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1

Kinetics of adaptive immunity to Haemophilus influenzae type b meningitis in transgenic mice: Evidence from diverse expression of double T1\\/T2 transgenes and Th1\\/Th2-related cytokines  

Microsoft Academic Search

To investigate the kinetic changes in adaptive immunity during experimental Haemophilus influenzae type b (Hib) meningitis, we established a murine meningitis model based on T1\\/T2 doubly transgenic mice. These mice carry two transgenes that express two distinct cell-surface markers: a human Thy1 transgene (hThy1) under the control of the murine IFN-? promoter, and a murine Thy1.1 transgene (mThy1.1) under the

Shyi-Jou Chen; Ching-Len Liao; Shie-Liang Hsieh; Mong-Ling Chu; Mei-Cho Fang; Huey-Kang Sytwu; Chih-Chien Wang

2006-01-01

2

Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.  

PubMed

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering. PMID:25239742

Holmes, Scott; Lyman, Suzanne; Hsu, Jen-Kang; Cheng, JrGang

2015-01-01

3

Accumulation of nickel in transgenic tobacco  

NASA Astrophysics Data System (ADS)

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.

Sidik, Nik Marzuki; Othman, Noor Farhan

2013-11-01

4

Case Study: Polycystic Livers in a Transgenic Mouse Line  

SciTech Connect

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.

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

2014-04-01

5

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

PubMed Central

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

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

6

Generation of Doubled Haploid Transgenic Wheat Lines by Microspore Transformation  

PubMed Central

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

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

2013-01-01

7

Generation of doubled haploid transgenic wheat lines by microspore transformation.  

PubMed

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

Brew-Appiah, Rhoda A T; Ankrah, Nii; Liu, Weiguo; Konzak, Calvin F; von Wettstein, Diter; Rustgi, Sachin

2013-01-01

8

Transgenic cell lines for detection of animal viruses.  

PubMed Central

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

Olivo, P D

1996-01-01

9

Transgenic Arabidopsis tester lines with dominant marker genes.  

PubMed

The map positions of a set of eight T-DNA insertions in the Arabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar), beta-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). The neo, hpt and bar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing the hpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites. PMID:8676880

Van Lijsebettens, M; Wang, X; Cnops, G; Boerjan, W; Desnos, T; Höfte, H; Van Montagu, M

1996-06-12

10

Case Study: Polycystic Livers in a Transgenic Mouse Line  

PubMed Central

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

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

2014-01-01

11

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

PubMed Central

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

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

2013-01-01

12

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

SciTech Connect

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.

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

1983-07-01

13

INFLUENCE OF COAT PROTEIN TRANSGENE COPY NUMBER ON RESISTANCE IN TRANSGENIC LINE 63-1 AGAINST PAPAYA RINGSPOT VIRUS ISOLATES  

Technology Transfer Automated Retrieval System (TEKTRAN)

Line 63-1 is a ‘Sunset’-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R1 plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thai...

14

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

PubMed

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

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

15

Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay.  

PubMed

The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection. PMID:25351627

Wang, Xianghong; Jiang, Daiming; Yang, Daichang

2015-01-01

16

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

PubMed

Combining two existing protocols of trangenesis, namely the REMI and the I-SceI meganuclease methods, we generated Xenopus leavis expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event. PMID:19404763

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

2009-10-01

17

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

PubMed

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. This article is protected by copyright. All rights reserved. PMID:25284137

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

2014-10-01

18

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

E-print Network

Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids Amir. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing

19

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

SciTech Connect

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.

Cheng, P.-C. [Institute of Tropical Medicine, National Yang-Ming University, Taipei, Taiwan (China); Hsu, C.-Y. [Institute of Molecular and Cellular Biology, National Tsing-Hua University, Hsinchu, Taiwan (China); Chen, C.-C. [Biotechnology Center, Grape King Inc., Chungli, Taiwan (China); Lee, K.-M. [Institute of Tropical Medicine, National Yang-Ming University, Taipei, Taiwan (China); Institute of Medical Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China)], E-mail: kmlee@ctust.edu.tw

2008-03-01

20

Impaired islet function in commonly used transgenic mouse lines due to human growth hormone minigene expression.  

PubMed

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on ? cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic ? cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the ? cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used. PMID:25470546

Brouwers, Bas; de Faudeur, Geoffroy; Osipovich, Anna B; Goyvaerts, Lotte; Lemaire, Katleen; Boesmans, Leen; Cauwelier, Elisa J G; Granvik, Mikaela; Pruniau, Vincent P E G; Van Lommel, Leentje; Van Schoors, Jolien; Stancill, Jennifer S; Smolders, Ilse; Goffin, Vincent; Binart, Nadine; in't Veld, Peter; Declercq, Jeroen; Magnuson, Mark A; Creemers, John W M; Schuit, Frans; Schraenen, Anica

2014-12-01

21

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

PubMed

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

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

22

Generation of Rab-based transgenic lines for in vivo studies of endosome biology in zebrafish  

PubMed Central

The Rab family of small GTPases function as molecular switches regulating membrane and protein trafficking. Individual Rab isoforms define and are required for specific endosomal compartments. To facilitate in vivo investigation of specific Rab proteins, and endosome biology in general, we have generated transgenic zebrafish lines to mark and manipulate Rab proteins. We also developed software to track and quantify endosome dynamics within time-lapse movies. The established transgenic lines ubiquitously express EGFP fusions of Rab5c (early endosomes), Rab11a (recycling endosomes), and Rab7 (late endosomes) to study localization and dynamics during development. Additionally, we generated UAS-based transgenic lines expressing constitutive active (CA) and dominant negative (DN) versions for each of these Rab proteins. Predicted localization and functional consequences for each line were verified through a variety of assays, including lipophilic dye uptake and Crumbs2a localization. In summary, we have established a toolset for in vivo analyses of endosome dynamics and functions. PMID:21976318

Clark, Brian S.; Winter, Mark; Cohen, Andrew R.; Link, Brian A.

2011-01-01

23

Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.  

PubMed

Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models. PMID:22455749

Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C

2012-09-20

24

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

PubMed Central

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

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

2012-01-01

25

lambda 5, but not mu, is required for B cell maturation in a unique gamma 2b transgenic mouse line  

PubMed Central

gamma 2b transgenic mice have a severe B cell defect, apparently caused by strong feedback inhibition of endogenous H-gene rearrangement coupled with an inability of gamma 2b to provide the survival/maturation functions of mu. A unique gamma 2b transgenic line, named the C line, was found to permit B cell development. When the C line is crossed with a mu-membrane knockout line, gamma 2b+ B cells develop in the homozygous knockout. In contrast, a transgenic line representative of all the other gamma 2b lines is completely B cell deficient when mu-mem is deleted. Strikingly, the C phenotype is dominant in C x other gamma 2b transgenic line crosses. There is no evidence for higher gamma 2b transgene expression or other position effects on the transgene in the C mouse. The sequences of the three gamma 2b transgene copies in the C line are identical to that of the original transgene. These results have led to the conclusion that in the C line the transgene integration constitutively induces a gene whose expression can replace mu. To more clearly delineate the stage at which the altered phenotype of the C line is expressed, C mice were crossed onto a lambda 5 knockout background. In the absence of lambda 5, the C line produces no B cells. Since it was also found that gamma 2b can associate with the surrogate light chain (sL; lambda 5/Vpre-B), the crosses between C line gamma 2b mice and lambda 5 knockout mice suggest that gamma 2b/sL is required for B cell maturation in this mouse line. Thus, gamma 2b alone is unable to replace mu for pre-B cell survival/maturation; however, in combination with an unknown factor and the sL, gamma 2b can provide these nurturing functions. PMID:7869028

1995-01-01

26

Use of a ROSA26:GFP transgenic line for long-term Xenopus fate-mapping studies  

PubMed Central

Widespread and persistent marker expression is a prerequisite for many transgenic applications, including chimeric transplantation studies. Although existing transgenic tools for the clawed frog, Xenopus laevis, offer a number of promoters that drive widespread expression during embryonic stages, obtaining transgene expression through metamorphosis and into differentiated adult tissues has been difficult to achieve with this species. Here we report the application of the murine ROSA26 promoter in Xenopus. GFP is expressed in every transgenic tissue and cell type examined at post-metamorphic stages. Furthermore, transgenic ROSA26:GFP frogs develop normally, with no apparent differences in growth or morphology relative to wild-type frogs. ROSA26 transgenes may be used as a reliable marker for embryonic fate-mapping of adult structures in Xenopus laevis. Utility of this transgenic line is illustrated by its use in a chimeric grafting study that demonstrates the derivation of the adult bony jaw from embryonic cranial neural crest. PMID:16928208

Gross, Joshua B; Hanken, James; Oglesby, Ericka; Marsh-Armstrong, Nicholas

2006-01-01

27

A transgenic insect cell line engineered to produce CMP–sialic acid and sialylated glycoproteins  

PubMed Central

We have previously engineered transgenic insect cell lines to express mammalian glycosyltransferases and showed that these cells can sialylate N-glycoproteins, despite the fact that they have little intracellular sialic acid and no detectable CMP–sialic acid. In the accompanying study, we presented evidence that these cell lines can salvage sialic acids for de novo glycoprotein sialylation from extracellular sialogly-coproteins, such as fetuin, found in fetal bovine serum. This finding led us to create a new transgenic insect cell line designed to synthesize its own sialic acid and CMP–sialic acid. SfSWT-1 cells, which encode five mammalian glycosyltransferases, were transformed with two additional mammalian genes that encode sialic acid synthase and CMP–sialic acid synthetase. The resulting cell line expressed all seven mammalian genes, produced CMP–sialic acid, and sialylated a recombinant glycoprotein when cultured in a serum-free growth medium supplemented with N-acetylmannosamine. Thus the addition of mammalian genes encoding two enzymes involved in CMP–sialic acid biosynthesis yielded a new transgenic insect cell line, SfSWT-3, that can sialylate recombinant glycoproteins in the absence of fetal bovine serum. This new cell line will be widely useful as an improved host for baculovirus-mediated recombinant glycoprotein production. PMID:12626399

Aumiller, Jared J.; Hollister, Jason R.; Jarvis, Donald L.

2013-01-01

28

Cadmium and zinc induction of ZnT-1 mRNA in an established carp cell line.  

PubMed

The cDNA of a zinc transporter-1 (ZnT-1) gene was cloned from an established cell line derived from common carp (Cyprinus carpio) using rapid amplification of cDNA ends (RACE). Using real-time quantitative PCR, we showed that both zinc (Zn) and cadmium (Cd) transiently upregulate ZnT-1 mRNA to comparable levels. The loosely bound cellular Zn pool, as estimated using the Zn-specific probe FluoZin-3, was increased threefold after exposure to 250 microM ZnCl(2). Correspondingly, the ZnT-1 mRNA level at 24 h was induced about fivefold, reflecting the need for more zinc export capacity. Total cellular zinc levels were not different from the controls after 72 h of exposure to 10, 50, or 250 microM ZnCl(2). A loss of total cellular Zn but little labile zinc changes were observed with up to 25 microM Cd. At 72 h, the total Zn was partially restored to the control levels, only 1 microM Cd allowed for a full recovery. Downregulation of ZnT-1 mRNA and partial loss of loosely bound Zn were observed with 50 microM Cd. Our results clearly show that although Zn and Cd can both regulate Zn export in EPC cells, the effects on the cellular Zn pools are quite different. PMID:16627006

Muylle, Frederik; Robbens, Johan; De Coen, Wim; Timmermans, Jean-Pierre; Blust, Ronny

2006-06-01

29

A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis.  

PubMed

The sea anemone, Nematostella vectensis, has become an attractive new model organism for comparative genomics and evolutionary developmental biology. Over the last few years, many genes have been isolated and their expression patterns studied to gain insight into their function. More recently, functional tools have been developed to manipulate gene function; however, most of these approaches rely on microinjection and are limited to early stages of development. Transgenic lines would significantly enhance the tractability of the system. In particular, the study of gene- or tissue-specific promoters would be most useful. Here we report the stable establishment of a transgenic line using the I-SceI meganuclease system to facilitate integration into the genome. We isolated a 1.6-kb fragment of the regulatory upstream region of the Myosin Heavy Chain1 (MyHC1) gene and found that the transgene is specifically expressed in the retractor and tentacle muscles of Nematostella polyps, faithfully reproducing the expression of the endogenous MyHC1 gene. This demonstrates that the 1.6-kb fragment contains all of the regulatory elements necessary to drive correct expression and suggests that retractor and tentacle muscles in Nematostella are distinct from other myoepithelial cells. The transgene is transmitted through the germline at high frequency, and G(1) transgenic polyps have only one integration site. The relatively high frequency of transgenesis, in combination with gene- or tissue-specific promoters, will foster experimental possibilities for studying in vivo gene functions in gene regulatory networks and developmental processes in the nonbilaterian sea anemone, Nematostella vectensis. PMID:20018670

Renfer, Eduard; Amon-Hassenzahl, Annette; Steinmetz, Patrick R H; Technau, Ulrich

2010-01-01

30

Promises and pitfalls of a Pannexin1 transgenic mouse line  

PubMed Central

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1tm1a(KOMP)Wtsi. Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1f/f mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions. PMID:23675350

Hanstein, Regina; Negoro, Hiromitsu; Patel, Naman K.; Charollais, Anne; Meda, Paolo; Spray, David C.; Suadicani, Sylvia O.; Scemes, Eliana

2013-01-01

31

Burial and Seed Survival in Brassica napus Subsp. oleifera and Sinapis arvensis Including a Comparison of Transgenic and Non-Transgenic Lines of the Crop  

Microsoft Academic Search

The creation of transgenic plants through genetic engineering has focused interest on how the fitness of a plant species may be altered by small changes in its genome. This study concentrates on a key component of fitness: persistence of seeds overwinter. Seeds of three lines of oilseed rape (Brassica napus subsp. oleifera DC Metzger) and of charlock (Sinapis arvensis L.)

