 was a sequence in the mouse calnexin gene, fragment R3-1 a portion of the Wdr1 gene, and fragment R2-4 a portion of the ect-2 protooncogene. These three genes were overexpressed in transformed cell lines. Fragment R1-2 was 90% homologous to a fragment of the DRIP/TRAP-80 (vitamin D receptor interacting protein/thyroid hormone receptor-activating protein 80) genes and was expressed in nontransformed but not in nickel-transformed cell lines. Specific insoluble carcinogenic nickel compounds are phagocytized into 10T1/2 cells and likely generate oxygen radicals, which would cause mutations in protooncogenes, and chromosome breakage, and mutations in tumor suppressor genes, inactivating them. These compounds also induce methylation of promoters of tumor suppressor genes, inactivating them. This could lead to permanent overexpresssion of the ect-2, calnexin, and Wdr1 genes and suppression of expression of the DRIP/TRAP-80 gene that we observed, which likely contribute to induction and maintenance of transformed phenotypes. PMID:12426144

  16. [Construction of mouse VCAM-1 expression vector and establishment of stably transfected MSC line C3H10T1/2].

    PubMed

    Chen, Hui; Zhu, Heng; Chu, Ya-Nan; Xu, Fen-Fen; Liu, Yuan-Lin; Tang, Bo; Li, Xi-Mei; Hu, Liang-Ding; Zhang, Yi

    2014-10-01

    This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study. PMID:25338596

  17. Successful derivation of EGFP-transgenic embryonic stem cell line from a genetically non-permissive FVB/N mouse

    PubMed Central

    Singh, Gurbind; Totiger, Tulasigeri M; Seshagiri, Polani B

    2012-01-01

    Derivation of embryonic stem (ES)-cell lines from genetically non-permissive mouse strains, such as FVB/N, has been difficult, despite this strain offering advantages for mouse transgenesis for developmental studies. We earlier generated β-actin promoter-driven enhanced green fluorescent protein (EGFP)-transgenic FVB/N mice, expressing EGFP in all cells. Here, by optimizing culture system and using RESGROTM ES-cell culture medium, we successfully derived EGFP-transgenic ES-cell line, ‘GS-2’ line, from F1 hybrid blastocysts, from wild-type 129/SvJ female X EGFP-transgenic homozygous FVB/N male. The GS-2 ES-cell line exhibited all defining criteria of a typical ES-cell line, including normal colony morphology and karyotype (40,XY), high replication-expansion efficiency (passages: >100), expression of pluripotent markers (Oct-4, Nanog, Sox-2, SSEA-1 and others) and, embryoid body (EB) development and EB differentiation to ecto-/meso-/endo-dermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively. GS-2 ES-cells formed (i) teratoma containing three germ lineage-derived cell types, (ii) chimeric blastocysts and fetuses, following their aggregation with wild-type 8-cell embryos, (iii) functional cardiac clusters and (iv) predominantly neural cell types when EBs were developed in KOSR-supplemented medium. Taken together, we derived a robust EGFP-transgenic GS-2 ES-cell line, from a non-permissive transgenic (FVB/N) mouse by a single cross to 129/SvJ wild-type mouse. The GS-2 ES-cell line exhibited full differentiation potential, in vitro/in vivo, providing enormous opportunity for stem cell research, including experimental cell transplantation studies. PMID:23671805

  18. 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

  19. Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

    PubMed Central

    Whitmer, Serap; Canel, Camilo; Hallard, Didier; Gonçalves, Cecilia; Verpoorte, Robert

    1998-01-01

    We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures. PMID:9490777

  20. CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.

    PubMed

    Auer, Thomas O; Duroure, Karine; Concordet, Jean-Paul; Del Bene, Filippo

    2014-12-01

    Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months. PMID:25393779

  1. Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection

    PubMed Central

    Zhang, Linlin; Cui, Zhengzhi; Zhou, Lei; Kang, Youmin; Li, Li; Li, Jinxiu; Dai, Yunping; Yu, Shuyang; Li, Ning

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens in the swine industry worldwide. Due to the lack of robust cell lines and small animal models, the pathogenesis of PRRSV infection and mechanism for protective vaccination are still not yet well understood. To obtain useful cell lines, several groups have attempted to construct different transgenic cell lines with three PRRSV receptors: CD163, CD169, and CD151. The results showed that CD163 is essential for PRRSV entry into target cells and replication, and both CD169 and CD151 play key roles during PRRSV infection. However, their interplay and combined effect remains unclear. In this study, we generated transgenic BHK-21 derived cell lines co-expressing different combinations of the three receptors, which were transfected with CD163 alone, or the combination of CD163 and CD169, or the combination of CD163 and CD151, or the combination of CD163, CD169, and CD151 using the PiggyBac transposon system. Our results showed that the synergistic interaction among the three receptors was important to improve the susceptibility of cells during PRRSV infection. Through a series of comparable analyses, we confirmed that the cell line co-expressing triple receptors sustained viral infection and replication, and was superior to the current cell platform used for the PRRSV study, MARC-145 cells. Moreover, we found that PRRSV infection of the transgenic cell lines could trigger IFN-stimulated gene responses similar to those of porcine alveolar macrophages and MARC-145 cells. In summary, we developed a stable transgenic cell line susceptible to PRRSV, which may not only provide a useful tool for virus propagation, vaccine development, and pathogenesis studies, but also establish the foundation for small animal model development. PMID:27182980

  2. Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression.

    PubMed

    Kanazawa, Ippei; Yamaguchi, Toru; Yano, Shozo; Yamauchi, Mika; Sugimoto, Toshitsugu

    2010-01-01

    Rho-kinase (ROK), downstream of the mevalonate pathway, is detrimental to vessels, and suppressing its activity is a target for the treatment of human disease such as coronary artery disease and pulmonary hypertension. Recent studies have shown that ROK has a crucial role in bone metabolism. However, the role of ROK in stromal cells is still unclear. The present study was undertaken to investigate the effect of a ROK inhibitor, fasudil hydrochloride, on stromal cell lines, C3H10T1/2 and ST2. In both cells, Fasudil significantly stimulated alkaline phosphatase activity and enhanced cell mineralization. Moreover, fasudil significantly increased the mRNA expression of collagen-I, osteocalcin, and bone morphogenetic protein-2 (BMP-2). Supplementation of noggin, a BMP-2 antagonist, significantly reversed the fasudil-induced collagen-I and osteocalcin mRNA expression in both cells. These findings suggest that fasudil induces the osteoblastic differentiation of stromal cells via enhancing BMP-2 expression, and that this drug might be beneficial for not only atherosclerosis but also osteoporosis by promoting bone formation. PMID:20154408

  3. Zebrafish Transgenic Line huORFZ Is an Effective Living Bioindicator for Detecting Environmental Toxicants

    PubMed Central

    Chu, Chien; Li, Hong-Ping; Tsai, Huai-Jen

    2014-01-01

    Reliable animal models are invaluable for monitoring the extent of pollution in the aquatic environment. In this study, we demonstrated the potential of huORFZ, a novel transgenic zebrafish line that harbors a human upstream open reading frame of the chop gene fused with GFP reporter, as an animal model for monitoring environmental pollutants and stress-related cellular processes. When huORFZ embryos were kept under normal condition, no leaked GFP signal could be detected. When treated with hazardous chemicals, including heavy metals and endocrine-disrupting chemicals near their sublethal concentrations (LC50), huORFZ embryos exhibited different tissue-specific GFP expression patterns. For further analysis, copper (Cu2+), cadmium (Cd2+) and Chlorpyrifos were applied. Cu2+ triggered GFP responses in skin and muscle, whereas Cd2+ treatment triggered GFP responses in skin, olfactory epithelium and pronephric ducts. Moreover, fluorescence intensity, as exhibited by huORFZ embryos, was dose-dependent. After surviving treated embryos were returned to normal condition, survival rates, as well as TUNEL signals, returned to pretreatment levels with no significant morphological defects observed. Such results indicated the reversibility of treatment conditions used in this study, as long as embryos survived such conditions. Notably, GFP signals decreased along with recovery, suggesting that GFP signaling of huORFZ embryos likely reflected the overall physiological condition of the individual. To examine the performance of the huORFZ line under real-world conditions, we placed huORFZ embryos in different river water samples. We found that the huORFZ embryos correctly detected the presence of various kinds of pollutants. Based on these findings, we concluded that such uORFchop-based system can be integrated into a first-line water alarm system monitoring the discharge of hazardous pollutants. PMID:24594581

  4. Two Types of Transgenic Lines for Doxycycline-Inducible, Cell-Specific Gene Expression in Zebrafish Ultraviolet Cone Photoreceptors

    PubMed Central

    West, Megan C.; Campbell, Leah J.; Willoughby, John J.; Jensen, Abbie M.

    2014-01-01

    Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On System to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTAflag). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16 hours of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTAflag) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2aIntraWT was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2aIntraWT), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTAflag) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease. PMID:24462722

  5. Characterization and chromosomal instability of novel derived cell lines from a wt-erbB-2 transgenic mouse model.

    PubMed

    Jeruss, Jacqueline S; Liu, Na Xin; Chung, Yongji; Magrane, Gregg; Waldman, Frederic; Edgerton, Susan; Yang, Xiaohe; Thor, Ann D

    2003-04-01

    Amplification and overexpression of the erbB-2 (HER-2/neu) proto-oncogene and exposure to the cell cycle mitogenic hormone estrogen (E2) have been associated with mammary tumorigenesis. Phytoestrogens found in soy act as selective estrogen receptor modulators and may also modify mammary carcinogenesis. We have used the wt-erbB-2 transgenic mouse model to study the effects of estrogen and dietary phytoestrogens on erbB-2-associated mammary tumorigenesis. Transgenic mice were treated with short-term E2 or placebo pellets during the early reproductive period and fed a casein or soy diet for life. Mammary tumors from the different treatment groups were used for the derivation of novel cell lines. Comparative genomic hybridization (CGH), flow cytometry, assays of cell proliferation and soft agar cloning were performed to study genomic instability and in vitro characteristics. CGH data were compared with corresponding parental tumors. Mammary tumors exhibited significantly fewer genetic changes than cell lines by CGH. Cell lines from soy-fed animals (that developed tumors with a longer latency) demonstrated the greatest frequency of chromosomal gain and loss. The E2-treated, casein-fed animals (that developed tumors with the shortest latency) had the fewest genetic changes in derived lines by CGH. Nonetheless, E2-associated tumors in vivo and lines in vitro had the most aggressive phenotypes. In addition, over 40% of all derived cell lines, and both tumors from the placebo-treated casein-fed mice, exhibited loss of chromosome 4 by CGH. In aggregate, our data suggest that estrogenic signaling influences mammary tumor development in this transgenic mouse model bearing the rat wt-erbB-2 gene. Once induced, tumors and derived lines demonstrate persistent phenotypic characteristics, including tumor aggression and shortened latency in E2-treated mice. Loss of chromosome 4 was commonly identified in derived lines and may have facilitated immortalization or passage in culture. PMID:12727793

  6. Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice.

    PubMed

    Rai, Mayank; Datta, Karabi; Parkhi, Vilas; Tan, Jing; Oliva, Norman; Chawla, Harvinder S; Datta, Swapan K

    2007-08-01

    Transgenics for the expression of beta-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and beta-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T(1) progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T(1) progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T(1) progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 mug/g of polished seeds. High variation in the carotenoid accumulation was observed within the T(1) progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the beta-carotene accumulation. PMID:17377795

  7. 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

  8. 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

  9. Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

    PubMed Central

    MacArthur, Chad C.; Xue, Haipeng; Van Hoof, Dennis; Lieu, Pauline T.; Dudas, Miroslav; Fontes, Andrew; Swistowski, Andrzej; Touboul, Thomas; Seerke, Rina; Laurent, Louise C.; Loring, Jeanne F.; German, Michael S.; Zeng, Xianmin; Rao, Mahendra S.; Lakshmipathy, Uma; Chesnut, Jonathan D.

    2012-01-01

    Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5′ and 3′ of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken β actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs. PMID:21699412

  10. Two step male release strategy using transgenic mosquito lines to control transmission of vector-borne diseases.

    PubMed

    Carvalho, Danilo Oliveira; Costa-da-Silva, André Luis; Lees, Rosemary Susan; Capurro, Margareth Lara

    2014-04-01

    Mosquitoes are responsible for the transmission of pathogens that cause devastating human diseases such as malaria and dengue. The current increase in mean global temperature and changing sea level interfere with precipitation frequency and some other climatic conditions which, in general, influence the rate of development of insects and etiologic agents causing acceleration as the temperature rises. The most common strategy employed to combat target mosquito species is the Integrated Vector Management (IVM), which comprises the use of multiple activities and various approaches to preventing the spread of a vector in infested areas. IVM programmes are becoming ineffective; and the global scenario is threatening, requiring new interventions for vector control and surveillance. Not surprisingly, there is a growing need to find alternative methods to combat the mosquito vectors. The possibility of using transgenic mosquitoes to fight against those diseases has been discussed over the last two decades and this use of transgenic lines to suppress populations or to replace them is still under investigation through field and laboratory trials. As an alternative, the available transgenic strategies could be improved by coupling suppression and substitution strategies. The idea is to first release a suppression line to significantly reduce the wild population, and once the first objective is reached a second release using a substitution line could be then performed. Examples of targeting this approach against vectors of malaria and dengue are discussed. PMID:24513036

  11. c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain

    PubMed Central

    Stevanato, Lara; Corteling, Randolph L; Stroemer, Paul; Hope, Andrew; Heward, Julie; Miljan, Erik A; Sinden, John D

    2009-01-01

    Background The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively. Results The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. Conclusion In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application. PMID:19622162

  12. 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

  13. Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.

    PubMed

    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

  14. Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

    PubMed

    Saibi, Walid; Zouari, Nabil; Masmoudi, Khaled; Brini, Faiçal

    2016-04-01

    Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases. PMID:26751399

  15. Developmental regulation of hepatitis B surface antigen expression in two lines of hepatitis B virus transgenic mice.

    PubMed Central

    DeLoia, J A; Burk, R D; Gearhart, J D

    1989-01-01

    Two lines of hepatitis B virus (HBV) transgenic mice, designated G7 and G26, show preferential expression of the 2.1-kilobase hepatitis B surface antigen (HBsAg) RNA transcript in liver and kidney tissues (R. D. Burk, J. A. DeLoia, M. K. ElAwady, and J. D. Gearhart, J. Virol 62:649-654, 1988). This transcript was first identified in transgenic mice at gestational day 14 and was detected at similar or increased levels through birth and early development. However, in contrast to 2.1-kilobase HBsAg mRNA levels, HBsAg protein levels in serum decreased shortly after birth. Thereafter, serum HBsAg increased 100-fold to adult levels, with a corresponding 5- to 10-fold increase in HBsAg mRNA levels. In addition, adult males have higher levels of HBsAg in serum than females. HBsAg in serum in males was reduced approximately 50% by surgical castration and was restored to near-normal levels by testosterone supplementation. Since both transgenic lines show similar patterns of gene expression, we suggest that HBsAg gene expression is determined by viral regulatory elements in response to host factors. Whether tissue specificity, developmental regulation, and sexual dimorphism of expression of the exogenous HBV sequences were determined by single or multiple HBV regulatory elements remains to be determined. Images PMID:2760988

  16. Assessment of Fetal Cell Chimerism in Transgenic Pig Lines Generated by Sleeping Beauty Transposition

    PubMed Central

    Garrels, Wiebke; Holler, Stephanie; Taylor, Ulrike; Herrmann, Doris; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A.

    2014-01-01

    Human cells migrate between mother and fetus during pregnancy and persist in the respective host for long-term after birth. Fetal microchimerism occurs also in twins sharing a common placenta or chorion. Whether microchimerism occurs in multiparous mammals such as the domestic pig, where fetuses have separate placentas and chorions, is not well understood. Here, we assessed cell chimerism in litters of wild-type sows inseminated with semen of transposon transgenic boars. Segregation of three independent monomeric transposons ensured an excess of transgenic over non-transgenic offspring in every litter. Transgenic siblings (n = 35) showed robust ubiquitous expression of the reporter transposon encoding a fluorescent protein, and provided an unique resource to assess a potential cell trafficking to non-transgenic littermates (n = 7) or mothers (n = 4). Sensitive flow cytometry, fluorescence microscopy, and real-time PCR provided no evidence for microchimerism in porcine littermates, or piglets and their mothers in both blood and solid organs. These data indicate that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during gestation. PMID:24811124

  17. 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.

  18. 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

  19. Assessment of genome integrity in cattle transgenic cell lines using array CGH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic cattle carrying multiple genomic modifications have been produced by serial rounds of somatic cell chromatin transfer (cloning) of sequentially genetically targeted somatic cells. However, cloning efficiency tends to decline with the increase of rounds of cloning. It is possible that mult...

  20. 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...

  1. Enhancement of spontaneous bursal lymphoma frequency by serotype 2 Marek's disease vaccine, SB-1, in transgenic and non-transgenic line 0 white leghorn chickens.

    PubMed

    Salter, D W; Payne, W; Kung, H J; Robinson, D; Ewert, D; Olson, W; Crittenden, L B; Fadly, A M

    1999-04-01

    A significant incidence of bursal lymphomas with long latencies was noted in transgenic breeders carrying a benign defective subgroup A avian leukosis provirus, ALVA6, in their germline and maintained free of exposure to avian retroviruses. Serotype 2 Marek's disease (MD) vaccine virus, strain SB-1, a component of the bivalent MD vaccine used to vaccinate the breeders, was suspected as a contributory factor in the increased bursal lymphoma incidence. Although these bursal lymphomas had several characteristics similar to retroviral-induced bursal lymphomas, we found no evidence of retroviral influence based on many virological, immunological and molecular tests that were performed on plasma and tumour cells. These tumours were therefore classified as spontaneous bursal lymphomas, similar to those reported for some specific pathogen-free (SPF) chicken lines. Long-term in vivo experiments in plastic isolators and carefully maintained pens with homozygous and hemizygous ALVA6 and ALVA6-free female chickens (line 0) that were either non-vaccinated, serotype 3 (herpesvirus of turkeys [HVT]; monovalent)-vaccinated, or HVT/SB-1 (bivalent) vaccinated, demonstrated that the incidence of spontaneous bursal lymphomas were significantly higher in those chickens that were vaccinated with the bivalent MD vaccine (P ⩽0.05). In addition, this incidence did not depend on the ALVA6 proviral insert since there was no significant difference in spontaneous bursal lymphoma incidence between bivalent vaccinated hemizygous ALVA6 and ALVA6-free line 0 female chickens. Thus, the increased incidence of spontaneous bursal lymphomas is correlated solely with the presence of SB-1 and is not dependent on the presence of ALVA6. PMID:26911501

  2. A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system

    PubMed Central

    Drane, Laurel; Ainsley, Joshua A.; Mayford, Mark R.; Reijmers, Leon G.

    2014-01-01

    Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; translating ribosome affinity purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types. PMID:25400545

  3. 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

  4. 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.

  5. Activation of the black seabream (Acanthopagrus schlegeli) somatolactin-alpha gene promoter by Pit-1c in the Hepa-T1 cell-line.

    PubMed

    Tian, Jing; Chan, King Ming

    2010-03-01

    Somatolactin (SL) is a pituitary hormone of the growth hormone (GH) gene family found only in fish. To understand the regulation of this hormone at the level of gene transcription, we obtained a SLalpha gene from black seabream (bsb), with its 5' flanking promoter region carrying several putative transcription factors including seven binding sites for pituitary-specific transcription factor 1 (Pit-1). To study the actions of Pit-1 on this gene promoter, we cloned three variants of bsbPit-1 (Pit-1a, Pit-1b and Pit-1c) derived from alternative splicing of mRNA or differential transcription start sites from black seabream pituitary. The deduced amino acid sequences of these Pit-1s contained 371 amino acids (aa), 333 and 311aa for the three Pit-1 variants, Pit-1a, Pit-1b and Pit-1c, respectively, with diverse regions of Pit-1 located at the transactivation domain. The actions of bsbPit-1 variants on the bsbSL gene promoter were investigated using a co-transfection assay, with a reporter gene using a transient expression assay in Hepa-T1 cells. The N-terminus truncated isoform bsbPit-1c showed the highest level of activity on SLalpha gene promoter activation in Hepa-T1 cells; however, neither Pit-1a nor Pit-1b activated the bsbSL gene promoter in the same study. PMID:19766121

  6. 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 γ-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

  7. A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.

    PubMed

    Marquès-Bueno, Maria Mar; Morao, Ana K; Cayrel, Anne; Platre, Matthieu P; Barberon, Marie; Caillieux, Erwann; Colot, Vincent; Jaillais, Yvon; Roudier, François; Vert, Grégory

    2016-01-01

    Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis. PMID:26662936

  8. 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

  9. Establishment of a human cell line (SKI-DLCL-1) with a t(1;14)(q21;q32) translocation from the ascites of a patient with diffuse large cell lymphoma.

    PubMed

    Goy, A; Gilles, F; Remache, Y; Filippa, D; Portlock, C S; Jhanwar, S C; Zelenetz, A D

    2001-01-01

    Cytogenetic abnormalities at chromosome 1q21 are among the most common second genetic events observed in Non-Hodgkin's Lymphomas and have prognostic significance. Recently, BCL9 has been cloned from a pre-B-cell lymphoblastic leukemia cell line, which carried a t(1:14)(q21;q32). However, among a panel of 39 B-cell malignancies with 1q21 translocation, only two cases showed rearrangement for the BCL9 gene. We report the establishment of a new lymphoma cell line from a patient with relapsed diffuse large cell lymphoma. This cell line SKI-DLCL-1 showed cell surface antigens identical to the original tumor and demonstrated the profile of a mature B-cell phenotype: CD19 and CD20 positive, CD5 and C10 negative. It carried a t(1;14)(q21;q32) translocation identical to the original tumor. Although the clinical presentation was an isolated effusion lymphoma, studies for HIV-1, HHV8 and EBV were all negative. Southern blot analysis demonstrated that BCL9 was not rearranged in the SKI-DLCL-1 cell line. In addition, the BCL9 gene was not over-expressed in SKI-DLCL-1 cell line. The identification of a new locus at 1q21 will help clarify the pathogenesis of B-cell malignancies with a translocation involving this locus. PMID:11426565

  10. 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

  11. Megaesophagus in a Line of Transgenic Rats: A Model of Achalasia

    PubMed Central

    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.

    2015-01-01

    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

  12. Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

    PubMed Central

    Chen, Chi-Fang; Chu, Che-Yu; Chen, Te-Hao; Lee, Shyh-Jye; Shen, Chia-Ning; Hsiao, Chung-Der

    2011-01-01

    Background Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. Methodology/Principal Findings This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. Conclusion/Significance The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo. PMID:21655190

  13. 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...

  14. 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.

  15. 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.

  16. 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

  17. Effect of vegetation of transgenic Bt rice lines and their straw amendment on soil enzymes, respiration, functional diversity and community structure of soil microorganisms under field conditions.

    PubMed

    Fang, Hua; Dong, Bin; Yan, Hu; Tang, Feifan; Wang, Baichuan; Yu, Yunlong

    2012-01-01

    With the development of transgenic crops, there is an increasing concern about the possible adverse effects of their vegetation and residues on soil environmental quality. This study was carried out to evaluate the possible effects of the vegetation of transgenic Bt rice lines Huachi B6 (HC) and TT51 (TT) followed by the return of their straw to the soil on soil enzymes (catalase, urease, neutral phosphatase and invertase), anaerobic respiration activity, microbial utilization of carbon substrates and community structure, under field conditions. The results indicated that the vegetation of the two transgenic rice lines (HC and TT) and return of their straw had few adverse effects on soil enzymes and anaerobic respiration activity compared to their parent and distant parent, although some transient differences were observed. The vegetation and subsequent straw amendment of Bt rice HC and TT did not appear to have a harmful effect on the richness, evenness and community structure of soil microorganisms. No different pattern of impact due to plant species was found between HC and TT. It could be concluded that the vegetation of transgenic Bt rice lines and the return of their straw as organic fertilizer may not alter soil microbe-mediated functions. PMID:23513447

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

    PubMed

    Soltsz, Alexandra; Smedley, Mark; Vashegyi, Ildik; Galiba, Gbor; Harwood, Wendy; Vgjfalvi, Attila

    2013-04-01

    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

  19. Transgenic indica rice lines, expressing Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1), exhibit enhanced resistance to major pathogens.

    PubMed

    Sadumpati, Vijayakumar; Kalambur, Muralidharan; Vudem, Dashavantha Reddy; Kirti, Pulugurtha Bharadwaja; Khareedu, Venkateswara Rao

    2013-07-10

    Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into commercial indica rice varieties by Agrobacterium-mediated genetic transformation. Transgenic rice plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in various transgenic rice lines. Transgenes NPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Homozygous transgenic rice lines expressing BjNPR1 protein displayed enhanced resistance to rice blast, sheath blight and bacterial leaf blight diseases. Rice transformants with higher levels of NPR1 revealed notable increases in plant height, panicle length, flag-leaf length, number of seeds/panicle and seed yield/plant as compared to the untransformed plants. The overall results amply demonstrate the profound impact of BjNPR1 in imparting resistance against major pathogens of rice. The multipotent BjNPR1, as such, seems promising as a prime candidate gene to fortify crop plants with durable resistance against various pathogens. PMID:23664883

  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. Inducible mutagenesis in TEPC 2372, a mouse plasmacytoma cell line that harbors the transgenic shuttle vector lambdaLIZ.

    PubMed

    Felix, K; Kovalchuk, A L; Park, S S; Coleman, A E; Ramsay, E S; Qian, M; Kelliher, K A; Jones, G M; Ried, T; Bornkamm, G W; Janz, S

    2001-01-25

    The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues. PMID:11166031

  2. Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.

    PubMed

    Kung, Yi-Jung; Yu, Tsong-Ann; Huang, Chiung-Huei; Wang, Hui-Chin; Wang, Shin-Lan; Yeh, Shyi-Dong

    2010-08-01

    Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids. PMID:19943109

  3. Chemically induced skin carcinogenesis in a transgenic mouse line (TG.AC) carrying a v-Ha-ras gene.

    PubMed

    Spalding, J W; Momma, J; Elwell, M R; Tennant, R W

    1993-07-01

    A transgenic mouse line (TG.AC) created in the FVB/N strain, carries a v-Ha-ras gene fused to a zeta-globin promoter gene. These trangenic mice have the properties of genetically initiated skin and have been shown to be sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-described promoter of skin papillomas in the two-stage mouse skin tumorigenesis model. It was of interest to determine whether the TG.AC mouse strain was also responsive to other known promoters. Groups of heterozygous or homozygous TG.AC mice were treated topically, 2x/week, for up to 20 weeks with benzoyl peroxide (BPO), 2-butanol peroxide (2-BUP), phenol (PH), acetic acid (AA), TPA and acetone (ACN), the vehicle control. Skin papillomas were induced in all groups treated with TPA, BPO and 2-BUP. Papillomas were observed in some treatment groups as early as 3 weeks. The relative activity of the promoters was TPA > 2-BUP > BPO > PH = AA = ACN. No papillomas were observed in any of the uninitiated FVB/N mice treated in a similar manner and which served as treatment control groups. Studies to determine the sensitivity of TG.AC mice to TPA, indicated that a total dose of 25-30 micrograms of TPA administered in 3 or 10 applications, was sufficient to induce an average incidence of 11-15 papillomas per mouse. The papilloma incidence continued to increase and was maintained up to 15 weeks after TPA treatment was terminated. The short latency period and high incidence of papilloma induction indicate that TG.AC mice have a high sensitivity to known skin promoters. The TG.AC line should prove to be a sensitive model for identifying putative tumor promoters or complete carcinogens. PMID:8330346

  4. Opposing effects of APP/PS1 and TrkB.T1 genotypes on midbrain dopamine neurons and stimulated dopamine release in vivo.

    PubMed

    Kärkkäinen, E; Yavich, L; Miettinen, P O; Tanila, H

    2015-10-01

    Brain derived neurotrophic factor (BDNF) signaling disturbances in Alzheimer׳s disease (AD) have been demonstrated. BDNF levels fall in AD, but the ratio between truncated and full-length BDNF receptors TrkB.T1 and TrkB.TK, respectively, increases in brains of AD patients and APPswe/PS1dE9 (APP/PS1) AD model mice. Dopaminergic (DAergic) system disturbances in AD and detrimental effects of BDNF signaling deficits on DAergic system functions have also been indicated. Against this, we investigated changes in nigrostriatal dopamine (DA) system in mice carrying APP/PS1 and/or TrkB.T1 transgenes, the latter line modeling the TrkB.T1/TK ratio change in AD. Employing in vivo voltammetry, we found normal short-term DA release in caudate-putamen of mice carrying APP/PS1 or TrkB.T1 transgenes but impaired capacity to recruit more DA upon prolonged stimulation. However, mice carrying both transgenes did not differ from wild-type controls. Immunohistochemistry revealed normal density of tyrosine hydroxylase positive axon terminals in caudate-putamen in all genotypes and intact presynaptic machinery for DA release and reuptake, as shown by unchanged levels of SNAP-25, α-synuclein and DA transporter. However, we observed increased DAergic neurons in substantia nigra of TrkB.T1 mice resulting in decreased tyrosine hydroxylase per neuron in TrkB.T1 mice. The finding of unchanged nigral DAergic neurons in APP/PS1 mice largely confirms earlier reports, but the unexpected increase in midbrain DA neurons in TrkB.T1 mice is a novel finding. We suggest that both APP/PS1 and TrkB.T1 genotypes disrupt DAergic signaling, but via separate mechanisms. PMID:26168899

  5. Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.).

    PubMed

    Goel, D; Singh, A K; Yadav, V; Babbar, S B; Bansal, K C

    2010-09-01

    Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants. PMID:20467880

  6. Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W.

    PubMed

    Yu, Tsong-Ann; Chiang, Chu-Hui; Wu, Hui-Wen; Li, Chin-Mei; Yang, Ching-Fu; Chen, Jun-Han; Chen, Yu-Wen; Yeh, Shyi-Dong

    2011-03-01

    Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV W) are major limiting factors for production of watermelon worldwide. For the effective control of these two viruses by transgenic resistance, an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes was transferred to commercial watermelon cultivars by Agrobacterium-mediated transformation. Using our protocol, a total of 27 putative transgenic lines were obtained from three cultivars of 'Feeling' (23 lines), 'China baby' (3 lines), and 'Quality' (1 line). PCR and Southern blot analyses confirmed that the chimeric construct was incorporated into the genomic DNA of the transformants. Greenhouse evaluation of the selected ten transgenic lines of 'Feeling' cultivar revealed that two immune lines conferred complete resistance to ZYMV and PRSV W, from which virus accumulation were not detected by Western blotting 4 weeks after inoculation. The transgenic transcript was not detected, but small interfering RNA (siRNA) was readily detected from the two immune lines and T(1) progeny of line ZW 10 before inoculation, indicating that RNA-mediated post-transcriptional gene silencing (PTGS) is the underlying mechanism for the double-virus resistance. The segregation ratio of T(1) progeny of the immune line ZW10 indicated that the single inserted transgene is nuclearly inherited and associated with the phenotype of double-virus resistance as a dominant trait. The transgenic lines derived from the commercial watermelon cultivars have great potential for control of the two important viruses and can be implemented directly without further breeding. PMID:21079966

  7. Monitoring changes in anthocyanin and steroid alkaloid glycoside content in lines of transgenic potato plants using liquid chromatography/mass spectrometry.