R. S. Hails; M. Rees; D. D. Kohn; M. J. Crawley

1997-01-01

32

Burial and seed survival in Brassica napus subsp. oleifera and Sinapis arvensis including a comparison of transgenic and non-transgenic lines of the crop  

Microsoft Academic Search

SUMMARY The creation of transgenic plants through genetic engineering has focused interest on how the fitness of a plant species may be altered by small changes in its genome. This study concentrates on a key component of fitness: persistence of seeds overwinter. Seeds of three lines of oilseed rape (Brassica napus subsp. oleifera DC Metzger) and of charlock (Sinapis aräensis

R. S. Hails; M. Rees; D. D. Kohn; M. J. Crawley

1997-01-01

33

Mannose binding protein is involved in first-line host defence: evidence from transgenic mice.  

PubMed Central

Mannose binding protein (MBP) is a calcium-dependent C-type lectin secreted by the liver which seems to be an important component of innate or natural immunity. We have investigated the effects of Candida albicans and thioglycolate injection into transgenic mice bearing the human MBP gene. The transgenes contained a 15 kb fragment of the MBP gene which included the complete coding sequence. Northern blot hybridization showed human MBP mRNA transcripts in the liver of two transgenic lines with low and high copy number respectively. Western blot analysis showed the presence in serum of human MBP which associated into the higher multimeric forms which are capable of activating complement. Enzyme-linked immunosorbent assays (ELISA) showed that serum human MBP concentrations in the transgenes (1.90 +/- 0.16 mg/l, mean +/- SEM) were about twice as high as the levels in man. The serum concentration of MBP A, which is the mouse homologue of MBP, (13.9 +/- 0.45 mg/l) was about seven times that of human MBP. Intravenous injection of Candida albicans caused the serum human MBP level to fall by more than 50% in the first hour and then slowly recover, but it did not return the initial value by 72 hr. Candida injection caused a 25% fall in serum mouse MBP A in the first hour which then rose to supranormal levels by 72 hr. Following Candida injection mouse MBP A mRNA concentrations increased over 72 hr in contrast to human MBP mRNA which remained constant in both transgenic lines. These data indicate that the human MBP gene fragment in the transgene did not include the regulatory elements of the gene. Total haemolytic complement activity and C3 concentrations also fell immediately after Candida and thioglycolate injection while the concentrations of mannose specific immunoglobulin G (IgG) and immunoglobulin M (IgM) did not fall. The data indicate that mannose binding protein plays an important role in the initial stages of defence against infection which, in this model, is quantitatively greater than that of mannose-specific IgG and IgM antibodies. Mannose binding protein is probably most important in defense of previously unexposed and non-immune hosts. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7635515

Tabona, P; Mellor, A; Summerfield, J A

1995-01-01

34

A transgenic medaka line with visible markers for genotypic and phenotypic sex.  

PubMed

Accurate genotyping of sex is required for correct interpretation in any in vivo assays with endocrine disrupting chemicals (EDCs). Visible markers for genotypic sex, if reliable, simplify assays because time-consuming PCR-based genotyping can be skipped. Here, we describe a line of Japanese medaka with a brain-expressed green fluorescent protein (GFP) transgene inserted near the sex-determining locus. When used with a white pigment cell marker, genotypic sex can be determined reliably as early as 3 days after fertilization (well before gonadal sex differentiation). No recombinants were found in more than 2000 progenies. We also introduced a strong ovarian GFP marker into the line with these genetic sex markers, so that phenotypic sex can also be determined reliably at 8 days after hatching. Well-known sex reversal protocols using exogenous steroid treatments of embryos were monitored by this transgenic line, demonstrating the line to be a useful tool for in vivo studies utilizing gonadal sex differentiation of the medaka, especially for screenings of potential estrogenic and androgenic EDCs. PMID:23638909

Kanamori, Akira; Toyama, Keiko

2013-06-18

35

Production and characterization of a pig line transgenic for human membrane cofactor protein.  

PubMed

A pig line transgenic for human membrane cofactor protein (hMCP) has been established. Offspring from the founder were produced by crossing the founder with pigs heterozygous for the human decay accelerating factor (hDAF) transgene. As a result, pigs transgenic for both hMCP and hDAF have been produced. Ribonuclease protection assay (RPA) indicated that hMCP was expressed in all the tissues analysed. In addition, immunohistochemical results indicated a high level of expression of hMCP on neural tissues and islets where hDAF was absent or weakly expressed. C3 fragment deposition and cytotoxicity assays indicated that hMCP expression alone on pig endothelial cells and peripheral blood lymphocytes (PBLs) provided protection against human complement mediated damage. However, we did not find that porcine endothelial cells expressing both hDAF and hMCP were better protected than those expressing hDAF alone. The expression of hMCP on tissues where hDAF is not expressed could provide these tissues with protection against human complement mediated lysis. PMID:11983016

Zhou, Chun-Yan; McInnes, Elizabeth; Parsons, Nicola; Langford, Gillian; Lancaster, Richard; Richards, Andrew; Pino-Chavez, Gilda; Dos Santos Cruz, Gabriela; Copeman, Laura; Carrington, Christine; Thompson, Simon

2002-05-01

36

[Investigation of neuroprotective activity of apolipoprotein E peptide mimetic Cog1410 in transgenic lines of Drosophila melanogaster].  

PubMed

The neuroprotective activity of apolipoprotein E (apoE) peptide mimetic Cog1410, containing amino acid sequence of the receptor-binding domain apoE, has been investigated in transgenic lines of Drosophila melanogaster expressing human APP and beta-secretase. Expression of two transgenes caused neuropathological processes attributed to Alzheimer's disease: neurodegeneration, cognitive abnormality and amyloid deposits formation in brain. It was shown that Cog 1410 reduces neurodegeneration in brain of transgenic flies and improves cognitive functions (odor recognition). These data suggest that Cog1410 is a potential neuroprotector that can be used in AD treatment. PMID:25249536

Latypova, E M; Timoshenko, S I; Kislik, G A; Vitek, M; Shvartsman, A L; Sarantseva, S V

2014-01-01

37

Modified expression of cysteine protease affects seed germination, vegetative growth and nodule development in transgenic lines of Medicago truncatula  

Microsoft Academic Search

Transgenic lines of Medicago truncatula (R108-1) were constructed to analyse Cyp15a (cysteine protease) gene functioning and localisation. The promoter region of PsCyp15a was fused to the coding sequence of uidA. GUS-analysis of transgenic plants containing this construct revealed strong expression from the Cyp15 promoter in cotyledonary leaves, senescent leaves, and root nodules. A seven-fold increase in GUS activity was observed

S. Sheokand; P. Dahiya; J. L. Vincent; N. J. Brewin

2005-01-01

38

Burial and seed survival in Brassica napus subsp. oleifera and Sinapis arvensis including a comparison of transgenic and non-transgenic lines of the crop.  

PubMed

The creation of transgenic plants through genetic engineering has focused interest on how the fitness of a plant species may be altered by small changes in its genome. This study concentrates on a key component of fitness: persistence of seeds overwinter. Seeds of three lines of oilseed rape (Brassica napus subsp. oleifera DC Metzger) and of charlock (Sinapis arvensis L.) were buried in nylon mesh bags at two depths in four habitats in each of three geographically separated sites: Cornwall, Berkshire and Sutherland. Seeds were recovered after 12 and 24 months. Charlock exhibited much greater seed survival (average 60% surviving the first year and 32.5% surviving the second year) than oilseed rape (1.5% surviving the first year and 0.2% surviving the second) at all sites. Charlock showed higher survival at 15 cm burial than 2 cm burial at certain sites, but oilseed rape showed no depth effect. Different genetic lines of oilseed rape displayed different rates of seed survival; non-transgenic rape showed greater survival (2%) than the two transgenic lines, one developed for tolerance to the antibiotic kanamycin (0.3%) and one for tolerance to both kanamycin and the herbicide glufosinate (0.25%). The absolute and relative performances of the different genetic lines of oilseed rape were context specific, illustrating the need to test hypotheses in a wide range of ecological settings. PMID:9061957

Hails, R S; Rees, M; Kohn, D D; Crawley, M J

1997-01-22

39

Mobility properties of the Hermes transposable element in transgenic lines of Aedes aegypti  

PubMed Central

The Hermes transposable element has been used to genetically transform a wide range of insect species, including the mosquito, Aedes aegypti, a vector of several important human pathogens. Hermes integrations into the mosquito germline are characterized by the non-canonical integration of the transposon and flanking plasmid and, once integrated, Hermes is stable in the presence of its transposase. In an effort to improve the post-integration mobility of Hermes in the germline of Ae. aegypti, a transgenic helper Mos1 construct expressing Hermes transposase under the control of a testis-specific promoter was crossed to a separate transgenic strain containing a target Hermes transposon. In less than 1% of the approximately 1,500 progeny from jumpstarter lines analyzed, evidence of putative Hermes germline remobilizations were detected. These recovered transposition events occur through an aberrant mechanism and provide insight into the non-canonical cut-and-paste transposition of Hermes in the germ line of Ae. aegypti. PMID:20596755

Smith, Ryan C.

2010-01-01

40

Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak Anne L'HOSTIS-GUIDET1  

E-print Network

a method based on the Restriction Enzyme Mediated Integration (REMI) developed in Dictyostelium discoideum simplified by omitting egg extracts and restriction enzyme to avoid the weakening of decondensed sperm nuclei-cell stage embryos of the enzyme and a plasmid carrying the transgene flanked by two restriction sites for I

Paris-Sud XI, Université de

41

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

PubMed Central

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

2012-01-01

42

De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment  

PubMed Central

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs. PMID:23620285

Olovnikov, Ivan; Ryazansky, Sergei; Shpiz, Sergey; Lavrov, Sergey; Abramov, Yuri; Vaury, Chantal; Jensen, Silke; Kalmykova, Alla

2013-01-01

43

De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment.  

PubMed

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs. PMID:23620285

Olovnikov, Ivan; Ryazansky, Sergei; Shpiz, Sergey; Lavrov, Sergey; Abramov, Yuri; Vaury, Chantal; Jensen, Silke; Kalmykova, Alla

2013-06-01

44

Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae).  

PubMed

Ten transgenic Bacillus thuringiensis Bt rice, Oryza sativa L., lines with different Bt genes (two Cry1Ac lines, three Cry2A lines, and five Cry9C lines) derived from the same variety Minghui 63 were evaluated in both the laboratory and the field. Bioassays were conducted by using the first instars of two main rice lepidopteran insect species: yellow stem borer, Scirpophaga incertulas (Walker) and Asiatic rice borer, Chilo suppressalis (Walker). All transgenic lines exhibited high toxicity to these two rice borers. Field evaluation results also showed that all transgenic lines were highly insect resistant with both natural infestation and manual infestation of the neonate larvae of S. incertulas compared with the nontransformed Minghui63. Bt protein concentrations in leaves of 10 transgenic rice lines were estimated by the sandwich enzyme-linked immunosorbent assay. The cry9C gene had the highest expression level, next was cry2A gene, and the cry1Ac gene expressed at the lowest level. The feeding behavior of 7-d-old Asiatic rice borer to three classes of Bt transgenic rice lines also was detected by using rice culm cuttings. The results showed that 7-d-old larvae of Asiatic rice borer have the capacity to distinguish Bt and non-Bt culm cuttings and preferentially fed on non-Bt cuttings. When only Bt culm cuttings with three classes of different Bt proteins (CrylAc, Cry2A, and Cry9C) were fed, significant distribution difference of 7-d-old Asiatic rice borer in culm cuttings of different Bt proteins also was found. In the current study, we evaluate different Bt genes in the same rice variety in both the laboratory and the field, and also tested feeding behavior of rice insect to these Bt rice. These data are valuable for the further development of two-toxin Bt rice and establishment of appropriate insect resistance management in the future. PMID:18330134

Chen, Hao; Zhang, Guoan; Zhang, Qifa; Lin, Yongjun

2008-02-01

45

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress  

PubMed Central

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the overexpression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlates with the induction of known stress-responsive genes, and suggests that putrescine may be directly or indirectly involved in ABA metabolism and gene expression. PMID:21330789

Alet, Analía I; Sanchez, Diego H; Cuevas, Juan C; del Valle, Secundino; Altabella, Teresa; Tiburcio, Antonio F; Marco, Francisco; Ferrando, Alejandro; Espasandín, Fabiana D; González, María E; Carrasco, Pedro

2011-01-01

46

zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish  

PubMed Central

The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology. PMID:24198277

Chen, Chen-Hui; Durand, Ellen; Wang, Jinhu; Zon, Leonard I.; Poss, Kenneth D.

2013-01-01

47

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

PubMed

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

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

2015-01-01

48

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

PubMed

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

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

2014-12-01

49

Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines  

PubMed Central

Sequencing across the junction between an integrated transfer DNA (T-DNA) and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression. PMID:24833334

Bartlett, Joanne G.; Smedley, Mark A.; Harwood, Wendy A.

2014-01-01

50

An ?-Smooth Muscle Actin (acta2/?sma) Zebrafish Transgenic Line Marking Vascular Mural Cells and Visceral Smooth Muscle Cells  

PubMed Central

Mural cells of the vascular system include vascular smooth muscle cells (SMCs) and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is ? smooth muscle actin (acta2; ?sma), which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels. PMID:24594685

Carter, Alyson D.; Rollins, Evvi-Lynn; Georgijevic, Sonja; Santoro, Massimo M.; Childs, Sarah J.

2014-01-01

51

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.  

PubMed Central

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest. Images Fig. 1 Fig. 2 Fig. 4 PMID:7724601

Efrat, S; Fusco-DeMane, D; Lemberg, H; al Emran, O; Wang, X

1995-01-01

52

Increased yield of heterologous viral glycoprotein in the seeds of homozygous transgenic tobacco plants cultivated underground.  

PubMed

The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 3020-3029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T(1)-2 and A24T(1)-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T(1)-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showedthat gB specific activity in A23T(1)-2 seeds was over 30-fold greater than the best T0 plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals. PMID:12834071

Tackaberry, Eilleen S; Prior, Fiona; Bell, Margaret; Tocchi, Monika; Porter, Suzanne; Mehic, Jelica; Ganz, Peter R; Sardana, Ravinder; Altosaar, Illimar; Dudani, Anil

2003-06-01

53

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

PubMed Central

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

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

2014-01-01

54

Transimmortalized proximal tubule and collecting duct cell lines derived from the kidneys of transgenic mice.  

PubMed

This review summarizes the strategy of cellular immortalization based on the principle of targeted oncogenesis in transgenic mice, used to establish models of transimmortalized renal proximal tubule cells, referred to as PKSV-PCT and PKSV-PR-cells, and collecting duct principal cells, referred to as mpkCCD(cl4) cells. These cell lines have maintained for long-term passages the main biochemical and functional properties of the parental cells from which they were derived. Proximal tubule PKSV-PCT and PKSV-PR cells have been proved to be suitable cell systems for toxicological and pharmacological studies. They also permitted the establishment of a model of multidrug-resistant (MDR) renal epithelial tubule cells, PKSV-PR(col50), which have served for the study of both MDR-dependent extrusion of chemotherapeutic drugs and inappropriate accumulation of weak base anthracyclines in intracellular acidic organelles. The novel collecting duct cell line mpkCCD(cl4), which has maintained the characteristics of tight epithelial cells, in particular Na(+) absorption stimulated by aldosterone, has been extensively used for pharmacological studies related to the regulation of ion transport. These cells have permitted the identification of several aldosterone-induced proteins playing a key role in the regulation of Na(+) absorption mediated by the epithelial Na(+) channel ENaC. Recent studies have also provided evidence that these cell lines represent valuable cell systems for the study of host-pathogen interactions and the analysis of the role of renal tubule epithelial cells in the induction of inflammatory response caused by uropathogens that may lead to severe renal damage. PMID:17219250

Chassin, C; Bens, M; Vandewalle, A

2007-07-01

55

Identification of new T1BL.1RS translocation lines derived from wheat ( Triticum aestivum L. cultivar “Xiaoyan No. 6”) and rye hybridization  

Microsoft Academic Search

Two new T1BL.1RS translocation lines, 48112 and 89121, derived from cross between common wheat (Triticum aestivum L.) cultivar “Xiaoyan No. 6” and rye (Secale cereale L.) cultivar “German White”, were developed and identified by using of molecular markers and cytogenetical methods, GISH\\u000a and FISH. PCR results of primers NOR-R1 specific for rye and Glu-B3 for 1BS detected the presence of

Jun Ji; Zhiguo Wang; Jiazhu Sun; Junming Li; Xiangqi Zhang; Daowen Wang; Aimin Zhang

2008-01-01

56

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

PubMed Central

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

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

2014-01-01

57

Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines  

PubMed Central

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a Knock-in of the same gene with less effort and shorter time of establishment. The ?-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. PMID:21732318

Hollenback, Steven M.; Lyman, Suzanne; Cheng, JrGang

2011-01-01

58

A transgenic tropical maize line generated by the direct transformation of the embryo-scutellum by A. tumefaciens  

Microsoft Academic Search

An efficient Agrobacterium-mediated transformation system, from which transgenic tropical maize plants were directly generated without previous crosses\\u000a with laboratory or temperate lines, was established. Experimental evaluations were focused on two main issues: (i) establishment\\u000a of appropriate tissue culture conditions, which induced somatic embryogenesis from the scutellum-cells, and (ii) the delivery\\u000a of T-DNA toward these cells. High rates of embryogenic-calli, mainly

Angel Valdez-Ortiz; Sergio Medina-Godoy; M. Elena Valverde; Octavio Paredes-López

2007-01-01

59

The thyrotropin beta-subunit gene is repressed by thyroid hormone in a novel thyrotrope cell line, mouse T alphaT1 cells.  

PubMed

TSH is expressed in two populations of thyrotropes in the pituitary: one in the pars distalis and a second in the pars tuberalis. Pars distalis thyrotropes exhibit classical endocrine inhibition of TSH by thyroid hormone, whereas pars tuberalis thyrotropes do not. The majority of our understanding of TSH subunit gene regulation has come from studies conducted in dispersed pituitary, dispersed thyrotropic tumors, or the GH3 somatolactotrope cell line. However, the dispersed pituitary model is limited because of its inherent heterogeneity, thyrotropic tumors are difficult to grow and maintain, and the GH3 cells lack endogenous TSH expression. The recent derivation of a clonal thyrotrope cell line, T alphaT1, that expresses thyrotrope-specific markers, overcomes these limitations. However, because it was not possible to distinguish whether the tumor from which the T alphaT1 cells are derived originated in the pars distalis or the pars tuberalis, it was necessary to define their cellular origin and thereby establish their status as representative thyrotrope cells for future molecular studies. In this study, we demonstrate that the T alphaT1 cells express thyroid hormone receptors (beta1 and beta2) and their heterodimeric partner, retinoid X receptor-gamma. Treatment with T3 causes a dose- and time-dependent decrease in the expression of the TSH beta-subunit messenger RNA. In contrast to previous reports in rat pituitary cultures, T3 does not alter TSH beta-subunit messenger RNA stability in the T alphaT1 cells. Based on these data and the presence of thyrotrope-specific isoforms of the transcription factor Pit-1, we conclude that the T alphaT1 cells represent differentiated thyrotropes of the pars distalis and will be a useful model system for future analysis of the cis- and trans-acting factors necessary for thyrotrope-specific and thyroid hormone-regulated TSH gene expression. PMID:9794455

Yusta, B; Alarid, E T; Gordon, D F; Ridgway, E C; Mellon, P L

1998-11-01

60

Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse.  

PubMed

Studies on cell lines have greatly improved our understanding of many important biological questions. Generation of cell lines is facilitated by the introduction of immortalizing oncogenes into cell types of interest. One gene known to immortalize many different cell types in vitro encodes the simian virus 40 (SV40) large tumor (T) antigen (TAg). To circumvent the need for gene insertion in vitro to generate cell lines, we created transgenic mice harboring the SV40 TAg gene. Since previous studies have shown that TAg expression in transgenic mice is associated with tumorigenesis and aberrant development, we utilized a thermolabile TAg [from a SV40 strain, tsA58, temperature sensitive (ts) for transformation] to reduce the levels of functional TAg present in vivo. To direct expression to a broad range of tissues, we used the mouse major histocompatibility complex H-2Kb promoter, which is both widely active and can be further induced by interferons. tsA58 TAg mRNA was expressed in tissues of all animals harboring the hybrid construct. Development of all tissues was macroscopically normal except for thymus, which consistently showed hyperplasia. Fibroblast and cytokeratin+ thymic epithelial cultures from these mice were readily established without undergoing crisis and were conditionally immortal in their growth; the degree of conditionality was correlated with the levels of tsA58 TAg detected. One strain of H-2Kb-tsA58 mice has been bred through several generations to homozygosity and transmits a functional copy of the transgene. PMID:1711218

Jat, P S; Noble, M D; Ataliotis, P; Tanaka, Y; Yannoutsos, N; Larsen, L; Kioussis, D

1991-06-15

61

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

PubMed Central

The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA), one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE)-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research. PMID:23251476

Jensen, Abbie M.

2012-01-01

62

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

PubMed

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

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

2013-01-01

63

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

PubMed Central

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

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

1989-01-01

64

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

SciTech Connect

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.

Orford, Michael; Mean, Richard; Lapathitis, George; Genethliou, Nicholas; Panayiotou, Elena; Panayi, Helen [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus)] [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus); Malas, Stavros, E-mail: smalas@cing.ac.cy [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus) [The Cyprus Institute of Neurology and Genetics, Airport Avenue, No. 6, Agios Dometios 2370, Nicosia (Cyprus); Department of Biological Sciences, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus)

2009-06-26

65

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...

66

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...

67

Adult onset cardiac dilatation in a transgenic mouse line with Gal?1,3GalNAc ?2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy  

PubMed Central

Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Gal?1,3GalNAc ?2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy. PMID:21986317

SUZUKI, Osamu; KANAI, Takao; NISHIKAWA, Toshio; YAMAMOTO, Yoshie; NOGUCHI, Akira; TAKIMOTO, Kazuhiro; KOURA, Minako; NOGUCHI, Yoko; UCHIO-YAMADA, Kozue; TSUJI, Shuichi; MATSUDA, Junichiro

2011-01-01

68

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

PubMed

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

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

2014-01-01

69

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

PubMed Central

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

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

2014-01-01

70

Production of Immortalized Distal Respiratory Epithelial Cell Lines from Surfactant Protien C\\/Simian Virus 40 Large Tumor Antigen Transgenic Mice  

Microsoft Academic Search

Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack of contact inhibition, and an

Kathryn A. Wikenheiser; Diane K. Vorbroker; Ward R. Rice; Jean C. Clark; Cindy J. Bachurski; Herbert K. Oie; Jeffrey A. Whitsett

1993-01-01

71

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

PubMed

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

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

2015-02-01

72

Establishment of transgenic lines for jumpstarter method using a composite transposon vector in the ladybird beetle, Harmonia axyridis.  

PubMed

In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, Harmonia axyridis, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G2 females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in Drosophila. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism. PMID:24959904

Kuwayama, Hisashi; Gotoh, Hiroki; Konishi, Yusuke; Nishikawa, Hideto; Yaginuma, Toshinobu; Niimi, Teruyuki

2014-01-01

73

Establishment of Transgenic Lines for Jumpstarter Method Using a Composite Transposon Vector in the Ladybird Beetle, Harmonia axyridis  

PubMed Central

In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, Harmonia axyridis, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G2 females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in Drosophila. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism. PMID:24959904

Kuwayama, Hisashi; Gotoh, Hiroki; Konishi, Yusuke; Nishikawa, Hideto; Yaginuma, Toshinobu; Niimi, Teruyuki

2014-01-01

74

Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene.  

PubMed

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases. PMID:17109643

Sacco, M G; Faggioli, F; Soldati, S; Gribaldo, L; Collotta, A; Pariselli, F; Malerba, I; Musio, A; Montagna, C; Catò, E Mira; Vezzoni, P

2006-12-01

75

Reduction in the formation of carcinogen-induced transformed foci by pencillin G sodium in the C3H/10T1/2 CL8 cell lines.  

PubMed

The C3H/10T1/2 CL8 cell line is being widely used to study mechanisms of malignant transformation in vitro. As currently employed, the standard assay system uses a combination of penicillin (100 I.U./ml) and streptomycin (50 micrograms/ml) to reduce the occurrence of bacterial contamination. The penicillin component of this mixture has been discovered to cause a reduction in the number of transformed foci which develop after exposure of cells to MCA, DMBA and X-rays. This reduction is dose dependent; 500 I.U./ml virtually eliminates transformation, while 100 I.U./ml causes an approximate 50% decrease in the number of foci. This effect does not appear to be due to overt toxicity and is largely reversible on removal of the antibiotic. Gentamicin (25 micrograms/ml) causes no reduction in the formation of transformed foci when compared to cultures maintained in antibiotic-free medium and offers the advantages of chemical stability, a wider spectrum of antibacterial activity in comparison with penicillin/streptomycin and, in addition, is active against many mycoplasma. It is suggested that future studies with this cell line should ideally be performed without antibiotics or should employ Gentamicin for antibacterial protection. PMID:116766

Bertram, J S

1979-09-01

76

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells  

Microsoft Academic Search

The production of subtle or conditional mu- tations in mice through the combined use of site-specific and homologous recombination has become an increasingly wide- spread experimental paradigm in mammalian genetics. Em- bryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify

STEPHEN O'GORMAN; NICOLE A. DAGENAIS; YELENA MARCHUK

1997-01-01

77

Induced expression of DREB transcriptional factor and study on its physiological effects of drought tolerance in transgenic wheat.  

PubMed

Expression vector pBAC128F, which carries DREB transcriptional factor gene driven by drought inducing promoter rd29B and bar gene driven by CaMV 35S promoter and maize Adh1 gene first intron, was transferred into the explants of immature inflorescence and immature embryos of hexaploid winter wheat cv. 8901, 5-98, 99-92 and 104 by particle bombardment. More than 70 resistant transgenic plants were obtained. Genomic PCR and RNA dot blotting analyses showed that DREB gene had been integrated into wheat genome of the transgenic plants (T0 and T1) and was well expressed in offspring seed of different transgenic lines. The content of proline in leaves and seeds of T2 transgenic lines was analyzed. Among 16 tested transgenic lines, 10 transgenic lines exhibited more than two fold of proline level in leaves as compared with CK plants. Under drought condition, after stopping water for 15 days the leaves of transgenic lines were still green, while CK were faded. After rewatering for 10 days, the leaves of transgenic lines maintained their green, while all CK plants were dead. Our research suggested that introducing a novel DREB transcriptional factor into wheat is an effective way to improve its drought-tolerance ability. PMID:16722342

Wang, Jun-Wei; Yang, Feng-Ping; Chen, Xu-Qing; Liang, Rong-Qi; Zhang, Li-Quan; Geng, Dong-Mei; Zhang, Xiao-Dong; Song, Ya-Zhen; Zhang, Gai-Sheng

2006-05-01

78

Expression of gamma-tocopherol methyltransferase transgene improves tocopherol composition in lettuce (Latuca sativa L.).  