    PubMed

    Stobiecki, Maciej; Matysiak-Kata, Iwona; Frański, Rafał; Skała, Jacek; Szopa, Jan

    2003-03-01

    Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized. PMID:12590123

  8. Effects of defoliating insect resistance QTLs and a cry1Ac transgene in soybean near-isogenic lines.

    PubMed

    Zhu, S; Walker, D R; Boerma, H R; All, J N; Parrott, W A

    2008-02-01

    The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a "Benning" genetic background. A set of 16 BC(6)F(2)-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes. PMID:18064435

  9. High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

    PubMed Central

    2010-01-01

    Background Although the complete genome sequence and annotation of Arabidopsis were released at the end of year 2000, it is still a great challenge to understand the function of each gene in the Arabidopsis genome. One way to understand the function of genes on a genome-wide scale is expression profiling by microarrays. However, the expression level of many genes in Arabidopsis genome cannot be detected by microarray experiments. In addition, there are many more novel genes that have been discovered by experiments or predicted by new gene prediction programs. Another way to understand the function of individual genes is to investigate their in vivo expression patterns by reporter constructs in transgenic plants which can provide basic information on the patterns of gene expression. Results A high throughput pipeline was developed to generate promoter-reporter (GFP) transgenic lines for Arabidopsis genes expressed at very low levels and to examine their expression patterns in vivo. The promoter region from a total of 627 non- or low-expressed genes in Arabidopsis based on Arabidopsis annotation release 5 were amplified and cloned into a Gateway vector. A total of 353 promoter-reporter (GFP) constructs were successfully transferred into Agrobacterium (GV3101) by triparental mating and subsequently used for Arabidopsis transformation. Kanamycin-resistant transgenic lines were obtained from 266 constructs and among them positive GFP expression was detected from 150 constructs. Of these 150 constructs, multiple transgenic lines exhibiting consistent expression patterns were obtained for 112 constructs. A total 81 different regions of expression were discovered during our screening of positive transgenic plants and assigned Plant Ontology (PO) codes. Conclusions Many of the genes tested for which expression data were lacking previously are indeed expressed in Arabidopsis during the developmental stages screened. More importantly, our study provides plant researchers with another resource of gene expression information in Arabidopsis. The results of this study are captured in a MySQL database and can be searched at http://www.jcvi.org/arabidopsis/qpcr/index.shtml. Transgenic seeds and constructs are also available for the research community. PMID:20687964

  10. Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target.

    PubMed

    Tewari, Rita; Patzewitz, Eva-Maria; Poulin, Benoit; Stewart, Lindsay; Baker, David A

    2014-01-01

    With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known. PMID:24805991

  11. Phenotype and Hierarchy of Two Transgenic T Cell Lines Targeting the Respiratory Syncytial Virus KdM282-90 Epitope Is Transfer Dose-Dependent

    PubMed Central

    Graham, Barney S.; Ruckwardt, Tracy J.

    2016-01-01

    In this study, we compared two lines of transgenic CD8+ T cells specific for the same KdM282-90 epitope of respiratory syncytial virus in the CB6F1 hybrid mouse model. Here we found that these two transgenic lines had similar in vivo abilities to control viral load after respiratory syncytial virus infection using adoptive transfer. Transfer of the TRBV13-2 line resulted in higher levels of IL-6 and MIP1-α in the lung than TRBV13-1 transfer. Interestingly, when large numbers of cells were co-transferred, the lines formed a hierarchy, with TRBV13-2 being immunodominant over TRBV13-1 in the mediastinal lymph node despite no identifiable difference in proliferation or apoptosis between the lines. This hierarchy was not established when lower cell numbers were transferred. The phenotype and frequency of proliferating cells were also cell transfer dose-dependent with higher percentages of CD127loCD62LloKLRG1lo and proliferating cells present when lower numbers of cells were transferred. These results illustrate the importance of cell number in adoptive transfer experiments and its influence on the phenotype and hierarchy of the subsequent T cell response. PMID:26752171

  12. Inducible responses to DNA damage in the mouse embryo fibroblasts cell line C3H/10T1/2 and its transformed counterpart C3H/MCA

    SciTech Connect

    Miller, L.S.

    1987-01-01

    Early passage mouse embryo fibroblasts cells (C3H/10T1/2) were treated with ultraviolet (UV) radiation of 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to determine whether such treatment induced DNA repair processes, measured as increased survival and mutagenesis of Herpes simplex (HSV-1). No enhanced host cell reactivation of UV-irradiated virus was observed following treatment of cells with UV-irradiation or TPA. Replication of undamaged virus in untreated C3H cells resulted in an increase over the background mutation frequency. When the cells were UV-irradiated and infected with unirradiated virus, a decrease in mutagenesis was observed. Decreased untargeted mutagenesis was shown to be dose- and time-dependent, reaching a minimum at a fluence of 5-7 Jm/sup /minus/2/ for 24 hours between irradiation and infection of cells. There was no change in mutagenesis of UV-irradiated virus grown in UV-irradiated cells compared to untreated cells. The repair capacity of methylcholanthrene-transformed C3H cells (MCA cells) was compared with untransformed C3H cells. The cell lines demonstrated similar cell survival curves following UV-irradiation but differed markedly in their ability to repair damaged HSV-1.

  13. The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes.

    PubMed

    Hirai, Tadayoshi; Kurokawa, Natsuko; Duhita, Narendra; Hiwasa-Tanase, Kyoko; Kato, Kazuhisa; Kato, Ko; Ezura, Hiroshi

    2011-09-28

    High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system. PMID:21861502

  14. 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...

  15. Effects of Defoliating Insect Resistance QTLs and a crylAc Transgene in Soybean Near-Isogenic Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Additional sources of resistance would be desirable to manage defoliating insect resistance to crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three soybean (G...

  16. CONDITIONAL PRODUCTION OF A FUNCTIONAL FISH GROWTH HORMONE IN THE TRANSGENIC LINE OF NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)(1).

    PubMed

    Chen, Hsin Liang; Li, Si Shen; Huang, Rang; Tsai, Huai-Jen

    2008-06-01

    Plasmid phr-YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a RUBISCO SSU 2 promoter, was transferred into the protoplast of marine microalga Nannochloropsis oculata (Droop) D. J. Hibberd by electroporation. Four transgenic clones were obtained in which the transferred phr-YPGHc was integrated into the genome and existed stably at least until the 50th generation. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in the amount of 0.42 to 0.27 μg · mL(-1) from the 50 mL of medium. We incubated artemia with the wildtype and transgenic N. oculata for 6 h and then fed these microalgae-treated artemia to red-tilapia larvae. After feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater (i.e., statistically significant: P < 0.05) than that of larvae that were fed artemia incubated with nontransgenic microalgae: 316% versus 104% in weight gain, and 217% versus 146% in body length increase, respectively. Therefore, the N. oculata enables production of functional GH, and we propose that it might be an excellent bioreactor material. PMID:27041435

  17. T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

    PubMed

    Ray, Durwood B; Merrill, Gerald A; Brenner, Frederic J; Lytle, Laurie S; Lam, Tan; McElhinney, Aaron; Anders, Joel; Rock, Tara Tauber; Lyker, Jennifer Kier; Barcus, Scott; Leslie, Kara Hust; Kramer, Jill M; Rubenstein, Eric M; Pryor Schanz, Karen; Parkhurst, Amy J; Peck, Michelle; Good, Kimberly; Granath, Kristi Lemke; Cifra, Nicole; Detweiler, Jessalee Wantz; Stevens, Laura; Albertson, Richard; Deir, Rachael; Stewart, Elisabeth; Wingard, Katherine; Richardson, Micah Rose; Blizard, Sarah B; Gillespie, Lauren E; Kriley, Charles E; Rzewnicki, Daniel I; Jones, David H

    2016-01-01

    Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7-10 days. T1-A, T-2A, T3-HA and T4-PA cells when injected i.v. into the tail produced local metastasis in non-nude or nude NIH/Swiss mice. T4-PA cells were more widely metastatic than T3-HA cells when injected i.v. into nude mice. Evaluation of the injected mice indicated a general increase in metastatic potential of each cell line in the progression as compared to the GhrasT-NIH/3T3 transformed cells. A new photomicrographic technique to follow growth rates within six preselected 2×2mm(2) grids per plate is described. Average doubling times of the transformed cells GhrasT-NIH/3T3 (17h), T1A (17.5h), T2A (15.5h), T3-HA (17.5h) and T4-PA (18.5h) (average 17.2h) were significantly faster (P=0.006) than NIH Swiss primary embryonic cells and NIH/3T3 cells (22 h each). This cell series is currently used in this lab for studies of cancer cell inhibitors, mitochondrial biogenesis and gene expression and is available for further study by other investigators for intra- and inter-laboratory comparisons of WGS, transcriptome sequencing, SKY and other analyses. The genome rearrangements in these cells together with their phenotypic properties may help provide more insights into how one tumorigenic progression occurred to produce the various cell lines that led to the highly metastatic T4-PA cell line. PMID:26254261

  18. Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice.

    PubMed Central

    Gaensler, K M; Kitamura, M; Kan, Y W

    1993-01-01

    Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8248258

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

    PubMed

    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. PMID:19371626

  20. 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.

  1. 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

  2. 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

  3. Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal.

    PubMed

    Anand, Ajith; Lei, Zhentian; Sumner, Lloyd W; Mysore, Kirankumar S; Arakane, Yasuyuki; Bockus, William W; Muthukrishnan, Subbaratnam

    2004-12-01

    A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack. PMID:15597736

  4. 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) δ-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

  5. Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

    PubMed Central

    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

    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) δ-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

  6. Transgenic Fish

    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) ...

  7. Ablation of cellular prion protein does not ameliorate abnormal neural network activity or cognitive dysfunction in the J20 line of human amyloid precursor protein transgenic mice.

    PubMed

    Cissé, Moustapha; Sanchez, Pascal E; Kim, Daniel H; Ho, Kaitlyn; Yu, Gui-Qiu; Mucke, Lennart

    2011-07-20

    Previous studies suggested that the cellular prion protein (PrP(c)) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Specifically, amyloid-β (Aβ) oligomers were proposed to cause synaptic and cognitive dysfunction by binding to PrP(c). To test this hypothesis, we crossed human amyloid precursor protein (hAPP) transgenic mice from line J20 onto a PrP(c)-deficient background. Ablation of PrP(c) did not prevent the premature mortality and abnormal neural network activity typically seen in hAPPJ20 mice. Furthermore, hAPPJ20 mice with or without PrP(c) expression showed comparably robust abnormalities in learning and memory and in other behavioral domains at 6-8 months of age. Notably, these abnormalities are not refractory to therapeutic manipulations in general: they can be effectively prevented by interventions that prevent Aβ-dependent neuronal dysfunction also in other lines of hAPP transgenic mice. Thus, at least in this model, PrP(c) is not an important mediator of Aβ-induced neurological impairments. PMID:21775587

  8. [Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

    PubMed

    Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

    2006-05-01

    The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement. PMID:16755923

  9. Transgenic tobacco plants expressing atzA exhibit resistance and strong ability to degrade atrazine.

    PubMed

    Wang, Huizhuan; Chen, Xiwen; Xing, Xuguang; Hao, Xiaohua; Chen, Defu

    2010-12-01

    Atrazine chlorohydrolase (AtzA) catalyzes hydrolytic dechlorination and can be used in detoxification of atrazine, a herbicide widely employed in the control of broadleaf weeds. In this study, to investigate the potential use of transgenic tobacco plants for phytoremediation of atrazine, atzA genes from Pseudomonas sp. strain ADP and Arthrobacter strain AD1 were transferred into tobacco. Three and four transgenic lines, expressing atzA-ADP and atzA-AD1, respectively, were produced by Agrobacterium-mediated transformation. Molecular characterization including PCR, RT-PCR and Southern blot revealed that atzA was inserted into the tobacco genome and stably inherited by and expressed in the progenies. Seeds of the T(1) transgenic lines had a higher germination percentage and longer roots than the untransformed plants in the presence of 40-150 mg/l atrazine. The T(2) transgenic lines grew taller, gained more dry biomass, and had higher total chlorophyll content than the untransformed plants after growing in soil containing 1 or 2 mg/kg atrazine for 90 days. No atrazine residue remained in the soil in which the T(2) transgenic lines were grown (except 401), while, in the case of the untransformed plants, 0.91 mg (81.3%) and 1.66 mg (74.1%) of the atrazine still remained in the soil containing 1 and 2 mg/kg of atrazine, respectively, indicating that the transgenic lines could degrade atrazine effectively. The transgenic tobacco lines developed could be useful for phytoremediation of atrazine-contaminated soil and water. PMID:20960204

  10. 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

  11. Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures

    PubMed Central

    Takács, Roland; Matta, Csaba; Somogyi, Csilla; Juhász, Tamás; Zákány, Róza

    2013-01-01

    Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models. PMID:23921684

  12. 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 Aβ 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

  13. Characterization of the BAC Id3-enhanced green fluorescent protein transgenic mouse line for in vivo imaging of astrocytes

    PubMed Central

    Lamantia, Cassandra; Tremblay, Marie-Eve; Majewska, Ania

    2014-01-01

    Abstract. Astrocytes are highly ramified glial cells with critical roles in brain physiology and pathology. Recently, breakthroughs in imaging technology have expanded our understanding of astrocyte function in vivo. The in vivo study of astrocytic dynamics, however, is limited by the tools available to label astrocytes and their processes. Here, we characterize the bacterial artificial chromosome transgenic Id3-EGFP knock-in mouse to establish its usefulness for in vivo imaging of astrocyte processes. Using fixed brain sections, we observed enhanced green fluorescent protein expression in astrocytes and blood vessel walls throughout the brain, although the extent and cell type specificity of expression depended on the brain area and developmental age. Using in vivo two-photon imaging, we visualized astrocytes in cortical layers 1–3 in both thin skull and window preparations. In adult animals, astrocytic cell bodies and fine processes could be followed over many hours. Our results suggest that Id3 mice could be used for in vivo imaging of astrocytes and blood vessels in development and adulthood. PMID:26157970

  14. Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor.

    PubMed

    Dong, Shanshan; Liu, Yan; Yu, Cigang; Zhang, Zhenhua; Chen, Ming; Wang, Changyong

    2016-01-01

    Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow. PMID:26975052

  15. Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor

    PubMed Central

    Dong, Shanshan; Liu, Yan; Yu, Cigang; Zhang, Zhenhua; Chen, Ming; Wang, Changyong

    2016-01-01

    Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow. PMID:26975052

  16. Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

    PubMed Central

    2011-01-01

    Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications. PMID:21595986

  17. Testing transgenic spring wheat and barley lines for reaction to Fusarium head blight: 2011 field nursery report.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2011 field screening nursery, with 56 wheat and 88 barley plots was located at UMore Park, Rosemount MN. Trial entries and untransformed controls were submitted by the University of North Texas (9+1 wheat), and USDA (17+2 barley). Lines with known reactions to Fusarium head blight (FHB) were a...

  18. 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...

  19. 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...

  20. The substantive equivalence of transgenic (Bt and Chi) and non-transgenic cotton based on metabolite profiles.

    PubMed

    Modirroosta, Bentol Hoda; Tohidfar, Masoud; Saba, Jalal; Moradi, Foad

    2014-03-01

    Compositional studies comparing transgenic with non-transgenic counterpart plants are almost universally required by governmental regulatory bodies. In the present study, two T(2) transgenic cotton lines containing chitinase (Line 11/57) and Bt lines (Line 61) were compared with non-transgenic counterpart. To do this, biochemical characteristics of leaves and seeds, including amino acids, fatty acids, carbohydrates, anions, and cations contents of the studied lines were analyzed using GC/MS, high-performance liquid chromatography (HPLC), and ion chromatography (IC) analyzers, respectively. polymerase chain reaction (PCR) and Western blot analyses confirmed the presence and expression of Chi and Bt genes in the studied transgenic lines. Although, compositional analysis of leaves contents confirmed no significant differences between transgenic and non-transgenic counterpart lines, but it was shown that glucose content of chitinase lines, fructose content of transgenic lines (Bt and chitinase) and asparagine and glutamine of chitinase lines were significantly higher than the non-transgenic counterpart plants. Both the transgenic lines (Bt and chitinase) showed significant decrease in the amounts of sodium in comparison to the non-transgenic counterpart plants. The experiments on the seeds showed that histidine, isoleucine, leucine, and phenylalanine contents of all transgenic and non-transgenic lines were the same, whereas other amino acids were significantly increased in the transgenic lines. Surprisingly, it was observed that the concentrations of stearic acid, myristic acid, oleic acid, and linoleic acid in the chitinase line were significantly different than those of non-transgenic counterpart plants, but these components were the same in both Bt line and its non-transgenic counterpart. It seems that more changes observed in the seed contents than leaves is via this point that seeds are known as metabolites storage organs, so they show greater changes in the metabolites contents comparing to the leaves. PMID:24374853

  1. Generation and Characterisation of a Pax8-CreERT2 Transgenic Line and a Slc22a6-CreERT2 Knock-In Line for Inducible and Specific Genetic Manipulation of Renal Tubular Epithelial Cells

    PubMed Central

    Espana-Agusti, Judit; Zou, Xiangang; Wong, Kim; Fu, Beiyuan; Yang, Fengtang; Tuveson, David A.; Adams, David J.; Matakidou, Athena

    2016-01-01

    Genetically relevant mouse models need to recapitulate the hallmarks of human disease by permitting spatiotemporal gene targeting. This is especially important for replicating the biology of complex diseases like cancer, where genetic events occur in a sporadic fashion within developed somatic tissues. Though a number of renal tubule targeting mouse lines have been developed their utility for the study of renal disease is limited by lack of inducibility and specificity. In this study we describe the generation and characterisation of two novel mouse lines directing CreERT2 expression to renal tubular epithelia. The Pax8-CreERT2 transgenic line uses the mouse Pax8 promoter to direct expression of CreERT2 to all renal tubular compartments (proximal and distal tubules as well as collecting ducts) whilst the Slc22a6-CreERT2 knock-in line utilises the endogenous mouse Slc22a6 locus to specifically target the epithelium of proximal renal tubules. Both lines show high organ and tissue specificity with no extrarenal activity detected. To establish the utility of these lines for the study of renal cancer biology, Pax8-CreERT2 and Slc22a6-CreERT2 mice were crossed to conditional Vhl knockout mice to induce long-term renal tubule specific Vhl deletion. These models exhibited renal specific activation of the hypoxia inducible factor pathway (a VHL target). Our results establish Pax8-CreERT2 and Slc22a6-CreERT2 mice as valuable tools for the investigation and modelling of complex renal biology and disease. PMID:26866916

  2. Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

    PubMed Central

    Dobie, K W; Lee, M; Fantes, J A; Graham, E; Clark, A J; Springbett, A; Lathe, R; McClenaghan, M

    1996-01-01

    Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event. Images Fig. 3 Fig. 4 PMID:8692874

  3. Cell Suspension Culture-Mediated Incorporation of the Rice Bel Gene into Transgenic Cotton

    PubMed Central

    Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang

    2012-01-01

    Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 µmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops. PMID:22768325

  4. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    PubMed

    Ke, Liping; Liu, RuiE; Chu, Bijue; Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang

    2012-01-01

    Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 µmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops. PMID:22768325

  5. [Treatment options of T1 glottic carcinoma].

    PubMed

    Wang, Qi; Fan, Guokang

    2016-01-01

    T1 glottic carcinoma is part of early laryngeal carcinoma which involves the vocal cords, including anterior commissure or posterior commissure. We analyzed the treatment options of T1 glottic carcinoma by reviewing the related literatures about T1 glottic carcinoma treated by conservative surgery (open surgery and laser microsurgery), radiotherapy, robot surgery, photodynamic treatment. PMID:27192922

  6. Enhanced Salt Tolerance Conferred by the Complete 2.3 kb cDNA of the Rice Vacuolar Na+/H+ Antiporter Gene Compared to 1.9 kb Coding Region with 5′ UTR in Transgenic Lines of Rice

    PubMed Central

    Amin, U. S. M.; Biswas, Sudip; Elias, Sabrina M.; Razzaque, Samsad; Haque, Taslima; Malo, Richard; Seraj, Zeba I.

    2016-01-01

    Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5′ UTR with CDS and the other of 2.3 kb, including 5′ UTR, CDS, and the 3′ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5′ and 3′ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance. PMID:26834778

  7. Expression and inheritance of nine transgenes in rice.

    PubMed

    Wu, Liying; Nandi, Somen; Chen, Lifang; Rodriguez, Raymond L; Huang, Ning

    2002-10-01

    A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R0) were generated from the transgenic cell lines and seeds of 32 transgenic R1 lines and 5 R2 lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R0, R1, and R2) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering. PMID:12437084

  8. 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

  9. ?-Cells with relative low HIMP1 overexpression levels in a transgenic mouse line enhance basal insulin production and hypoxia/hypoglycemia tolerance.

    PubMed

    Zhang, Xiaoping; Degenstein, Linda; Cao, Yun; Stein, Jeffrey; Osei, Kwame; Wang, Jie

    2012-01-01

    Rodent pancreatic ?-cells that naturally lack hypoglycemia/hypoxia inducible mitochondrial protein 1 (HIMP1) are susceptible to hypoglycemia and hypoxia influences. A linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 is suggested in a recent study using transformed ?-cells lines. To further illuminate this linkage, we applied mouse insulin 1 gene promoter (MIP) to control HIMP1-a isoform cDNA and have generated three lines (L1 to L3) of heterozygous HIMP1 transgenic (Tg) mice by breeding of three founders with C57BL/6J mice. In HIMP1-Tg mice/islets, we performed quantitative polymerase chain reaction (PCR), immunoblot, histology, and physiology studies to investigate HIMP1 overexpression and its link to ?-cell function/survival and body glucose homeostasis. We found that the HIMP1 level increased steadily in ?-cells of L1 to L3 heterozygous HIMP1-Tg mice. HIMP1 overexpression at relatively lower levels in L1 heterozygotes results in a negligible decline in blood glucose concentrations and an insignificant elevation in blood insulin levels, while HIMP1 overexpression at higher levels are toxic, causing hyperglycemia in L2/3 heterozygotes. Follow-up studies in 5-30-week-old L1 heterozygous mice/islets found that HIMP1 overexpression at relatively lower levels in ?-cells has enhanced basal insulin biosynthesis, basal insulin secretion, and tolerances to low oxygen/glucose influences. The findings enforced the linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 in ?-cells, and show a potential value of HIMP1 overexpression at relatively lower levels in modulating ?-cell function and survival. PMID:22470529

  10. 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.…

  11. 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

  12. An inbred line of transgenic mice expressing an internally deleted gene for type II procollagen (COL2A1). Young mice have a variable phenotype of a chondrodysplasia and older mice have osteoarthritic changes in joints.

    PubMed Central

    Helminen, H J; Kiraly, K; Pelttari, A; Tammi, M I; Vandenberg, P; Pereira, R; Dhulipala, R; Khillan, J S; Ala-Kokko, L; Hume, E L

    1993-01-01

    Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis. Images PMID:8349798

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

    PubMed

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

    2014-04-01

    A marker-free Bt transgenic rice line, mfb-MH86, was recently developed in China, which contains a cry1Ab gene driven by a ubiquitin promoter. This Bt gene confers resistance to a range of lepidopteran species, including the striped stem borer, Chilo suppressalis (Walker). The expression of Cry1Ab protein in mfb-MH86 leaves, stems and leaf sheaths (hereinafter referred to as stems), and roots was evaluated throughout the rice-growing season using an enzyme-linked immunosorbent assay. In addition, mfb-MH86 resistance to C. suppressalis, a major pest of rice, was evaluated in a laboratory bioassay with field-collected rice stems. Cry1Ab protein levels of mfb-MH86 were highest in leaves (9.71-34.09 μg/g dry weight [DW]), intermediate in stems (7.66-18.51 μg/g DW), and lowest in roots (1.95-13.40 μg/g DW). In all tissues, Cry1Ab levels in mfb-MH86 were higher in seedling and tillering stages than in subsequent growth stages. In the laboratory bioassay, mortality of C. suppressalis after 6 d of feeding on mfb-MH86 stems was 100% throughout the rice-growing season; mortality of C. suppressalis when feeding on stems of the nontransformed isoline, MH86, ranged from 15.0 to 38.3%. The results indicate that Cry1Ab protein levels in mfb-MH86 stems are sufficient to protect plants against C. suppressalis throughout the rice-growing season. Although our results are promising, further comprehensive evaluations of mfb-MH86, including field surveys, will be needed before commercial use. PMID:24495566

  14. 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.

  15. 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.

  16. 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...

  17. High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

    PubMed

    Banguela, Alexander; Arrieta, Juan G; Rodríguez, Raisa; Trujillo, Luis E; Menéndez, Carmen; Hernández, Lázaro

    2011-06-10

    Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan. PMID:21540065

  18. 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

  19. Morphological analysis of the early development of telencephalic and diencephalic gonadotropin-releasing hormone neuronal systems in enhanced green fluorescent protein-expressing transgenic medaka lines.

    PubMed

    Takahashi, Akiko; Islam, M Sadiqul; Abe, Hideki; Okubo, Kataaki; Akazome, Yasuhisa; Kaneko, Takeshi; Hioki, Hiroyuki; Oka, Yoshitaka

    2016-03-01

    Teleosts possess two or three paralogs of gonadotropin-releasing hormone (GnRH) genes: gnrh1, gnrh2, and gnrh3. Some species have lost the gnrh1 and/or gnrh3 genes, whereas gnrh2 has been completely conserved in the teleost species analyzed to date. In most teleosts that possess gnrh1, GnRH1 peptide is the authentic GnRH that stimulates gonadotropin release, whereas GnRH2 and GnRH3, if present, are neuromodulatory. Progenitors of GnRH1 and GnRH3 neurons originate from olfactory placodes and migrate to their destination during early development. However, because of the relatively low affinity/specificity of generally available antibodies that recognize GnRH1 or GnRH3, labeling of these neurons has only been possible using genetic manipulation. We used a model teleost, medaka, which possesses all three paralogous gnrh genes, to analyze development of forebrain GnRH neurons composed of GnRH1 and GnRH3 neurons. Here, we newly generated transgenic medaka lines that express enhanced green fluorescent protein under the control of promoters for gnrh1 or gnrh3, to detect GnRH neurons and facilitate immunohistochemical analysis of the neuronal morphology. We used a combination of immunohistochemistry and three-dimensional confocal microscopy image reconstructions to improve identification of neurites from GnRH1 or GnRH3 neuronal populations with greater precision. This led us to clearly identify the hypophysiotropic innervation of GnRH1 neurons residing in the ventral preoptic area (vPOA) from as early as 10 days post hatching. Furthermore, these analyses also revealed retinopetal projections of nonhypophysiotropic GnRH1 neurons in vPOA, prominent during early developmental stages, and multiple populations of GnRH3 neurons with different origins and migratory pathways. J. Comp. Neurol. 524:896-913, 2016. © 2015 Wiley Periodicals, Inc. PMID:26287569

  20. A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics

    PubMed Central

    Neelam, Anil; Cassol, Tatiana; Mehta, Roshni A.; Abdul-Baki, Aref A.; Sobolev, Anatoli P.; Goyal, Ravinder K.; Abbott, Judith; Segre, Anna L.; Handa, Avtar K.; Mattoo, Autar K.

    2008-01-01

    Genetic modification of crop plants to introduce desirable traits such as nutritional enhancement, disease and pest resistance, and enhanced crop productivity is increasingly seen as a promising technology for sustainable agriculture and boosting food production in the world. Independently, cultural practices that utilize alternative agriculture strategies including organic cultivation subscribe to sustainable agriculture by limiting chemical usage and reduced tillage. How the two together affect fruit metabolism or plant growth in the field or whether they are compatible has not yet been tested. Fruit-specific yeast S-adenosylmethionine decarboxylase (ySAMdc) line 579HO, and a control line 556AZ were grown in leguminous hairy vetch (Vicia villosa Roth) (HV) mulch and conventional black polyethylene (BP) mulch, and their fruit analysed. Significant genotype×mulch-dependent interactions on fruit phenotype were exemplified by differential profiles of 20 fruit metabolites such as amino acids, sugars, and organic acids. Expression patterns of the ySAMdc transgene, and tomato SAMdc, E8, PEPC, and ICDHc genes were compared between the two lines as a function of growth on either BP or HV mulch. HV mulch significantly stimulated the accumulation of asparagine, glutamate, glutamine, choline, and citrate concomitant with a decrease in glucose in the 556AZ fruits during ripening as compared to BP. It enables a metabolic system in tomato somewhat akin to the one in higher polyamine-accumulating transgenic fruit that have higher phytonutrient content. Finally, synergism was found between HV mulch and transgenic tomato in up-regulating N:C indicator genes PEPC and ICDHc in the fruit. PMID:18469323

  1. Strategies for designing transgenic DNA constructs.

    PubMed

    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

  2. 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

  3. Conspiracy of silence among repeated transgenes.

    PubMed

    Henikoff, S

    1998-07-01

    Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredictable from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of expression in vertebrate transgene arrays might result from interactions between repeats within the array. A recent experiment using transgenic mouse lines demonstrates that reduction in copy number of silenced transgenes within an array leads to a striking increase in expression, demonstrating that silencing is intrinsic to the array, and is not attributable to position effects of nearby sequences. This work calls into question functions that have been attributed to vertebrate locus control regions and boundaries, and draws attention to the notion that repeat-induced gene silencing is a system for protection of eukaryotic genomes against threatening sequence elements. PMID:9723001

  4. Transgenic mouse offspring generated by ROSI.

    PubMed

    Moreira, Pedro; Pérez-Cerezales, Serafín; Laguna, Ricardo; Fernández-Gonzalez, Raúl; Sanjuanbenito, Belén Pintado; Gutiérrez-Adán, Alfonso

    2016-02-20

    The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes. PMID:26498042

  5. Transgenic mouse offspring generated by ROSI

    PubMed Central

    MOREIRA, Pedro; PÉREZ-CEREZALES, Serafín; LAGUNA, Ricardo; FERNÁNDEZ-GONZALEZ, Raúl; SANJUANBENITO, Belén Pintado; GUTIÉRREZ-ADÁN, Alfonso

    2015-01-01

    The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes. PMID:26498042

  6. 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. ...

  7. Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis)

    PubMed Central

    de Oliveira, Raquel S.; Oliveira-Neto, Osmundo B.; Moura, Hudson F. N.; de Macedo, Leonardo L. P.; Arraes, Fabrício B. M.; Lucena, Wagner A.; Lourenço-Tessutti, Isabela T.; de Deus Barbosa, Aulus A.; da Silva, Maria C. M.; Grossi-de-Sa, Maria F.

    2016-01-01

    Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests. PMID:26925081

  8. Transgenic Cotton Plants Expressing Cry1Ia12 Toxin Confer Resistance to Fall Armyworm (Spodoptera frugiperda) and Cotton Boll Weevil (Anthonomus grandis).

    PubMed

    de Oliveira, Raquel S; Oliveira-Neto, Osmundo B; Moura, Hudson F N; de Macedo, Leonardo L P; Arraes, Fabrício B M; Lucena, Wagner A; Lourenço-Tessutti, Isabela T; de Deus Barbosa, Aulus A; da Silva, Maria C M; Grossi-de-Sa, Maria F

    2016-01-01

    Gossypium hirsutum (commercial cooton) is one of the most economically important fibers sources and a commodity crop highly affected by insect pests and pathogens. Several transgenic approaches have been developed to improve cotton resistance to insect pests, through the transgenic expression of different factors, including Cry toxins, proteinase inhibitors, and toxic peptides, among others. In the present study, we developed transgenic cotton plants by fertilized floral buds injection (through the pollen-tube pathway technique) using an DNA expression cassette harboring the cry1Ia12 gene, driven by CaMV35S promoter. The T0 transgenic cotton plants were initially selected with kanamycin and posteriorly characterized by PCR and Southern blot experiments to confirm the genetic transformation. Western blot and ELISA assays indicated the transgenic cotton plants with higher Cry1Ia12 protein expression levels to be further tested in the control of two major G. hirsutum insect pests. Bioassays with T1 plants revealed the Cry1Ia12 protein toxicity on Spodoptera frugiperda larvae, as evidenced by mortality up to 40% and a significant delay in the development of the target insects compared to untransformed controls (up to 30-fold). Also, an important reduction of Anthonomus grandis emerging adults (up to 60%) was observed when the insect larvae were fed on T1 floral buds. All the larvae and adult insect survivors on the transgenic lines were weaker and significantly smaller compared to the non-transformed plants. Therefore, this study provides GM cotton plant with simultaneous resistance against the Lepidopteran (S. frugiperda), and the Coleopteran (A. grandis) insect orders, and all data suggested that the Cry1Ia12 toxin could effectively enhance the cotton transgenic plants resistance to both insect pests. PMID:26925081

  9. 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.