PubMed

A cDNA encoding gamma-tocopherol methyltransferase (gamma-TMT) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce (T0) containing the gamma-TMT transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and gamma-TMT activities. The ratio of alpha-/gamma-tocopherol content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the T0 plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 T1 progenies of the T0 transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and gamma-TMT activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in T2 progenies of T1 plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis gamma-TMT transgene in lettuce results in a higher enzyme activity and the conversion of the gamma-tocopherol pool to alpha-tocopherol in transgenic lettuce. PMID:15750335

Cho, Eun Ae; Lee, Chong Ae; Kim, Young Soo; Baek, So Hyeon; de los Reyes, Benildo G; Yun, Song Joong

2005-02-28

79

Down-Regulation of Transmembrane Carbonic Anhydrases in Renal Cell Carcinoma Cell Lines by Wild-Type von Hippel-Lindau Transgenes  

Microsoft Academic Search

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact

Sergey V. Ivanov; Igor Kuzmin; Ming-Hui Wei; Svetlana Pack; Laura Geil; Bruce E. Johnson; Eric J. Stanbridge; Michael I. Lerman

1998-01-01

80

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

PubMed

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

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

2014-01-01

81

Development of a Transgenic Plasmodium berghei Line (Pbpfpkg) Expressing the P. falciparum cGMP-Dependent Protein Kinase, a Novel Antimalarial Drug Target  

PubMed Central

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

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

2014-01-01

82

Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions.  

PubMed

Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanum lycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was "HighCaro (HC)," a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to beta-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to beta-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place. PMID:17096211

Giorio, Giovanni; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

2007-02-01

83

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

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.

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

1986-12-01

84

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

PubMed

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

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

85

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) ...

86

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

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

Khareedu, Venkateswara Rao; Vudem, Dashavantha Reddy

2014-01-01

87

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

SciTech Connect

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, the authors characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire [beta]-globin locus. They 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[prime] 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.

Gaensler, K.M.L.; Kitamura, M.; Kan, Yuet Wai (Univ. of California, San Francisco, CA (United States))

1993-11-15

88

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...

89

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

SciTech Connect

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.

Pohjanvirta, Raimo [Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O. Box 66, FI-00014 University of Helsinki (Finland); National Institute for Health and Welfare, Laboratory of Toxicology, P.O. Box 95, FI-70701 Kuopio (Finland); Finnish Food Safety Authority EVIRA, Kuopio Research Unit, P.O. Box 92, FI-70701 Kuopio (Finland)], E-mail: raimo.pohjanvirta@helsinki.fi

2009-04-15

90

Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana  

PubMed Central

Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels led to increased growth, as well as to smaller abscisic acid levels and reduced damage to PSII (as indicated by Fv/Fm ratios) during the reproductive stages in rosette leaves of NahG transgenic lines and sid2 mutants, compared with wild-type plants. Furthermore, salicylic acid deficiency highly influenced seed yield and composition. Seed production increased by 4.4-fold and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to the wild type. Salicylic acid deficiency also improved seed composition in terms of antioxidant vitamin concentrations, seeds of salicylic acid-deficient plants showing higher levels of ?- and ?-tocopherol (vitamin E) and ?-carotene (pro-vitamin A) than seeds of wild-type plants. Seeds of salicylic acid-deficient plants also showed higher nitrogen concentrations than seeds of wild-type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of growth, senescence, and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the concentrations of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants. PMID:19188277

Abreu, Maria Elizabeth; Munné-Bosch, Sergi

2009-01-01

91

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24.  

PubMed

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T1 lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T1 plants. Resistance to stripe rust in transgenic T2 and T3 XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27-36 %) was recorded for transgenic T2 and T3 XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat. PMID:23529204

Huang, Xuan; Wang, Jian; Du, Zhen; Zhang, Chen; Li, Lan; Xu, Ziqin

2013-10-01

92

Transgenic cell lines as a useful tool to study the biochemistry of down-regulation of an endogenous rice gene using a heterologous diamine-oxidase cDNA  

Microsoft Academic Search

Transgenic rice (Oryza sativa L.) cell lines engineered with the pea diamine oxidase cDNA in antisense orientation under the control of two different promoters were recovered using particle bombardment. Plasmids p35Sdaoa and pEdaoa contained the pea diamine oxidase cDNA driven by the CaMV35S and the pea ENOD12 nodulin promoter, respectively. Molecular analyses confirmed the stable integration of the transgene and

Ludovic Bassie; Matthiew Noury; Jean Pierre Wisniewski; Lola Topsom; Paul Christou; Teresa Capell

2000-01-01

93

A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase ( SOD1 ) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis  

Microsoft Academic Search

About 15–20% of patients with familial amyotrophic lateral sclerosis (ALS) carry one of several missense mutations in the\\u000a gene for Cu,Zn superoxide dismutase (SOD1). We have previously reported on an animal model of this disease produced by the transgenic expression of a mutant form of\\u000a human SOD1 containing a Gly93?Ala amino acid substitution. Several lines of transgenic mice were produced,

M. C. Dal Canto; Mark E. Gurney

1997-01-01

94

Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker  

Microsoft Academic Search

To generate transgenic planarians we used a set of versatile vectors for animal transgenesis based on the promiscuous transposons, mariner, Hermes and piggyBac, and a universal enhanced GFP (EGFP) marker system with three Pax6 dimeric binding sites, the 3xP3-EGFP developed by Berghammer et al. [Berghammer, A. J., Klinger, M. & Wimmer, E. A. (1999) Nature 402, 370-371]. This marker is

C. González-Estévez; T. Momose; W. J. Gehring; E. Saló

2003-01-01

95

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

Microsoft Academic Search

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,\\u000a Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance\\/resistance\\u000a to crystal proteins by defoliating insects and to sustain the deployment of Bt crops.

S. Zhu; D. R. Walker; H. R. Boerma; W. A. Parrott

2008-01-01

96

DISEASE RESISTANCE IN TRANSGENIC COTTONS  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

97

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

PubMed Central

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

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

2014-01-01

98

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

PubMed Central

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

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

2014-01-01

99

Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS)  

Microsoft Academic Search

Two different lines of mice, G1 and G20, carrying a transgene for a mutant form of Cu,Zn SOD, found in a family with familial amyotrophic lateral sclerosis (FALS), develop clinical and pathological changes which are, in their late stages, strikingly similar to those in human disease. We have analyzed the distribution and characteristics of lesions in the central and peripheral

Mauro C. Dal Canto; Mark E. Gurney

1995-01-01

100

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.  

PubMed

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants. PMID:7659750

Bagnall, D J; King, R W; Whitelam, G C; Boylan, M T; Wagner, D; Quail, P H

1995-08-01

101

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression  

PubMed Central

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

Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

2014-01-01

102

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene.  

PubMed

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations. PMID:21947755

Panter, S; Chu, P G; Ludlow, E; Garrett, R; Kalla, R; Jahufer, M Z Z; de Lucas Arbiza, A; Rochfort, S; Mouradov, A; Smith, K F; Spangenberg, G

2012-06-01

103

T-1 Training Area  

ScienceCinema

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.

None

2015-01-09

104

T-1 Training Area  

SciTech Connect

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.

None

2014-11-07

105

Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.  

PubMed

Intestinal mucosal cells have proved difficult to culture in vitro. Many attempts have been made to develop long-term cultures of these cells either by direct culturing or by attempting to immortalize these cells by using a range of transforming viral genes, but with little success. The recent development of a transgenic mouse bearing a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) has enabled us to initiate conditionally immortalized cultures of epithelial cells from both small intestinal and colonic mucosa of adult mice. Crypts were isolated from either the small intestines or colons of young adult mice and cultured at the permissive temperature (33 degrees C) in medium containing conditioned medium from a human colon carcinoma cell line, LIM1863. Crypts from both tissues yielded cultures of epithelial cells that have now been in culture for more than 12 months with regular passaging. The epithelial nature of the cells has been confirmed by staining with anti-keratin antibodies. The intestinal origin of the cells was demonstrated by the ability of the cells to synthesize low levels of both brush border peptidases and a disaccharidase. The levels of expression of these enzymes were modulated by the addition of sodium butyrate or phorbol myristate acetate to the medium, which resulted in an increase in the synthesis of the peptidases and a decrease in the synthesis of the disaccharidase. The cells proliferate continuously at the permissive temperature (33 degrees C), but proliferation ceases at the nonpermissive temperature (39.5 degrees C). To our knowledge, this is the first description of the establishment of epithelial cell lines from both small intestine and colon of the same mouse strain. The success reported here indicates that this transgenic mouse will be a useful source of tissue for the study of the mechanisms that control the proliferation and eventual differentiation and senescence of the cells of the intestinal mucosa. These mice will also be a useful source of cells for attempts to culture cells from other tissues that have proved difficult to culture in vitro. PMID:7678459

Whitehead, R H; VanEeden, P E; Noble, M D; Ataliotis, P; Jat, P S

1993-01-15

106

Production of marker-free and RSV-resistant transgenic rice using a twin T-DNA system and RNAi.  

PubMed

A twin T-DNA system is a convenient strategy for creating selectable marker-free transgenic plants. The standard transformation plasmid, pCAMBIA 1300, was modified into a binary vector consisting of two separate T-DNAs, one of which contained the hygromycin phosphotransferase (hpt) marker gene. Using this binary vector, we constructed two vectors that expressed inverted-repeat (IR) structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene. Transgenic rice lines were obtained via Agrobacterium-mediated transformation. Seven independent clones harbouring both the hpt marker gene and the target genes (RSV CP or SP) were obtained in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T1 plants were 8.72 percent for pDTRSVCP and 12.33 percent for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were strongly resistant to RSV. A molecular analysis of the resistant transgenic plants confirmed the stable integration and expression of the target genes. The resistant transgenic plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, suggesting that RNAi induced the viral resistance. PMID:23938389

Jiang, Yayuan; Sun, Lin; Jiang, Mingsong; Li, Kaidong; Song, Yunzhi; Zhu, Changxiang

2013-09-01

107

Transgenic Animals.  

ERIC Educational Resources Information Center

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.…

Jaenisch, Rudolf

1988-01-01

108

Establishment and characterization of a colonic epithelial cell line MCE301 from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene.  

PubMed

We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33 degrees C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39 degrees C). The cells expressed large T-antigen at 33 degrees C but not at 39 degrees C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33 degrees C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium. PMID:11235898

Tabuchi, Y; Ohta, S; Arai, Y; Kawahara, M; Ishibashi, K; Sugiyama, N; Horiuchi, T; Furusawa, M; Obinata, M; Fuse, H; Takeguchi, N; Asano, S

2000-10-01

109

Genomic stability and long-term transgene expression in poplar.  

PubMed

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression. PMID:23740206

Fladung, Matthias; Hoenicka, Hans; Raj Ahuja, M

2013-12-01

110

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

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

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

2015-01-01

111

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

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

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

2015-01-01

112

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...

113

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

PubMed

Maize (Zea mays L.), as one of the most important crops in the world, is deficient in lysine and tryptophan. Environmental conditions greatly impact plant growth, development and productivity. In this study, we used particle bombardment mediated co-transformation to obtain marker-free transgenic maize inbred X178 lines harboring a lysine-rich protein gene SBgLR from potato and an ethylene responsive factor (ERF) transcription factor gene, TSRF1, from tomato. Both of the target genes were successfully expressed and showed various expression levels in different transgenic lines. Analysis showed that the protein and lysine content in T1 transgenic maize seeds increased significantly. Compared to non-transformed maize, the protein and lysine content increased by 7.7% to 24.38% and 8.70% to 30.43%, respectively. Moreover, transgenic maize exhibited more tolerance to salt stress. When treated with 200 mM NaCl for 48 h, both non-transformed and transgenic plant leaves displayed wilting and losing green symptoms and dramatic increase of the free proline contents. However, the degree of control seedlings was much more serious than that of transgenic lines and much more increases of the free proline contents in the transgenic lines than that in the control seedlings were observed. Meanwhile, lower extent decreases of the chlorophyll contents were detected in the transgenic seedlings. Quantitative RT-PCR was performed to analyze the expression of ten stress-related genes, including stress responsive transcription factor genes, ZmMYB59 and ZmMYC1, proline synthesis related genes, ZmP5CS1 and ZmP5CS2, photosynthesis-related genes, ZmELIP, ZmPSI-N, ZmOEE, Zmrbcs and ZmPLAS, and one ABA biosynthesis related gene, ZmSDR. The results showed that with the exception of ZmP5CS1 and ZmP5CS2 in line 9-10 and 19-11, ZmMYC1 in line 19-11 and ZmSDR in line 19-11, the expression of other stress-related genes were inhibited in transgenic lines under normal conditions. After salt treatment, the expressions of the ten stress-related genes were significantly induced in both wild-type (WT) and transgenic lines. However, compared to WT, the increases of ZmP5CS1 in all these three transgenic lines and ZmP5CS2 in line 9-10 were less than WT plants. This study provides an effective approach of maize genetic engineering for improved nutritive quality and salt tolerance. PMID:23629675

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

2013-01-01

114

Strategies for Designing Transgenic DNA Constructs  

PubMed Central

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

Liu, Chengyu

2013-01-01

115

Herbicide sensitivity of transgenic multiple herbicide-tolerant oilseed rape.  

PubMed

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

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

2002-04-01

116

Mutation spectrum of 1,2-dibromo-3-chloropropane, an endocrine disruptor, in the lacI transgenic Big Blue Rat2 fibroblast cell line.  

PubMed

1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant against nematodes, has been extensively studied for genotoxicity, carcinogenicity and damage to male reproduction-related organs, as a possible endocrine disruptor. However, the precise mechanisms involved in DBCP-induced mutagenesis and carcinogenesis are as yet unknown. Thus, in this study the mutagenicity and mutation spectrum of DBCP was determined using the lacI transgenic Big Blue Rat2 fibroblast cell line. In determining the optimal concentration of DBCP in Big Blue Rat2 fibroblast cells, the 50% inhibition concentration was calculated to be 0.75 mM. When cells were exposed to DBCP concentrations of 0.21, 0.39 and 0.75 mM, the respective relative survival rates were approximately 80, 70 and 50%. The mean mutant frequencies (MFs) (x 10(-5), +/- SEM) of the medium and 1% DMSO solvent controls were determined as 6.43 +/- 0.616 and 5.28 +/- 1.086, respectively. The MFs (x 10(-5), +/- SEM) of cells exposed to 0.21, 0.39 and 0.75 mM DBCP were 8.09 +/- 1.02, 10.86 +/- 2.17 and 12.26 +/- 0.79, respectively, with a dose-dependent effect (ANOVA, P = 0.007). Moreover, MF values for the 0.75 and 0.39 mM DBCP-treated groups were statistically significant (ANOVA, P < 0.05). The majority of recovered mutations (31/40, 77.5%) were single base pair substitutions in the DBCP-induced groups. Among 31 single base pair substitutions, 25 (62.5%) occurred at G:C base pairs, while six (15%) were at A:T base pairs. The predominant mutation was G:C-->A:T transitions (16/40, 40%), followed by G:C-->T:A transversions (9/40, 22.5%). We conclude that DBCP is a possible base substitution mutagen, especially at guanine bases. PMID:12110625

Ryu, Jae-Chun; Kim, Youn-Jung; Chai, Young-Gyu

2002-07-01

117

[Transgenic plants].  

PubMed

Advances in molecular genetics and recombinant DNA technology have revolutionized our understanding of the pathogenesis as well as the diagnosis, therapy and prevention of human diseases. Similar developments characterize plant biotechnology with the production of plant derived biomedical as well as health products. Apart from the fundamentals of molecular plant genetics, the production of transgenic plants as well as the clinical relevance, benefits, limitations and potential problems of plant biotechnology will be reviewed in some detail. It is a particular challenge to physicians in an increasingly informed environment to be informed about the new developments in molecular biology and recombinant DNA technology and to have a qualified opinion about their clinical relevance. PMID:12523178

Blum, H E

2002-12-01

118

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

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 genotypexmulch-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

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

119

Production of transgenic livestock: promise fulfilled.  

PubMed

The introduction of specific genes into the genome of farm animals and its stable incorporation into the germ line has been a major technological advance in agriculture. Transgenic technology provides a method to rapidly introduce "new" genes into cattle, swine, sheep, and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Methods to produce transgenic animals have been available for more than 20 yr, yet recently lines of transgenic livestock have been developed that have the potential to improve animal agriculture and benefit producers and/or consumers. There are a number of methods that can be used to produce transgenic animals. However, the primary method to date has been the microinjection of genes into the pronuclei of zygotes. This method is one of an array of rapidly developing transgenic methodologies. Another method that has enjoyed recent success is that of nuclear transfer or "cloning." The use of this technique to produce transgenic livestock will profoundly affect the use of transgenic technology in livestock production. Cell-based, nuclear transfer or cloning strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition, and increased disease resistance. One practical application of transgenics in swine production is to improve milk production and/or composition. To address the problem of low milk production, transgenic swine over-expressing the milk protein bovine alpha-lactalbumin were developed and characterized. The outcomes assessed were milk composition, milk yield, and piglet growth. Our results indicate that transgenic overexpression of milk proteins may provide a means to improve swine lactation performance. PMID:15000404

Wheeler, M B

2003-01-01

120

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.  

PubMed Central

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications. PMID:9218515

He, H; Giordano, F J; Hilal-Dandan, R; Choi, D J; Rockman, H A; McDonough, P M; Bluhm, W F; Meyer, M; Sayen, M R; Swanson, E; Dillmann, W H

1997-01-01

121

Augmented B Lymphocyte Response to Antigen in the Absence of Antigen-Induced B Lymphocyte Signaling in an IgG-Transgenic Mouse Line  

PubMed Central

IgG-containing B cell antigen receptor (IgG-BCR), the BCR mostly expressed on memory B cells, contains a distinct signaling function from IgM-BCR or IgD-BCR expressed on naïve B cells. Because naïve B cells transgenic for IgG exhibit augmented response to antigens similar to memory B cells, the distinct signaling function of IgG-BCR appears to play a role in augmented antibody responses of memory B cells. However, how IgG-BCR signaling augments B cell responses is not yet well understood. Here we demonstrate that B cells from IgG-transgenic mice are anergic with defect in generation of BCR signaling upon BCR ligation. However, these IgG-transgenic B cells generate markedly augmented antibody response to a T cell-dependent antigen, probably due to hyper-responsiveness to a T cell-derived signal through CD40. Both BCR signaling defect and augmented response to CD40 ligation are partially restored in xid IgG-transgenic mice in which BCR signaling is down-modulated due to a loss-of-function mutation in the tyrosine kinase Btk crucial for BCR signaling. Thus, IgG-BCR induces augmented B cell responses in the absence of antigen-induced BCR signaling probably through high ligand-independent BCR signaling that may “idle” B cells to make them ready to respond to T cell help. This finding strongly suggests a crucial role of ligand-independent signaling in receptor function. PMID:20098688

Komatsu, Emi; Tsubata, Takeshi

2010-01-01

122

Transgenic Mice Carrying a Human Mutant Superoxide Dismutase Transgene Develop Neuronal Cytoskeletal Pathology Resembling Human Amyotrophic Lateral Sclerosis Lesions  

Microsoft Academic Search

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 --> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not.

Pang-Hsien Tu; Pramod Raju; Kathryn A. Robinson; Mark E. Gurney; John Q. Trojanowski; Virginia M.-Y. Lee

1996-01-01

123

[Progress in transgenic fish techniques and application].  

PubMed

Transgenic technique provides a new way for fish breeding. Stable lines of growth hormone gene transfer carps, salmon and tilapia, as well as fluorescence protein gene transfer zebra fish and white cloud mountain minnow have been produced. The fast growth characteristic of GH gene transgenic fish will be of great importance to promote aquaculture production and economic efficiency. This paper summarized the progress in transgenic fish research and ecological assessments. Microinjection is still the most common used method, but often resulted in multi-site and multi-copies integration. Co-injection of transposon or meganuclease will greatly improve the efficiency of gene transfer and integration. "All fish" gene or "auto gene" should be considered to produce transgenic fish in order to eliminate misgiving on food safety and to benefit expression of the transferred gene. Environmental risk is the biggest obstacle for transgenic fish to be commercially applied. Data indicates that transgenic fish have inferior fitness compared with the traditional domestic fish. However, be-cause of the genotype-by-environment effects, it is difficult to extrapolate simple phenotypes to the complex ecological interactions that occur in nature based on the ecological consequences of the transgenic fish determined in the laboratory. It is critical to establish highly naturalized environments for acquiring reliable data that can be used to evaluate the environ-mental risk. Efficacious physical and biological containment strategies remain to be crucial approaches to ensure the safe application of transgenic fish technology. PMID:21586396

Ye, Xing; Tian, Yuan-Yuan; Gao, Feng-Ying

2011-05-01

124

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...

125

Improved cre reporter transgenic Xenopus.  

PubMed

We have produced and characterized improved transgenic reporter lines for detection of Cre recombinase activity during Xenopus development. Improvements include choice of fluorophores, which make these Cre reporter lines generally suitable for lineage tracing studies. We also include data for several new parameters affecting survival and transgenesis efficiency using the recently developed meganuclease method of frog transgenesis. These transgenic frogs express cyan fluorescent protein (CFP) under control of the ubiquitous promoter CMV, where CFP is replaced by DsRed2 (a red fluorescent protein) in the presence of Cre. Three independent, high expression, Cre-sensitive lines have been identified that maintain robust fluorophore expression across generations and lack DsRed2 expression in the absence of Cre. A novel use of these lines is to indelibly mark embryonic blastomeres by Cre mRNA injection for permanent fate mapping. Similarly, transgenically expressed Cre under control of tissue-specific promoters will allow detailed analysis of cell lineage relationships throughout embryogenesis, metamorphosis, and adulthood. PMID:19653309

Rankin, Scott A; Hasebe, Takashi; Zorn, Aaron M; Buchholz, Daniel R

2009-09-01

126

Chicken embryonic stem cells and transgenic strategies.  

PubMed

The production of transgenic birds is an important goal for both fundamental and applied biology. Different methods have been employed to generate transgenic chickens, including microinjection, use of retroviruses and transfection of primordial germ or embryonic germ cells. In this review we will briefly describe these techniques and our efforts to obtain genetically modified avian embryonic stem (ES) cells using liposomes. This latter technique should allow us to modify chicken ES cells with a high efficiency, permitting the rapid generation of transgenic bird lines. PMID:10592393

Pain, B; Chenevier, P; Samarut, J

1999-01-01

127

Somatic Generation of Hybrid Antibody H Chain Genes in Transgenic Mice via Interchromosomal Gene Conversion  

Microsoft Academic Search

Summary We have constructed lines of mice with transgenes containing an antibody heavy (H) chain variable region (VHDJ.) 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

Angela M. Giusti; Tim Manser

1994-01-01

128

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

E-print Network

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

Feng, Guoping

129

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

PubMed

The frequency of possible "horizontal" 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 "natural" 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(-2) under optimal control conditions to a calculated 2.0 x 10(-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. PMID:9636282

Schlüter, K; Fütterer, J; Potrykus, I

1995-10-01

130

Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart.  

PubMed

Iron is an essential micronutrient for human nutrition and polished rice contains very low amount of iron. Rice with high iron content in seed endosperm has been developed by insertion of soybean ferritin gene under the control of the endosperm specific glutelin promoter into the genome of indica rice line IR68144. The nutritional composition of the brown and milled rice grain has been compared with that of the non-transgenic rice of the same variety. In this study, the nutritional components, as well as the anti-nutrient levels, were measured. Our studies established that apart from the increased level of iron and zinc in transgenic seeds, the nutritional quality of both the brown and milled rice grains from the transgenic line was substantially equivalent to that of the non-transgenic rice plants. The result clearly shows that the measured amounts of the nutritional components are well within the range of values reported for other commercial lines. PMID:23411185

Gayen, Dipak; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

2013-06-01

131

Transgenic labeling of hair cells in the zebrafish acousticolateralis system.  

PubMed

The zebrafish provides a useful experimental system for investigations of aural development. To permit the controlled expression of transgenes in developing hair cells, we isolated the genomic control regions of the parvalbumin 3a (pvalb3a) and parvalbumin 3b (pvalb3b) genes. Deletion analysis and somatic-cell transgenesis restricted the cis-acting control regions for hair cells to as little as 484base pairs for pvalb3a and 650base pairs for pvalb3b. Using both meganuclease-mediated and standard methods, we produced transgenic animals that transmit transgenes through their germ lines. These fish express GFP in hair cells in the inner ear and lateral line. Two stable transgenic lines express GFP prior to hair-bundle formation, so the associated promoter constructs are suitable for manipulating gene expression during bundle development. We additionally identified a transgenic line that offers variable labeling of supporting cells. PMID:20085825

McDermott, Brian M; Asai, Yukako; Baucom, Jessica M; Jani, Shraddha D; Castellanos, Yaneth; Gomez, Gustavo; McClintock, James M; Starr, Catherine J; Hudspeth, A J

2010-01-01

132

Generation of transgenic Hydra by embryo microinjection.  

PubMed

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost. PMID:25285460

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

2014-01-01

133

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

PubMed Central

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

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

2014-01-01

134

Characterization of a Maize Wip1 Promoter in Transgenic Plants  

PubMed Central

The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip1 promoters led to strong GUS activities in both transgenic Arabidopsis and tobacco leaves, whereas low GUS activity was detected in transgenic rice leaves; (2) the Wip1 promoter was not wound-induced in transgenic tobacco leaves, but was induced by wounding in transgenic rice leaves; (3) the truncated Wip1 promoter had different activity in different organs of transgenic tobacco plants; (4) the transgenic plant leaves containing different truncated Wip1 promoters had low GUS transcripts, even though high GUS protein level and GUS activities were observed; (5) there was one transcription start site of Wip1 gene in maize and two transcription start sites of GUS in Wip1::GUS transgenic lines; (6) the adjacent 35S promoter which is present in the transformation vectors enhanced the activity of the truncated Wip1 promoters in transgenic tobacco leaves, but did not influence the disability of truncated Wip1231 promoter to respond to wounding signals. We speculate that an ACAAAA hexamer, several CAA trimers and several elements similar to ACAATTAC octamer in the 5?-untranslated region might contribute to the strong GUS activity in Wip1231 transgenic lines, meanwhile, compared to the 5?-untranslated region from Wip1231 transgenic lines, the additional upstream open reading frames (uORFs) in the 5?-untranslated region from Wip1737 transgenic lines might contribute to the lower level of GUS transcript and GUS activity. PMID:24322445

Zhang, Shengxue; Lian, Yun; Liu, Yan; Wang, Xiaoqing; Liu, Yunjun; Wang, Guoying

2013-01-01

135

Complementation of null CF mice with a human CFTR YAC transgene  

Microsoft Academic Search

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well,

Ania L. Manson; Ann E. O. Trezise; Lesley J. MacVinish; Kristin D. Kasschau; Nina Birchall; Vasso Episkopou; Georges Vassaux; Martin J. Evans; William H. Colledge; Alan W. Cuthbert; Clare Huxley

1997-01-01

136

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

PubMed

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

Jackson, Kathryn A; Berg, Jolene M; Murray, James D; Maga, Elizabeth A

2010-12-01

137

Segregation of transgenes in maize.  