  10. Position effect variegation and imprinting of transgenes in lymphocytes.

    PubMed

    Williams, Adam; Harker, Nicola; Ktistaki, Eleni; Veiga-Fernandes, Henrique; Roderick, Kathleen; Tolaini, Mauro; Norton, Trisha; Williams, Keith; Kioussis, Dimitris

    2008-04-01

    Sequences proximal to transgene integration sites are able to deregulate transgene expression resulting in complex position effect phenotypes. In addition, transgenes integrated as repeated arrays are susceptible to repeat-induced gene silencing. Using a Cre recombinase-based system we have addressed the influence of transgene copy number (CN) on expression of hCD2 transgenes. CN reduction resulted in a decrease, increase or no effect on variegation depending upon the site of integration. This finding argues that repeat-induced gene silencing is not the principle cause of hCD2 transgene variegation. These results also suggest that having more transgene copies can be beneficial at some integration sites. The transgenic lines examined in this report also exhibited a form of imprinting, which was manifested by decreased levels of expression and increased levels of variegation, upon maternal transmission; and this correlated with DNA hypermethylation and a reduction in epigenetic chromatin modifications normally associated with active genes. PMID:18296483

  11. {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.

  12. Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

    PubMed

    Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

    2011-02-01

    Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields. PMID:21241190

  13. 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

  14. 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

  15. Comparison of Trifecta and Pentafecta Outcomes between T1a and T1b Renal Masses following Robot-Assisted Partial Nephrectomy (RAPN) with Minimum One Year Follow Up: Can RAPN for T1b Renal Masses Be Feasible?

    PubMed Central

    Kim, Dae Keun; Kim, Lawrence H. C.; Raheem, Ali Abdel; Shin, Tae Young; Alabdulaali, Ibrahim; Yoon, Young Eun; Han, Woong Kyu; Rha, Koon Ho

    2016-01-01

    Purpose/Objectives To investigate the feasibility of RAPN on T1b renal mass by assessment of Trifecta and Pentafecta rate between T1a and T1b renal mass. Materials/Methods We retrospectively reviewed the medical records of 277 cases of RPN performed from 2006 to 2015. Sixty patients with clinically T1b renal masses (> 4cm and ≤ 7 cm) were identified, and from 180 patients with clinically T1a renal mass, 60 patients were matched with T1b renal mass by propensity score. Tumor complexity was investigated according to R.E.N.A.L nephrometry score. “Pentafecta” was defined as achievement of Trifecta (negative surgical margin, no postoperative complications and warm ischemia time of ≤ 25 minutes) with addition of over 90% estimated GFR preservation and no chronic kidney disease stage upgrading at 1 year postoperative period. Propensity score matching was performed by OneToManyMTCH. Logistic regression models were used to identify the variables which predict the Trifecta, and Pentafecta ac. Results Preoperative variables (age, sex, body mass index, ASA score) were similar between T1a and T1b after propensity score matching. The median R.E.N.A.L. nephrometry score was 8 vs 9 for T1a and T1b respectively (p<0.001). The median warm ischemia time was 20.1 min vs 26.2 min (p<0.001). Positive surgical margin rate was 5% vs 6.6% (p = 0.729) and overall complication rate of 13.3%. vs 15% (p = 0.793). The rate of achievement of Trifecta rate were 65.3% vs 43.3% (p = 0.017) and Pentafecta rate were 38.3% vs 26.7% (p = 0.172). For achievement of Pentafecta, R.E.N.A.L nephrometry score (HR 0.80; 95% CI (0.67–0.97); p = 0.031) was significant predictor of achieving Pentafecta. Subanalyis to assess the component of R.E.N.A.L nephrometry score, L component (location relative to the polar lines, HR 0.63; 95% CI (0.38–1.03); P = 0.064) was relatively important component for Pentafecta achievement. Conclusions The rate of Pentafecta after RAPN was comparable between T1a and T1b renal masses. RAPN is a feasible modality with excellent long term outcome for patients with larger renal mass (cT1b). PMID:26987069

  16. 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...

  17. Inorganic nanoparticle-based T1 and T1/T2 magnetic resonance contrast probes.

    PubMed

    Hu, Fengqin; Zhao, Yong Sheng

    2012-10-21

    Magnetic resonance imaging (MRI) yields high spatially resolved contrast with anatomical details for diagnosis, deeper penetration depth and rapid 3D scanning. To improve imaging sensitivity, adding contrast agents accelerates the relaxation rate of water molecules, thereby greatly increasing the contrast between specific issues or organs of interest. Currently, the majority of T(1) contrast agents are paramagnetic molecular complexes, typically Gd(iii) chelates. Various nanoparticulate T(1) and T(1)/T(2) contrast agents have recently been investigated as novel agents possessing the advantages of both the T(1) contrast effect and nanostructural characteristics. In this minireview, we describe the recent progress of these inorganic nanoparticle-based MRI contrast agents. Specifically, we mainly report on Gd and Mn-based inorganic nanoparticles and ultrasmall iron oxide/ferrite nanoparticles. PMID:22971876

  18. [Evaluation of Salt Tolerance of Transgenic Tobacco Plants Bearing with P5CS1 Gene of Arabidopsis thaliana].

    PubMed

    Ibragimova, S M; Trifonova, E A; Filipenko, E A; Shymny, V K

    2015-12-01

    Arabidopsis thaliana delta1-pyrroline-5-carhoxylate synthase 1 gene (P5CS1) cDNA was cloned under the control of the potent constitutive 35S RNA promoter of the cauliflower mosaic virus and transferred into genome of tobacco cv. Petit Havana SR-1 (Nicotiana tabacum L.) plants. It is shown that the constitutive level of proline in the transgenic plants T0 exceeds that of the SR1 reference line by 1.5 to 4 times. Under conditions of salt stress (200, 300 mM NaCl) T1-generation transgenic plants in early stages of development formed a large biomass, developed more quickly, and had a higher rate of root growth compared to the control, which confirms the involvement of the P5CS1 gene in molecular mechanisms of stress resistance in plants. PMID:27055296

  19. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco.

    PubMed

    Senthilkumar, Rajendran; Cheng, Chiu-Ping; Yeh, Kai-Wun

    2010-01-01

    Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens. PMID:20055959

  20. Neuroanatomy and transgenic technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This is a short review that introduces recent advances of neuroanatomy and transgenic technologies. The anatomical complexity of the nervous system remains a subject of tremendous fascination among neuroscientists. In order to tackle this extraordinary complexity, powerful transgenic technologies a...

  1. Expression of the Shpx2 peroxidase gene of Stylosanthes humilis in transgenic tobacco leads to enhanced resistance to Phytophthora parasitica pv. nicotianae and Cercospora nicotianae.

    PubMed

    Way, H M; Kazan, K; Goulter, K C; Birch, R G; Manners, J M

    2000-07-01

    Abstract Previous research indicated that the constitutive expression of a pathogen-inducible peroxidase gene (Shpx6a) from Stylosanthes humilis in transgenic plants resulted in enhanced resistance to fungal pathogens ( Kazan, K., Goulter, K.C., Way, H.M. and Manners, J.M. (1998) Expression of a pathogenesis-related peroxidase of Stylosanthes humilis in transgenic tobacco and canola and its effect on disease development. Plant Sci. 136, 207-217). We have now investigated another pathogen-inducible peroxidase gene of S. humilis, termed Shpx2, which is highly divergent from Shpx6a. Constitutive expression of the Shpx2 cDNA was obtained in tobacco using the 35S CaMV promoter, and up to a 12-fold increase in total peroxidase activity was observed in the leaves of transgenic plants compared to nontransgenic controls. Disease development was evaluated after inoculations in T(1) and T(2) transgenic lines expressing Shpx2. Lesion expansion was significantly (P < 0.05) reduced by up to 25% and 50% on leaves and stems, respectively, of transgenic plants expressing high levels of peroxidase compared to nontransgenic controls, following inoculation with Phytophthora parasitica pv. nicotianae, the cause of black shank disease. In addition, plant survival and recovery were greatly enhanced in transgenic plants following stem inoculations of plants with this plant pathogen. A significant (55%, P < 0.05) reduction in lesion number was observed in transgenic plants with high levels of peroxidase activity following inoculation with the fungus Cercospora nicotianae, the cause of frog-eye disease. No significant differences in disease development were observed between the lines expressing Shpx2 and controls following inoculation with the bacterium Pseudomonas syringae pv. tabaci, the cause of wildfire disease. These results provide evidence for a role for this peroxidase gene in plant defence, and suggest that diverse peroxidase genes may be employed as components of strategies aimed at the engineering of disease resistance. PMID:20572969

  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. SirT1 gain of function increases energy efficiency and prevents diabetes in mice.

    PubMed

    Banks, Alexander S; Kon, Ning; Knight, Colette; Matsumoto, Michihiro; Gutiérrez-Juárez, Roger; Rossetti, Luciano; Gu, Wei; Accili, Domenico

    2008-10-01

    In yeast, worms, and flies, an extra copy of the gene encoding the Sirtuin Sir2 increases metabolic efficiency, as does administration of polyphenols like resveratrol, thought to act through Sirtuins. But evidence that Sirtuin gain of function results in increased metabolic efficiency in mammals is limited. We generated transgenic mice with moderate overexpression of SirT1, designed to mimic the Sirtuin gain of function that improves metabolism in C. elegans. These mice exhibit normal insulin sensitivity but decreased food intake and locomotor activity, resulting in decreased energy expenditure. However, in various models of insulin resistance and diabetes, SirT1 transgenics display improved glucose tolerance due to decreased hepatic glucose production and increased adiponectin levels, without changes in body weight or composition. We conclude that SirT1 gain of function primes the organism for metabolic adaptation to insulin resistance, increasing hepatic insulin sensitivity and decreasing whole-body energy requirements. These findings have important implications for Sirtuin-based therapies in humans. PMID:18840364

  4. Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells

    PubMed Central

    Pohlkamp, Theresa; Steller, Laura; May, Petra; Günther, Thomas; Schüle, Roland; Frotscher, Michael

    2014-01-01

    We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo. PMID:24950299

  5. An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

    PubMed

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K; Clemente, Tom E; Long, Stephen P

    2016-04-01

    Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

  6. P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.

    PubMed

    Ronsseray, S; Boivin, A; Anxolabéhère, D

    2001-12-01

    In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed. PMID:11779802

  7. MOLLI and AIR T1 mapping pulse sequences yield different myocardial T1 and ECV measurements.

    PubMed

    Hong, KyungPyo; Kim, Daniel

    2014-11-01

    Both post-contrast myocardial T1 and extracellular volume (ECV) have been reported to be associated with diffuse interstitial fibrosis. Recently, the cardiovascular magnetic resonance (CMR) field is recognizing that post-contrast myocardial T1 is sensitive to several confounders and migrating towards ECV as a measure of collagen volume fraction. Several recent studies using widely available Modified Look-Locker Inversion-recovery (MOLLI) have reported ECV cutoff values to distinguish between normal and diseased myocardium. It is unclear if these cutoff values are translatable to different T1 mapping pulse sequences such as arrhythmia-insensitive-rapid (AIR) cardiac T1 mapping, which was recently developed to rapidly image patients with cardiac rhythm disorders. We sought to evaluate, in well-controlled canine and pig experiments, the relative accuracy and precision, as well as intra- and inter-observer variability in data analysis, of ECV measured with AIR as compared with MOLLI. In 16 dogs, as expected, the mean T1 was significantly different (p < 0.001) between MOLLI (891 ± 373 ms) and AIR (1071 ± 503 ms), but, surprisingly, the mean ECV between MOLLI (21.8 ± 2.1%) and AIR (19.6 ± 2.4%) was also significantly different (p < 0.001). Both intra- and inter-observer agreements in T1 calculations were higher for MOLLI than AIR, but intra- and inter-observer agreements in ECV calculations were similar between MOLLI and AIR. In six pigs, the coefficient of repeatability (CR), as defined by the Bland-Altman analysis, in T1 calculation was considerably lower for MOLLI (32.5 ms) than AIR (82.3 ms), and the CR in ECV calculation was also lower for MOLLI (1.8%) than AIR (4.5%). In conclusion, this study shows that MOLLI and AIR yield significantly different T1 and ECV values in large animals and that MOLLI yields higher precision than AIR. Findings from this study suggest that CMR researchers must consider the specific pulse sequence when translating published ECV cutoff values into their own studies. PMID:25323070

  8. Age-dependent silencing of globin transgenes in the mouse.

    PubMed Central

    Robertson, G; Garrick, D; Wilson, M; Martin, D I; Whitelaw, E

    1996-01-01

    Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation. PMID:8628679

  9. Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature.

    PubMed

    Sharma, Vijendra K; Monostori, Tamas; Göbel, Cornelia; Hänsch, Robert; Bittner, Florian; Wasternack, Claus; Feussner, Ivo; Mendel, Ralf R; Hause, Bettina; Schulze, Jutta

    2006-02-01

    Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence. PMID:16376956

  10. Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

    PubMed Central

    Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

    2009-01-01

    Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida–mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies. PMID:19549857

  11. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  12. 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

  13. Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-β and bone morphogenetic protein signaling

    PubMed Central

    SAWADA, SHUNSUKE; CHOSA, NAOYUKI; TAKIZAWA, NAOKI; YOKOTA, JUN; IGARASHI, YASUYUKI; TOMODA, KOICHI; KONDO, HISATOMO; YAEGASHI, TAKASHI; ISHISAKI, AKIRA

    2016-01-01

    Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG-2, -3, and -5 from the bone marrow of green fluorescent protein (GFP)-transgenic mice. These cell lines clearly expressed mouse MSC markers Sca-1 and CD44, and SG-2 and -5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)-β superfamily. By contrast, SG-3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG-3 cells, which underwent osteogenesis in response to BMP, while TGF-β receptor II was most highly expressed in SG-3 and -5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF-β1 in SG-2 cells but not in SG-3 or -5 cells. Furthermore, TGF-β1 clearly induced the expression of Smad-interacting transcription factor CCAAT/enhancer binding protein-β in SG-2 but not in SG-3 or -5 cells. These results demonstrated the establishment of TGF-β-responsive SG-2 MSCs, BMP-responsive SG-3 MSCs and TGF-β/BMP-unresponsive SG-5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF-β superfamily affects the proliferation and differentiation status of MSCs in vivo. PMID:26781600

  14. Studies of an expanded trinucleotide repeat in transgenic mice

    SciTech Connect

    Bingham, P.; Wang, S.; Merry, D.

    1994-09-01

    Spinal and bulbar muscular atrophy (SBMA) is a progressive motor neuron disease caused by expansion of a trinucleotide repeat in the androgen receptor gene (AR{sup exp}). AR{sup exp} repeats expand further or contract in approximately 25% of transmissions. Analogous {open_quotes}dynamic mutations{close_quotes} have been reported in other expanded trinucleotide repeat disorders. We have been developing a mouse model of this disease using a transgenic approach. Expression of the SBMA AR was documented in transgenic mice with an inducible promoter. No phenotypic effects of transgene expression were observed. We have extended our previous results on stability of the expanded trinucleotide repeat in transgenic mice in two lines carrying AR{sup exp}. Tail DNA was amplified by PCR using primers spanning the repeat on 60 AR{sup exp} transgenic mice from four different transgenic lines. Migration of the PCR product through an acrylamide gel showed no change of the 45 CAG repeat length in any progeny. Similarly, PCR products from 23 normal repeat transgenics showed no change from the repeat length of the original construct. Unlike the disease allele in humans, the expanded repeat AR cDNA in transgenic mice showed no change in repeat length with transmission. The relative stability of CAG repeats seen in the transgenic mice may indicate either differences in the fidelity of replicative enzymes, or differences in error identification and repair between mice and humans. Integration site or structural properties of the transgene itself might also play a role.

  15. Wheat storage proteins in transgenic rice endosperm.

    PubMed

    Oszvald, Mária; Balázs, Gábor; Pólya, Sára; Tömösközi, Sándor; Appels, Rudi; Békés, Ferenc; Tamás, László

    2013-08-01

    Transgenic rice seed expressing wheat HMW glutenin subunit was characterized to study the effects of the wheat prolamin on the protein expression pattern and protein size distribution in the endosperm and the functional and rheological properties of the rice flour and dough. Significant differences were found in the protein expression pattern between the transgenic and wild type samples. Comparing the protein expression profiles of transgenic and nontransgenic plants, combined with proteomic-based studies, indicated increased protein disulfide isomerase (PDI) levels in the transgenic rice lines. The accurate molecular size of HMW-GS in rice endosperm was identified by MALDI-TOF-MS analysis. The expressed wheat HMW (subunit 1Dx5) GS showed a positive effect on the functional properties of rice dough by significantly increasing the size distribution of the polymeric protein fraction and modifying the dough mixing parameters. PMID:23802557

  16. Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpin Xooc-encoding hrf2 gene.

    PubMed

    Huo, Rong; Wang, Yu; Ma, Ling-Li; Qiao, Jun-Qing; Shao, Min; Gao, Xue-Wen

    2010-10-01

    hrf2 gene is a member of the harpin-encoding gene family of rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola. In our previous studies, we observed that harpin(Xooc) could elicit hypersensitive cell death in non-host plants, induce disease and insect resistance in plants, and enhance plant growth. In this study, the rapeseed cultivar, Yangyou 4, was genetically engineered via Agrobacterium-mediated transformation to express the hrf2 gene. Polymerase chain reaction (PCR) and southern blot analyses of T(1) generation of transgenic rapeseed revealed stable integration and expression of the inserted gene hrf2. In addition, the resistance to Sclerotinia sclerotiorum was greatly enhanced. A comparison between agronomic characters of transgenic and control lines displayed significant differences in terms of plant height, stem width, number of pods per plant, number of seeds per pod, 1,000-seed weight, and seed yield per plant. Among lines with resistance to S. sclerotiorum, T(1)1 had improved agronomic traits compared with controls with a 22.7% seed yield increase. These results suggest that the introduction of the hrf2 gene into rapeseed can be an effective strategy for enhancing resistance to S. sclerotiorum. PMID:20107894

  17. Sampling Submicron T1 Bacteriophage Aerosols

    PubMed Central

    Harstad, J. Bruce

    1965-01-01

    Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 μ were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity. Images Fig. 1 Fig. 4 PMID:5866038

  18. Sampling submicron T1 bacteriophage aerosols.

    PubMed

    Harstad, J B

    1965-11-01

    Liquid impingers, filter papers, and fritted bubblers were partial viable collectors of radioactive submicron T1 bacteriophage aerosols at 30, 55, and 85% relative humidity. Sampler differences for viable collection were due to incomplete physical collection (slippage) and killing of phage by the samplers. Dynamic aerosols of a mass median diameter of 0.2 mu were produced with a Dautrebande generator from concentrated aqueous purified phage suspensions containing extracellular soluble radioactive phosphate as a physical tracer. There was considerable destruction of phage by the Dautrebande generator; phage titers of the Dautrebande suspension decreased exponentially, but there was a progressive (linear) increase in tracer titers. Liquid impingers recovered the most viable phage but allowed considerable (30 to 48%) slippage, which varies inversely with the aerosol relative humidity. Filter papers were virtually complete physical collectors of submicron particles but were the most destructive. Fritted bubbler slippage was more than 80%. With all samplers, phage kill was highest at 85% relative humidity and lowest at 55% relative humidity. An electrostatic precipitator was used to collect aerosol samples for particle sizing with an electron microscope. The particle size was slightly larger at 85% relative humidity than at 30 or 55% relative humidity. PMID:5866038

  19. Hepatic steatosis in transgenic mice overexpressing human histone deacetylase 1

    SciTech Connect

    Wang, Ai-Guo; Seo, Sang-Beom; Moon, Hyung-Bae; Shin, Hye-Jun; Kim, Dong Hoon; Kim, Jin-Man; Lee, Tae-Hoon; Kwon, Ho Jeong; Yu, Dae-Yeul . E-mail: dyyu10@kribb.re.kr; Lee, Dong-Seok . E-mail: lee10@kribb.re.kr

    2005-05-06

    It is generally thought that histone deacetylases (HDACs) play important roles in the transcriptional regulation of genes. However, little information is available concerning the specific functions of individual HDACs in disease states. In this study, two transgenic mice lines were established which harbored the human HDAC1 gene. Overexpressed HDAC1 was detected in the nuclei of transgenic liver cells, and HDAC1 enzymatic activity was significantly higher in the transgenic mice than in control littermates. The HDAC1 transgenic mice exhibited a high incidence of hepatic steatosis and nuclear pleomorphism. Molecular studies showed that HDAC1 may contribute to nuclear pleomorphism through the p53/p21 signaling pathway.

  20. Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew.

    PubMed

    Latha, A Madhavi; Rao, K V; Reddy, T P; Reddy, V D

    2006-09-01

    Transgenic pearl millet lines expressing pin gene--exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola--were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T(0), 2T(0), and 3T(0) showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T(1) progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T(1) plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T(1) plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T(2) progenies of 1T(0) conformed to dihybrid segregation of 15 resistant:1 susceptible plants. PMID:16607531

  1. Transgenic Mouse Technology: Principles and Methods

    PubMed Central

    Kumar, T. Rajendra; Larson, Melissa; Wang, Huizhen; McDermott, Jeff; Bronshteyn, Illya

    2014-01-01

    Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now referred to as transgenic mouse technology has revolutionized virtually all fields of biology and provided new genetic approaches to model many human diseases in a whole animal context. Several hundreds of transgenic lines with expression of foreign genes specifically targeted to desired organelles/cells/tissues have been characterized. Further, the ability to spatio-temporally inactivate or activate gene expression in vivo using the “Cre-lox” technology has recently emerged as a powerful approach to understand various developmental processes including those relevant to molecular endocrinology. In this chapter, we will discuss the principles of transgenic mouse technology, and describe detailed methodology standardized at our Institute. PMID:19763515

  2. Generation of Transgenic Mice

    PubMed Central

    Cho, Andrew; Haruyama, Naoto; Kulkarni, Ashok B.

    2009-01-01

    This unit describes detailed step-by-step protocols, reagents, and equipment required for successful generation of transgenic mice using pronuclear injection. The experimental methods and practical tips given here will help guide beginners in understanding what is required and what to avoid in these standard protocols for efficiently generating transgenic mice. PMID:19283729

  3. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 c...

  5. Dose correction for post-contrast T1 mapping of the heart: the MESA study.

    PubMed

    Gai, Neville D; Sandfort, Veit; Liu, Songtao; Lima, João A C; Bluemke, David A

    2016-02-01

    Post-contrast myocardial T1 (T1myo,c) values have been shown to be sensitive to myocardial fibrosis. Recent studies have shown differences in results obtained from T1myo,c and extracellular volume fraction (ECV) with respect to percentage fibrosis. By exploring the relationship between blood plasma volume and T1myo,c, the underlying basis for the divergence can be explained. Furthermore, dose administration based on body mass index (BMI), age and gender can mitigate the divergence in results. Inter-subject comparison of T1myo,c required adjustment for dose (in mmol/kg), time and glomerular filtration rate. Further adjustment for effective dose based on lean muscle mass reflected by blood/plasma volume was performed. A test case of 605 subjects from the MESA study who had undergone pre- and post-contrast T1 mapping was studied. T1myo,c values were compared between subjects with and without metabolic syndrome (MetS), between smoking and non-smoking subjects, and subjects with and without impaired glucose tolerance, before and after dose adjustment based on plasma volume. Comparison with ECV (which is dose independent), pre-contrast myocardial T1 and blood normalized myocardial T1 values was also performed to validate the correction. There were significant differences in T1myo,c (post plasma volume correction) and ECV between current and former smokers (p value 0.017 and 0.01, respectively) but not T1myo,c prior to correction (p = 0.12). Prior to dose adjustment for plasma volume, p value was <0.001 for T1myo,c between MetS and non-MetS groups and was 0.13 between subjects with and without glucose intolerance; after adjustment for PV, p value was 0.63 and 0.99. Corresponding ECV p values were 0.44 and 0.99, respectively. Overall, ECV results showed the best agreement with PV corrected T1myo,c (mean absolute difference in p values = 0.073) and pre-contrast myocardial T1 in comparison with other measures (T1myo,c prior to correction, blood/plasma T1 value normalized myocardium). Weight-based contrast dosing administered in mmol/kg results in a bias in T1 values which can lead to erroneous conclusions. After adjustment for lean muscle mass based on plasma volume, results from T1myo,c were in line with ECV derived results. Furthermore, the use of a modified equivalent dose adjusted for BMI, age, sex and hematocrit can be adopted for quantitative imaging. PMID:26362875

  6. 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...

  7. Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits

    PubMed Central

    Jackson, Kathryn A.; Berg, Jolene M.; Murray, James D.

    2010-01-01

    While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their mammary gland to evaluate the reproductive fitness and growth and development of these animals compared to their non-transgenic counterparts and the impact of consuming a transgenic food product, lysozyme-containing milk. In males, none of the parameters of semen quality, including semen volume and concentration, total sperm per ejaculate, sperm morphology, viability and motility, were significantly different between transgenic bucks and non-transgenic full-sib controls. Likewise, transgenic females of this line did not significantly differ in the reproductive traits of gestation length and litter size compared to their non-transgenic counterparts. To evaluate growth, transgenic and non-transgenic kid goats received colostrum and milk from either transgenic or non-transgenic does from birth until weaning. Neither the presence of the transgene nor the consumption of milk from transgenic animals significantly affected birth weight, weaning weight, overall gain and post-wean gain. These results indicate that the analyzed reproductive and growth traits were not regularly or substantially impacted by the presence or expression of the transgene. The evaluation of these general parameters is an important aspect of defining the safety of applying transgenic technology to animal agriculture. PMID:20135222

  8. L-Theanine elicits umami taste via the T1R1 + T1R3 umami taste receptor.

    PubMed

    Narukawa, Masataka; Toda, Yasuka; Nakagita, Tomoya; Hayashi, Yukako; Misaka, Takumi

    2014-06-01

    L-Theanine is a unique amino acid present in green tea. It elicits umami taste and has a considerable effect on tea taste and quality. We investigated L-theanine activity on the T1R1 + T1R3 umami taste receptor. L-Theanine activated T1R1 + T1R3-expressing cells and showed a synergistic response with inosine 5'-monophosphate. The site-directed mutagenesis analysis revealed that L-theanine binds to L-amino acid binding site in the Venus flytrap domain of T1R1. This study shows that L-theanine elicits an umami taste via T1R1 + T1R3. PMID:24633359

  9. [A study on introduction of chitinase gene and beta-1,3-glucanase gene into restorer line of dian-type hybrid rice (Oryza sativa L.) and enhanced resistance to blast (Magnaporthe grisea)].

    PubMed

    Xu, Ming-Hui; Tang, Zuo-Shun; Tan, Ya-Ling; Tian, Ying-Chuan; Li, Cheng-Yun; Zhang, Shu-Hua; Chen, Zheng-Hua; Tian, Wen-Zhong

    2003-04-01

    Plasmid pBLGC containing chitinase gene from Phaseolus limensis and beta-1,3-glucanase gene from Nicotiana tabacum was bombarded into the restorer line "Nan29" of Dian-type hybrid rice (Oryza sativa L. ssp. japonica) from Yunnan province of South-west China. 93 regenerants were obtained from the calli that were resistant to G418 (100 to 150 mg/L) on NB medium. Using beta-1,3 glucanase gene as the probe, 17 of the regenerants were identified to be transgenic lines by dot blotting and the foreign genes construction were integrated into the genomes of T1 lines by Southern blotting hybridization. Two foreign genes were inherited stably to T4 generation according to PCR results of the lines. The resistance to rice blast of six transgenic lines were evaluated by inoculating four violent biological races of Magnaporthe grisea from Yunnan province and inducing the disease in the field. The results indicated that the resistance to rice blast of transgenic lines were enhanced to varying degrees compared with the receptor line and the transgenic lines could be used in rice blast resistant breeding. PMID:12812057

  10. The natural degeneration course in the T1rho values of normal knee cartilage.

    PubMed

    Goto, Hajimu; Iwama, Yuki; Fujii, Masahiko; Aoyama, Nobukazu; Kubo, Seiji; Kuroda, Ryosuke; Ohno, Yoshiharu; Sugimura, Kazuro

    2011-01-01

    The purpose of our study is to investigate whether there is an age-related change in T1 rho values and to evaluate the effects of weight bearing on age-related increase in T1 rho values of normal cartilage. Thirty-two asymptomatic patients were examined using a 3.0T MRI to determine knee cartilage T1 rho values. Femorotibial and patella cartilage was defined as weight-bearing cartilage (WB-C) and non-weight-bearing cartilage (NWB-C), respectively. The femoral cartilage was divided into weight-bearing (WB-P) and less-weight-bearing (LWB-P) portions. Pearson's correlation coefficient and single regression analysis were used to assess the relationship between cartilage T1 rho values and age. The slopes of the regression lines of cartilage T1 rho values and age were compared between WB-C and NWB-C and between WB-P and LWB-P. Cartilage T1 rho values correlated positively with aging for all cartilage regions and all age groups (p<0.001). In the medial femoral cartilage, the age-related increase in T1 rho values was significantly greater for WB-P than for NWB-P (p<0.05). For several cartilage regions, this increase was greater for WB-C than for LWB-C (p<0.05). The T1 rho value is very sensitive to age-related cartilage degeneration and weight bearing-related degeneration, and hence may be a very sensitive and useful measure for the early diagnosis of osteoarthritis. PMID:22971986

  11. 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, α-, γ-, 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

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

    PubMed

    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, α-, γ-, 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

  13. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

    PubMed

    Tang, Wei; Newton, Ronald J; Weidner, Douglas A

    2007-01-01

    An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies. PMID:17158108

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. 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

  16. A transgenic rabbit model for human hypertrophic cardiomyopathy

    PubMed Central

    Marian, Ali J.; Wu, Yun; Lim, Do-Sun; McCluggage, Meghan; Youker, Keith; Yu, Qun-tao; Brugada, Ramon; DeMayo, Francesco; Quinones, Miguel; Roberts, Robert

    1999-01-01

    Certain mutations in genes for sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). We have developed a transgenic rabbit model for HCM caused by a common point mutation in the β-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human β-MyHC cDNAs were cloned 3′ to a 7-kb murine β-MyHC promoter. We injected purified transgenes into fertilized zygotes to generate two lines each of the wild-type and mutant transgenic rabbits. Expression of transgene mRNA and protein were confirmed by Northern blotting and 2-dimensional gel electrophoresis followed by immunoblotting, respectively. Animals carrying the mutant transgene showed substantial myocyte disarray and a 3-fold increase in interstitial collagen expression in their myocardia. Mean septal thicknesses were comparable between rabbits carrying the wild type transgene and their nontransgenic littermates (NLMs) but were significantly increased in the mutant transgenic animals. Posterior wall thickness and left ventricular mass were also increased, but dimensions and systolic function were normal. Premature death was more common in mutant than in wild-type transgenic rabbits or in NLMs. Thus, cardiac expression of β-MyHC-Q403 in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to β-MyHC-Q403. J. Clin. Invest. 104:1683–1692 (1999). PMID:10606622

  17. Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor).