PubMed

Progeny recovered from backcrossed transgenic maize tissue culture regenerants (R0) were analyzed to determine the segregation, expression, and stability of the introduced genes. Transgenic A188 x B73 R0 plants (regenerated from embryogenic suspension culture cells transformed by microprojectile bombardment; see [9]) were pollinated with nontransformed B73 pollen. Inheritance of a selectable marker gene, bar, and a nonselectable marker gene, uidA, was analyzed in progeny (R1) representing four independent transformation events. Activity of the bar gene product, phosphinothricin acetyltransferase (PAT), was assessed in plants comprising the four R1 populations. The number of R1 plants containing PAT activity per total number of R1 plants recovered for each population was 2/7, 19/34, 3/14 and 73/73. Molecular analysis confirmed the segregation of bar in three R1 populations and the lack of segregation in one R1 population. Cosegregation analysis indicated genetic linkage of bar and uidA in all four R1 populations. Analysis of numerous R2 plants derived from crossing transformed R1 plants with nontransformed inbreds revealed 1:1 segregation of PAT activity in three of four lines, including the line that failed to segregate in the R1 generation. Integrated copies of bar in one line appeared to be unstable or poorly transmitted. PMID:1731983

Spencer, T M; O'Brien, J V; Start, W G; Adams, T R; Gordon-Kamm, W J; Lemaux, P G

1992-01-01

138

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...

139

Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control.  

PubMed

The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis. This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin. R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis. Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs. PMID:9636319

Wünn, J; Klöti, A; Burkhardt, P K; Biswas, G C; Launis, K; Iglesias, V A; Potrykus, I

1996-02-01

140

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene  

Microsoft Academic Search

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5’ and 3’ RACE of a partial sequence

Indira Sivaraman; Neelakantan Arumugam; Yashpal Singh Sodhi; Vibha Gupta; Arundhati Mukhopadhyay; Akshay K. Pradhan; Pradeep Kumar Burma; Deepak Pental

2004-01-01

141

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

PubMed

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

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

2010-03-01

142

[Enhancement of artemisinin biosynthesis in transgenic Artemisia annua L. by overexpressed HDR and ADS genes].  

PubMed

Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin. PMID:25518337

Wang, Ya-Xiong; Long, Shi-Ping; Zeng, Li-Xia; Xiang, Li-En; Lin, Zhi; Chen, Min; Liao, Zhi-Hua

2014-09-01

143

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

PubMed Central

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

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

2014-01-01

144

Institute for Systems Biology: T1DBase  

NSDL National Science Digital Library

T1DBase was created jointly by the Institute for Systems Biology, the Juvenile Diabetes Research Foundation International, and the Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory to support the work of Type 1 diabetes (T1D) researchers. T1DBase currently "includes annotated genomic sequences for suspected T1D susceptibility regions; microarray data; functional annotation of genes active in beta cells; and 'global' datasets, generally from the literature, that are useful for systems biology studies." Information is available for human, rat, and mouse Type 1 diabetes candidate regions. The site also offers several tools including Gbrowse, Cytoscape, Beta Cell Gene Expression Bank, and Kegg Pathways. T1DBase was created as a community resource, and the website invites researchers to contribute both ideas and data.

145

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

PubMed

We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene. PMID:18514005

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

2008-01-01

146

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

PubMed Central

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacIR, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacIR protein. PMID:24755679

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

2014-01-01

147

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...

148

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

PubMed Central

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

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

2014-01-01

149

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

PubMed Central

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

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

2012-01-01

150

Viable transgenic goats derived from skin cells.  

PubMed

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

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

2004-06-01

151

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)  

Microsoft Academic Search

BACKGROUND: The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution. DESCRIPTION: The information gathered during the course of systematic investigation

Benjamin GH Choo; Igor Kondrichin; Sergey Parinov; Alexander Emelyanov; William Go; Wei-chang Toh; Vladimir Korzh

2006-01-01

152

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...

153

Expression of an osmotin-like protein from Solanum nigrum confers drought tolerance in transgenic soybean.  

PubMed

BackgroundDrought is by far the most important environmental factor contributing to yield losses in crops, including soybeans [Glycine max (L.) Merr.]. To address this problem, a gene that encodes an osmotin-like protein isolated from Solanum nigrum var. americanum (SnOLP) driven by the UBQ3 promoter from Arabidopsis thaliana was transferred into the soybean genome by particle bombardment.ResultsTwo independently transformed soybean lines expressing SnOLP were produced. Segregation analyses indicated single-locus insertions for both lines. qPCR analysis suggested a single insertion of SnOLP in the genomes of both transgenic lines, but one copy of the hpt gene was inserted in the first line and two in the second line. Transgenic plants exhibited no remarkable phenotypic alterations in the seven analyzed generations. When subjected to water deficit, transgenic plants performed better than the control ones. Leaf physiological measurements revealed that transgenic soybean plants maintained higher leaf water potential at predawn, higher net CO2 assimilation rate, higher stomatal conductance and higher transpiration rate than non-transgenic plants. Grain production and 100-grain weight were affected by water supply. Decrease in grain productivity and 100-grain weight were observed for both transgenic and non-transgenic plants under water deficit; however, it was more pronounced for non-transgenic plants. Moreover, transgenic lines showed significantly higher 100-grain weight than non-transgenic plants under water shortage.ConclusionsThis is the first report showing that expression of SnOLP in transgenic soybeans improved physiological responses and yield components of plants when subjected to water deficit, highlighting the potential of this gene for biotechnological applications. PMID:25492565

Weber, Ricardo; Wiebke-Strohm, Beatriz; Bredemeier, Christian; Margis-Pinheiro, Márcia; de Brito, Giovani; Rechenmacher, Ciliana; Bertagnolli, Paulo; de Sá, Maria; Campos, Magnólia; de Amorim, Regina; Beneventi, Magda; Margis, Rogério; Grossi-de-Sa, Maria; Bodanese-Zanettini, Maria

2014-12-10

154

Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle  

Microsoft Academic Search

In order to evaluate pea lectin as a resistance factor against the pollen beetle (Meligethes aeneus), transgenic oilseed rape (Brassica napus) has been produced wherein the pea lectin gene expression is driven by a pollen-specific promoter. The aim of the present study was to identify and characterise non-segregating transgenic and non-transgenic lines to be compared in various future tests for

Inger M. Åhman; Nadiya I. Kazachkova; Irene M. Kamnert; Per A. Hagberg; Christophe I. Dayteg; G. Monica Eklund; L. Johan O. Meijer; Barbara Ekbom

2006-01-01

155

Transgenic silver birch (Betula pendula) expressing sugarbeet chitinase 4 shows enhanced resistance to Pyrenopeziza betulicola  

Microsoft Academic Search

A sugar beet chitinase gene driven by the (42) CaMV 35S promoter was introduced into silver birch (Betula pendula) through Agrobacterium-mediated transformation. Transgenic shoots were regenerated and grown on WPM medium supplemented with 150 mg\\/ml kanamycin. From a total of 220 explants, 52 transgenics were obtained and 13 transgenic lines were randomly taken for molecular analysis to confirm the presence

A. Pappinen; Y. Degefu; L. Syrjälä; K. Keinonen; K. von Weissenberg

2002-01-01

156

Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene  

PubMed Central

Transgenic animals are extensively used to model human disease. Typically, the transgene copy number is estimated, but the exact integration site and configuration of the foreign DNA remains uncharacterized. When transgenes have been closely examined, some unexpected configurations have been found. Here, we describe a method to recover transgene insertion sites and assess structural rearrangements of host and transgene DNA using microarray hybridization and targeted sequence capture. We used information about the transgene insertion site to develop a polymerase chain reaction genotyping assay to distinguish heterozygous from homozygous transgenic animals. Although we worked with a bacterial artificial chromosome transgenic mouse line, this method can be used to analyse the integration site and configuration of any foreign DNA in a sequenced genome. PMID:23314155

DuBose, Amanda J.; Lichtenstein, Stephen T.; Narisu, Narisu; Bonnycastle, Lori L.; Swift, Amy J.; Chines, Peter S.; Collins, Francis S.

2013-01-01

157

Generation of transgenic frogs.  

PubMed

The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed. PMID:19504064

Loeber, Jana; Pan, Fong Cheng; Pieler, Tomas

2009-01-01

158

Transgenic fish systems and their application in ecotoxicology.  

PubMed

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

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

2014-11-14

159

Gene silencing results in instability of antibody production in transgenic plants.  

PubMed

The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed. PMID:9928938

De Neve, M; De Buck, S; De Wilde, C; Van Houdt, H; Strobbe, I; Jacobs, A; Van Montagu; Depicker, A

1999-01-01

160

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

PubMed

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

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

2014-01-01

161

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

PubMed Central

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

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

2014-01-01

162

Transgenic algae engineered for higher performance  

DOEpatents

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.

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

2014-10-21

163

Integration of reporter transgenes into Schistosoma mansoni chromosomes mediated by pseudotyped murine leukemia virus  

Microsoft Academic Search

The recent release of draft genome se- quences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome- scale investigation of transgene integration into schis- tosome chromosomes. Retrovirus-mediated transduc- tion offers a means to establish transgenic lines of schistosomes, to elucidate schistosome

Kristine J. Kines; Maria E. Morales; Victoria H. Mann; Geoffrey N. Gobert; Paul J. Brindley

2008-01-01

164

EXPRESSION AND CHARACTERIZATION OF ACIDOTHERMUS CELLULOLYTICUS E1 ENDOGLUCANASE IN TRANSGENIC DUCKWEED LEMNA MINOR 8627  

Technology Transfer Automated Retrieval System (TEKTRAN)

Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627. The selected transgenic duckweed line Cel25IX-15 generated using Agrobacterium-mediated transformation exhibited no phen...

165

Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP  

Microsoft Academic Search

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the

Guoping Feng; Rebecca H. Mellor; Michael Bernstein; Cynthia Keller-Peck; Quyen T. Nguyen; Mia Wallace; Jeanne M. Nerbonne; Jeff W. Lichtman; Joshua R. Sanes

2000-01-01

166

Two Amyloid Precursor Protein Transgenic Mouse Models with Alzheimer Disease-Like Pathology  

Microsoft Academic Search

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (Abeta ) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on

Christine Sturchler-Pierrat; Dorothee Abramowski; Mairead Duke; Karl-Heinz Wiederhold; Claudia Mistl; Sabin Rothacher; Brigit Ledermann; Kurt Burki; Peter Frey; Paolo A. Paganetti; Caroline Waridel; Michael E. Calhoun; Mathias Jucker; Alphonse Probst; Matthias Staufenbiel; Bernd Sommer

1997-01-01

167

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 ...

168

RNase T1 mimicking artificial ribonuclease  

PubMed Central

Recently, artificial ribonucleases (aRNases)—conjugates of oligodeoxyribonucleotides and peptide (LR)4-G-amide—were designed and assessed in terms of the activity and specificity of RNA cleavage. The conjugates were shown to cleave RNA at Pyr-A and G–X sequences. Variations of oligonucleotide length and sequence, peptide and linker structure led to the development of conjugates exhibiting G–X cleavage specificity only. The most efficient catalyst is built of nonadeoxyribonucleotide of unique sequence and peptide (LR)4-G-NH2 connected by the linker of three abasic deoxyribonucleotides (conjugate pep-9). Investigation of the cleavage specificity of conjugate pep-9 showed that the compound is the first single-stranded guanine-specific aRNase, which mimics RNase T1. Rate enhancement of RNA cleavage at G–X linkages catalysed by pep-9 is 108 compared to non-catalysed reaction, pep-9 cleaves these linkages only 105-fold less efficiently than RNase T1 (kcat_RNase T1/kcat_pep-9 = 105). PMID:17389642

Mironova, N. L.; Pyshnyi, D. V.; Shtadler, D. V.; Fedorova, A. A.; Vlassov, V. V.; Zenkova, M. A.

2007-01-01

169

Recombination technologies for enhanced transgene stability in bioengineered insects  

PubMed Central

Transposon-based vectors currently provide the most suitable gene transfer systems for insect germ-line transformation and are used for molecular improvement of the Sterile Insect Technique. However, the long time stability of genome-integrated transposon constructs depends on the absence of transposase activity that could remobilize the transposon-embedded transgenes. To achieve transgene stability transposon vectors are usually non-autonomous, lacking a functional transposase gene, and chosen so that endogenous or related transposon activities are not present in the host. Nevertheless, the non-autonomous transposon-embedded transgenes could become unstable by the unintended presence of a mobilizing transposase that may have been undetected or subsequently entered the host species by horizontal gene transfer. Since the field release of transgenic insects will present environmental concerns relating to large populations and high mobility, it will be important to ensure that transgene constructs are stably integrated for maintaining strain integrity and eliminating the possibility for unintentional transfer into the genome of another organism. Here we review efficient methods to delete or rearrange terminal repeat sequences of transposons necessary for their mobility, subsequent to their initial genomic integration. These procedures should prevent transposase-mediated remobilization of the transgenes, ensuring their genomic stability. PMID:20844938

Schetelig, Marc F.; Götschel, Frank; Viktorinová, Ivana; Handler, Alfred M.