    PubMed

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

    2013-12-27

    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

  18. Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

    PubMed

    Tamás, Cecília; Kisgyörgy, Boglárka N; Rakszegi, Mariann; Wilkinson, Mark D; Yang, Moon-Sik; Láng, László; Tamás, László; Bedo, Zoltán

    2009-07-01

    An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein. PMID:19466426

  19. Transgenes are dispensable for the RNA degradation step of cosuppression

    PubMed Central

    Palauqui, Jean-Christophe; Vaucheret, Hervé

    1998-01-01

    Cosuppression results in the degradation of RNA from host genes and homologous transgenes after transcription in the nucleus. By using grafting experiments, we have shown previously that a systemic signal mediates the propagation of cosuppression of Nia host genes and 35S-Nia2 transgenes from silenced 35S-Nia2 transgenic stocks to nonsilenced 35S-Nia2 transgenic scions but not to wild-type scions. Here, we examined the requirements for triggering and maintenance of cosuppression in various types of scions. Grafting-induced silencing occurred in 35S-Nia2 transgenic lines over-accumulating Nia mRNA whether they are able to spontaneously trigger cosuppression or not and in 35S-Nia2 transgene-free plants over-accumulating host Nia mRNA caused by metabolic derepression. When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in the 35S-Nia2 transgene-free plants and in the 35S-Nia2 transgenic lines that are not able to trigger cosuppression spontaneously. Conversely, silencing was maintained in the 35S-Nia2 transgenic lines that are able to trigger cosuppression spontaneously. Our results indicate that the presence of a 35S-Nia2 transgene is dispensable for the RNA degradation step of posttranscriptional silencing when host Nia mRNA over-accumulate above the level of wild-type plants. They also suggest that grafting-induced RNA degradation does not result in the production of the systemic silencing signal required for spontaneous triggering and maintenance. PMID:9689140

  20. T1 Relaxation Time in Lungs of Asymptomatic Smokers

    PubMed Central

    Alamidi, Daniel F.; Kindvall, Simon S. I.; Hubbard Cristinacce, Penny L.; McGrath, Deirdre M.; Young, Simon S.; Naish, Josephine H.; Waterton, John C.; Wollmer, Per; Diaz, Sandra; Olsson, Marita; Hockings, Paul D.; Lagerstrand, Kerstin M.; Parker, Geoffrey J. M.; Olsson, Lars E.

    2016-01-01

    Purpose Interest in using T1 as a potential MRI biomarker of chronic obstructive pulmonary disease (COPD) has recently increased. Since tobacco smoking is the major risk factor for development of COPD, the aim for this study was to examine whether tobacco smoking, pack-years (PY), influenced T1 of the lung parenchyma in asymptomatic current smokers. Materials and Methods Lung T1 measurements from 35 subjects, 23 never smokers and 12 current smokers were retrospectively analyzed from an institutional review board approved study. All 35 subjects underwent pulmonary function test (PFT) measurements and lung T1, with similar T1 measurement protocols. A backward linear model of T1 as a function of FEV1, FVC, weight, height, age and PY was tested. Results A significant correlation between lung T1 and PY was found with a negative slope of -3.2 ms/year (95% confidence interval [CI] [-5.8, -0.6], p = 0.02), when adjusted for age and height. Lung T1 shortens with ageing among all subjects, -4.0 ms/year (95%CI [-6.3, -1.7], p = 0.001), and among the never smokers, -3.7 ms/year (95%CI [-6.0, -1.3], p = 0.003). Conclusions A correlation between lung T1 and PY when adjusted for both age and height was found, and T1 of the lung shortens with ageing. Accordingly, PY and age can be significant confounding factors when T1 is used as a biomarker in lung MRI studies that must be taken into account to detect underlying patterns of disease. PMID:26958856

  1. [Inheritance and expression stability of transgene in transgenic animals].

    PubMed

    Kong, Qing-Ran; Liu, Zhong-Hua

    2011-05-01

    Transgenic technology is one of the most hotspots in biology. In the past decade, the progress in animal cloning has provided an alternative method to improve transgenic efficiency. Many kinds of transgenic animals have been successfully produced via the combination of transfection and nuclear transfer. However, the ultimate aim of transgenesis is not to produce several transgenic animals, but to service for the needs of human. In animal production, transgenic technology has been used to breed new livestock, which has received a lot of attention in China. It has been evidenced that inheritance and expression instability of transgene in transgenic animals is still the major limitation, which is attributed to position effect, epigenetic modification, and hereditary efficiency of transgene. In this review, we discussed the three points for promoting the industrialization of animal transgenic breeding. PMID:21586397

  2. Irreversible change in the T1 temperature dependence with thermal dose using the PRF-T1 Technique

    PubMed Central

    Diakite, Mahamadou; Payne, Allison; Todd, Nick; Parker, Dennis L.

    2012-01-01

    Denaturation of macromolecules within the tissues is believed to be the major factor contributing to the damage of tissues upon hyperthermia. As a result, the value of the spin-lattice relaxation time T1 of the tissue water, which is related to the translational and rotational rates of water, represents an intrinsic probe for investigating structural changes in tissues at high temperature. Therefore, the goal of the present work is to investigate whether the simultaneous measurement of temperature and T1 using a hybrid PRF-T1 measurement technique, can be used to detect irreversible changes in T1 that might be indicative of tissue damage. A new hybrid PRF-T1 sequence was implemented based on the variable flip angle DESPOT1 method from a standard 3D segmented EPI sequence by alternating two flip angles from measurement to measurement. The structural changes of the heated tissue volumes were analyzed based on the derived T1 values and the corresponding PRF temperatures. Using the hybrid PRF-T1 technique, we demonstrate that the change of spin lattice relaxation time T1 is reversible with temperature for low thermal dose (thermal dose ≤ 240 CEM43°C) and irreversible with temperature after significant accumulation of thermal dose in ex vivo chicken breast tissue. These results suggest that the hybrid PRF-T1 method may be a potentially powerful tool to investigate the extent and mechanism of heat damage of biological tissues. PMID:22576265

  3. Sarcosine based indandione hGlyT1 inhibitors.

    PubMed

    Thomson, Christopher G; Duncan, Karen; Fletcher, Stephen R; Huscroft, Ian T; Pillai, Gopalan; Raubo, Piotr; Smith, Alison J; Stead, Darren

    2006-03-01

    A series of sarcosine based indandione hGlyT1 inhibitors has been developed. Optimization of substitution around the indandione and sarcosine moieties has led to highly potent inhibitors at hGlyT1, which show selectivity over a number of other receptors. PMID:16321523

  4. 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

  5. Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T(1) seedlings of tomato exposed to metal ions.

    PubMed

    Kamaladini, Hossein; Nor Akmar Abdullah, Siti; Aziz, Maheran Abdul; Ismail, Ismanizan Bin; Haddadi, Fatemeh

    2013-02-15

    Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 μM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions. PMID:23290536

  6. Systematic T1 improvement for hyperpolarized 129xenon

    NASA Astrophysics Data System (ADS)

    Repetto, Maricel; Babcock, Earl; Blmler, 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 /T1Xel -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.

  7. Dynamic Quantitative T1 Mapping in Orthotopic Brain Tumor Xenografts.

    PubMed

    Herrmann, Kelsey; Erokwu, Bernadette O; Johansen, Mette L; Basilion, James P; Gulani, Vikas; Griswold, Mark A; Flask, Chris A; Brady-Kalnay, Susann M

    2016-04-01

    Human brain tumors such as glioblastomas are typically detected using conventional, nonquantitative magnetic resonance imaging (MRI) techniques, such as T2-weighted and contrast enhanced T1-weighted MRI. In this manuscript, we tested whether dynamic quantitative T1 mapping by MRI can localize orthotopic glioma tumors in an objective manner. Quantitative T1 mapping was performed by MRI over multiple time points using the conventional contrast agent Optimark. We compared signal differences to determine the gadolinium concentration in tissues over time. The T1 parametric maps made it easy to identify the regions of contrast enhancement and thus tumor location. Doubling the typical human dose of contrast agent resulted in a clearer demarcation of these tumors. Therefore, T1 mapping of brain tumors is gadolinium dose dependent and improves detection of tumors by MRI. The use of T1 maps provides a quantitative means to evaluate tumor detection by gadolinium-based contrast agents over time. This dynamic quantitative T1 mapping technique will also enable future quantitative evaluation of various targeted MRI contrast agents. PMID:27084431

  8. Transgenic mouse model of cutaneous adnexal tumors

    PubMed Central

    Kito, Yusuke; Saigo, Chiemi; Atsushi, Kurabayashi; Mutsuo, Furihata; Tamotsu, Takeuchi

    2014-01-01

    TMEM207 was first characterized as being an important molecule for the invasion activity of gastric signet-ring cell carcinoma cells. In order to unravel the pathological properties of TMEM207, we generated several transgenic mouse lines, designated C57BL/6-Tg (ITF-TMEM207), in which murine TMEM207 was ectopically expressed under a truncated (by ~200 bp) proximal promoter of the murine intestinal trefoil factor (ITF) gene (also known as Tff3). Unexpectedly, a C57BL/6-Tg (ITF-TMEM207) mouse line exhibited a high incidence of spontaneous intradermal tumors with histopathological features that resembled those of various human cutaneous adnexal tumors. These tumors were found in ~14% female and 13% of male 6- to 12-month-old mice. TMEM207 immunoreactivity was found in hair follicle bulge cells in non-tumorous skin, as well as in cutaneous adnexal tumors of the transgenic mouse. The ITF-TMEM207 construct in this line appeared to be inserted to a major satellite repeat sequence at chromosome 2, in which no definite coding molecule was found. In addition, we also observed cutaneous adnexal tumors in three other C57BL/6-Tg (ITF-TMEM207) transgenic mouse lines. We believe that the C57BL/6-Tg (ITF-TMEM207) mouse might be a useful model to understand human cutaneous adnexal tumors. PMID:25305140

  9. Overexpression of mouse follistatin causes reproductive defects in transgenic mice.

    PubMed

    Guo, Q; Kumar, T R; Woodruff, T; Hadsell, L A; DeMayo, F J; Matzuk, M M

    1998-01-01

    Follistatin is an activin-binding protein that can act as an activin antagonist in vitro. Follistatin also binds heparin sulfate proteoglycans and may function as a reservoir for activins in vivo. In the mouse, follistatin mRNA is first detected in the deciduum on embryonic day 5.5 and later in the developing hindbrain, somites, vibrissae, teeth, epidermis, and muscle. We have previously shown that follistatin-deficient mice have numerous embryonic defects including shiny, taut skin, growth retardation, and cleft palate leading to death within hours of birth. To further define the roles of follistatin during mammalian reproduction and development, we created gain-of-function mutant mice in which mouse follistatin is overexpressed. The mouse metallothionein (MT)-I promoter was placed upstream of the six-exon mouse follistatin (FS) gene. To distinguish wild-type and transgenic follistatin mRNA, the 3'-untranslated region of the mouse follistatin gene was replaced with the SV40 untranslated and polyA sequences. Three male and two female founder transgenic mice were produced, were fertile, and transmitted the transgene to offspring. Northern blot analysis demonstrated that the transgene mRNA was expressed at varying levels in the livers of offspring from four of five of the transgenic lines and was expressed in the testes in all five lines. In MT-FS line 4, which had the highest expression of the transgene mRNA in the liver, the transgene transcripts were also present in multiple other tissues. Phenotypically, the MT-FS transgenic lines had defects in the testis, ovary, and hair. Mice from MT-FS lines 7 and 10 had slightly decreased testis size, whereas mice from lines 4, 5, and 9 had much smaller testes and shiny, somewhat irregular, fur. Histological analysis of the adult testes from line 5 and 9 males showed variable degrees of Leydig cell hyperplasia, an arrest of spermatogenesis, and seminiferous tubular degeneration leading to infertility. Female transgenic mice from lines 4 and 9 had thin uteri and small ovaries due to a block in folliculogenesis at various stages. Many of the line 9 female mice eventually became infertile, and all of the line 4 female mice were infertile. Suppressed serum FSH levels were seen in only the line 4 transgenic male and female mice, the line with widespread expression of the transgene. Serum FSH levels were not significantly different in gonadectomized wild-type and line 5 transgenic male mice despite high levels of the follistatin transgene mRNA in the liver of these transgenic mice. These results suggest that follistatin exerts its effects at the levels of the gonads and pituitary as a local regulator of activin and possibly other transforming growth factor-beta family members. PMID:9440814

  10. A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass, precinct vegetation and the microbial community.

    PubMed

    Oguchi, Taichi; Kashimura, Yuko; Mimura, Makiko; Yu, Xiang; Matsunaga, Etsuko; Nanto, Kazuya; Shimada, Teruhisa; Kikuchi, Akira; Watanabe, Kazuo N

    2014-10-01

    A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial. PMID:24927812

  11. Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants.

    PubMed

    Udawat, Pushpika; Jha, Rajesh K; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and [Formula: see text] radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

  12. Overexpression of a Cytosolic Abiotic Stress Responsive Universal Stress Protein (SbUSP) Mitigates Salt and Osmotic Stress in Transgenic Tobacco Plants

    PubMed Central

    Udawat, Pushpika; Jha, Rajesh K.; Sinha, Dinkar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    The universal stress protein (USP) is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologs of intron less SbUSP gene which encodes for salt and osmotic responsive USP. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control [wild-type (WT) and vector control (VC)] plants under different abiotic stress condition. Transgenic lines (T1) exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability, and lower electrolyte leakage and lipid peroxidation (malondialdehyde content) under stress treatments than control (WT and VC) plants. Lower accumulation of H2O2 and O2− radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant. PMID:27148338

  13. Design and Management of Field Trials of Transgenic Cereals

    NASA Astrophysics Data System (ADS)

    Bedő, Zoltán; Rakszegi, Mariann; Láng, László

    The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment.

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

    PubMed

    Faria, Josias C; Carneiro, Geraldo E S; Aragão, 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

  15. Design and management of field trials of transgenic cereals.

    PubMed

    Bedo, Zoltán; Rakszegi, Mariann; Láng, László

    2009-01-01

    The development of gene transformation systems has allowed the introgression of alien genes into plant genomes, thus providing a mechanism for broadening the genetic resources available to plant breeders. The design and the management of field trials vary according to the purpose for which transgenic cereals are developed. Breeders study the phenotypic and genotypic stability of transgenic plants, monitor the increase in homozygosity of transgenic genotypes under field conditions, and develop backcross generations to transfer the introduced genes into secondary transgenic cereal genotypes. For practical purposes, they may also multiply seed of the transgenic lines to produce sufficient amounts of grain for the detailed analysis of trait(s) of interest, to determine the field performance of transgenic lines, and to compare them with the non-transformed parental genotypes. Prior to variety registration, the Distinctness, Uniformity and Stability (DUS) tests and Value for Cultivation and Use (VCU) experiments are carried out in field trials. Field testing includes specific requirements for transgenic cereals to assess potential environmental risks. The capacity of the pollen to survive, establish and disseminate in the field test environment, the potential for gene transfer, the effects of products expressed by the introduced sequences and phenotypic and genotypic instability that might cause deleterious effects must all be specifically monitored, as required by EU Directives 2003/701/EC (1) on the release of genetically modified higher plants in the environment. PMID:19009453

  16. Irreversible change in the T1 temperature dependence with thermal dose using the proton resonance frequency-T1 technique.

    PubMed

    Diakite, Mahamadou; Payne, Allison; Todd, Nick; Parker, Dennis L

    2013-04-01

    Denaturation of macromolecules within the tissues is believed to be the major factor contributing to the damage of tissues upon hyperthermia. As a result, the value of the spin-lattice relaxation time T1 of the tissue water, which is related to the translational and rotational rates of water, represents an intrinsic probe for investigating structural changes in tissues at high temperature. Therefore, the goal of this work is to investigate whether the simultaneous measurement of temperature and T1 using a hybrid proton resonance frequency (PRF)-T1 measurement technique can be used to detect irreversible changes in T1 that might be indicative of tissue damage. A new hybrid PRF-T1 sequence was implemented based on the variable flip angle driven-equilibrium single-pulse observation (DESPOT)1 method from a standard three dimensional segmented echo-planar imaging sequence by alternating two flip angles from measurement to measurement. The structural changes of the heated tissue volumes were analyzed based on the derived T1 values and the corresponding PRF temperatures. Using the hybrid PRF-T1 technique, we demonstrate that the change of spin lattice relaxation time T1 is reversible with temperature for low thermal dose (thermal dose ≤ 240 cumulative equivalent minutes [CEM] 43°C) and irreversible with temperature after significant accumulation of thermal dose in ex vivo chicken breast tissue. These results suggest that the hybrid PRF-T1 method may be a potentially powerful tool to investigate the extent and mechanism of heat damage of biological tissues. PMID:22576265

  17. Free-Breathing Post-Contrast Three-dimensional T1 Mapping: Volumetric Assessment of Myocardial T1 Values

    PubMed Central

    Weingrtner, Sebastian; Akakaya, Mehmet; Roujol, Sbastien; Basha, Tamer; Stehning, Christian; Kissinger, Kraig V.; Goddu, Beth; Berg, Sophie; Manning, Warren J.; Nezafat, Reza

    2014-01-01

    Purpose: To develop a 3D free-breathing myocardial T1 mapping sequence for assessment of left ventricle diffuse fibrosis after contrast administration. Methods: In the proposed sequence, multiple 3D inversion recovery images are acquired in an interleaved manner. A mixed prospective/retrospective navigator scheme is used to obtain the 3D Cartesian k-space data with fully sampled center and randomly undersampled outer k-space. The resulting undersampled 3D k-space data are then reconstructed using compressed sensing. Subsequently, T1 maps are generated by voxel-wise curve-fitting of the individual interleaved images. In a phantom study, the accuracy of the 3D sequence was evaluated against modified 2D Look-Locker inversion recovery (MOLLI) and spin-echo sequences. In-vivo T1 times of the proposed method were compared to 2D multi-slice MOLLI T1 mapping. Subsequently, the feasibility of high-resolution 3D T1 mapping with spatial resolution of 1.71.74mm3 was demonstrated. Results: The proposed method shows good agreement with 2D MOLLI and the spin-echo reference in phantom. No significant difference was found in the in-vivo T1 times estimated using the proposed sequence and the 2D MOLLI technique (myocardium: 330 66ms vs. 319 93 ms, blood-pools: 211 68 ms vs. 210 98 ms). However, improved homogeneity, as measured using standard deviation of the T1 signal, was observed in the 3D T1 maps. Conclusion: The proposed sequence enables high-resolution 3D T1 mapping after contrast injection during free-breathing with volumetric LV coverage. PMID:24554395

  18. 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...

  19. A thermoalkaliphilic lipase of Geobacillus sp. T1.

    PubMed

    Leow, Thean Chor; Rahman, Raja Noor Zaliha Raja Abd; Basri, Mahiran; Salleh, Abu Bakar

    2007-05-01

    A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra. PMID:17426920

  20. Introgression of the SbASR-1 gene cloned from a halophyte Salicornia brachiate enhances salinity and drought endurance in transgenic groundnut (arachis hypogaea)and acts as a transcription factor [corrected].

    PubMed

    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

  1. 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

  2. Transgenic Crops for Herbicide Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their introduction in 1995, crops made resistant to the broad-spectrum herbicides glyphosate and glufosinate with transgenes are widely available and used in much of the world. As of 2008, over 80% of the transgenic crops grown world-wide have this transgenic trait. This technology has had m...

  3. Substrate upregulation of the human small intestinal peptide transporter, hPepT1

    PubMed Central

    Walker, Dianne; Thwaites, David T; Simmons, Nicholas L; Gilbert, Harry J; Hirst, Barry H

    1998-01-01

    Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl-l-glutamine (Gly-Gln) of the proton-coupled peptide transporter, hPepT1, in the Caco-2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl-[14C]sarcosine was increased 1.64 (± 0.34)-fold after incubation of Caco-2 cells for 3 days in a peptide-rich medium (4 mm Gly-Gln replacing 4 mm Gln). A full-length Caco-2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile-662 to Val. Transcript sizes, on Northern blots of Caco-2 poly(A)+ RNA probed with a 630 bp 5′ hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide-induced increase in substrate transport was accompanied by a parallel increase of 1.92 (± 0.30)-fold (n = 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half-life from 8.9 ± 1.1 to 12.5 ± 1.6 h (n = 3), but the increase in half-life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide-mediated increase in hPepT1 transcription. Anti-hPepT1-specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco-2 cells. Gly-Gln supplementation of media resulted in a 1.72 (± 0.26)-fold (n = 5) increase in staining intensity of Caco-2 cells. We conclude that Caco-2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly-Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control. PMID:9508831

  4. Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells

    PubMed Central

    Phang, Wai-Mei; Tan, Aik-Aun; Gopinath, Subash C.B.; Hashim, Onn H.; Kiew, Lik Voon; Chen, Yeng

    2016-01-01

    Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer. PMID:27226773

  5. 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...

  6. Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco

    PubMed Central

    Tizaoui, Kalthoum; Kchouk, Mohamed Elyes

    2012-01-01

    Transgene integration into plant genomes is a complex process accompanied by molecular rearrangements. Classic methods that are normally used to study transgenic population genetics are generally inadequate for assessing such integration. Two major characteristics of transgenic populations are that a transgenic genome may harbor many copies of the transgene and that molecular rearrangements can create an unstable transgenic locus. In this work, we examined the segregation of T1, T2 and T3 transgenic tobacco progenies. Since transfer DNA (T-DNA) contains the NptII selectable marker gene that confers resistance to kanamycin, we used this characteristic in developing a method to estimate the number of functional inserts integrated into the genome. This approach was based on calculation of the theoretical segregation ratios in successive generations. Mendelian ratios of 3:1, 15:1 and 63:1 were confirmed for five transformation events whereas six transformation events yielded non-segregating progenies, a finding that raised questions about causal factors. A second approach based on a maximum likelihood method was performed to estimate recombination frequencies between linked inserts. Recombination estimates varied among transformation events and over generations. Some transgenic loci were unstable and evolved continuously to segregate independently in the T3 generation. Recombination and amplification of the transgene and filler DNA yielded additional transformed genotypes. PMID:23055804

  7. 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...

  8. Generation of transgenic papaya with double resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus.

    PubMed

    Kung, Yi-Jung; Bau, Huey-Jiunn; Wu, Yi-Ling; Huang, Chiung-Huei; Chen, Tsui-Miao; Yeh, Shyi-Dong

    2009-11-01

    During the field tests of coat protein (CP)-transgenic papaya lines resistant to Papaya ringspot virus (PRSV), another Potyvirus sp., Papaya leaf-distortion mosaic virus (PLDMV), appeared as an emerging threat to the transgenic papaya. In this investigation, an untranslatable chimeric construct containing the truncated CP coding region of the PLDMV P-TW-WF isolate and the truncated CP coding region with the complete 3' untranslated region of PRSV YK isolate was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterium-mediated transformation to generate transgenic plants with resistance to PLDMV and PRSV. Seventy-five transgenic lines were obtained and challenged with PRSV YK or PLDMV P-TW-WF by mechanical inoculation under greenhouse conditions. Thirty-eight transgenic lines showing no symptoms 1 month after inoculation were regarded as highly resistant lines. Southern and Northern analyses revealed that four weakly resistant lines have one or two inserts of the construct and accumulate detectable amounts of transgene transcript, whereas nine resistant lines contain two or three inserts without significant accumulation of transgene transcript. The results indicated that double virus resistance in transgenic lines resulted from double or more copies of the insert through the mechanism of RNA-mediated posttranscriptional gene silencing. Furthermore, three of nine resistant lines showed high levels of resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. Our transgenic lines have great potential for controlling a number of PRSV strains and PLDMV in Taiwan and elsewhere. PMID:19821736

  9. 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

  10. 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.

  11. T1ρ MR Imaging of Human Musculoskeletal System

    PubMed Central

    Wang, Ligong; Regatte, Ravinder R.

    2014-01-01

    Magnetic resonance imaging (MRI) offers the direct visualization of human musculoskeletal (MSK) system, especially all diarthrodial tissues including cartilage, bone, menisci, ligaments, tendon, hip, synovium etc. Conventional MR imaging techniques based on T1- and T2-weighted, proton density (PD) contrast are inconclusive in quantifying early biochemically degenerative changes in MSK system in general and articular cartilage in particular. In recent years, quantitative MR parameter mapping techniques have been used to quantify the biochemical changes in articular cartilage with a special emphasis on evaluating joint injury, cartilage degeneration, and soft tissue repair. In this article, we will focus on cartilage biochemical composition, basic principles of T1ρ MR imaging, implementation of T1ρ pulse sequences, biochemical validation, and summarize the potential applications of T1ρ MR imaging technique in MSK diseases including osteoarthritis (OA), anterior cruciate ligament (ACL) injury, and knee joint repair. Finally, we will also review the potential advantages, challenges, and future prospects of T1ρ MR imaging for widespread clinical translation. PMID:24935818

  12. Nitrogen 15 T1 measurements of semicrystalline nylon 6

    NASA Astrophysics Data System (ADS)

    Powell, Douglas G.; Mathias, Lon J.

    1989-05-01

    The solid state 15N NMR characterization of nylon 6 is reported. Nylon 6 (20 percent 15N enriched) was prepared by anionic polymerization of isotopically enriched caprolactam. The samples were prepared by three different treatments: quenched from the melt, slowly cooled and annealed, and artificially plasticized with excess caprolactam. CP/MAS spectra of the 15N enriched samples showed a single sharp peak (alpha crystal form) at 84.2ppm (relative to glycine) and a broader resonance at 87.2 ppm. Relaxation experiments were conducted to determine the spin lattice relaxation time T1N for each sample at 300K. The crystalline resonance was found to have T1N's of 125-416s, consistent with a rigid, crystalline component. The downfield peak (87.2 ppm) had two measurable T1N's: a short component with T1 of 1-3s and a second component with a longer T1 of 19-29s.

  13. 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

  14. 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

  15. 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

  16. 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

  17. Supersymmetric consistent truncations of IIB on T 1, 1

    NASA Astrophysics Data System (ADS)

    Bena, Iosif; Giecold, Gregory; Graña, Mariana; Halmagyi, Nick; Orsi, Francesco

    2011-04-01

    We study consistent Kaluza-Klein reductions of type IIB supergravity on T 1,1 down to five-dimensions. We find that the most general reduction containing singlets under the global SU(2) × SU(2) symmetry of T 1,1 is mathcal{N} = 4 gauged supergravity coupled to three vector multiplets with a particular gauging due to topological and geometric flux. Key to this reduction is several modes which have not been considered before in the literature and our construction allows us to easily show that the Papadopoulos -Tseytlin ansatz for IIB solutions on T 1,1 is a consistent truncation. This explicit reduction provides an organizing principle for the linearized spectrum around the warped deformed conifold as well as the baryonic branch and should have applications to the physics of flux compactifications with warped throats.

  18. Management of colorectal T1 carcinoma treated by endoscopic resection.

    PubMed

    Saitoh, Yusuke; Inaba, Yuhei; Sasaki, Takahiro; Sugiyama, Ryuji; Sukegawa, Ryuji; Fujiya, Mikihiro

    2016-04-01

    As a result of recent advances in endoscopic therapeutic technology, the number of endoscopic resections carried out in the treatment of early colorectal carcinomas with little risk of lymph node metastasis has increased. There are no reports of lymph node metastasis in intramucosal (Tis) carcinomas, whereas lymph node metastasis occurs in 6.8-17.8% of submucosal (T1) carcinomas. Three clinical guidelines have been published in Japan and the management strategy for early colorectal tumors has been demonstrated. According to the 2014 Japanese Society for Cancer of the Colon and Rectum (JSCCR) Guidelines for the Treatment of Colorectal Cancer, additional surgery should be done in cases of endoscopically resected T1 carcinoma with a histologically diagnosed positive vertical margin. Additional surgery may also be considered when one of the following histological findings is detected: (i) SM invasion depth ≥1000 µm; (ii) histological type por., sig., or muc.; (iii) grade 2-3 tumor budding; and (iv) positive vascular permeation. A resected lesion that is histologically diagnosed as a T1 carcinoma without any of the above-mentioned findings can be followed up without additional surgery. As for the prognosis of endoscopically resected T1 carcinomas, the relapse ratio of approximately 3.4% (44/1312) is relatively low. However, relapse is associated with a poor prognosis, with 72 cancer-related deaths reported out of 134 relapsed cases (54%). A more detailed stratification of the lymph node metastasis risk after endoscopic resection for T1 carcinomas and the prognosis of relapsed cases will be elucidated through prospective studies. Thereafter, the appropriate indications and safe and secure endoscopic resection for T1 carcinomas will be established. PMID:26076802

  19. Visualization and genetic manipulation of adult neurogenesis using transgenic mice.

    PubMed

    Dhaliwal, Jagroop; Lagace, Diane C

    2011-03-01

    Many laboratories have focused efforts on the creation of transgenic mouse models to study adult neurogenesis. In the last decade several constitutive reporter, as well as inducible transgenic lines have been published that allowed for visualization, tracking and alteration of specific neurogenic cell populations in the adult brain. Given the popularity of this approach, multiple mouse lines are available, and this review summarizes the differences in the basic techniques that have been used to create these mice, highlighting the different constructs and reporter proteins used, as well as the strengths and limitations of each of these models. Representative examples from the literature demonstrate some of the diverse and seminal findings that have come to fruition through the laborious, yet highly rewarding work of creating transgenic mouse lines for adult neurogenesis research. PMID:21395845

  20. A novel enterocin T1 with anti-Pseudomonas activity produced by Enterococcus faecium T1 from Chinese Tibet cheese.

    PubMed

    Liu, Hui; Zhang, Lanwei; Yi, Huaxi; Han, Xue; Gao, Wei; Chi, Chunliang; Song, Wei; Li, Haiying; Liu, Chunguang

    2016-02-01

    An enterocin-producing Enterococcus faecium T1 was isolated from Chinese Tibet cheese. The enterocin was purified by SP-Sepharose and reversed phase HPLC. It was identified as unique from other reported bacteriocins based on molecular weight (4629 Da) and amino acid compositions; therefore it was subsequently named enterocin T1. Enterocin T1 was stable at 80-100 °C and over a wide pH range, pH 3.0-10.0. Protease sensitivity was observed to trypsin, pepsin, papain, proteinase K, and pronase E. Importantly, enterocin T1 was observed to inhibit the growth of numerous Gram-negative and Gram-positive bacteria including Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes. Take together, these results suggest that enterocin T1 is a novel bacteriocin with the potential to be used as a bio-preservative to control Pseudomonas spp. in food. PMID:26745981

  1. 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.

  2. Gene expression profile in liver of hB1F transgenic mice

    PubMed Central

    Wang, Shui-Liang; Yang, Hua; Xie, You-Hua; Wang, Yuan; Li, Jian-Zhong; Wang, Long; Wang, Zhu-Gang; Fu, Ji-Liang

    2004-01-01

    AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hB1F transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of the differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hB1F were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hB1F transgene may cause changes of gene expression profiles in the liver of transgenic mice. PMID:15378783

  3. Transgenic control of perforin gene expression

    SciTech Connect

    Lichtenheld, M.G.; Podack, E.R.; Levy, R.B.

    1995-03-01

    Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murin perforin 5{prime} flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Transgene expression occurred in all cells that physiologically are able to express perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8{sup +}, as well as on CD4{sup +} cells. Also targeted were Thy-1.2{sup +} {gamma}{delta} T cells, but not Thy-1.2{sup -} {gamma}{delta} T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4{sup -}CD8{sup -}) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5{prime} flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or Nk cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation. 43 refs., 7 figs., 1 tab.

  4. Simplifying transgene locus structure through Cre-lox recombination.

    PubMed

    Srivastava, Vibha; Ow, David W

    2015-01-01

    Transgene silencing is often associated with multicopy integrations, which occur frequently during plant transformation. Transgene expression can be restored in a number of multicopy loci by converting them to single copy. This chapter describes a plant transformation protocol based on use of the Cre-lox system, which allows conversion of a multicopy transgene locus into single copy. The strategy is based on designing a transformation vector with lox sites, developing transgenic lines, and introducing Cre activity to initiate Cre-lox recombination, which leads to the simplification of a multicopy locus to a single- or low-copy state. This method is compatible with both gene gun and Agrobacterium-mediated gene delivery and should be particularly useful for crops that are difficult to transform. PMID:25740358

  5. [Features of development and reproduction of transgenic flax].