2010-01-01

170

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

PubMed

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

Singh, Natwar; Mishra, Avinash; Jha, Bhavanath

2014-08-15

171

Transgenic Mice Secreting Coronavirus Neutralizing Antibodies into the Milk  

PubMed Central

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 104 by radioimmunoassay (RIA) and neutralized virus infectivity up to 104-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 106 as determined by RIA, neutralized virus infectivity by 106-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic ?-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and ?-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of ?-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens. PMID:9557658

Sola, Isabel; Castilla, Joaquín; Pintado, Belén; Sánchez-Morgado, José M.; Whitelaw, C. Bruce A.; Clark, A. John; Enjuanes, Luis

1998-01-01

172

Pyramiding transgenes for multiple resistance in rice against bacterial blight, yellow stem borer and sheath blight.  

PubMed

Here we describe the development of transgene-pyramided stable elite rice lines resistant to disease and insect pests by conventional crossing of two transgenic parental lines transformed independently with different genes. The Xa21 gene (resistance to bacterial blight), the Bt fusion gene (for insect resistance) and the chitinase gene (for tolerance of sheath blight) were combined in a single rice line by reciprocal crossing of two transgenic homozygous IR72 lines. F4 plant lines carrying all the genes of interest stably were identified using molecular methods. The identified lines, when exposed to infection caused by Xanthomonas oryzae pv oryzae, showed resistance to bacterial blight. Neonate larval mortality rates of yellow stem borer ( Scirpophaga incertulas) in an insect bioassay of the same identified lines were 100%. The identified line pyramided with different genes to protect against yield loss showed high tolerance of sheath blight disease caused by Rhizoctonia solani. PMID:12582865

Datta, K; Baisakh, N; Thet, K Maung; Tu, J; Datta, S K

2002-12-01

173

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...

174

Next-generation transgenic mice for optogenetic analysis of neural circuits  

E-print Network

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

Asrican, Brent

175

Transgenic Mosquitoes Expressing a Phospholipase A2 Gene Have a Fitness Advantage When Fed Plasmodium falciparum-Infected Blood  

PubMed Central

Background Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. Results We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2) into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. Conclusions Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control. PMID:24098427

Smith, Ryan C.; Kizito, Christopher; Rasgon, Jason L.; Jacobs-Lorena, Marcelo

2013-01-01

176

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

PubMed Central

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

Ting, Jonathan T.; Feng, Guoping

2014-01-01

177

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

PubMed

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

Byeon, Yeong; Back, Kyoungwhan

2014-05-01

178

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

PubMed Central

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

2014-01-01

179

The Seeds of Lotus japonicus Lines Transformed with Sense, Antisense, and Sense/Antisense Galactomannan Galactosyltransferase Constructs Have Structurally Altered Galactomannans in Their Endosperm Cell Walls1  

PubMed Central

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1?6)-?-galactose (Gal) substitution of the (1?4)-?-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense (“hairpin loop”) constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase. PMID:14988472

Edwards, Mary E.; Choo, Tze-Siang; Dickson, Cathryn A.; Scott, Catherine; Gidley, Michael J.; Reid, J.S. Grant

2004-01-01

180

T1D in School (Type 1 Diabetes)  

MedlinePLUS

... for throughout the year. JDRF’s Position on T1D Management in Schools JDRF believes that students with type ... provide them with an opportunity to make a difference by raising money for T1D research. Our program ...

181

Dealing with a New T1D Diagnosis in College  

MedlinePLUS

... New T1D Diagnosis in College Dealing with a New T1D Diagnosis in College A diabetes diagnosis is shocking at any point in life, but a new diagnosis can be especially difficult in college. Most ...

182

Acquired immunity of transgenic torenia plants overexpressing agmatine coumaroyltransferase to pathogens and herbivore pests  

PubMed Central

We investigated the ability of transgenic torenia (Scrophulariaceae) plants to resist fungi and arthropod herbivores. Torenia hybrida cv. Summerwave Blue was manipulated to produce Arabidopsis agmatine coumaroyltransferase (AtACT). This catalyses the last step in the biosynthesis of hydroxycinnamic acid amides (HCAAs) which function in defence. Transgenic plants accumulated substantial HCAAs, predominantly p-coumaroylagmatine, and the HCAAs were isomerized from the trans-form to the cis-form in planta. The transgenic line, accumulated the highest amount of endogenous HCAAs (CouAgm at 32.2 µM and total HCAAs at 47.5 µM) and this line was resistant to the necrotrophic fungus, Botrytis cinerea. There was no resistance, however, in their wild-type progenitors or in other transgenic lines accumulating low HCAA amounts. In contrast, the transformants were not significantly resistant to three representative herbivores, Frankliniella occidentalis, Aphis gossypii, and Tetranychus ludeni. PMID:23008754

Muroi, Atsushi; Matsui, Kenji; Shimoda, Takeshi; Kihara, Hirotomo; Ozawa, Rika; Ishihara, Atsushi; Nishihara, Masahiro; Arimura, Gen-ichiro

2012-01-01

183

Efficient discovery of ASCL1 regulatory sequences through transgene pooling  

PubMed Central

Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64 kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application. PMID:20206680

McGaughey, David M.

2010-01-01

184

Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase.  

PubMed

A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well-known bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene. PMID:24433490

Byeon, Yeong; Park, Sangkyu; Lee, Hyoung Yool; Kim, Young-Soon; Back, Kyoungwhan

2014-04-01

185

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression  

PubMed Central

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo ‘gain-of-function’ and ‘loss-of-function’ studies. PMID:20880997

Ohtsuka, Masato; Ogiwara, Sanae; Miura, Hiromi; Mizutani, Akiko; Warita, Takayuki; Sato, Masahiro; Imai, Kenji; Hozumi, Katsuto; Sato, Takehito; Tanaka, Masafumi; Kimura, Minoru; Inoko, Hidetoshi

2010-01-01

186

SirT1 modulates the estrogen–insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice  

PubMed Central

Introduction Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-?B, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice. Methods SirT1-deficient (SirT1ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region. Results Both male and female SirT1ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1ko/ko mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1ko/ko mammary tissues, but not that of I?B? expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNF? treatment enhanced the level of the newly synthesized I?B? in SirT1ko/ko cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals. Conclusion SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells. PMID:17201918

Li, Hongzhe; Rajendran, Grace K; Liu, Ninning; Ware, Carol; Rubin, Brian P; Gu, Yansong

2007-01-01

187

A 90-day toxicology study of high-amylose transgenic rice grain in Sprague–Dawley rats  

Microsoft Academic Search

A transgenic rice line (TRS) with high amylose level has been developed by antisense RNA inhibition of starch branching enzymes. Compositional analysis of TRS demonstrated that the content of resistant starch (RS) was significantly higher compared to conventional non-transgenic rice. High level of RS is an important raw material in food industry and has various physiological effects for human health.

Xing Hua Zhou; Ying Dong; Xiang Xiao; Yun Wang; Yong Xu; Bin Xu; Wei Dong Shi; Yi Zhang; Li Jia Zhu; Qiao Quan Liu

2011-01-01

188

Transgenic maize plants expressing a fungal phytase gene.  

PubMed

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

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

2008-08-01

189

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

PubMed Central

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

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

1998-01-01

190

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

PubMed Central

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

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

1991-01-01

191

Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants  

PubMed Central

Background Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear. Results Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. Conclusions The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits. PMID:23837904

2013-01-01

192

A postimplantation lethal mutation induced by transgene insertion on mouse chromosome 8  

SciTech Connect

We have produced three lines of transgenic mice that contain additional copies of the mouse phosphoglycerate kinase 1 (Pgk1) gene. Two of these lines, 94-A and 94-K, which are descendants of a common founder, did not produce liveborn progeny carrying two copies of these transgenes (i.e., A/A, K/K, or A/K). Genotyping of midgestation embryos showed that A/K embryos are dead by Embryonic Day 10. Comparison of the level of transgene expression in the three transgenic lines ruled out PGK1 toxicity as the cause of death of A/A, A/K, and K/K embryos. The death of A/A, K/K, and A/K transgenic mice was therefore attributed to an insertional mutation disrupting a gene or genes essential for normal embryogenesis. Analysis of the structure of the 94-A and 94-K transgenes indicated that they differ in the number of tandem repeats and in the positions of the transgene-cellular DNA junctions. To determine if the two transgenes represent a single integration followed by a rearrangement or two independent integration events, we cloned the endogenous sequences surrounding the 94-A and 94-K transgene insertion sites. Restriction analysis of the isolated genomic clones indicated that the endogenous sequences abutting the 3{prime} ends of the 94-A and 94-K transgenes are separated by less than 20 kb, providing strong support for the single integration model. Further analysis indicated that the 94-A transgene is associated with a deletion of at least 18 kb and is located in the vicinity of a widely transcribed endogenous gene. Chromosomal mapping of the endogenous sequences flanking the 94-A and 94-K transgene insertions using mouse -hamster somatic cell hybrids and a (C57BL/6J X SPRET/Ei)F1 X SPRET/Ei backcross panel allowed us to assign the 94-A(K) transgene insertion to the subcentral region of mouse chromosome 8. 100 refs., 10 figs., 5 tabs.

Pravtcheva, D.D.; Wise, T.L. [Saint Louis Univ., MO (United States)] [Saint Louis Univ., MO (United States)

1995-12-10

193

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

PubMed

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

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

2014-06-01

194

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

PubMed

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

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

2011-09-01

195

Amino Acids Regulate Transgene Expression in MDCK Cells  

PubMed Central

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

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

2014-01-01

196

How To Produce and Characterize Transgenic Plants.  

ERIC Educational Resources Information Center

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)

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

2002-01-01

197

Native T1 Mapping of the Heart – A Pictorial Review  

PubMed Central

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.

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

2014-01-01

198

Next-generation transgenic mice for optogenetic analysis of neural circuits  

PubMed Central

Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice. PMID:24324405

Asrican, Brent; Augustine, George J.; Berglund, Ken; Chen, Susu; Chow, Nick; Deisseroth, Karl; Feng, Guoping; Gloss, Bernd; Hira, Riichiro; Hoffmann, Carolin; Kasai, Haruo; Katarya, Malvika; Kim, Jinsook; Kudolo, John; Lee, Li Ming; Lo, Shun Qiang; Mancuso, James; Matsuzaki, Masanori; Nakajima, Ryuichi; Qiu, Li; Tan, Gregory; Tang, Yanxia; Ting, Jonathan T.; Tsuda, Sachiko; Wen, Lei; Zhang, Xuying; Zhao, Shengli

2013-01-01

199

Synergistic activity of chitinases and ?-1,3-glucanases enhances fungal resistance in transgenic tomato plants  

Microsoft Academic Search

Summary  Simultaneous expression of a tobacco class I chitinase and a class I ?-1,3-glucanase gene in tomato resulted in increased\\u000a fungal resistance, whereas transgenic tomato plants expressing either one of these genes were not protected against fungal\\u000a infection. After infection with Fusarium oxysporum f.sp. lycopersici, a 36% to 58% reduction in disease severity was observed in resistant tomato lines. Two transgenic

Erik Jongedijk; Henk Tigelaar; Jeroen S. C. van Roekel; Sandra A. Bres-Vloemans; Ilma Dekker; Ben J. C. Cornelissen; Leo S. Melchers

1995-01-01

200

Improved Carica papaya Tolerance to Carmine Spider Mite by the Expression of Manduca sexta chitinase Transgene  

Microsoft Academic Search

Papaya plants producing the tobacco hornworm (Manduca sexta) chitinase protein were obtained following microprojectile bombardment of embryogenic calli derived from the hypocotyls of\\u000a the cultivar Kapoho. Polymerase chain reaction (PCR) was carried out to confirm the presence of the transgene. RT-PCR and\\u000a a quantitative chitinase assay showed increased levels of chitinase activity in every selected transgenic line. Insect bioassays\\u000a in

Heather R. K. McCafferty; Paul H. Moore; Yun J. Zhu

2006-01-01

201

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)...

202

T1R receptors mediate mammalian sweet and umami taste.  

PubMed

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

Li, Xiaodong

2009-09-01

203

Mouse neutrophils express functional umami taste receptor T1R1/T1R3  

PubMed Central

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

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

2014-01-01

204

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

PubMed

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

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

2013-12-27

205

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

PubMed Central

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

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

2013-01-01

206

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.  

PubMed Central

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

207

Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene  

PubMed Central

Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

2012-01-01

208

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

PubMed Central

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

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

2013-01-01

209

Divergent Phenotypes in Mutant TDP-43 Transgenic Mice Highlight Potential Confounds in TDP-43 Transgenic Modeling  

PubMed Central

The majority of cases of frontotemporal lobar degeneration and amyotrophic lateral sclerosis are pathologically defined by the cleavage, cytoplasmic redistribution and aggregation of TAR DNA binding protein of 43 kDa (TDP-43). To examine the contribution of these potentially toxic mechanisms in vivo, we generated transgenic mice expressing human TDP-43 containing the familial amyotrophic lateral sclerosis-linked M337V mutation and identified two lines that developed neurological phenotypes of differing severity and progression. The first developed a rapid cortical neurodegenerative phenotype in the early postnatal period, characterized by fragmentation of TDP-43 and loss of endogenous murine Tdp-43, but entirely lacking aggregates of ubiquitin or TDP-43. A second, low expressing line was aged to 25 months without a severe neurodegenerative phenotype, despite a 30% loss of mouse Tdp-43 and accumulation of lower molecular weight TDP-43 species. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal, but rather were derived from a central portion of human TDP-43. Thus we find that aggregation is not required for cell loss, loss of murine Tdp-43 is not necessarily sufficient in order to develop a severe neurodegenerative phenotype and lower molecular weight TDP-43 positive species in mouse models should not be inherently assumed to be representative of human disease. Our findings are significant for the interpretation of other transgenic studies of TDP-43 proteinopathy. PMID:24466128

D’Alton, Simon; Altshuler, Marcelle; Cannon, Ashley; Dickson, Dennis W.; Petrucelli, Leonard; Lewis, Jada

2014-01-01

210

Transgenic Unit Standard operating procedures  

E-print Network

Transgenic Unit Standard operating procedures Handling Immuno-compromised mice Purpose disease to mice. General procedures All equipment and caging is wrapped and autoclaved prior to using by autoclaving .the cage will be replaced as a unit weekly. Autoclaved Feed must be replaced weekly to prevent

Shihadeh, Alan

211

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...