    PubMed

    Lemesh, V A; Samatadze, T E; Guzenko, E V; Zhelezniakova, E V; Amosova, A V; Zelenin, A V; Muravenko, O V

    2014-01-01

    Primary transformants carrying a genetic construct with the chimeric gfp-tua6 gene were obtained using biolistic transformation of hypocotyl explants of flax variety Vasilek. Viable modified plants were used as a basis for the production of inbred lines with confirmed inheritance of introduced genetic construct in three generations. The characteristics of phenological growth stages, plant height, number of bolls and meiosis were studied for transgenic plants. A comparison of transformed lines based on reproduction years revealed a significant decrease of seed production in one line. Meiotic analysis of this line at metaphase I and anaphase I stages was conducted. The percentage of cells with impaired meiosis was highest in transgenic plants of the line with the lowest seed production. Thus, the nonspecific incorporation of genetic construct into the flax genome using biolistic transformation impairs meiosis to a different extent and it is the main reason for unequal reproducibility of transgenic flax. The production of stably reproducing transgenic lines requires systematic analysis of meiosis. PMID:25739298

  6. 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

  7. Airway-Specific Inducible Transgene Expression Using Aerosolized Doxycycline

    PubMed Central

    Tata, Purushothama Rao; Pardo-Saganta, Ana; Prabhu, Mythili; Vinarsky, Vladimir; Law, Brandon M.; Fontaine, Benjamin A.; Tager, Andrew M.

    2013-01-01

    Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter–driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell–specific transgene expression using a cytokeratin 5 (also known as keratin 5)–driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype. PMID:23848320

  8. Generation and characterization of JSRV envelope transgenic mice in FVB background.

    PubMed

    Chitra, Ebenezer; Yu, Shu-Ling; Hsiao, Kuang-Nan; Shao, Hsiao-Yun; Sia, Charles; Chen, I-Hua; Hsieh, Shih-Yang; Chen, Jen-Hao; Chow, Yen-Hung

    2009-10-10

    Jaagsiekte sheep retrovirus (JSRV) that causes contagious ovine pulmonary adenocarcinoma (OPA) in sheep carries an oncogenic Envelope gene (Env), which is capable of transforming target cells in vitro and in vivo. We cloned full-length JSRV Env cDNA into an expression vector, SPC/SV40, where the transgene was driven by lung-specific surfactant protein C (SPC) promoter, to obtain SPC-JSRV Env construct. SPC-JSRV Env was microinjected into immunocompetent FVB/N mice embryos to generate Env transgenic mice. We obtained two lines of transgenic mice, both of which were capable of developing spontaneous lung tumors from 1 month onwards and the tumor incidence rate was about 56% at the age of 7 months in Env Transgenic line 1 and about 71% at the age of 6 months in Env Transgenic line 2. We were able to correlate higher tumor incidence rate and tumorigenicity in Env Transgenic line 2 to higher level of expression of Env transgene compared to Env Transgenic line 1. Immunohistochemical analysis showed that the tumor was primarily composed of type II pneumocytes where SPC promoter is known to be active similar to natural infection of JSRV in sheep. Analysis of cellular mitogenic signal transduction pathways revealed significant induction of p44/42 ERK pathway in the transgenic mice lungs with tumors compared to the lungs from non-transgenic FVB/N mice. Tumors in our transgenic mice pose similarities to human lung adenocarcinoma and therefore our mice could serve as a model system for evaluating the mechanisms of lung tumorigenesis in vivo. PMID:19695657

  9. Obesity-related abnormalities couple environmental triggers with genetic susceptibility in adult-onset T1D.

    PubMed

    Nguyen, K Hoa; Ande, Sudharsana R; Mishra, Suresh

    2016-01-29

    The incidence of adult-onset T1D in low-risk non-HLA type has increased several folds, whereas the contemporaneous incidence in high-risk HLA-type remains stable. Various factors behind this selective increase in T1D in young adults remain unclear. Obesity and its associated abnormalities appear to be an important determinant; however, the underlying mechanism involved is not understood. Recently, we have developed two novel transgenic obese mice models, Mito-Ob and m-Mito-Ob, by expressing a pleiotropic protein prohibitin (PHB) and a phospho mutant form of PHB (Y114F-PHB or m-PHB) from the aP2 gene promoter, respectively. Both mice models develop obesity in a sex-neutral manner, independent of diet; but obesity associated chronic low-grade inflammation and insulin resistance in a male sex-specific manner. Interestingly, on a high fat diet (HFD) only male m-Mito-Ob mice displayed marked mononuclear cell infiltration in pancreas and developed insulitis that mimic adult-onset T1D. Male Mito-Ob mice that share the metabolic phenotype of male m-Mito-Ob mice, and female m-Mito-Ob that harbor m-PHB similar to male m-Mito-Ob mice, did not develop insulitis. Thus, insulitis development in male m-Mito-Ob in response to HFD requires both, obesity-related abnormalities and m-PHB. Collectively, this data provides a proof-of-concept that obesity-associated abnormalities couple environmental triggers with genetic susceptibility in adult-onset T1D and reveals PHB as a potential susceptibility gene for T1D. PMID:26766792

  10. 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; Aragão, 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

  11. Transgenic algae engineered for higher performance

    DOEpatents

    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.

  12. Difference between ferritin genes overexpressing in transgenic tobacco.

    PubMed

    Tang, X; Zou, D; Zhang, L; Yang, C; Jiang, T

    2014-01-01

    With the development of molecular biology techniques, intron was known as playing an imperative role in gene's expression and regulation. Transgenic tobacco IN lines overexpressing InFer1 gene and NT lines overexpressing NtFer1 cDNA gene were obtained, and the exogenous gene expression were confirmed by molecular test. Then for iron content of transgenic tobacco lines and non-transformants as a physiological indicator in status of different iron concentration were measured, and results indicated that the iron content of transgenic tobacco was more than that of non-transformants. In high Fe (II) condition, the NT lines showed higher level in plant height, fresh weight and iron content then that of IN lines, while NT lines showed lower in Malondialdehyde content then IN lines. In soil condition, IN lines showed higher level in plant height, fresh weight, chlorophyll, photosynthesis rate, iron content then that of NT lines. It indicates that intron could play a vital role for improved protective enzyme activity level while reducing reactive oxygen damage and also could help to inhibit the absorption of aborting iron. PMID:24841649

  13. 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

  14. Allergenicity assessment of the Papaya ringspot virus coat protein expressed in transgenic Rainbow papaya

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The virus-resistant, transgenic commercial papaya cultivars Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland US and Canada since their release to planters in Hawaii in 1998. These cultivars are derived from transgenic papaya line 55-1 and carry ...

  15. Transgene expression after rep-mediated site-specific integration into chromosome 19.

    PubMed

    Philpott, Nicola J; Gomos, Janette; Falck-Pedersen, Erik

    2004-01-01

    We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks). PMID:14965377

  16. Removal of Reprogramming Transgenes Improves the Tissue Reconstitution Potential of Keratinocytes Generated From Human Induced Pluripotent Stem Cells

    PubMed Central

    Kokubu, Chikara; Yusa, Kosuke; Horie, Kyoji; Yoshimura, Yasuhide; Yamauchi, Kaori; Suemori, Hirofumi; Yokozeki, Hiroo; Toyoda, Masashi; Kiyokawa, Nobutaka; Okita, Hajime; Miyagawa, Yoshitaka; Akutsu, Hidenori; Umezawa, Akihiro; Katayama, Ichiro

    2014-01-01

    Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes. PMID:25024429

  17. Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.

    PubMed

    Igawa, Ken; Kokubu, Chikara; Yusa, Kosuke; Horie, Kyoji; Yoshimura, Yasuhide; Yamauchi, Kaori; Suemori, Hirofumi; Yokozeki, Hiroo; Toyoda, Masashi; Kiyokawa, Nobutaka; Okita, Hajime; Miyagawa, Yoshitaka; Akutsu, Hidenori; Umezawa, Akihiro; Katayama, Ichiro; Takeda, Junji

    2014-09-01

    Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes. PMID:25024429

  18. Tandem constructs to mitigate transgene persistence: tobacco as a model.

    PubMed

    Al-Ahmad, Hani; Galili, Shmuel; Gressel, Jonathan

    2004-03-01

    Some transgenic crops can introgress genes into other varieties of the crop, to related weeds or themselves remain as 'volunteer' weeds, potentially enhancing the invasiveness or weediness of the resulting offspring. The presently suggested mechanisms for transgene containment allow low frequency of gene release (leakage), requiring the mitigation of continued spread. Transgenic mitigation (TM), where a desired primary gene is tandemly coupled with mitigating genes that are positive or neutral to the crop but deleterious to hybrids and their progeny, was tested as a mechanism to mitigate transgene introgression. Dwarfism, which typically increases crop yield while decreasing the ability to compete, was used as a mitigator. A construct of a dominant ahasR (acetohydroxy acid synthase) gene conferring herbicide resistance in tandem with the semidominant mitigator dwarfing Delta gai (gibberellic acid-insensitive) gene was transformed into tobacco (Nicotiana tabacum). The integration and the phenotypic stability of the tandemly linked ahasR and Delta gai genomic inserts in later generations were confirmed by polymerase chain reaction. The hemizygous semidwarf imazapyr-resistant TM T1 (= BC1) transgenic plants were weak competitors when cocultivated with wild type segregants under greenhouse conditions and without using the herbicide. The competition was most intense at close spacings typical of weed offspring. Most dwarf plants interspersed with wild type died at 1-cm, > 70% at 2.5-cm and 45% at 5-cm spacing, and the dwarf survivors formed no flowers. At 10-cm spacing, where few TM plants died, only those TM plants growing at the periphery of the large cultivation containers formed flowers, after the wild type plants terminated growth. The highest reproductive TM fitness relative to the wild type was 17%. The results demonstrate the suppression of crop-weed hybrids when competing with wild type weeds, or such crops as volunteer weeds, in seasons when the selector (herbicide) is not used. The linked unfitness would be continuously manifested in future generations, keeping the transgene at a low frequency. PMID:14871372

  19. Recent Results of IRAN-T1 Tokamak

    NASA Astrophysics Data System (ADS)

    Dorranian, D.; Ghoranneviss, M.; Salem, M. K.; Mahmoodi D., M.; Arvin, R.; Talebitaher, Alireza; Abhari, Ali; Khorshid, P.; Hojabri, A.

    2006-12-01

    In this article after introducing the IR-T1 tokamak and its diagnostic systems a brief discussion on the range of grossly stable operating conditions of its plasma by Hugill diagram is presented. Hard disruption instability is studied experimentally in the next part, which confirms that MHD behavior in small tokamaks can be characterized by a single parameter q(a), safety factor at plasma edge. Finally the characteristics of the new regime of IR-T1 are reported. By our new model of triggering different fields (toroidal, ohmic and vertical), the plasma duration time is increased up to 35 ms with Ip of about 25 kA. By modifying capacitance and charging voltage of ohmic and vertical fields the spike oscillations which was appeared in the plasma behavior is taken out. The role of cleaning the vacuum chamber and using heavier gas for glow discharge and the effect of base pressure is described in detail.

  20. [Management of T1a vocal fold carcinoma].

    PubMed

    Reiter, R; Brosch, S; Smith, E; Pickhard, A

    2013-12-01

    About 2/3 of the larynx carcinomas affect the vocal chords. The main risk factor is smoking. Carcinomas in this localisation often arise from leukoplakias with dysplasia. A typical symptom is dysphonia. Arrest of vibration in microlaryngostroboscopy is a hint that a carcinoma could be present. Transoral laser cordectomy or radiotherapy show equivalent oncological results and results in quality of voice in the treatment of vocal fold carcinoma (T1a). As lymph node and distant metastasis are very rare, follow-up can concentrate on microlaryngoscopy. In case of a suspicious area on the vocal fold, biopsy of the affected tissue is needed to plan correct treatment. The prognosis of the T1 vocal chord carcinoma is quite good with a 5-year survival rate of almost 100%. PMID:23929210

  1. 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

  2. Transverse CAT of the myocardium with 201 T1.

    PubMed

    Maublant, J; Jourde, M; Veyre, A; Meyniel, G

    1979-12-01

    A new computerized axial tomographic (CAT) scanner (J&P Tomoscanner) which enables the transverse section viewing of any organ labelled by one of the monophotonic gamma-ray emitting racers commonly used in Nuclear Medicine, has been recently commercialized. Its abilities in visualising the 201 T1 labelled myocardium are evaluated with normal hearts and patients with documented infarctions. Positive results have been obtained, allowing an interesting approach to the estimation of the anatomical extent of necrosis. PMID:520362

  3. Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea).

    PubMed

    Singh, Natwar; Mishra, Avinash; Jha, Bhavanath

    2014-08-15

    Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T0 transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T1 plants were raised. Transgenic plants (T1) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T1) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture. PMID:24954532

  4. Cardiac and neurological abnormalities in v-fps transgenic mice.

    PubMed Central

    Yee, S P; Mock, D; Maltby, V; Silver, M; Rossant, J; Bernstein, A; Pawson, T

    1989-01-01

    Transgenic mice that widely express the v-fps protein-tyrosine kinase develop several independent pathological conditions, in addition to a high tumor incidence. v-fps expression and protein-tyrosine kinase activity in the heart were directly correlated with cardiac enlargement. This cardiomegaly was accompanied by severe myocardial and endocardial damage, which was concentrated in the left ventricular wall, and characterized by a progressive atrophy and necrosis of cardiac muscle fibers with concomitant fibrosis. This pathology was associated with congestive heart failure. Mice from five lines developed a marked trembling, correlated with expression of the v-fps transgene in the brain, and two lines showed a striking bilateral enlargement of the trigeminal nerves. Unlike tumor formation, these cardiac and neurological phenotypes were evident shortly after birth and showed 100% penetrance. The pleiotropic effects of the v-fps transgene suggest the involvement of protein-tyrosine kinases in mammalian neural development and cardiac function. Images PMID:2788278

  5. Difficulties of T1 brain MRI segmentation techniques

    NASA Astrophysics Data System (ADS)

    Atkins, M. Stella; Siu, Kevin; Law, Benjamin; Orchard, Jeffery J.; Rosenbaum, Wilfred L.

    2002-05-01

    This paper looks at the difficulties that can confound published T1-weighted Magnetic Resonance Imaging (MRI) brain segmentation methods, and compares their strengths and weaknesses. Using data from the Internet Brain Segmentation Repository (IBSR) as a gold standard, we ran three different segmentation methods with and without correcting for intensity inhomogeneity. We then calculated the similarity index between the brain masks produced by the segmentation methods and the mask provided by the IBSR. The intensity histograms under the segmented masks were also analyzed to see if a Bi-Gaussian model could be fit onto T1 brain data. Contrary to our initial beliefs, our study found that intensity based T1-weighted segmentation methods were comparable or even superior to, methods utilizing spatial information. All methods appear to have parameters that need adjustment depending on the data set used. Furthermore, it seems that the methods we tested for intensity inhomogeneity did not improve the segmentations due to the nature of the IBSR data set.

  6. Is mortalin a candidate gene for T1DM ?

    PubMed

    Johannesen, Jesper; Pie, Angeles; Karlsen, Allan Ertmann; Larsen, Zenia Marian; Jensen, Allan; Vissing, Henrik; Kristiansen, Ole Peter; Pociot, Flemming; Nerup, Jrn

    2004-01-01

    Mortalin has been found to be up-regulated by 2D-protein gel analysis in isolated rodent islets exposed to cytokines. In islets from two rat strains with different sensitivity to the toxic effects of cytokines we observed a significant difference in IL-1beta mediated mortalin expression. Constitutive over-expression of rat mortalin in NIH3T3 cells reduced cellular survival in accordance with mortalin being associated to cellular senescence. Hence we consider the gene encoding for mortalin at chromosome 5q31.1 a putative candidate gene in cytokine induced beta-cell destruction. We scanned the human mortalin gene for polymorphisms and identified three novel polymorphisms. Neither the SNPs individually nor as constructed haplotypes showed disease association tested by (E)TDT in a Danish type 1 diabetes (T1DM) population. Furthermore, we tested the D5S500 microsatelite located close to 5q31.1 without finding linkage to (T1DM). In conclusion, the functional data identifying a difference in mortalin expression in IL-1beta stimulated islets between two rat strains and over-expression of mortalin in NIH3T3 cells associated with decreased viability suggests a functional role for mortalin in cytokine mediated beta cell destruction; however, the identified polymorphisms did not reveal any association in the presence of linkage disequilibrium of mortalin to T1DM in the Danish population. PMID:15621568

  7. 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...

  8. 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

  9. 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.

  10. Mosquito transgenic technologies to reduce Plasmodium transmission.

    PubMed

    Fuchs, Silke; Nolan, Tony; Crisanti, Andrea

    2013-01-01

    The ability to introduce genetic constructs of choice into the genome of Anopheles mosquitoes provides a valuable tool to study the molecular interactions between the Plasmodium parasite and its insect host. In the long term, this technology could potentially offer new ways to control vector-borne diseases through the suppression of target mosquito populations or through the introgression of traits that preclude pathogen transmission. Here, we describe in detail protocols for the generation of transgenic Anopheles gambiae mosquitoes based on germ-line transformation using either modified transposable elements or the site-specific PhiC31 recombinase. PMID:22990807

  11. [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

  12. Identification and quantification of anthocyanins in transgenic purple tomato.

    PubMed

    Su, Xiaoyu; Xu, Jianteng; Rhodes, Davina; Shen, Yanting; Song, Weixing; Katz, Benjamin; Tomich, John; Wang, Weiqun

    2016-07-01

    Anthocyanins are natural pigments derived from the phenylpropanoid pathway. Most tomatoes produce little anthocyanins, but the transgenic purple tomato biosynthesizes a high level of anthocyanins due to expression of two transcription factors (Del and Ros1). This study was to identify and quantify anthocyanins in this transgenic tomato line. Seven anthocyanins, including two new anthocyanins [malvidin-3-(p-coumaroyl)-rutinoside-5-glucoside and malvidin-3-(feruloyl)-rutinoside-5-glucoside], were identified by LC-MS/MS. Petunidin-3-(trans-coumaroyl)-rutinoside-5-glucoside and delphinidin-3-(trans-coumaroyl)-rutinoside-5-glucoside were the most abundant anthocyanins, making up 86% of the total anthocyanins. Compared to undetectable anthocyanins in the wild type, the contents of anthocyanins in the whole fruit, peel, and flesh of the Del/Ros1-transgenic tomato were 5.2±0.5, 5.1±0.5, and 5.8±0.3g/kg dry matter, respectively. Anthocyanins were undetectable in the seeds of both wide-type and transgenic tomato lines. Such novel and high levels of anthocyanins obtained in this transgenic tomato may provide unique functional products with potential health benefits. PMID:26920283

  13. Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

    PubMed

    Juárez, Paloma; Presa, Silvia; Espí, Joaquín; Pineda, Benito; Antón, María T; Moreno, Vicente; Buesa, Javier; Granell, Antonio; Orzaez, Diego

    2012-04-01

    Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins. PMID:22070155

  14. 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...

  15. Probing pineal-specific gene expression with transgenic zebrafish.

    PubMed

    Kojima, Daisuke; Dowling, John E; Fukada, Yoshitaka

    2008-01-01

    The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis-acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P(20)-rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P(20)-rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N-ethyl-N-nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P(20)-rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland. PMID:18466202

  16. Dissection of a Synthesized Quantitative Trait to Characterize Transgene Interactions

    PubMed Central

    Nap, J. P.; Conner, A. J.; Mlynarova, L.; Stiekema, W. J.; Jansen, R. C.

    1997-01-01

    Six transgenic tobacco lines, each homozygous for the β-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits. PMID:9286691

  17. Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene.

    PubMed

    Singh, Deepali; Ambroise, Annick; Haicour, Robert; Sihachakr, Darasinh; Rajam, Manchikatla Venkat

    2014-04-01

    The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5-7 days in the disease development as compared to wild-type plants. PMID:24757318

  18. Identification, cloning and characterization of a plasma membrane zinc efflux transporter, TrZnT-1, from fugu pufferfish (Takifugu rubripes)

    PubMed Central

    Balesaria, Sara; Hogstrand, Christer

    2005-01-01

    An orthologue of the mammalian ZnT-1 (zinc transporter-1) gene was cloned from the intestine of the torafugu pufferfish (Takifugu rubripes), demonstrating that this gene predates the evolution of land-living vertebrates. TrZnT-1 (T. rubripes ZnT-1) shares overall topology with other members of the ZnT-1 family of zinc transporters, with six TMs (transmembrane domains) including a large histidine-rich intracellular loop between TM IV and V and intracellular C- and N-termini. Expression of TrZnT-1 in a metallothionein acquiescent cell line suggested that this protein reduces intracellular Zn2+ levels. Manipulation of the transporting media showed that several externally applied hydrominerals had no effect on TrZnT-1 activity. However, addition of N-ethylmaleimide increased TrZnT-1-mediated transport, possibly by increasing intracellular free Zn2+ levels by Zn2+ release from carrier proteins. Generation of a specific antibody and subsequent immunocytochemistry on fixed cells overexpressing TrZnT-1 indicated that the protein is localized to the plasma membrane in these cells. The genomic organization of TrZnT-1 is the same as that in mammals with two exons. The upstream regulatory region of the TrZnT-1 gene contains several putative cis-acting elements, including metal-response elements and an Sp1 site. Analysis of the DNA contigs surrounding the TrZnT-1 gene reveal limited synteny between corresponding regions in the rat, mouse and human; however, this was very low, with only two syntenic genes, ZnT-1 and NEK2 (never in mitosis gene A-related kinase). PMID:16212555

  19. Exercise and type 1 diabetes (T1DM).

    PubMed

    Galassetti, Pietro; Riddell, Michael C

    2013-07-01

    Physical exercise is firmly incorporated in the management of type 1 diabetes (T1DM), due to multiple recognized beneficial health effects (cardiovascular disease prevention being preeminent). When glycemic values are not excessively low or high at the time of exercise, few absolute contraindications exist; practical guidelines regarding amount, type, and duration of age-appropriate exercise are regularly updated by entities such as the American Diabetes Association and the International Society for Pediatric and Adolescent Diabetes. Practical implementation of exercise regimens, however, may at times be problematic. In the poorly controlled patient, specific structural changes may occur within skeletal muscle fiber, which is considered by some to be a disease-specific myopathy. Further, even in well-controlled patients, several homeostatic mechanisms regulating carbohydrate metabolism often become impaired, causing hypo- or hyperglycemia during and/or after exercise. Some altered responses may be related to inappropriate exogenous insulin administration, but are often also partly caused by the "metabolic memory" of prior glycemic events. In this context, prior hyperglycemia correlates with increased inflammatory and oxidative stress responses, possibly modulating key exercise-associated cardio-protective pathways. Similarly, prior hypoglycemia correlates with impaired glucose counterregulation, resulting in greater likelihood of further hypoglycemia to develop. Additional exercise responses that may be altered in T1DM include growth factor release, which may be especially important in children and adolescents. These multiple alterations in the exercise response should not discourage physical activity in patients with T1DM, but rather should stimulate the quest for the identification of the exercise formats that maximize beneficial health effects. PMID:23897688

  20. [Transgenics without Manichaeism].

    PubMed

    Valle, S

    2000-01-01

    We live in an era characterized by the hegemony of science and technology, an era fraught with questions awaiting answers which would enable a safe and sustainable future for humankind. The development of agro-industrial processes - food products in particular - through recombinant DNA technology has enhanced the profit prospects of the few big biotechnology companies and of large-scale farmers who have access to the latest technological developments. We thus oppose a moratorium on recombinant DNA technology. Moreover, hasty statements about risk-free transgenics may be misleading in the absence of extensive safety tests. There is a pressing need for the establishment of biosafety policy in this country involving the organized civil society and every government agency responsible for monitoring such matters. There is also the need to put in place a bio-surveillance and a code of ethics regarding genetic manipulation. PMID:16680900

  1. 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

  2. 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.

  3. Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple.

    PubMed

    Schfer, Tina; Hanke, Magda-Viola; Flachowsky, Henryk; Knig, Stephan; Peil, Andreas; Kaldorf, Michael; Polle, Andrea; Buscot, Franois

    2012-04-01

    This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF). We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova) and transgenic lines (M9/T386 and M9/T389) were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities, AMF root colonization and biomass. PMID:22888297

  4. Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple

    PubMed Central

    Schfer, Tina; Hanke, Magda-Viola; Flachowsky, Henryk; Knig, Stephan; Peil, Andreas; Kaldorf, Michael; Polle, Andrea; Buscot, Franois

    2012-01-01

    This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF). We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova) and transgenic lines (M9/T386 and M9/T389) were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities, AMF root colonization and biomass. PMID:22888297

  5. Ultrastructural and cytochemical characterization of T1 and T2 secretory bodies from the tegument of Echinostoma paraensei.

    PubMed

    Gonçalves, Júlia P; Oliveira-Menezes, Aleksandra; Maldonado Junior, Arnaldo; Carvalho, Técia M U; de Souza, Wanderley

    2016-01-01

    Trematodes are lined by a syncytial layer that is named the tegument and contains small mitochondria and two different kinds of secretory inclusions. The structure and size of these bodies differs among genera and species. In a previous study, we observed many secretory bodies in the tegument of Echinostoma paraensei and named these bodies the T1 and T2 secretory bodies. No previous studies analyzed the secretory bodies of trematodes from the genus Echinostoma. Thus, the aim of this work was to use electron microscopy and cytochemistry to characterize these secretory bodies and to provide a detailed ultrastructural and morphological picture of these bodies, which are found in the tegument of E. paraensei. After ultrastructural cytochemistry analysis, we showed that both the T1 and T2 secretory bodies of E. paraensei were formed by glycoconjugates. 3D reconstruction confirmed the ovoid form of T1 secretory bodies and the biconcave and thin form of T2 secretory bodies. PMID:26451647

  6. Transgenic mice: beyond the knockout

    PubMed Central

    2011-01-01

    Transgenic mice have had a tremendous impact on biomedical research. Most researchers are familiar with transgenic mice that carry Cre recombinase (Cre) and how they are used to create conditional knockouts. However, some researchers are less familiar with many of the other types of transgenic mice and their applications. For example, transgenic mice can be used to study biochemical and molecular pathways in primary cultures and cell suspensions derived from transgenic mice, cell-cell interactions using multiple fluorescent proteins in the same mouse, and the cell cycle in real time and in the whole animal, and they can be used to perform deep tissue imaging in the whole animal, follow cell lineage during development and disease, and isolate large quantities of a pure cell type directly from organs. These novel transgenic mice and their applications provide the means for studying of molecular and biochemical events in the whole animal that was previously limited to cell cultures. In conclusion, transgenic mice are not just for generating knockouts. PMID:21068085

  7. An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield.

    PubMed

    Byeon, Yeong; Back, Kyoungwhan

    2014-05-01

    No previous reports have described the effects of an increase in endogenous melatonin levels on plant yield and reproduction. Here, the phenotypes of melatonin-rich transgenic rice plants overexpressing sheep serotonin N-acetyltransferase were investigated under field conditions. Early seedling growth of melatonin-rich transgenic rice was greatly accelerated, with enhanced biomass relative to the wild type (WT). However, flowering was delayed by 1 wk in the transgenic lines compared with the WT. Grain yields of the melatonin-rich transgenic lines were reduced by 33% on average. Other phenotypes also varied among the transgenic lines. For example, the transgenic line S1 exhibited greater height and biomass than the WT, while the S10 transgenic line showed diminished height and an increase in panicle numbers per plant. The expression levels of Oryza sativa homeobox1 (OSH1) and TEOSINTE BRANCHED1 (TB1) genes, two key regulators of meristem initiation and maintenance, were not altered in the transgenic lines. These data demonstrate that an alteration of endogenous melatonin levels leads to pleiotropic effects such as height, biomass, panicle number, flowering time, and grain yield, indicating that melatonin behaves as a signaling molecule in plant growth and reproduction. PMID:24571270

  8. Perspectives on the state of insect transgenics.

    PubMed

    O'Brochta, David A; Handler, Alfred M

    2008-01-01

    Genetic transformation is a critical component to the fundamental genetic analysis of insect species and holds great promise for establishing strains that improve population control and behavior for practical application. This is especially so for insects that are disease vectors, many of which are currently subject to genomic sequence analysis, and intensive population control measures that must be improved for better efficacy and cost-effectiveness. Transposon-mediated germ-line transformation has been the ultimate goal for most fundamental and practical studies, and impressive strides have been made in recent development of transgene vector and marker systems for several mosquito species. This has resulted in rapid advances in functional genomic sequence analysis and new strategies for biological control based on conditional lethality. Importantly, advances have also been made in our ability to use these systems more effectively in terms of enhanced stability and targeting to specific genomic loci. Nevertheless, not all insects are currently amenable to germ-line transformation techniques, and thus advances in transient somatic expression and paratransgenesis have also been critical, if not preferable for some applications. Of particular importance is how this technology will be used for practical application. Early ideas for population replacement of indigenous pests with innocuous transgenic siblings by transposon-vector spread, may require reevaluation in terms of our current knowledge of the behavior of transposons currently available for transformation. The effective implementation of any control program using released transgenics, will also benefit from broadening the perspective of these control measures as being more mainstream than exotic. PMID:18510010

  9. Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: Progress and prospects for behavioral neuroscience

    PubMed Central

    Ting, Jonathan T.; Feng, Guoping

    2014-01-01

    Here we review the rapidly growing toolbox of transgenic mice and rats that exhibit functional expression of engineered opsins for neuronal activation and silencing with light. Collectively, these transgenic animals are enabling neuroscientists to access and manipulate the many diverse cell types in the mammalian nervous system in order to probe synaptic and circuitry connectivity, function, and dysfunction. The availability of transgenic lines affords important advantages such as stable and heritable transgene expression patterns across experimental cohorts. As such, the use of transgenic lines precludes the need for other costly and labor-intensive procedures to achieve functional transgene expression in each individual experimental animal. This represents an important consideration when large cohorts of experimental animals are desirable as in many common behavioral assays. We describe the diverse strategies that have been implemented for developing transgenic mouse and rat lines and highlight recent advances that have led to dramatic improvements in achieving functional transgene expression of engineered opsins. Furthermore, we discuss considerations and caveats associated with implementing recently developed transgenic lines for optogenetics-based experimentation. Lastly, we propose strategies that can be implemented to develop and refine the next generation of genetically modified animals for behaviorally-focused optogenetics-based applications. PMID:23473879

  10. Enhanced UV-Induced Skin Carcinogenesis in Transgenic Mice Overexpressing Proprotein Convertases1

    PubMed Central

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

    2013-01-01

    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

  11. Somatic generation of hybrid antibody H chain genes in transgenic mice via interchromosomal gene conversion

    PubMed Central

    1994-01-01

    We have constructed lines of mice with transgenes containing an antibody heavy (H) chain variable region (VHDJH) gene and various amounts of natural immunoglobulin (Ig) and plasmid flanking DNA. In these lines, recombination of the transgene and the endogenous Igh locus takes place in B cells, leading to the expression of functional H chains partially encoded by the transgenic VHDJH gene. Here, we demonstrate that the transgenic VHDJH gene, and various amounts of flanking sequence are recombined with Igh locus DNA via interchromosomal gene conversion. The structures of the resulting "hybrid" transgene-Igh H chain loci are consistent with the 3' end of the conversion occurring in regions of sequence identity, and the 5' end taking place between regions of little or no homology. This mode of antibody transgene recombination with the Igh locus is fundamentally different from the previously reported "trans H chain class switching" that results in reciprocal translocations. In contrast, this recombination resembles events previously observed in mammalian tissue culture cells between adjacent homologous chromosomal sequences, or transfected DNA and a homologous chromosomal target. Our data indicate that this recombination takes place at a low frequency, and that the frequency is influenced by both the length and extent of homology between the transgene and the Igh locus, but is not greatly affected by transgene copy number. This recombination pathway provides a novel approach for the subtle alteration of the clonal composition of the mouse B cell compartment in vivo using VH genes with defined structures and functions. PMID:8270869

  12. [Development of a hepatitis B virus carrier transgenic mice model].

    PubMed

    Caner, Müge; Arat, Sezen; Bircan, Rifat

    2008-01-01

    The studies for the development of transgenic mice models which provide important profits for the studies concerning immunopathogenesis of hepatitis B virus (HBV) infections are in progress since 20 years. For this purpose different lineages bearing whole HBV genome or selected viral genes have been developed and their usage in clarifying the HBV replication and pathogenesis mechanisms have been emphasized. The aim of this study was to develop and breed a HBV carrier mice model. In the study the full HBV genome has been transferred to mouse embryos by microinjection procedure. Following transgenic manipulation, the HBV carriers among the daughter mice have been detected by molecular methods in which HBV-DNA replication and expression have been shown. The manipulations for transgene transfers have been performed in TUBITAK Marmara Research Center Transgene Laboratory, Gebze, Istanbul. The HBV-DNA carrier mice have been demonstrated by polymerase chain reaction (PCR) using the DNA samples obtained from tail tissues and also by dot-blot hybridization of the mice sera. Integrated HBV-DNA has been detected by applying in-situ hybridization to the liver tissue sections. HBV-DNA expression has been shown by reverse transcriptase PCR method with total RNA molecules that have been isolated from the liver tissues of the HBV-DNA carrier mice. HBsAg has been detected in the liver by immunohistochemical method, and HBsAg and HBeAg have additionally been demonstrated by ELISA. HBV genome, expression of the genome and the expression products have been determined in approximately 10% of the mice of which HBV-DNA have been transferred. By inbreeding heterozygote carrier mice, homozygote HBV transgenic mice line have been obtained. These HBV transgenic mice are the first lineages developed in our country. It is hopefully thought that this HBV carrier transgenic mouse model may contribute to the studies on the pathogenesis of HBV infections which are important health problems in the world as well as in Turkey. PMID:18444564

  13. Effects of HCV proteins in current HCV transgenic models.

    PubMed

    Jiao, Jian; Wang, Jiangbin; Sallberg, Matii

    2010-02-01

    Hepatits C virus (HCV) is an enveloped virus with positive-sense single-stranded RNA genome that causes both acute and persistent infections associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma, which needs fully functional human hepatocytes for its development. Due to the strict human tropism of HCV, only human and higher primates such as chimpanzees have been receptive to HCV infection and development, cognition about pathophysiololgy and host immune responses of HCV infection is limited by lacking of simple laboratory models of infection for a long time. During the past decade, gene transfer approaches have been helpful to the understanding of the molecular basis of human disease. Transgenic cell lines, chimeric and transgenic animal models were developed and had been demonstrated their invaluable benefits. This review focuses on the existing HCV transgenic models and summarize the relative results about probable pathophysical changes induced by HCV proteins. PMID:19788690

  14. [Traumatic fracture of the thoracic spine T1-T10].

    PubMed

    Falavigna, Asdrubal; Righesso Neto, Orlando; Ferraz, Fernando Antonio Patriani; Boniatti, Mrcio Manozzo

    2004-12-01

    We describe the incidence, causes, management and prognosis of traumatic fractures of the thoracic spine from T1 to T10 in surgical cases of traumatic fractures of spine during the period from June 1994 to June 2003 studied retrospectively. The type of fracture was determined according to the Gertzbein classification, and the degree of stability using the Denis classification. The neurological picture at admission and 30 days after surgery was evaluated using the ASIA/IMSOP classification. Surgery was performed in patients with complete spinal cord injury (n=7) for the purpose of stabilization using the posterior approach. In cases without spinal cord injury or incomplete injury (n=12), the surgical procedure was performed aiming to decompress the nerve tissue, to correct the alignment of the spine and to stabilize the spine. PMID:15608977

  15. Effect of Air Ions on Submicron T1 Bacteriophage Aerosols

    PubMed Central

    Happ, John W.; Harstad, J. Bruce; Buchanan, Lee M.

    1966-01-01

    The effect of a high concentration of ionized air molecules on sampling T1 phage aerosols of submicron particle size was evaluated by comparing the phage recoveries of all-glass impingers (AGI-4) and type 6 filter papers. Sampler recoveries of all ionized aerosols were less than the recoveries of nonionized control aerosols. These reductions in recovery were greater with positive ions than with negative ions or ions of mixed polarity. The AGI-4 allowed considerable slippage, which was not affected by the air ions. Type 6 filter paper recoveries were less than AGI-4 recoveries. The air ions did not appear to affect the aerosol particle size as determined by an electron microscope. Images Fig. 1 Fig. 3 PMID:16349691

  16. 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.

  17. FAIR exempting separate T (1) measurement (FAIREST): a novel technique for online quantitative perfusion imaging and multi-contrast fMRI.

    PubMed

    Lai, S; Wang, J; Jahng, G H

    2001-01-01

    A new pulse sequence, dubbed FAIR exempting separate T(1) measurement (FAIREST) in which a slice-selective saturation recovery acquisition is added in addition to the standard FAIR (flow-sensitive alternating inversion recovery) scheme, was developed for quantitative perfusion imaging and multi-contrast fMRI. The technique allows for clean separation between and thus simultaneous assessment of BOLD and perfusion effects, whereas quantitative cerebral blood flow (CBF) and tissue T(1) values are monitored online. Online CBF maps were obtained using the FAIREST technique and the measured CBF values were consistent with the off-line CBF maps obtained from using the FAIR technique in combination with a separate sequence for T(1) measurement. Finger tapping activation studies were carried out to demonstrate the applicability of the FAIREST technique in a typical fMRI setting for multi-contrast fMRI. The relative CBF and BOLD changes induced by finger-tapping were 75.1 +/- 18.3 and 1.8 +/- 0.4%, respectively, and the relative oxygen consumption rate change was 2.5 +/- 7.7%. The results from correlation of the T(1) maps with the activation images on a pixel-by-pixel basis show that the mean T(1) value of the CBF activation pixels is close to the T(1) of gray matter while the mean T(1) value of the BOLD activation pixels is close to the T(1) range of blood and cerebrospinal fluid. PMID:11746944

  18. Silibinin and Paclitaxel Cotreatment Significantly Suppress the Activity and Lung Metastasis of Triple Negative 4T1 Mammary Tumor Cell in Mice

    PubMed Central

    Ho, Bing-Ying; Lin, Chun-Hung; Apaya, Maria Karmella; Chao, Wen-Wan; Shyur, Lie-Fen

    2012-01-01

    The in vitro and in vivo bioactivities of silibinin (SB), paclitaxel (PTX) and SB and PTX in combination (SB+PTX) against murine metastatic mammary 4T1 cancer cell line were investigated. Isobologram and combination index (CI) analyses showed that SB and PTX can function synergistically in the inhibition of 4T1 cell proliferation with a CI value < 1. Both SB and PTX alone or SB+PTX treatment inhibited 4T1 cell migration and motility possibly through downregulation of the serpin protease nexin-1 (PN-1) and N-cadherin expression, inhibition of matrix metalloprotease (MMP)-9 activity, and upregulation of E-cadherin. Flow cytometry and Western blot analyses demonstrated that both drugs deregulated cell-cycle mediators and induced apoptosis in 4T1 cells. A real-time in vivo bioluminescence imaging system to monitor the breast cancer cell metastasis in syngeneic BALB/c mice was established using a stable 4T1pGL-COX-2/Luc cell clone carrying a COX-2 promoter driven-luciferase reporter gene. In vivo study using the allograft 4T1pGL-COX-2/Luc metastatic mouse model indicated that SB co-treated with PTX can significantly suppress lung metastasis of 4T1 cells likely through inhibiting cell proliferation and angiogenesis. Together, this study demonstrates that SB could act synergistically with PTX in 4T1 cells, providing a therapeutic option for highly metastatic triple negative breast cancer. PMID:24716145

  19. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

    PubMed

    Sanderson, N; Factor, V; Nagy, P; Kopp, J; Kondaiah, P; Wakefield, L; Roberts, A B; Sporn, M B; Thorgeirsson, S S

    1995-03-28

    Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine. PMID:7708687

  20. Expression and inheritance of multiple transgenes in rice plants.

    PubMed

    Chen, L; Marmey, P; Taylor, N J; Brizard, J P; Espinoza, C; D'Cruz, P; Huet, H; Zhang, S; de Kochko, A; Beachy, R N; Fauquet, C M

    1998-11-01

    The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation. PMID:9831036

  1. CgT1: a non-LTR retrotransposon with restricted distribution in the fungal phytopathogen Colletotrichum gloeosporioides.

    PubMed

    He, C; Nourse, J P; Kelemu, S; Irwin, J A; Manners, J M

    1996-09-13

    Two genetically distinct biotypes (A and B) of Colletotrichum gloeosporioides that cause different anthracnose diseases on the legumes Stylosanthes spp. have been identified in Australia. A DNA sequence that was present in biotype B and absent in biotype A was isolated by differential hybridisation of a genomic library using total genomic DNA of each biotype as hybridisation probes. This sequence also failed to hybridise to DNA of three biotypes of C. gloeosporioides from other host species and to DNA of three other species of Colletotrichum. This clone was used to isolate two cosmid clones of biotype B. Sequence analysis of these clones revealed a repetitive element of approximately 5.7 kb in length. This element, termed CgT1, was dispersed in the genome and present in about 30 copies. The element contained open reading frames encoding deduced sequence motifs homologous to gag-like proteins, reverse transcriptase and RNase H domains of non-LTR retrotransposons. The termini of CgT1 lacked long terminal repeats (LTRs) but contained a 3' A-rich domain. The insertion site of one copy of the element was flanked by short 13-bp direct repeats. These characteristics of the termini, taken together with the overall structure and sequence homologies, indicate that CgT1 belongs to the non-LTR, LINE-like retrotransposon class of elements that are present in many eukaryotes. PCR primers designed to amplify regions of CgT1 can be used to distinguish biotypes A and B in Australia. DNA fingerprinting analysis of genomic DNA using hybridisation probes derived from the terminal regions of CgT1 revealed that Australian isolates of biotype B are monomorphic. CgT1 was not detected in some isolates causing Type B disease from other countries and when CgT1 was present there was considerable polymorphism in CgT1 organisation in the genome. CgT1 is the first transposon-like element to be identified in the genus Colletotrichum and has considerable potential as a tool for the study of population structure, genome dynamics and evolution in C. gloeosporioides. PMID:8842152

  2. [Inheritance and expression of the maize pepc gene in progenies of transgenic rice bred by crossing].

    PubMed

    Wang, De-Zheng; Wang, Shou-Hai; Wu, Shuang; Li, Cheng-Quan; Jiao, De-Mao; Luo, Yan-Chang; Wang, Xiu-Feng; Du, Shi-Yun

    2004-02-01

    By cross breeding, the maize pepc gene in the pepc transgenic rice was successfully incorporated into the parents of two-, three-line hybrid rice, including sterile lines (Peiai64S, 2302S, 2304S, 2306S and Shuangjiu A) and restorer lines (5129, 02428 and Wanjing97) to breed the high-photosynthetic efficiency parents of hybrid rice and utilize heterosis between C4 and C4/C3 rice. Some lines of pepc transgenic rice (LPTR) have been developed. The study on the generations of LPTR suggests the following: (1) The segregation observed in F2 and BC1 progenies demonstrated that pepc transgene inherited as a single dominant gene in the progenies of LPTR. (2) The maize pepc gene is actively expressed at high level in LPTR, and changes of pepc gene expression in the progenies of LPTR may be related to position effect, difference of gene copy number and environmental factors. (3) Through the selection method of soaking seeds into hygromycin solution to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities, the segregation of the pepc transgene in LPTR was controlled effectively. Based on the above strategy three pepc transgene lines, H1596, H1597 and Y1470, have been selected. The result suggests that it is possible to breed practical, stable and high-expression pepc transgenic rice by conventional crossing. PMID:15473312

  3. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum

    PubMed Central

    Shin, Sanghyun; Mackintosh, Caroline A.; Lewis, Janet; Heinen, Shane J.; Radmer, Lorien; Dill-Macky, Ruth; Baldridge, Gerald D.; Zeyen, Richard J.; Muehlbauer, Gary J.

    2008-01-01

    Fusarium head blight (FHB; scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat worldwide. FHB causes yield reductions and contamination of grains with trichothecene mycotoxins such as deoxynivalenol (DON). The genetic variation in existing wheat germplasm pools for FHB resistance is low and may not provide sufficient resistance to develop cultivars through traditional breeding approaches. Thus, genetic engineering provides an additional approach to enhance FHB resistance. The objectives of this study were to develop transgenic wheat expressing a barley class II chitinase and to test the transgenic lines against F. graminearum infection under greenhouse and field conditions. A barley class II chitinase gene was introduced into the spring wheat cultivar, Bobwhite, by biolistic bombardment. Seven transgenic lines were identified that expressed the chitinase transgene and exhibited enhanced Type II resistance in the greenhouse evaluations. These seven transgenic lines were tested under field conditions for percentage FHB severity, percentage visually scabby kernels (VSK), and DON accumulation. Two lines (C8 and C17) that exhibited high chitinase protein levels also showed reduced FHB severity and VSK compared to Bobwhite. One of the lines (C8) also exhibited reduced DON concentration compared with Bobwhite. These results showed that transgenic wheat expressing a barley class II chitinase exhibited enhanced resistance against F. graminearum in greenhouse and field conditions. PMID:18467324

  4. PsT-1: a new tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei.

    PubMed

    Wang, Ran; Chen, Tianbao; Zhou, Mei; Wang, Lei; Shaw, Chris

    2013-06-10

    The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologs, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy. PMID:23518460

  5. Generation of insulin-producing cells from C3H10T1/2 mesenchymal progenitor cells.

    TOXLINE Toxicology Bibliographic Information

    Jian RL; Mao LB; Xu Y; Li XF; Wang FP; Luo XG; Zhou H; He HP; Wang N; Zhang TC

    2015-05-10

    Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate ? cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes.

  6. Generation of insulin-producing cells from C3H10T1/2 mesenchymal progenitor cells.

    PubMed

    Jian, Ruo-Lei; Mao, Li-Bin; Xu, Yao; Li, Xiao-Fan; Wang, Feng-Po; Luo, Xue-Gang; Zhou, Hao; He, Hong-Peng; Wang, Nan; Zhang, Tong-Cun

    2015-05-10

    Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate ? cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes. PMID:25724395

  7. 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

  8. 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 α-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

  9. [Marker-assisted selection and pyramiding for three blast resistance genes, Pi-d(t)1, Pi-b, Pi-ta2, in rice].

    PubMed

    Chen, Xue-Wei; Li, Shi-Gui; Ma, Yu-Qing; Li, Han-Yun; Zhou, Kai-Da; Zhu, Li-Huang

    2004-09-01

    G46B is a promising holding line used for three-lines breeding strategy in hybrid rice, but it is susceptible to blast disease caused by Pyricularia grisea. To improve its blast resistance, three rice varieties, Digu, BL-1, and Pi-4, with blast resistance genes, Pi-d(t), Pi-b, and Pi-ta2, respectively, were used to be crossed with G46B, and 15 plants with these three blast resistance genes, Pi-d(t)1, Pi-b, and Pi-ta2, were selected from their F2 and B1C1 populations via a marker-aided crossing procedure. Among them, four plants were heterozygotes in the three resistance genes, with the genotype of Pi-d(t)1 pi-d(t)/Pi-b pi-b/ Pi-ta2 pi-ta2; ten plants were heterozygotes in two of the three resistance genes, of which six with the genotype of Pi-d(t)1 Pi-d(t)1/Pi-b pi-b/Pi-ta2 pi-ta2, three with the genotype of Pi-d(t)1 pi-d(t)1/Pi-b pi-b/Pi-ta2 Pi-ta2, and one with the genotype of Pi-d(t)1pi-d(t)1/Pi-b Pi-b/Pi-ta2 pi-ta2; and only one plant was homozygote in two of the three resistance genes with the genotype of Pi-d(t)1 Pi-d(t)/Pi-b pi-b/Pi-ta2 Pi-ta2. These results demonstrate the capacity of maker-assisted selection (MAS) in gene pyramiding for rice blast resistance and its enhancement for the efficiency in rice resistance breeding. PMID:15973995

  10. A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

    PubMed Central

    2014-01-01

    Background Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. Results To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. Conclusions This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription. PMID:24985467

  11. 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.

  12. Generation of transgenic energy cane plants with integration of minimal transgene expression cassette.

    PubMed

    Fouad, Walid M; Hao, Wu; Xiong, Yuan; Steeves, Cody; Sandhu, Surinder K; Altpeter, Fredy

    2015-01-01

    Lignocellulosic biomass has the potential to serve as feedstock and direct replacement for petrochemicals in the fuel, chemical, pharmaceutical and material industries. Energy cane has been identified by the U.S. Department of Energy (DOE) as prime lignocellulosic feedstock as it produces record biomass yields and is able to grow on low-value land with reduced inputs. Molecular improvement of energy cane is an essential step toward the development of a high-value crop and may contribute to improved biomass conversion to value added products. Such improvements require a development of an efficient regeneration and transformation system for the vegetatively propagated energy cane varieties. In this report, an efficient biolistic gene delivery protocol for energy canes (genotype L 79-1002 and Ho 00-961) has been established with immature leaf rolls as explants. Embryonic calli, developed approximately 6 weeks after culture initiation and was used as target for biolistic transfer of a minimum expression cassette of P-ubi::nptII::35S polyA derived from plasmid pJFNPTII. Putative transgenic clones of callus were obtained after selection on callus induction medium supplemented with 30 mg l(-1) geneticin. Regeneration was carried out on NB medium, which is modified from MS supplemented with 1.86 mg l(-1) naphthaleneacetic acid (NAA) and 0.1mg l(-1), 6- benzylaminopurine (BAP) and 20mg l(-1) paromomycin. Shoots growing on selection media were transferred to hormone free medium with 20 mg l(-1) paromomycin. Putative transgenic lines were first analyzed by PCR. Transgene integration was confirmed by Southern blot analysis. ELISA (Enzyme-Linked Immunosorbent Assay) and Immunochromathography assays confirmed transgene expression. PMID:25751171

  13. Transgenic models of human cancer.

    PubMed

    Hinrichs, S H; Fontes, J D; Bills, N D; Schneider, P D

    1991-01-01

    Transgenic animal technology has been useful for the direct demonstration of the tumorigenic potential of oncogenes in vivo. Over the past eight years a wide variety of oncogenes and proto-oncogenes from viral and cellular sources have been inserted into the germline of mice with subsequent development of neoplasia. Many of the published reports describe similarities between morphologic features of the transgenic mice tumors and those occurring naturally in humans. We discuss the morphologic features of selected transgenic models carrying viral genes and review their applicability to investigations directed toward understanding cancer in general and specifically gastric cancer, neurofibromatosis and leukemia. Examples of the impact of nutrition, interaction with growth factors and initiation with chemical carcinogens are presented. In one of the models functional similarities to the mechanism of oncogenesis in human T-cell leukemia virus type-1 (HTLV-1) lymphoma may exist with activation of cytokine production and subsequent autocrine stimulation. The transgenic model of proximal gastric cancer demonstrates features similar to those seen in carcinogen-induced neoplasia. These studies underscore the vast potential of transgenic models for inquiry into the genetic and epigenetic basis of human carcinogenesis. However, many features of transgenic cancer models differ from cancer in humans and the specific criteria for judging the value of transgenic models remain unclarified. For example, although the tumors arising in the HTLV-1 Tax transgenic mice show numerous similarities to human neurofibromatosis including development of lesions of the iris, the similarities do not necessarily extend to the molecular involvement of neurofibromatosis-1 (NF-1), a gene with structural and functional homology to GTPase activating proteins. Transgenic experiments of the future will ask questions beyond whether a particular gene is capable of initiating the neoplastic process. The ability to construct systems in vivo with a defined starting point that facilitate further controlled manipulation of events resulting in cancer provide great opportunities to dissect the various molecular pathways involved in such a process. Therefore, gene knockout experiments and disruption of gene function will further enhance our ability to understand the multi-factorial process of tumor development.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1668887

  14. Quality and agronomic effects of three high-molecular-weight glutenin subunit transgenic events in winter wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quality and agronomic effects of three transgenic high-molecular-weight glutenin subunit (HMWGS) events were characterized in advanced-generation breeding lines of hard winter wheat (Triticum aestivum L.) in three Nebraska (U.S.A.) crop years. Two of the transgenic events studied, Dy10-E and B52a-6...

  15. YpT1-2N0 rectal cancer after neoadjuvant chemoradiation has lower survival compared with pT1-2N0 rectal cancer

    PubMed Central

    Wan, Jue-feng; Zhu, Ji; Li, Gui-chao; Sun, Wen-jie; Zhang, Zhen

    2015-01-01

    Pathologic T1-2N0 rectal cancer shows an excellent prognosis without preoperative or postoperative chemoradiation. However, oncologic outcome of ypT1-2N0 remains unclear and undetermined. Thus, the aim of this study was to compare the survival of ypT1-2 and pT1-2 rectal cancer patients after radical resection and identify risk factors of ypT1-2 rectal cancer in Surveillance, Epidemiology, and End Results Program (SEER)-registered rectal cancer patients. The results showed that ypT1-2N0 rectal cancer after neoadjuvant chemoradiation has lower survival compared with pT1-2N0 rectal cancer and mucinous/signet-ring cancer and less than 12 lymph nodes retrieval were two risk factors in ypT1-2 patients. These results suggest that ypT1-2 patients with one or two risk factors may benefit from postoperative adjuvant chemotherapy. PMID:26517674

  16. 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

  17. Transgenic mice expressing a chimaeric anti-E. coli immunoglobulin alpha heavy chain gene.

    PubMed

    Kooyman, D L; Pinkert, C A

    1994-05-01

    To induce constitutive immunity against a pathogenic strain of Escherichia coli (K99), a rearranged immunoglobulin (Ig) heavy chain (HC) gene was constructed. Because the route of E. coli infection is enteric, an IgA transgene was desirable. A chimaeric gene construct was cloned that coded for a HC that recognized a specific E. coli pilus antigen. The construct comprised a kappa gene promoter, murine VDJ, and bovine alpha-HC constant region. Following microinjection of the HC construct into murine zygotes, of 50 liveborn mice, three were identified as transgenic. In all three transgenic founders, transgene-encoded mRNA expression was detected by northern blot. The transgenic founders were analysed for transgene-encoded RNA expression in splenic tissue before and after challenge with pathogenic E. coli. Founder 4-3 male expressed transgene-encoded RNA both before and after challenge; expression was detected in the other two founders only post-challenge. As no differences were found when sera were analysed for bovine IgA in control and transgenic mice, protein expression was assessed by challenge of HC founders with K99 E. coli by gavage. Control mice challenged with K99 E. coli were moribund within 24 h post-gavage, but there was no observable affect in the three transgenic founders. Unfortunately, after obtaining offspring from all founders, no transgenic offspring were identified (0/108). The low yield of transgenic founders, coupled with the apparent germ-line mosaicism may point to either mechanical or critical developmental anomalies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8025595

  18. Genetic marking of sex using a W chromosome-linked transgene.

    PubMed

    Ma, Sanyuan; Wang, Xiaogang; Fei, Jitao; Liu, Yuanyuan; Duan, Jianping; Wang, Feng; Xu, Hanfu; Zhao, Ping; Xia, Qingyou

    2013-12-01

    Many species belonging to the order Lepidoptera are major pests in agriculture and arboriculture. The sterile insect technique (SIT) is an eco-friendly and highly efficient genetically targeted pest management approach. In many cases, it is preferable to release only sterile males in an SIT program, and efficient sexing strategies are crucial to the successful large-scale implementation of SIT. In the present study, we established 160 transgenic silkworm (Bombyx mori) lines to test the possibility of genetic sexing using a W chromosome-linked transgene, which is thought to be the best sexing strategy for lepidopteran species. One transgenic line with a female-specific expression pattern of reporter gene was obtained. The expression level of the W-linked transgene was comparable with autosomal insertions and was stable for 17 continuous generations. Molecular characterization showed this line contained a single copy of the reporter gene on the W chromosome, and the integration site was TTAG in contig W-BAC-522N19-C9. The feasibility of using a W chromosome-linked transgene demonstrated here and the possible improvements discussed will provide valuable information for other lepidopteran pests. The novel W chromosome-linked transgenic line established in this study will serve as an important resource for fundamental research with the silkworm B. mori. PMID:24036279

  19. Transgenic trait deployment using designed nucleases.

    PubMed

    Petolino, Joseph F; Kumar, Sandeep

    2016-02-01

    The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation. PMID:26332789

  20. 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

  1. [Transgenic animals and animal welfare

    PubMed

    Reinhardt, Christoph

    1998-01-01

    Under the pressure of a public vote in Switzerland (7 June 1998) on an initiative to ban the production, use and patenting of transgenic animals, their value for biomedical research and development is intensely debated. In addition, the Swiss legislation has adopted (1992) a constitutional obligation to "take into account the dignity of creatures". The term "dignity of creatures", however, can be interpreted in anthropocentric or biocentric ways. The government has now formulated the legal implications of this term for transgenic animals and plants in various laws including the animal and environmental protection laws. This paper gives arguments for a fair evaluation of trangenic animals from an animal welfare point of view where not only the costs of animal suffering must be considered but also the probability of potential benefit for man. A self-confident research community should allow such an evaluation procedure even in view of an outcome which could ban many uses of transgenic animals PMID:11208266

  2. Transgenic Zebrafish Using Transposable Elements

    PubMed Central

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

    2012-01-01

    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, this data should enable the ready generation of transgenic zebrafish for scientific inquiry. PMID:21924161

  3. Rapid localization of transgenes in mouse chromosomes with a combined Spectral Karyotyping/FISH technique.

    PubMed

    Matsui, Sei-ichi; Sait, Sheila; Jones, Craig A; Nowak, Norma; Gross, Kenneth W

    2002-12-01

    We explored the feasibility of combined Spectral Karyotyping (SKY) and Fluorescence In Situ Hybridization (FISH) as means to rapidly map a chromosomally integrated renin/green fluorescent protein (GFP) fusion gene construct (Ren-GFP) in the transgenic mouse, Tg(Ren-GFP)1Kwg. A sequential hybridization with SKY probes followed by FISH gave consistently satisfactory results, demonstrating that multiple copies of the Ren-GFP transgene in this transgenic mouse line are integrated into a single chromosomal site of Chromosome (Chr) 4, most probably in the juxta-centromeric euchromatic region consisting of the A2-A3 domain. Chr 4 as a sole carrier of the transgene also was confirmed by co-hybridization to p1 BAC clone DNA containing telomeric sequences specific for mouse Chr 4 and the Ren-GFP construct in pGEM4Z vector. The hemizygosity of the Ren-GFP transgene is maintained not only in bone marrow cells, but also in lung cells proliferating in vitro, indicating that stable integration of the Ren-GFP transgene into chromosomal DNA was established at a very early embryonic stage. We conclude that the SKY/FISH technique is a reliable and facile method for establishing the integration site of a transgene. As such, this protocol has obvious advantages over traditional backcross methods in terms of time, cost and labor for determining the chromosomal location of transgenes. PMID:12514745

  4. Hypoxia-induced in vivo sickling of transgenic mouse red cells.

    PubMed Central

    Rubin, E M; Witkowska, H E; Spangler, E; Curtin, P; Lubin, B H; Mohandas, N; Clift, S M

    1991-01-01

    To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions. Images PMID:1991848

  5. Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes.

    PubMed

    Ivo, Nayche L; Nascimento, Cristina P; Vieira, Lívia S; Campos, Francisco A P; Aragão, Francisco J L

    2008-09-01

    We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws. PMID:18587583

  6. The choline oxidase gene codA confers salt tolerance to transgenic Eucalyptus globulus in a semi-confined condition.

    PubMed

    Yu, Xiang; Kikuchi, Akira; Matsunaga, Etsuko; Morishita, Yoshihiko; Nanto, Kazuya; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa; Watanabe, Kazuo N

    2013-06-01

    The performance of tree species is influenced by environmental factors and growth stages. To evaluate the practical performance of transgenic tree species, it is insufficient to grow small, young trees under controlled conditions, such as in a growth chamber. Three transgenic Eucalyptus globulus lines, carrying the choline oxidase gene, were investigated for their salt tolerance and expression of the transgene at the young plantlet stage in a special netted-house. To clarify the characteristics at the young as well during the later stages, salt tolerance and the properties of the transgenic lines at large juvenile and adult stages were evaluated in the special netted-house. All transgenic lines showed high glycinebetaine content, particularly in young leaves. Trees of the transgenic line 107-1 showed low damage because of salinity stress based on the results from the chlorophyll analysis and malondialdehyde content, and they survived the high-salt-shock treatment at the large juvenile and adult stages. Only this line showed salt tolerance at all stages in the special netted-house. In this evaluation in the special netted-house, the tolerant line among young plantlets might perform better at all stages. Since evaluation in these special netted-house mimics field evaluation, line 107-1 is a potential tolerant line. PMID:22752644

  7. 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

  8. Sensing of amino acids by the gut-expressed taste receptor T1R1-T1R3 stimulates CCK secretion.

    PubMed

    Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew; Marcello, Marco; Ninomiya, Yuzo; Shirazi-Beechey, Soraya P

    2013-02-01

    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 Ca(2+)-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

  9. PbWoxT1 mRNA from pear (Pyrus betulaefolia) undergoes long-distance transport assisted by a polypyrimidine tract binding protein.

    PubMed

    Duan, Xuwei; Zhang, Wenna; Huang, Jing; Hao, Li; Wang, Shengnan; Wang, Aide; Meng, Dong; Zhang, Qiulei; Chen, Qiuju; Li, Tianzhong

    2016-04-01

    Little is known about the mechanisms by which mRNAs are transported over long distances in the phloem between the rootstock and the scion in grafted woody plants. We identified an mRNA in the pear variety 'Du Li' (Pyrus betulaefolia) that was shown to be transportable in the phloem. It contains a WUSCHEL-RELATED HOMEOBOX (WOX) domain and was therefore named Wox Transport 1 (PbWoxT1). A 548-bp fragment of PbWoxT1 is critical in long-distance transport. PbWoxT1 is rich in CUCU polypyrimidine domains and its mRNAs interact with a polypyrimidine tract binding protein, PbPTB3. Furthermore, the expression of PbWoxT1 significantly increased in the stems of wild-type (WT) tobacco grafted onto the rootstocks of PbWoxT1 or PbPTB3 co-overexpressing lines, but this was not the case in WT plants grafted onto PbWoxT1 overexpressing rootstocks, suggesting that PbPTB3 mediates PbWoxT1 mRNA long-distance transport. We provide novel information that adds a new mechanism with which to explain the noncell-autonomous manner of WOX gene function, which enriches our understanding of how WOX genes work in fruit trees and other species. PMID:26661583

  10. Gene flow from transgenic rice to red rice (Oryza sativa L.) in the field.

    PubMed

    Busconi, M; Baldi, G; Lorenzoni, C; Fogher, C

    2014-01-01

    In this study, we simulate a transgenic rice crop highly infested with red rice to examine transgene transfer from a transgenic line (A2504) resistant to glufosinate ammonium to cohabitant red rice. The red rice was sown along with the transgenic line at the highest density found in naturally infested crops in the region. Agricultural practices similar to those used to control red rice infestation in northern Italy rice fields were used to reproduce the local rice production system. During the first 2 years, the field was treated with herbicide at the appropriate time; in the first year the dosage of herbicide was three times the recommended amount. In this first year, detectable red rice plants that escaped herbicide treatment were manually removed. Nevertheless, two herbicide-resistant hybrid plants (named 101 and 104) were identified in the experimental field during the second year of cultivation. Phenotypic and molecular characterisation suggests the hybrid nature of these two plants, deriving from crossing events involving A2504, respectively, with red rice (plant 101) and the buffer cultivar Gladio (plant 104). The progeny of two subsequent generations of the two plants were examined and the presence of the transgene detected, indicating stable transfer of the transgene across generations. In conclusion, despite control methods, red rice progeny tolerant to the herbicide can be expected following use of transgenic rice and, consequently, difficulties in controlling this weed with chemicals will emerge in a relatively short time. PMID:23590388

  11. Application of Arabidopsis AGAMOUS second intron for the engineered ablation of flower development in transgenic tobacco.

    PubMed

    Wang, Hui-Zhong; Hu, Bin; Chen, Guan-Ping; Shi, Nong-Nong; Zhao, Yan; Yin, Qi-Cai; Liu, Jun-Jun

    2008-02-01

    To explore a new approach to generating reproductive sterility in transgenic plants, the barnase gene from Bacillus amyloliquefaciens was placed under the control of an 1853-bp nucleotide sequence from the 3'end of the second intron of Arabidopsis AGAMOUS and CaMV 35S (-60) minimal promoter [AG-I-35S (-60)::Barnase], and was introduced into tobacco through transformation mediated by Agrobacterium tumefaciens. All AG-I-35S (-60)::Barnase transgenic plants showed normal vegetative growth and 28% of the transgenic lines displayed complete ablation of flowering. Two transgenic lines, Bar-5 and Bar-15, were 98.1 and 98.4% sterile, respectively, as determined by seed production and germination. When controlled by AG-I-35S (-60) chimeric promoter, barnase mRNA was detected in the reproductive tissues of transgenic tobacco plants, but not in vegetative parts. This study presents the first application of an AG intron sequence in the engineered ablation of sexual reproduction in plants. The AG-I-35S (-60)::Barnase construct can be useful in diminishing pollen and seed formation in plants, providing a novel bisexual sterility strategy for interception of transgene escape and has other potentially commercial use for transgenic engineering. PMID:17934737

  12. Untranslatable tospoviral NSs fragment coupled with L conserved region enhances transgenic resistance against the homologous virus and a serologically unrelated tospovirus.

    PubMed

    Yazhisai, Uthaman; Rajagopalan, Prem Anand; Raja, Joseph A J; Chen, Tsung-Chi; Yeh, Shyi-Dong

    2015-08-01

    Tospoviruses cause severe damages to important crops worldwide. In this study, Nicotiana benthamiana transgenic lines carrying individual untranslatable constructs comprised of the conserved region of the L gene (denoted as L), the 5' half of NSs coding sequence (NSs) or the antisense fragment of whole N coding sequence (N) of Watermelon silver mottle virus (WSMoV), individually or in combination, were generated. A total of 15-17 transgenic N. benthamiana lines carrying individual transgenes were evaluated against WSMoV and the serologically unrelated Tomato spotted wilt virus (TSWV). Among lines carrying single or chimeric transgenes, the level of resistance ranged from susceptible to completely resistant against WSMoV. From the lines carrying individual transgenes and highly resistant to WSMoV (56-63% of lines assayed), 30% of the L lines (3/10 lines assayed) and 11% of NSs lines (1/9 lines assayed) were highly resistant against TSWV. The chimeric transgenes provided higher degrees of resistance against WSMoV (80-88%), and the NSs fragment showed an additive effect to enhance the resistance to TSWV. Particularly, the chimeric transgenes with the triple combination of fragments, namely L/NSs/N or HpL/NSs/N (a hairpin construct), provided a higher degree of resistance (both 50%, with 7/14 lines assayed) against TSWV. Our results indicate that the untranslatable NSs fragment is able to enhance the transgenic resistance conferred by the L conserved region. The better performance of L/NSs/N and HpL/NSs/N in transgenic N. benthamiana lines suggests their potential usefulness in generating high levels of enhanced transgenic resistance against serologically unrelated tospoviruses in agronomic crops. PMID:25721329

  13. Amino acids regulate transgene expression in MDCK cells.

    PubMed

    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 eIF2α 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

  14. 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 α-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

  15. 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 eIF2α 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

  16. Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization.

    PubMed

    Newhouse, Andrew E; Schrodt, Franziska; Liang, Haiying; Maynard, Charles A; Powell, William A

    2007-07-01

    The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm. PMID:17310333

  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. Taste information derived from T1R-expressing taste cells in mice.

    PubMed

    Yoshida, Ryusuke; Ninomiya, Yuzo

    2016-03-01

    The taste system of animals is used to detect valuable nutrients and harmful compounds in foods. In humans and mice, sweet, bitter, salty, sour and umami tastes are considered the five basic taste qualities. Sweet and umami tastes are mediated by G-protein-coupled receptors, belonging to the T1R (taste receptor type1) family. This family consists of three members (T1R1, T1R2 and T1R3). They function as sweet or umami taste receptors by forming heterodimeric complexes, T1R1+T1R3 (umami) or T1R2+T1R3 (sweet). Receptors for each of the basic tastes are thought to be expressed exclusively in taste bud cells. Sweet (T1R2+T1R3-expressing) taste cells were thought to be segregated from umami (T1R1+T1R3-expressing) taste cells in taste buds. However, recent studies have revealed that a significant portion of taste cells in mice expressed all T1R subunits and responded to both sweet and umami compounds. This suggests that sweet and umami taste cells may not be segregated. Mice are able to discriminate between sweet and umami tastes, and both tastes contribute to behavioural preferences for sweet or umami compounds. There is growing evidence that T1R3 is also involved in behavioural avoidance of calcium tastes in mice, which implies that there may be a further population of T1R-expressing taste cells that mediate aversion to calcium taste. Therefore the simple view of detection and segregation of sweet and umami tastes by T1R-expressing taste cells, in mice, is now open to re-examination. PMID:26912569

  19. Accelerated and Navigator-Gated Look-Locker Imaging for Cardiac T1 Estimation (ANGIE): Development and Application to T1 Mapping of the Right Ventricle

    PubMed Central

    Mehta, Bhairav B.; Chen, Xiao; Bilchick, Kenneth C.; Salerno, Michael; Epstein, Frederick H.

    2014-01-01

    Purpose: To develop a method for high-resolution cardiac T1 mapping. Methods: A new method, accelerated and navigator-gated look-locker imaging for cardiac T1 estimation (ANGIE), was developed. An adaptive acquisition algorithm that accounts for the interplay between navigator gating and undersampling patterns well-suited for compressed sensing was used to minimize scan time. Computer simulations, phantom experiments, and imaging of the left ventricle (LV) were used to optimize and evaluate ANGIE. ANGIE’s high spatial resolution was demonstrated by T1 mapping of the right ventricle (RV). Comparisons were made to modified Look-Locker imaging (MOLLI). Results: Retrospective reconstruction of fully sampled datasets demonstrated the advantages of the adaptive algorithm. For the LV, ANGIE measurements of T1 were in good agreement with MOLLI. For the RV, ANGIE achieved a spatial resolution of 1.2 × 1.2 mm2 with a scan time of 157±53 s per slice, and measured RV T1 values of 980±96 ms versus 1076±157 ms for lower-resolution MOLLI. ANGIE provided lower intrascan variation in the RV T1 estimate compared with MOLLI (P<0.05). Conclusion: ANGIE enables high-resolution cardiac T1 mapping in clinically reasonable scan times. ANGIE opens the prospect of quantitative T1 mapping of thin cardiovascular structures such as the RV wall. PMID:24515952

  20. Diabetes-Associated Dry Eye Syndrome in a New Humanized Transgenic Model of Type 1 Diabetes

    PubMed Central

    Imam, Shahnawaz; Elagin, Raya B.

    2013-01-01

    Purpose Patients with Type 1 Diabetes (T1D) are at high risk of developing lacrimal gland dysfunction. We have developed a new model of human T1D using double-transgenic mice carrying HLA-DQ8 diabetes-susceptibility haplotype instead of mouse MHC-class II and expressing the human beta cell autoantigen Glutamic Acid Decarboxylase in pancreatic beta cells. We report here the development of dry eye syndrome (DES) after diabetes induction in our humanized transgenic model. Methods Double-transgenic mice were immunized with DNA encoding human GAD65, either naked or in adenoviral vectors, to induce T1D. Mice monitored for development of diabetes developed lacrimal gland dysfunction. Results Animals developed lacrimal gland disease (classically associated with diabetes in Non Obese Diabetic [NOD] mice and with T1D in humans) as they developed glucose intolerance and diabetes. Animals manifested obvious clinical signs of dry eye syndrome (DES), from corneal erosions to severe keratitis. Histological studies of peri-bulbar areas revealed lymphocytic infiltration of glandular structures. Indeed, infiltrative lesions were observed in lacrimal/Harderian glands within weeks following development of glucose intolerance. Lesions ranged from focal lymphocytic infiltration to complete acinar destruction. We observed a correlation between the severity of the pancreatic infiltration and the severity of the ocular disease. Conclusions Our results demonstrate development of DES in association with antigen-specific insulitis and diabetes following immunization with clinically relevant human autoantigen concomitantly expressed in pancreatic beta cells of diabetes-susceptible mice. As in the NOD mouse model and as in human T1D, our animals developed diabetes-associated DES. This specific finding stresses the relevance of our model for studying these human diseases. We believe our model will facilitate studies to prevent/treat diabetes-associated DES as well as human diabetes. PMID:23805032

  1. 26 CFR 31.3402(t)-1 - Withholding requirement on certain payments made by government entities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... made by government entities. 31.3402(t)-1 Section 31.3402(t)-1 Internal Revenue INTERNAL REVENUE... EMPLOYMENT TAXES AND COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source 31.3402(t)-1... 31.3402(t)-3(b) and 31.3402(t)-4, the Government of the United States, every State, every...

  2. Manipulating transgenes using a chromosome vector

    PubMed Central

    Ikeno, Masashi; Suzuki, Nobutaka; Hasegawa, Yoshinori; Okazaki, Tsuneko

    2009-01-01

    Recent technological advances have enabled us to visualize the organization and dynamics of local chromatin structures; however, the comprehensive mechanisms by which chromatin organization modulates gene regulation are poorly understood. We designed a human artificial chromosome vector that allowed manipulation of transgenes using a method for delivering chromatin architectures into different cell lines from human to fish. This methodology enabled analysis of de novo construction, epigenetic maintenance and changes in the chromatin architecture of specific genes. Expressive and repressive architectures of human STAT3 were established from naked DNA in mouse embryonic stem cells and CHO cells, respectively. Delivery of STAT3 within repressive architecture to embryonic stem cells resulted in STAT3 activation, accompanied by changes in DNA methylation. This technology for manipulating a single gene with a specific chromatin architecture could be utilized in applied biology, including stem cell science and regeneration medicine. PMID:19223328

  3. WP1: transgenic opto-animals

    NASA Astrophysics Data System (ADS)

    UŻarowska, E.; Czajkowski, Rafał; Konopka, W.

    2014-11-01

    We aim to create a set of genetic tools where permanent opsin expression (ChR or NpHR) is precisely limited to the population of neurons that express immediate early gene c-fos during a specific temporal window of behavioral training. Since the c-fos gene is only expressed in neurons that form experience-dependent ensemble, this approach will result in specific labeling of a small subset of cells that create memory trace for the learned behavior. To this end we employ two alternative inducible gene expression systems: Tet Expression System and Cre/lox System. In both cases, the temporal window for opsin induction is controlled pharmacologically, by doxycycline or tamoxifen, respectively. Both systems will be used for creating lines of transgenic animals.

  4. 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

  5. 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)...

  6. Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

    PubMed

    Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping; Que, Youxiong

    2016-01-01

    We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines. PMID:27093437

  7. Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

    PubMed Central

    Gao, Shiwu; Yang, Yingying; Wang, Chunfeng; Guo, Jinlong; Zhou, Dinggang; Wu, Qibin; Su, Yachun; Xu, Liping

    2016-01-01

    We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines. PMID:27093437

  8. Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

    PubMed Central

    Kong, Qingran; Wu, Meiling; Huan, Yanjun; Zhang, Li; Liu, Haiyan; Bou, Gerelchimeg; Luo, Yibo; Mu, Yanshuang; Liu, Zhonghua

    2009-01-01

    Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs. PMID:19688097

  9. 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

  10. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

    PubMed Central

    2011-01-01

    Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV) movement protein (MP) gene and the partial Cucumber mosaic virus (CMV) replication protein (Rep) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58) immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32) immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃) did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection. PMID:21269519

  11. 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 invivo. 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

  12. Human health and transgenic crops

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Under the joint auspices of the Agrochemical and the Agricultural and Food Chemistry Divisions of the American Chemical Society, we organized a short symposium on “Human Health and Transgenic Crops” at the 244th ACS national meeting, held August 19-23, 2012 in Philadelphia, PA, to examine an array o...

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

    PubMed Central

    2012-01-01

    Background The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes β-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. Results The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. Conclusions Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest. PMID:22794278

  14. B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice

    SciTech Connect

    Bucchini, D.; Desbois, P.; Pictet, R.; Jami, J. ); Madsen, O. )

    1989-02-01

    Transgenic mouse lines carrying the human insulin gene were previously shown to express it in pancreas but not in other tissues. The present study reports evidence that the expression of the transgene is restricted to a single category of cells. Immunofluorescence staining of frozen pancreas sections showed that the human C-peptide was present in pancreatic islets only, and more precisely in the B cells of the islets. Human insulin transcripts were initiated correctly in mouse pancreas at the same site as in human pancreas. Three different transgenic lines with different insertion sites and various copy numbers of the human insulin transgene had the same high levels of the transgene transcripts corresponding to a well-balanced contribution in insulin gene expression.

  15. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish

    PubMed Central

    Ellett, Felix; Pase, Luke; Hayman, John W.; Andrianopoulos, Alex

    2011-01-01

    Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology. PMID:21084707

  16. Dynamic Smad-mediated BMP signaling revealed through transgenic zebrafish

    PubMed Central

    Collery, Ross F.; Link, Brian A.

    2011-01-01

    BMP signaling is fundamental to development, injury response, and homeostasis. We have developed transgenic zebrafish that report Smad-mediated BMP signaling in embryos and adults. These lines express either eGFP, destabilized eGFP, or destabilized KO2 under the well-characterized ‘BMP Response Element’ (BRE). These fluorescent proteins were found to be expressed dynamically in regions of known BMP signaling including the developing tailbud, hematopoietic lineage, dorsal eye, brain structures, heart, jaw, fins, and somites, as well as other tissues. Responsiveness to changes in BMP signaling was confirmed by observing fluorescence after activation in an hsp70:bmp2b transgenic background or by inhibition in an hsp70:nog3 background. We further demonstrated faithful reportage by the BRE transgenic lines following chemical repression of BMP signaling using an inhibitor of BMP receptor activity, dorsomorphin. Overall, these lines will serve as valuable tools to explore the mechanisms and regulation of BMP signal during embryogenesis, in tissue maintenance, and during disease. PMID:21337469

  17. Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values.

    PubMed

    Livshits, V A; Marsh, D

    2005-08-01

    The use of 90 degrees-out-of-phase first-harmonic absorption (V1'-) EPR to resolve the spectra from nitroxide spin labels with differing T1-relaxation times is described. Non-linear V1'-EPR spectra recorded under moderate saturation have sharper lines compared with the in-phase V1-EPR spectra, and amplitudes that preferentially enhance components with longer T1-relaxation. Discrimination between V1'-spectral components can be increased further by means of selective paramagnetic relaxation enhancement agents. Examples are given of biophysical applications to double labelling in single-component membranes and phase separation in two-component membranes, to lipid-peptide complexes, and to binding of spin-labelled reagents. It is concluded that optimal resolution in V1'-EPR spectroscopy is obtained at relatively low Zeeman modulation frequencies (20-30 kHz) and moderate saturation (H1 approximately 0.2-0.3 G). PMID:15946873

  18. [Effects of transgenic Bt rice on soil dissolved organic carbon and nitrogen contents and microbiological properties].

    PubMed

    Li, Xiu-Qiang; Chen, Fa-Jun; Liu, Man-Qiang; Hu, Feng

    2012-01-01

    A two-year field experiment (2009 and 2010) was conducted to evaluate the effects of three transgenic Bt rice lines (KMD, HH1, and BtSY63) and their non-Bt lines (XSD, MH63, and SY63) on soil dissolved organic carbon (DOC) and nitrogen (DON) and microbiological properties. All the measured indices changed significantly with sampling time. Comparing with their corresponding non-Bt lines, the test transgenic Bt lines had little effects on the soil DOC, DON, and microbial biomass nitrogen (MBN). The transgenic Bt lines had significant effects on the soil microbial biomass carbon (MBC), basal respiration (BR), and microbial metabolic quotient (qCO2) in certain periods of time in the first year, but no effects in the second year. Among the soils planted with the three non-Bt rice lines, no difference was observed in the DOC, DON, and microbiological properties, whereas in the soil planted with BtSY63, the MBC and BR were significantly higher, but the qCO2 was significantly lower, as compared with those in the soils planted with KMD and HH1. In sum, two years' planting transgenic Bt rice had little effects on the soil DOC, DON, and microbiological properties, but the differences of soil microbiological properties induced by the planting of different transgenic Bt rice lines were larger than those induced by the planting of different non-Bt lines, implying that long term monitoring would help to reveal the effects of transgenic Bt rice on the structure and function of soil ecosystem. PMID:22489485

  19. The a”MAZE”ing world of lung specific transgenic mice

    PubMed Central

    Rawlins, Emma L.; Perl, Anne-Karina

    2013-01-01

    The purpose of this review is to give a comprehensive overview of transgenic mouse lines suitable for studying gene function and cellular lineage relationships in lung development, homeostasis, injury and repair. Many of the mouse strains reviewed in this article have been widely shared within the lung research community and new strains are continuously being developed. There are many useful transgenic lines that work to target subsets of lung cells, but it remains a challenge for investigators to select the correct transgenic modules for their experiment. This review covers both the tetracycline and tamoxifen inducible systems and will primarily focus on conditional lines that target the epithelial cells. We point out the limitations of each strain so investigators can choose the system that will work best for their scientific question. Current mesenchymal and endothelial lines are limited by the fact that they are not lung specific. These lines will be summarized in a brief overview. In addition, useful transgenic reporter mice for studying lineage relationships, promoter activity and signaling pathways will complete our lung specific conditional transgenic mouse-shopping list. PMID:22180870

  20. T1ρ magnetic resonance: basic physics principles and applications in knee and intervertebral disc imaging

    PubMed Central

    Zhang, Qinwei; Li, Xiaojuan; Chen, Weitian; Ahuja, Anil; Yuan, Jing

    2015-01-01

    T1ρ relaxation time provides a new contrast mechanism that differs from T1- and T2-weighted contrast, and is useful to study low-frequency motional processes and chemical exchange in biological tissues. T1ρ imaging can be performed in the forms of T1ρ-weighted image, T1ρ mapping and T1ρ dispersion. T1ρ imaging, particularly at low spin-lock frequency, is sensitive to B0 and B1 inhomogeneity. Various composite spin-lock pulses have been proposed to alleviate the influence of field inhomogeneity so as to reduce the banding-like spin-lock artifacts. T1ρ imaging could be specific absorption rate (SAR) intensive and time consuming. Efforts to address these issues and speed-up data acquisition are being explored to facilitate wider clinical applications. This paper reviews the T1ρ imaging’s basic physic principles, as well as its application for cartilage imaging and intervertebral disc imaging. Compared to more established T2 relaxation time, it has been shown that T1ρ provides more sensitive detection of proteoglycan (PG) loss at early stages of cartilage degeneration. T1ρ has also been shown to provide more sensitive evaluation of annulus fibrosis (AF) degeneration of the discs. PMID:26807369

  1. Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

    PubMed Central

    Moreau-Gachelin, F; Wendling, F; Molina, T; Denis, N; Titeux, M; Grimber, G; Briand, P; Vainchenker, W; Tavitian, A

    1996-01-01

    Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages. PMID:8628313

  2. Transgenic plants: from first successes to future applications.

    PubMed

    Van Lijsebettens, Mieke; Angenon, Geert; De Block, Marc

    2013-01-01

    This dialogue was held between the Guest Editors of the Special Issue on "Plant Transgenesis" of the Int. J. Dev. Biol. and Marc De Block. He was one of the first scientists worldwide to obtain transgenic plants transformed with the chimeric selectable marker genes encoding neomycin phosphotransferase and bialaphos that confer resistance against the antibiotic kanamycin and the herbicide Basta®/glufosinate, respectively at the Department of Genetics of Ghent University and, later on, at the spin-off company, Plant Genetic Systems. Today, these two genes are still the most frequently utilized markers in transgene technology. Marc De Block chose to work on the improvement of crops in an industrial environment to help realize the production of superior seeds or products. He was part of the team that developed the male sterility/restorer system in canola (Brassica napus var. napus) that led to the first hybrid lines to be commercialized as successful products of transgene technology. In more than 30 years of research, he developed transformation procedures for numerous crops, designed histochemical, biochemical and physiological assays to monitor plant performance, and made original and innovative contributions to plant biology. Presently, he considers transgenic research part of the toolbox for plant improvement and essential for basic plant research. PMID:24166429

  3. Oncogene-induced liver neoplasia in transgenic mice.

    PubMed

    Sandgren, E P; Quaife, C J; Pinkert, C A; Palmiter, R D; Brinster, R L

    1989-06-01

    Models of hepatocarcinogenesis were generated by directing the expression of SV40 T-antigens, an oncogenic mutant of c-H-ras, or c-myc to the liver of transgenic mice using the albumin enhancer/promoter. The majority of mice carrying the ras transgene (group A) were born with enlarged livers and atypical hepatic architecture, and these all died within several days of birth. The remaining ras transgenic mice (group B) had lower levels of hepatic ras expression, exhibited mild hepatic dysplasia but no liver enlargement, and all ultimately died from development of lung tumors. In contrast, the livers of mice expressing T-antigens were relatively normal at birth, by one month displayed marked dysplasia, and by three to seven months developed multiple nodular adenomas and carcinomas. Myc expression caused mild to severe hepatic dysplasia in young mice, and focal hepatic adenomas in some mice over fifteen months of age. Lines of mice expressing ras (group B), T-antigen, or myc were established and crossed with each other to generate dual transgenic mice expressing oncogene pairs. Each combination resulted in accelerated tumor development, suggesting that these oncoproteins can cooperate with one another during multistep hepatic transformation. PMID:2543942

  4. 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 μT. Using our ULF MRI system with adjustable static field of typically 55 to 240 μT, 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.

  5. 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

  6. The Organic Composition of Comet C/1999 T1 (McNaught-Hartley)

    NASA Astrophysics Data System (ADS)

    DiSanti, M. A.; Mumma, M. J.; Dello Russo, N.; Magee-Sauer, K.; Novak, R.

    2001-11-01

    The volatile organic composition of the long-period comet C/1999 T1 (McNaught-Hartley) was studied at infrared wavelengths on several dates post-perihelion using CSHELL at the IRTF and NIRSPEC at Keck 2. The heliocentric distance varied from R= 1.27 AU on UT 2001 January 13 to 1.71 AU on March 05. High-resolution spectra were obtained (lambda / Delta-lambda 15,000 - 20,000), revealing emissions from water, carbon monoxide, ethane, methane, methanol, and hydrogen cyanide, as well as OH prompt emission. Preliminary results indicate a substantial CO mixing ratio (CO:H2O = 0.15 - 0.20). Owing to the relatively small nod distance between A and B beams ( 12 - 15 arc-seconds along slit), this is primarily a measure of the native CO production rate. This CO abundance is similar to the native CO mixing ratio we find for C/1996 B2 (Hyakutake), and somewhat higher than that measured for C/1995 O1 (Hale-Bopp), 0.12. It is substantially higher than that measured for 3 other Oort Cloud comets contained in our sample (C/1999 H1 Lee, C/1999 S4 Linear, and C/2001 A2 Linear), for which CO:H2O was a few percent or less. Mixing ratios measured for other species in C/1999 T1 were more closely in line with those found in the other comets sampled. A summary of production rates, and implications for the formation of C/McNaught-Hartley will be presented. This work was supported by the NASA Planetary Astronomy Program through Grants NAG5-7905 and RTOP 344-32-30-07.

  7. Orosensory detection of sucrose, maltose, and glucose is severely impaired in mice lacking T1R2 or T1R3, but Polycose sensitivity remains relatively normal

    PubMed Central

    Treesukosol, Yada

    2012-01-01

    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 EC50 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

  8. Characterization of FLOWERING LOCUS T1 (FT1) Gene in Brachypodium and Wheat

    PubMed Central

    Han, Xiuli; Wang, Shuyun; Ni, Fei; Li, Kun; Pearce, Stephen; Wu, Jiajie; Dubcovsky, Jorge; Fu, Daolin

    2014-01-01

    The phase transition from vegetative to reproductive growth is a critical event in the life cycle of flowering plants. FLOWERING LOCUS T (FT) plays a central role in the regulation of this transition by integrating signals from multiple flowering pathways in the leaves and transmitting them to the shoot apical meristem. In this study, we characterized FT homologs in the temperate grasses Brachypodium distachyon and polyploid wheat using transgenic and mutant approaches. Downregulation of FT1 by RNAi was associated with a significant downregulation of the FT-like genes FT2 and FT4 in Brachypodium and FT2 and FT5 in wheat. In a transgenic wheat line carrying a highly-expressed FT1 allele, FT2 and FT3 were upregulated under both long and short days. Overexpression of FT1 caused extremely early flowering during shoot regeneration in both Brachypodium and hexaploid wheat, and resulted in insufficient vegetative tissue to support the production of viable seeds. Downregulation of FT1 transcripts by RNA interference (RNAi) resulted in non-flowering Brachypodium plants and late flowering plants (2–4 weeks delay) in wheat. A similar delay in heading time was observed in tetraploid wheat plants carrying mutations for both FT-A1 and FT-B1. Plants homozygous only for mutations in FT-B1 flowered later than plants homozygous only for mutations in FT-A1, which corresponded with higher transcript levels of FT-B1 relative to FT-A1 in the early stages of development. Taken together, our data indicate that FT1 plays a critical role in the regulation of flowering in Brachypodium and wheat, and that this role is associated with the simultaneous regulation of other FT-like genes. The differential effects of mutations in FT-A1 and FT-B1 on wheat heading time suggest that different allelic combinations of FT1 homoeologs could be used to adjust wheat heading time to improve adaptation to changing environments. PMID:24718312

  9. 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...

  10. 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.

  11. Rapid Acquisition Strategy for Functional T1ρ Mapping of the Brain

    PubMed Central

    Johnson, Casey P.; Heo, Hye-Young; Thedens, Daniel R.; Wemmie, John A.; Magnotta, Vincent A.

    2014-01-01

    Functional T1ρ mapping has been proposed as a method to assess pH and metabolism dynamics in the brain with high spatial and temporal resolution. The purpose of this work is describe and evaluate a variant of the spin-locked echo-planar imaging (SLEPI) sequence for functional T1ρ mapping at 3T. The proposed sequence rapidly acquires a time series of T1ρ maps with 4.0 sec temporal resolution and 10 slices of volumetric coverage. Simulation, phantom, and in vivo experiments are used to evaluate many aspects of the sequence and its implementation including fidelity of measured T1ρ dynamics, potential confounds to the T1 response, imaging parameter tradeoffs, time series analysis approaches, and differences compared to BOLD fMRI. It is shown that the high temporal resolution and volumetric cove of the sequence are obtained with some expense including underestimation of the T1ρ response, sensitivity to T1 dynamics, and reduced signal-to-noise ratio. In vivo studies using a flashing checkerboard fMRI paradigm suggest differences between T1ρ and BOLD activation pattern Possible sources of the functional T1ρ response and potential sequence improvements are discussed. The capability of T1ρ to map whole-brain pH and metabolism dynamics with high temporal and spatial resolution is potentially unique and warrants further investigation and development. PMID:25093630

  12. Improved neuronal tract tracing using manganese enhanced magnetic resonance imaging with fast T(1) mapping.

    PubMed

    Chuang, Kai-Hsiang; Koretsky, Alan

    2006-03-01

    There has been growing interest in using manganese-enhanced MRI (MEMRI) to detect neuronal activation, neural architecture, and neuronal connections. Usually Mn(2+) produces a very wide range of T(1) change. In particular, in neuronal tract tracing experiments the site of Mn(2+) injection can have very short T(1) while distant regions have small T(1) reductions, primarily due to dilution of Mn(2+). Most MEMRI studies use T(1)-weighted sequences, which can only give optimal contrast for a narrow range of T(1) changes. To improve sensitivity to the full extent of Mn(2+) concentrations and to optimize detection of low concentrations of Mn(2+), a fast T(1) mapping sequence based on the Look and Locker technique was implemented. Phantom studies demonstrated less than 6.5% error in T(1) compared to more conventional T(1) measurements. Using center-out segmented EPI, whole-brain 3D T(1) maps with 200-microm isotropic resolution were obtained in 2 h from rat brain. Mn(2+) transport from the rat olfactory bulb through appropriate brain structures could be detected to the amygdala in individual animals. The method reliably detected less than 7% reductions in T(1). With this quantitative imaging it should be possible to study more extensive pathways using MEMRI and decrease the dose of Mn(2+) used. PMID:16470592

  13. Cytosine methylation of an Sp1 site contributes to organ-specific and cell-specific regulation of expression of the lung epithelial gene t1alpha.

    PubMed Central

    Cao, Y X; Jean, J C; Williams, M C

    2000-01-01

    Several recent observations have suggested that cytosine methylation has a role in the in vivo transcriptional regulation of cell-specific genes in normal cells. We hypothesized that methylation regulates T1alpha, a gene expressed primarily in lung in adult rodents. In fetuses T1alpha is expressed in several organs, including the entire nervous system, but during development its expression is progressively restricted to lung alveolar type I epithelial cells, some osteoblasts and choroid plexus. Here we report that T1alpha is methylated at a key Sp1 site in the proximal promoter in cells and organs, including brain, where no gene expression is detectable. Conversely, in T1alpha-expressing cells, these sites are not methylated. In embryonic brain T1alpha is unmethylated and expressed; in adult brain the gene is methylated and not expressed. In lung epithelial cell lines, methylation of the T1alpha promoter in vitro decreases expression by approx. 50% (the maximum suppression being 100%). Analysis of mutated promoter constructs indicates that a single Sp1 site in the proximal promoter provides all or most of the methylation-sensitive gene silencing. We conclude that, in addition to regulation by transcription factors, cytosine methylation has a role in the complex expression patterns of this gene in intact animals and primary cells. PMID:10970805

  14. Distinct Contributions of T1R2 and T1R3 Taste Receptor Subunits to the Detection of Sweet Stimuli

    SciTech Connect

    Nie,Y.; Vigues, S.; Hobbs, J.; Conn, G.; Munger, S.

    2005-01-01

    The molecular mechanisms by which G protein-coupled receptor (GPCR)-type chemosensory receptors of animals selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. This study showed that each of the two subunits in the mammalian heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli, though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.

  15. Multiplicative or t1 Noise in NMR Spectroscopy

    SciTech Connect

    Granwehr, Josef

    2005-01-25

    The signal in an NMR experiment is highly sensitive to fluctuations of the environment of the sample. If, for example, the static magnetic field B{sub 0}, the amplitude and phase of radio frequency (rf) pulses, or the resonant frequency of the detection circuit are not perfectly stable and reproducible, the magnetic moment of the spins is altered and becomes a noisy quantity itself. This kind of noise not only depends on the presence of a signal, it is in fact proportional to it. Since all the spins at a particular location in a sample experience the same environment at any given time, this noise primarily affects the reproducibility of an experiment, which is mainly of importance in the indirect dimensions of a multidimensional experiment, when intense lines are suppressed with a phase cycle, or for difference spectroscopy techniques. Equivalently, experiments which are known to be problematic with regard to their reproducibility, like flow experiments or experiments with a mobile target, tend to be affected stronger by multiplicative noise. In this article it is demonstrated how multiplicative noise can be identified and characterized using very simple, repetitive experiments. An error estimation approach is developed to give an intuitive, yet quantitative understanding of its properties. The consequences for multidimensional NMR experiments are outlined, implications for data analysis are shown, and strategies for the optimization of experiments are summarized.

  16. 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.

  17. A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development.

    PubMed

    Uchiyama, Koji; Watanabe, Daisuke; Hayasaka, Michiko; Hanaoka, Kazunori

    2014-12-01

    A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1 alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid-gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re-established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely-flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non-CpG cytosines of the retroelement promoter were highly methylated especially in the transgene-active mid-gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid-gestational embryo. PMID:25389047

  18. Enhanced Virus Resistance in Transgenic Maize Expressing a dsRNA-Specific Endoribonuclease Gene from E. coli

    PubMed Central

    Liu, He; Tian, Lanzhi; Zhang, Aihong; Zhang, Yanjing; Shi, Lindan; Guo, Bihong; Xu, Jin; Duan, Xifei; Wang, Xianbing; Han, Chenggui; Miao, Hongqin; Yu, Jialin; Li, Dawei

    2013-01-01

    Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection. PMID:23593318

  19. Nucleocapsid Gene-Mediated Transgenic Resistance Provides Protection Against Tomato spotted wilt virus Epidemics in the Field.

    PubMed

    Herrero, S; Culbreath, A K; Csinos, A S; Pappu, H R; Rufty, R C; Daub, M E

    2000-02-01

    ABSTRACT Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in 'Burley 21' than in 'KY-14' or 'K-326', but highly resistant 'Burley 21' transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R(3) and R(4)) transgenic resistant lines of 'Burley 21' and a 'K-326' F(1) hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in 'Burley 21' susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic 'Burley 21' lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the 'Burley 21' transgenic lines compared with the susceptible controls. The 'K-326' F(1) hybrid was as susceptible as the 'K-326' nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant 'Burley 21' transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance. PMID:18944602

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

    PubMed Central

    2011-01-01

    Background Tomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance. Results The open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV. Conclusion In all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance. PMID:22014312

  1. Enhanced virus resistance in transgenic maize expressing a dsRNA-specific endoribonuclease gene from E. coli.

    PubMed

    Cao, Xiuling; Lu, Yingui; Di, Dianping; Zhang, Zhiyan; Liu, He; Tian, Lanzhi; Zhang, Aihong; Zhang, Yanjing; Shi, Lindan; Guo, Bihong; Xu, Jin; Duan, Xifei; Wang, Xianbing; Han, Chenggui; Miao, Hongqin; Yu, Jialin; Li, Dawei

    2013-01-01

    Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection. PMID:23593318

  2. 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

  3. 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

  4. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    PubMed

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-07-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. PMID:26771455

  5. Hyperplasia and tumours in lung, breast and other tissues in mice carrying a RAR beta 4-like transgene.

    PubMed Central

    Bérard, J; Gaboury, L; Landers, M; De Repentigny, Y; Houle, B; Kothary, R; Bradley, W E

    1994-01-01

    Transgenic mice were generated which express a truncated nuclear retinoic acid receptor beta (RAR beta), closely resembling the natural isoform RAR beta 4, under the control of the MMTV promoter. The transgene was expressed in salivary gland, testis, lung and mammary tissue in two different lines. At approximately 11-14 months virtually all the transgenic mice showed hyperplasia of the lung alveolar epithelium with an excess of type II pneumocytes. Hyperplasia of the mammary alveoli and terminal ducts was also seen in some females. Salivary glands and some sebaceous glands were hyperplastic in most male transgenic mice, but only rarely in females or in non-transgenics. Primary benign and malignant tumours were more numerous in transgenic mice than in controls, with a total of 23 in 43 mice versus two in 33 non-transgenic animals. Treatment with dexamethasone to increase transgene expression resulted in exaggerated versions of the above phenotypes. Overexpression of RAR beta 4 therefore appears to predispose various tissues to hyperplasia and neoplasia, and this by contrast to the RAR beta 2 isoform, which has tumour suppressor activity. A survey of ratios of RAR beta 4:RAR beta 2 expression in human lung tumour cell lines showed an increase compared with normal lung tissue, suggesting that RAR beta 4 may play a similar role in human tumorigenesis. Images PMID:7988554

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

    PubMed

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

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes. PMID:23241066

  7. 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

  8. Insertional mutagenesis in transgenic mice

    SciTech Connect

    Woychik, R.P.; Bultman, S.J.; Schrick, J.J.; Moyer, J.H.; Beatty, B.R.; Godfrey, V.L.; Selby, P.B. )

    1991-03-11

    The molecular analysis of mutations in the mouse is an ideal way of establishing structure-function relationships between individual genes and complex developmental phenotypes. Insertional mutations, caused by the integration of a transgene, are advantageous because the mutant locus is tagged' with the exogenously added DNA and can be accessed with existing cloning and molecular analysis techniques. The authors have implemented a large-scale insertional mutagenesis program at the Oak Ridge National Laboratory in an effort to identify numerous mutations with developmental phenotypes. Several of these mutations are currently being subjected to an in-depth molecular analysis involving the characterization of the structure and expression of the mutant locus. Additionally, the authors are mapping transgene integration sites with in situ hybridization procedures, which will result in additional well-characterized molecular access points for the mouse genome.

  9. 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

  10. 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.

  11. 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

  12. Development and utilization of transgenic New World screwworm, Cochliomyia hominivorax.

    PubMed

    Handler, A M; Allen, M L; Skoda, S R

    2009-06-01

    The New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), was the first insect to be effectively controlled using the sterile insect technique (SIT). Recent efforts to improve SIT control of this species have centred on the development of genetically transformed strains using the piggyBac transposon vector system. Eight transgenic strains were produced incorporating an enhanced green fluorescent protein (EGFP) marker gene under polyubiquitin regulation that has the potential for use as a genetic marking system for released males. The transgenic strains were genetically and phenotypically characterized, including for life fitness parameters and mating competitiveness. These characteristics were unique for each strain and thus some strains were deemed suitable for incorporation into SIT eradication programmes. The strain with the best attributes is designated 'CLAY'. Four of the strains, including CLAY, have been successfully cryopreserved so that their original characteristics should be unchanged when further evaluation is required. With the demonstration of efficient germ-line transformation in NWS, allowing production of fit and competitive transformants, it is now possible to consider further transgenic strain development to improve SIT that are currently being tested in other dipteran species. This includes strains that allow genetic marking with fluorescent proteins, genetic sexing by female lethality, male-specific fluorescent sorting and male sterility by testis-specific lethality. The SIT may also be improved upon by new strategies resulting in lethality of offspring of released insects using conditional lethal systems based upon temperature-dependent or dietary tetracycline regulation of lethal gene expression. Both the creation of new NWS transgenic strains and the ecological safety of their release will be enhanced by new vector systems that allow specific genomic targeting of vector constructs and their subsequent immobilization, ensuring transgene and strain stability. PMID:19335836

  13. 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.

  14. Restriction of neuroblastoma to the prostate gland in transgenic mice.

    PubMed

    Skalnik, D G; Dorfman, D M; Williams, D A; Orkin, S H

    1991-09-01

    Male transgenic mice that carry a construct containing 5'-flanking sequences of the gp91-phox gene linked to the early region of the simian virus 40 (SV40) genome reproducibly develop tumors arising from the prostate gland. As gp91-phox is expressed exclusively in terminally differentiating hematopoietic cells of the myelomonocytic lineage, the induction of tumors arising from the prostate gland was unexpected. These lesions appear to be due to a novel transcription signal that was generated during the construction of the transgene. Surprisingly, the histopathological and biochemical properties of the tumor are diagnostic of neuroblastoma rather than of adenocarcinoma of the prostate gland. Tumors produce SV40 T antigen and isoforms of neural cell adhesion molecule characteristic of neuronal cells, and they occur in a testosterone-independent manner. Microscopic examination of prostate glands from young transgenic mice reveals the presence of small lesions arising outside of the prostate gland epithelium, which is consistent with the diagnosis of neuroblastoma and further distinguishes this tumor from prostatic adenocarcinoma. Prostate gland tumors occur in all male animals of susceptible lines carrying the gp91-phox promoter/SV40 early-region transgene. However, variability in the time at which gross tumors appear and the presence of cells expressing T antigen prior to tumorigenesis suggest that somatic events in addition to T-antigen production are required for the development of a malignancy. The extraordinary restriction of the site of tumorigenesis in these animals indicates the presence in the prostate gland of a novel, tissue-specific neuroectodermal cell of origin. These transgenic animals provide a model system for the study of neuroectodermal malignancies. PMID:1652058

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

    PubMed

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

    2013-02-01

    We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans. PMID:23176559

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

    SciTech Connect

    Callow, M.J.; Stoltzfus, L.J.; Rubin, E.M.; Lawn, R.M.

    1994-03-15

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

  17. A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N-acetyltransferase expressed in transgenic rice plants.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Lee, Kyungjin; Back, Kyoungwhan

    2014-09-01

    Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter. PMID:24920304

  18. 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

  19. Anticancer Activity of Saponins from Allium chinense against the B16 Melanoma and 4T1 Breast Carcinoma Cell.

    PubMed

    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

  20. 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

  1. Inheritance and expression of chimeric genes in the progeny of transgenic maize plants.

    PubMed

    Fromm, M E; Morrish, F; Armstrong, C; Williams, R; Thomas, J; Klein, T M

    1990-09-01

    We obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film. PMID:1366794

  2. Increased native T1-values at the interventricular insertion regions in precapillary pulmonary hypertension.

    PubMed

    Spruijt, Onno A; Vissers, Loek; Bogaard, Harm-Jan; Hofman, Mark B M; Vonk-Noordegraaf, Anton; Marcus, J Tim

    2016-03-01

    Cardiac magnetic resonance imaging of the pressure overloaded right ventricle (RV) of precapillary pulmonary hypertension (PH) patients, exhibits late gadolinium enhancement at the interventricular insertion regions, a phenomenon which has been linked to focal fibrosis. Native T1-mapping is an alternative technique to characterize myocardium and has the advantage of not requiring the use of contrast agents. The aim of this study was to characterize the myocardium of idiopathic pulmonary arterial hypertension (IPAH), systemic scleroderma related PH (PAH-Ssc) and chronic thromboembolic PH (CTEPH) patients using native T1-mapping and to see whether native T1-values were related to disease severity. Furthermore, we compared native T1-values between the different precapillary PH categories. Native T1-mapping was performed in 46 IPAH, 14 PAH-SSc and 10 CTEPH patients and 10 control subjects. Native T1-values were assessed using regions of interest at the RV and LV free wall, interventricular septum and interventricular insertion regions. In PH patients, native T1-values of the interventricular insertion regions were significantly higher than the native T1-values of the RV free wall, LV free wall and interventricular septum. Native T1-values at the insertion regions were significantly related to disease severity. Native T1-values were not different between IPAH, PAH-Ssc and CTEPH patients. Native T1-values of the interventricular insertion regions are significantly increased in precapillary PH and are related to disease severity. Native T1-mapping can be developed as an alternative technique for the characterization of the interventricular insertion regions and has the advantage of not requiring the use of contrast agents. PMID:26472581

  3. Activation of latent transgenes in Arabidopsis using a hybrid transcription factor.

    PubMed Central

    Guyer, D; Tuttle, A; Rouse, S; Volrath, S; Johnson, M; Potter, S; Görlach, J; Goff, S; Crossland, L; Ward, E

    1998-01-01

    A hybrid transcription factor comprising a fusion of the DNA-binding domain of Saccharomyces cerevisiae GAL4 and the transcription activation domain of maize C1 was expressed in stably transformed Arabidopsis. Additional transgenic lines were created containing test genes controlled by a synthetic promoter consisting of concatemeric copies of the cis-acting site recognized by GAL4 (UASG) fused to a minimal promoter. The GAL4/C1 effector line was crossed to two lines containing a synthetic promoter/GUS fusion. Both histochemical staining and GUS activity assays indicate strong activation of GUS expression was achieved only after crossing. The GAL4/C1 effector line was also crossed to 15 lines containing a synthetic promoter/antisense adenylosuccinate synthetase gene. Severely retarded growth, and in some cases lethality, was observed in 40% of the F1 lines. This system of activation by crossing is generally useful for activating expression of test transgenes. PMID:9611179

  4. Polyhydroxybutyrate synthesis in transgenic flax.

    PubMed

    Wróbel, Magdalena; Zebrowski, Jacek; Szopa, Jan

    2004-01-01

    Flax (Linum usitatissimum L.) is an annual plant species widely cultivated in temperate climates for bast fibres and linseed oil. Apart from traditional textile use, the fibres are fast becoming an integral part of new composite materials utilized in automobile and constructive industry. Especially attractive for environmental safety demands are biodegradable and renewable biocomposities based on polyhydroxybutyrate (PHB) polymer as a matrix and reinforced with the flax fibres. Manufacturing of PHB by bacteria fermentation is however substantially more expansive as compared to technologies producing conventional plastics. We report for the first time generation of transgenic plants which produce both components of flax/PHB composites, i.e. the fibres and the thermoplastic matrix in the same plant organ of a crop. The flax (cv. Nike) plants were transformed using constructs bearing either single cDNA, encoding the beta-ketothiolase enzyme (C plants), or all three of the genes necessary for poly-beta-hydroxybutyrate (PHB) synthesis (M plants). Both constructs contained a plastidial targeting sequence. The amount of PHB produced by the transgenic plants was up to over 70-fold higher than in wild-type plants, when analysed using the gas chromatography/mass spectrometry (GC-MS method). The PHB accumulation in plastids caused change both in their shape and size. The use of a stem-specific promoter for transgene expression protected the transgenic plant from growth retardation and also provided higher PHB synthesis than in the case of constructs governed by the 35S CaMV constitutive promoter. None toxic effects that could lead to stunted growth or the loss of fertility were observed, when 14-3-3 promoter was used as the stem-specific. Significant modifications in stem mechanical properties were accompanied to the PHB accumulation in growing cell of fibres in the transgenic plants. The Young's modulus E, the average measure of stem tissues resistance to tensile loads increased up to twice in M plants as compared to a single gene transformed ones. However, a wide range of E values, from 24.1 to 54.4 MPa, was observed in dependence of tested strain. Potential commercial significance of the genetic manipulation approach enabling synthesis of thermoplastic in crops cultivated for fibres is discussed. PMID:14687970

  5. [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

  6. Change in the Proton T1 of Fat and Water in Mixture

    PubMed Central

    Hu, Houchun H.; Nayak, Krishna S.

    2010-01-01

    This work describes observed changes in the proton T1 relaxation time of both water and lipid when they are in relatively homogeneous mixtures. Results obtained from vegetable oil–water emulsions, pork kidney and lard mixtures, and excised samples of white and brown adipose tissues are presented to demonstrate this change in T1 as a function of mixture fat fraction. As an initial proof of concept, a simpler acetone-water experiment was performed to take advantage of complete miscibility between acetone and water and both components’ single chemical shift peaks. Single-voxel MR spectroscopy was used to measure the T1 of predominant methylene spins in fat and the T1 of water spins in each setup. In the vegetable oil–water emulsions, the T1 of fat varied by as much as 3-fold when water was the dominant mixture component. The T1 of pure lard increased by 170 msec (+37%) when it was blended with lean kidney tissue in a 16% fatty mixture. The fat T1 of lipid-rich white adipose tissue was 312 msec. In contrast, the fat T1 of leaner brown adipose tissue (fat fraction 53%) was 460 msec. A change in the water T1 from that of pure water was also observed in the experiments. PMID:19918888

  7. Optimized efficient liver T1ρ mapping using limited spin lock times

    NASA Astrophysics Data System (ADS)

    Yuan, Jing; Zhao, Feng; Griffith, James F.; Chan, Queenie; Wang, Yi-Xiang J.

    2012-03-01

    T1ρ relaxation has recently been found to be sensitive to liver fibrosis and has potential to be used for early detection of liver fibrosis and grading. Liver T1ρ imaging and accurate mapping are challenging because of the long scan time, respiration motion and high specific absorption rate. Reduction and optimization of spin lock times (TSLs) are an efficient way to reduce scan time and radiofrequency energy deposition of T1ρ imaging, but maintain the near-optimal precision of T1ρ mapping. This work analyzes the precision in T1ρ estimation with limited, in particular two, spin lock times, and explores the feasibility of using two specific operator-selected TSLs for efficient and accurate liver T1ρ mapping. Two optimized TSLs were derived by theoretical analysis and numerical simulations first, and tested experimentally by in vivo rat liver T1ρ imaging at 3 T. The simulation showed that the TSLs of 1 and 50 ms gave optimal T1ρ estimation in a range of 10-100 ms. In the experiment, no significant statistical difference was found between the T1ρ maps generated using the optimized two-TSL combination and the maps generated using the six TSLs of [1, 10, 20, 30, 40, 50] ms according to one-way ANOVA analysis (p = 0.1364 for liver and p = 0.8708 for muscle).

  8. Measurement of T1 of human arterial and venous blood at 7T.

    PubMed

    Rane, Swati D; Gore, John C

    2013-04-01

    Techniques for measuring cerebral perfusion require accurate longitudinal relaxation (T1) of blood, an 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 37C, the T1 of blood at arterial pO2 was 2.290.1s and 2.070.12 at venous pO2. The in vivo T1 of venous blood, in three subjects, was slightly longer at 2.450.11s. 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

  9. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains

    PubMed Central

    Nango, Eriko; Akiyama, Shuji; Maki-Yonekura, Saori; Ashikawa, Yuji; Kusakabe, Yuko; Krayukhina, Elena; Maruno, Takahiro; Uchiyama, Susumu; Nuemket, Nipawan; Yonekura, Koji; Shimizu, Madoka; Atsumi, Nanako; Yasui, Norihisa; Hikima, Takaaki; Yamamoto, Masaki; Kobayashi, Yuji; Yamashita, Atsuko

    2016-01-01

    Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states. PMID:27160511

  10. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains.

    PubMed

    Nango, Eriko; Akiyama, Shuji; Maki-Yonekura, Saori; Ashikawa, Yuji; Kusakabe, Yuko; Krayukhina, Elena; Maruno, Takahiro; Uchiyama, Susumu; Nuemket, Nipawan; Yonekura, Koji; Shimizu, Madoka; Atsumi, Nanako; Yasui, Norihisa; Hikima, Takaaki; Yamamoto, Masaki; Kobayashi, Yuji; Yamashita, Atsuko

    2016-01-01

    Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states. PMID:27160511

  11. A transgenic approach to study argininosuccinate synthetase gene expression

    PubMed Central

    2014-01-01

    Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. Conclusions We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene. PMID:24884799

  12. Constitutive expression of McCHIT1-PAT enhances resistance to rice blast and herbicide, but does not affect grain yield in transgenic glutinous rice.

    PubMed

    Zeng, Xiao-Fang; Li, Lei; Li, Jian-Rong; Zhao, De-Gang

    2016-02-01

    To produce new rice blast- and herbicide-resistant transgenic rice lines, the McCHIT1 gene encoding the class I chitinase from Momordica charantia and the herbicide resistance gene PAT were introduced into Lailong (Oryza sativa L. ssp. Japonica), a glutinous local rice variety from Guizhou Province, People's Republic of China. Transgenic lines were identified by ß-glucuronidase (GUS) histochemical staining, PCR, and Southern blot analyses. Agronomic traits, resistance to rice blast and herbicide, chitinase activities, and transcript levels of McCHIT1 were assessed in the T2 progeny of three transgenic lines (L1, L8, and L10). The results showed that the introduction of McCHIT1-PAT into Lailong significantly enhanced herbicide and blast resistance. After infection with the blast fungus Magnaporthe oryzae, all of the T2 progeny exhibited less severe lesion symptoms than those of wild type. The disease indices were 100% for wild type, 65.66% for T2 transgenic line L1, 59.69% for T2 transgenic line L8, and 79.80% for T2 transgenic line L10. Transgenic lines expressing McCHIT1-PAT did not show a significant difference from wild type in terms of malondialdehyde (MDA) content, polyphenol oxidase (PPO) activity, and superoxide dismutase (SOD) activity in the leaves. However, after inoculation with M. oryzae, transgenic plants showed significantly higher SOD and PPO activities and lower MDA contents in leaves, compared with those in wild-type leaves. The transgenic and the wild-type plants did not show significant differences in grain yield parameters including plant height, panicles per plant, seeds per panicle, and 1000-grain weight. Therefore, the transgenic plants showed increased herbicide and blast resistance, with no yield penalty. PMID:25639923

  13. The TaDREB3 transgene transferred by conventional crossings to different genetic backgrounds of bread wheat improves drought tolerance.

    PubMed

    Shavrukov, Yuri; Baho, Manahil; Lopato, Sergiy; Langridge, Peter

    2016-01-01

    Drought tolerance of the wheat cultivar Bobwhite was previously enhanced by transformation with a construct containing the wheat DREB3 gene driven by the stress-inducible maize Rab17 promoter. Progeny of a single T2 transgenic line were used as pollinators in crosses with four elite bread wheat cultivars from Western Australia: Bonnie Rock, IGW-2971, Magenta and Wyalkatchem, with the aim of evaluating transgene performance in different genetic backgrounds. The selected pollinator line, BW8-9-10-3, contained multiple transgene copies, had significantly improved drought tolerance compared with wild-type plants and showed no growth and development penalties or abnormalities. A single hybrid plant was selected from each cross-combination for three rounds of backcrossing with the corresponding maternal wheat cultivar. The transgene was detected in all four F1 BC3 combinations, but stress-inducible transgene expression was found in only three of the four combinations. Under well-watered conditions, the phenotypes and grain yield components of the F2 BC3 transgene-expressing lines were similar to those of corresponding recurrent parents and null-segregants. Under severe drought conditions, the backcross lines demonstrated 12-18% higher survival rates than the corresponding control plants. Two from four F3 BC3 transgenic lines showed significantly higher yield (18.9% and 21.5%) than control plants under limited water conditions. There was no induction of transgene expression under cold stress, and therefore, no improvement of frost tolerance observed in the progenies of drought-tolerant F3 BC3 lines. PMID:25940960

  14. 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

  15. High Resolution T1ρ Mapping of In Vivo Human Knee Cartilage at 7T

    PubMed Central

    Singh, Anup; Haris, Mohammad; Cai, Kejia; Kogan, Feliks; Hariharan, Hari; Reddy, Ravinder

    2014-01-01

    Purpose Spin lattice relaxation time in rotating frame (T1ρ) mapping of human knee cartilage has shown promise in detecting biochemical changes during osteoarthritis. Due to higher field strength, MRI at 7T has advantages in term of SNR compared to clinical MR scanners and this can be used to increase in image resolution. Objective of current study was to evaluate the feasibility of high resolution T1ρ mapping of in vivo human knee cartilage at 7T MR scanner. Materials and Methods In this study we have used a T1ρ prepared GRE pulse sequence for obtaining high resolution (in plan resolution  = 0.2 mm2) T1ρ MRI of human knee cartilage at 7T. The effect of a global and localized reference frequency and reference voltage setting on B0, B1 and T1ρ maps in cartilage was evaluated. Test-retest reliability results of T1ρ values from asymptomatic subjects as well as T1ρ maps from abnormal cartilage of two human subjects are presented. These results are compared with T1ρ MRI data obtained from 3T. Results Our approach enabled acquisition of 3D-T1ρ data within allowed SAR limits at 7T. SNR of cartilage on T1ρ weighted images was greater than 90. Off-resonance effects present in the cartilage B0, B1 and T1ρ maps obtained using global shim and reference frequency and voltage setting, were reduced by the proposed localized reference frequency and voltage setting. T1ρ values of cartilage obtained with the localized approach were reproducible. Abnormal knee cartilage showed elevated T1ρ values in affected regions. T1ρ values at 7T were significantly lower (p<0.05) compared to those obtained at 3T. Conclusion In summary, by using proposed localized frequency and voltage setting approach, high-resolution 3D-T1ρ maps of in vivo human knee cartilage can be obtained in clinically acceptable scan times (<30 min) and SAR constraints, which provides the ability to characterize cartilage molecular integrity. PMID:24830386

  16. T1- Thresholds in Black Holes Increase Clinical-Radiological Correlation in Multiple Sclerosis Patients

    PubMed Central

    Thaler, Christian; Faizy, Tobias; Sedlacik, Jan; Holst, Brigitte; Stellmann, Jan-Patrick; Young, Kim Lea; Heesen, Christoph; Fiehler, Jens; Siemonsen, Susanne

    2015-01-01

    Background Magnetic Resonance Imaging (MRI) is an established tool in diagnosing and evaluating disease activity in Multiple Sclerosis (MS). While clinical-radiological correlations are limited in general, hypointense T1 lesions (also known as Black Holes (BH)) have shown some promising results. The definition of BHs is very heterogeneous and depends on subjective visual evaluation. Objective We aimed to improve clinical-radiological correlations by defining BHs using T1 relaxation time (T1-RT) thresholds to achieve best possible correlation between BH lesion volume and clinical disability. Method 40 patients with mainly relapsing-remitting MS underwent MRI including 3-dimensional fluid attenuated inversion recovery (FLAIR), magnetization-prepared rapid gradient echo (MPRAGE) before and after Gadolinium (GD) injection and double inversion-contrast magnetization-prepared rapid gradient echo (MP2RAGE) sequences. BHs (BHvis) were marked by two raters on native T1-weighted (T1w)-MPRAGE, contrast-enhancing lesions (CE lesions) on T1w-MPRAGE after GD and FLAIR lesions (total-FLAIR lesions) were detected separately. BHvis and total-FLAIR lesion maps were registered to MP2RAGE images, and the mean T1-RT were calculated for all lesion ROIs. Mean T1 values of the cortex (CTX) were calculated for each patient. Subsequently, Spearman rank correlations between clinical scores (Expanded Disability Status Scale and Multiple Sclerosis Functional Composite) and lesion volume were determined for different T1-RT thresholds. Results Significant differences in T1-RT were obtained between all different lesion types with highest T1 values in visually marked BHs (BHvis: 1453.3±213.4 ms, total-FLAIR lesions: 1394.33±187.38 ms, CTX: 1305.6±35.8 ms; p<0.05). Significant correlations between BHvis/total-FLAIR lesion volume and clinical disability were obtained for a wide range of T1-RT thresholds. The highest correlation for BHvis and total-FLAIR lesion masks were found at T1-RT>1500 ms (Expanded Disability Status Scale vs. lesion volume: rBHvis = 0.442 and rtotal-FLAIR = 0.497, p<0.05; Multiple Sclerosis Functional Composite vs. lesion volume: rBHvis = -0.53 and rtotal-FLAIR = -0.627, p<0.05). Conclusion Clinical-radiological correlations in MS patients are increased by application of T1-RT thresholds. With the short acquisition time of the MP2RAGE sequences, quantitative T1 maps could be easily established in clinical studies. PMID:26659852

  17. COPD Patients Have Short Lung Magnetic Resonance T1 Relaxation Time.

    PubMed

    Alamidi, Daniel F; Morgan, Alexandra R; Hubbard Cristinacce, Penny L; Nordenmark, Lars H; Hockings, Paul D; Lagerstrand, Kerstin M; Young, Simon S; Naish, Josephine H; Waterton, John C; Maguire, Niall C; Olsson, Lars E; Parker, Geoffrey J M

    2016-04-01

    Magnetic resonance imaging (MRI) may provide attractive biomarkers for assessment of pulmonary disease in clinical trials as it is free from ionizing radiation, minimally invasive and allows regional information. The aim of this study was to characterize lung MRI T1 relaxation time as a biomarker of chronic obstructive pulmonary disease (COPD); and specifically its relationship to smoking history, computed tomography (CT), and pulmonary function test (PFT) measurements in comparison to healthy age-matched controls. Lung T1 and inter-quartile range (IQR) of T1 maps from 24 COPD subjects and 12 healthy age-matched non-smokers were retrospectively analyzed from an institutional review board approved study. The subjects underwent PFTs and two separate MR imaging sessions at 1.5 tesla to test T1 repeatability. CT scans were performed on the COPD subjects. T1 repeatability (intraclass correlation coefficient) was 0.72 for repeated scans acquired on two visits. The lung T1 was significantly shorter (p < 0.0001) and T1 IQR was significantly larger (p = 0.0002) for the COPD subjects compared to healthy controls. Lung T1 significantly (p = 0.001) correlated with lung density assessed with CT. Strong significant correlations (p < 0.0001) between lung T1 and all PFT measurements were observed. Cigarette exposure did not correlate with lung T1 in COPD subjects. In conclusion, lung MRI T1 mapping shows potential as a repeatable, radiation free, non-invasive imaging technique in the evaluation of COPD. PMID:26488310

  18. Can T1-rho MRI detect acetabular cartilage degeneration in femoroacetabular impingement?: a pilot study.

    PubMed

    Rakhra, K S; Lattanzio, P-J; Cárdenas-Blanco, A; Cameron, I G; Beaulé, P E

    2012-09-01

    Advanced MRI cartilage imaging such as T(1)-rho (T1ρ) for the diagnosis of early cartilage degradation prior to morpholgic radiological changes may provide prognostic information in the management of joint disease. This study aimed first to determine the normal T1ρ profile of cartilage within the hip, and secondly to identify any differences in T1ρ profile between the normal and symptomatic femoroacetabular impingement (FAI) hip. Ten patients with cam-type FAI (seven male and three female, mean age 35.9 years (28 to 48)) and ten control patients (four male and six female, mean age 30.6 years (22 to 35)) underwent 1.5T T1ρ MRI of a single hip. Mean T1ρ relaxation times for full thickness and each of the three equal cartilage thickness layers were calculated and compared between the groups. The mean T1ρ relaxation times for full cartilage thickness of control and FAI hips were similar (37.17 ms (SD 9.95) and 36.71 ms (SD 6.72), respectively). The control group demonstrated a T1ρ value trend, increasing from deep to superficial cartilage layers, with the middle third having significantly greater T1ρ relaxation values than the deepest third (p = 0.008). The FAI group demonstrated loss of this trend. The deepest third in the FAI group demonstrated greater T1ρ relaxation values than controls (p = 0.028). These results suggest that 1.5T T1ρ MRI can detect acetabular hyaline cartilage changes in patients with FAI. PMID:22933489

  19. Serial changes in the T1 magnetic relaxation parameter after myocardial infarction in man.

    PubMed Central

    Been, M; Smith, M A; Ridgway, J P; Douglas, R H; de Bono, D P; Best, J J; Muir, A L

    1988-01-01

    A low field resistive nuclear magnetic resonance imaging system (0.08 Tesla) was used to study the in vivo changes in the relaxation parameter T1 of the left ventricular myocardium from the first day to six months after acute myocardial infarction in 41 consecutive patients admitted to a coronary care unit. T1 maps were constructed from transverse and coronal images at various times after infarction. Thrombolytic treatment had been successful in 28 patients. Thirty three of the 34 patients studied within two weeks of infarction had a significantly increased T1 value but this developed only after the third day in four. At day 1-3 the mean (1 SD) maximum T1 was 413 (29) ms (n = 23) compared with 430 (41) ms (n = 22) at day 4-7, 433 (35) ms (n = 24) at day 8-14, 420 (34) at one month (n = 22), 388 (39) (n = 20) at three months, and 361 (24) (n = 14) at six months. The number of regions of interest with an increased T1 followed a similar time course. Although the increase in T1 measured at three months correlated with the initial maximum creatine kinase and with the left ventricular ejection fraction measured at one month, the number of regions with abnormal T1 from day 4 through to one month correlated best with left ventricular ejection fraction. There was no significant difference in T1 between patients with or without reperfusion. The rise in T1 over the first few days together with the prolonged time course of T1 increase suggests that the increase in T1 may reflect cellular infiltration as much or more than tissue oedema. Images Fig 3 PMID:3342143

  20. Effect of the Citrus Lycopene β-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 β-cyclase (LYCB) is the key enzyme for the synthesis of β-carotene, a valuable component of the human diet. In this study, tomato constitutively express Lycb-1 was engineered. The β-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 α-branch metabolic fluxes were repressed during the three developmental stages while α-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

  1. 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

  2. DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

    PubMed Central

    Flatau, E; Gonzales, F A; Michalowsky, L A; Jones, P A

    1984-01-01

    A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines. PMID:6209556

  3. Repressible transgenic model of NRAS oncogene-driven mast cell disease in the mouse.

    PubMed

    Wiesner, Stephen M; Jones, Jamie M; Hasz, Diane E; Largaespada, David A

    2005-08-01

    To create a model in which to study the effects of RAS dysregulation in hematopoietic disease, we developed separate founder lines of transgenic mice, with the tetrac