212

Transgenic plants: from first successes to future applications.  

PubMed

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

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

2013-01-01

213

Ultrastructural and behavioural changes precede amyloid deposition in a transgenic model of Alzheimer’s disease  

Microsoft Academic Search

We describe the thorough characterisation of a new transgenic mouse line overexpressing the 695-amino acid isoform of human amyloid precursor protein harbouring the Swedish double familial Alzheimer’s disease mutation. This line, referred to as TAS10, exhibits neuropathological features and cognitive deficits that are closely correlated to the accumulation of A? in their brain and that are reminiscent of those observed

J. C Richardson; C. E Kendal; R Anderson; F Priest; E Gower; P Soden; R Gray; S Topps; D. R Howlett; D Lavender; N. J Clarke; J. C Barnes; R Haworth; M. G Stewart; H. T. R Rupniak

2003-01-01

214

6, 1296712999, 2006 The T1-T2 study  

E-print Network

City15 over T1 and T2 occurred. Organic and elemental carbon concentrations at T1 showed diurnal cycles 2 Desert Research Institute, Reno Nevada, USA 3 Sunset Laboratory, Inc., Tigard, Oregon, USA 4 carbon as it aged and became coated with compounds such as sulfate and organic carbon, evolving from

Boyer, Edmond

215

Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct.  

PubMed

Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish. PMID:17406394

Rembold, Martina; Lahiri, Kajori; Foulkes, Nicholas S; Wittbrodt, Joachim

2006-01-01

216

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

PubMed

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

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

2014-09-01

217

Exon 1 of the HD Gene with an Expanded CAG Repeat Is Sufficient to Cause a Progressive Neurological Phenotype in Transgenic Mice  

Microsoft Academic Search

Huntington's disease (HD) is one of an increasing number of neurodegenerative disorders caused by a CAG\\/polyglutamine repeat expansion. Mice have been generated that are transgenic for the 5? end of the human HD gene carrying (CAG)115–(CAG)150 repeat expansions. In three lines, the transgene is ubiquitously expressed at both mRNA and protein level. Transgenic mice exhibit a progressive neurological phenotype that

Laura Mangiarini; Kirupa Sathasivam; Mary Seller; Barbara Cozens; Alex Harper; Colin Hetherington; Martin Lawton; Yvon Trottier; Hans Lehrach; Stephen W. Davies; Gillian P. Bates

1996-01-01

218

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

PubMed

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

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

219

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

PubMed Central

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

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

220

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

PubMed

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

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

221

Diversity of transgenic mouse models for selective targeting of midbrain dopamine neurons.  

PubMed

Ventral tegmental area (VTA) dopamine (DA) neurons have been implicated in reward, aversion, salience, cognition, and several neuropsychiatric disorders. Optogenetic approaches involving transgenic Cre-driver mouse lines provide powerful tools for dissecting DA-specific functions. However, the emerging complexity of VTA circuits requires Cre-driver mouse lines that restrict transgene expression to a precisely defined cell population. Because of recent work reporting that VTA DA neurons projecting to the lateral habenula release GABA, but not DA, we performed an extensive anatomical, molecular, and functional characterization of prominent DA transgenic mouse driver lines. We find that transgenes under control of the tyrosine hydroxylase, but not the dopamine transporter, promoter exhibit dramatic non-DA cell-specific expression patterns within and around VTA nuclei. Our results demonstrate how Cre expression in unintentionally targeted cells in transgenic mouse lines can confound the interpretation of supposedly cell-type-specific experiments. This Matters Arising paper is in response to Stamatakis et al. (2013), published in Neuron. See also the Matters Arising Response paper by Stuber et al. (2015), published concurrently with this Matters Arising in Neuron. PMID:25611513

Lammel, Stephan; Steinberg, Elizabeth E; Földy, Csaba; Wall, Nicholas R; Beier, Kevin; Luo, Liqun; Malenka, Robert C

2015-01-21

222

Restriction of neuroblastoma to the prostate gland in transgenic mice.  

PubMed Central

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. Images PMID:1652058

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

1991-01-01

223

Functional screening of an asthma QTL in YAC transgenic mice  

SciTech Connect

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.

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

1999-07-02

224

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

PubMed

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

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

225

Cardiac magnetic resonance T1 mapping of left atrial myocardium  

PubMed Central

BACKGROUND Cardiac magnetic resonance (CMR) T1 mapping is an emerging tool for objective quantification of myocardial fibrosis. OBJECTIVES To (a) establish the feasibility of left atrial (LA) T1 measurements, (b) determine the range of LA T1 values in patients with atrial fibrillation (AF) vs healthy volunteers, and (c) validate T1 mapping vs LA intracardiac electrogram voltage amplitude measures. METHODS CMR imaging at 1.5 T was performed in 51 consecutive patients before AF ablation and in 16 healthy volunteers. T1 measurements were obtained from the posterior LA myocardium by using the modified Look-Locker inversion-recovery sequence. Given the established association of reduced electrogram amplitude with fibrosis, intracardiac point-by-point bipolar LA voltage measures were recorded for the validation of T1 measurements. RESULTS The median LA T1 relaxation time was shorter in patients with AF (387 [interquartile range 364–428] ms) compared to healthy volunteers (459 [interquartile range 418–532] ms; P < .001) and was shorter in patients with AF with prior ablation compared to patients without prior ablation (P = .035). In a generalized estimating equations model, adjusting for data clusters per participant, age, rhythm during CMR, prior ablation, AF type, hypertension, and diabetes, each 100-ms increase in T1 relaxation time was associated with 0.1 mV increase in intracardiac bipolar LA voltage (P = .025). CONCLUSIONS Measurement of the LA myocardium T1 relaxation time is feasible and strongly associated with invasive voltage measures. This methodology may improve the quantification of fibrotic changes in thin-walled myocardial tissues. PMID:23643513

Beinart, Roy; Khurram, Irfan M.; Liu, Songtao; Yarmohammadi, Hirad; Halperin, Henry R.; Bluemke, David A.; Gai, Neville; van der Geest, Rob J.; Lima, Joao A.C.; Calkins, Hugh; Zimmerman, Stefan L.; Nazarian, Saman

2013-01-01

226

A thermoalkaliphilic lipase of Geobacillus sp. T1  

Microsoft Academic Search

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\\u000a solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase\\u000a was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U\\/mg and 51.5%,

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

2007-01-01

227

Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels.  

PubMed

Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra-high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research. PMID:19271958

Bradeen, James M; Iorizzo, Massimo; Mollov, Dimitre S; Raasch, John; Kramer, Lara Colton; Millett, Benjamin P; Austin-Phillips, Sandra; Jiang, Jiming; Carputo, Domenico

2009-04-01

228

NINDS GENSAT BAC Transgenic Project  

NSDL National Science Digital Library

This website from Rockefeller University in New York contains "a gene expression atlas of the central nervous system of the mouse based on bacterial artificial chromosomes (BACs)." GENSAT, or the Gene Expression Nervous System Atlas, contains brain slice images of BAC transgenic mice at the embryonic, postnatal (7 days old), and adult stages, stained to show areas of gene activity. The website comes with a detailed and helpful tutorial that recreates GENSAT's user interface and demonstrates how to manipulate search results.

2008-09-11

229

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

PubMed Central

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

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

2014-01-01

230

Caloric restriction attenuates Abeta-deposition in Alzheimer transgenic models.  

PubMed

Dietary influences on Alzheimer disease (AD) are gaining recognition. Because many aging processes are attenuated in laboratory mammals by caloric restriction (CR), we examined the effects of short-term CR in two AD-transgenic mice, APP(swe/ind) (J20) and APP(swe) + PS1(M146L) (APP + PS1). CR substantially decreased the accumulation of Abeta-plaques in both lines: by 40% in APP(swe/ind) (CR, 6 weeks), and by 55% in APP + PS1 (CR, 14 weeks). CR also decreased astrocytic activation (GFAP immunoreactivity). These influences of CR on AD-transgenic mice are consistent with epidemiological reports that show that high caloric diets associate with the risk of AD, and suggest that dietary interventions in adult life might slow disease progression. PMID:15748777

Patel, Nilay V; Gordon, Marcia N; Connor, Karen E; Good, Robert A; Engelman, Robert W; Mason, Jerimiah; Morgan, David G; Morgan, Todd E; Finch, Caleb E

2005-07-01

231

Conditional Transgenic System for Mouse Aurora A Kinase: Degradation by the Ubiquitin Proteasome Pathway Controls the Level of the Transgenic Protein  

Microsoft Academic Search

Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G2-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system

Tomokazu Fukuda; Yuji Mishina; Michael P. Walker; Richard P. DiAugustine

2005-01-01

232

Determining gene flow in transgenic cotton.  

PubMed

Gene flow is one of the major concerns associated with the release of transgenic plants into the environment. Unrestricted gene flow can results in super weeds, reduction in species fitness and genetic diversity, and contamination of traditional plants and foods. Thus, it is important and also necessary to evaluate the extent of gene flow in the field for transgenic plants already released or being considered for a release. Transgenic cotton is among the first transgenic crops for commercialization, which are widely cultivated around the world. In this chapter, we use transgenic insect resistant cotton and herbicide-tolerant cotton as two examples to present a field practice method for determining transgene flow in cotton. The procedure includes three major sections: (1) field design, (2) seed collection, and (3) field and lab bioassay. PMID:23143499

Pan, Xiaoping

2013-01-01

233

MAIN-BELT COMET P/2012 T1 (PANSTARRS)  

E-print Network

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 ...

Hsieh, Henry H.

234

Lesions of the ovary with T1-hypersignal.  

PubMed

Pelvic magnetic resonance imaging (MRI) has been used successfully to diagnose ovarian masses that cannot be adequately evaluated using pelvic ultrasound. T1 hyperintensity is a common finding in an ovarian mass and has important diagnostic implications. The aims of this review are to list the substances that may produce high signal intensity on T1-weighted MRI, to explain the physical basis for causes of the high T1 signal intensity, and to describe common and uncommon T1 hyperintense ovarian lesions. The morphological findings, the imaging features of the additional sequences, and clinical variables can help in the differential diagnosis, and in some cases, enable a specific diagnosis, leading to appropriate management of the patient. PMID:25017450

Chabrol, A; Rousset, P; Charlot, M; Chateau, F; Cotton, F; Golfier, F; Rety, F

2014-10-01

235

Polyhydroxybutyrate synthesis in transgenic flax.  

PubMed

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

Wróbel, Magdalena; Zebrowski, Jacek; Szopa, Jan

2004-01-01

236

Promoter Sequences for Defining Transgene Expression  

NASA Astrophysics Data System (ADS)

The design of reverse genetic experiments that utilize transgenic approaches often requires transgenes to be expressed in a predefined pattern and there is limited information regarding the gene expression profile for specific promoters. It is important that expression patterns are predetermined in the specific genotype targeted for transformation because the same promoter-transgene construct can produce different expression patterns in different host species. This chapter compares constitutive, targeted, or inducible promoters that have been characterized in specific cereal species.

Jones, Huw D.; Sparks, Caroline A.

237

Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice.  

PubMed

The cultivation of rice (Oryza sativa L.), a major food crop, requires ample water (30 % of the fresh water available worldwide), and its productivity is greatly affected by drought, the most significant environmental factor. Much research has focussed on identifying quantitative trait loci, stress-regulated genes and transcription factors that will contribute towards the development of climate-resilient/tolerant crop plants in general and rice in particular. The transcription factor DREB1A, identified from the model plant Arabidopsis thaliana, has been reported to enhance stress tolerance against drought stress. We developed transgenic rice plants with AtDREB1A in the background of indica rice cultivar Samba Mahsuri through Agrobacterium-mediated transformation. The AtDREB1A gene was stably inherited and expressed in T1 and T2 plants and in subsequent generations, as indicated by the results of PCR, Southern blot and RT-PCR analyses. Expression of AtDREB1A was induced by drought stress in transgenic rice lines, which were highly tolerant to severe water deficit stress in both the vegetative and reproductive stages without affecting their morphological or agronomic traits. The physiological studies revealed that the expression of AtDREB1A was associated with an increased accumulation of the osmotic substance proline, maintenance of chlorophyll, increased relative water content and decreased ion leakage under drought stress. Most of the homozygous lines were highly tolerant to drought stress and showed significantly a higher grain yield and spikelet fertility relative to the nontransgenic control plants under both stressed and unstressed conditions. The improvement in drought stress tolerance in combination with agronomic traits is very essential in high premium indica rice cultivars, such as Samba Mahsuri, so that farmers can benefit in times of seasonal droughts and water scarcity. PMID:24398893

Ravikumar, G; Manimaran, P; Voleti, S R; Subrahmanyam, D; Sundaram, R M; Bansal, K C; Viraktamath, B C; Balachandran, S M

2014-06-01

238

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

239

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

PubMed Central

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

2014-01-01

240

Transgenic animals in inflammatory disease models.  

PubMed

Inflammatory diseases affect a significant portion of the population worldwide and have been intensely studied for several decades. The advent of transgenic technology has allowed researchers to study individual gene contributions to the pathogenesis of these diseases. This has been done using standard inflammatory disease models in transgenic animals and by identifying novel models through the spontaneous generation of disease in the transgenic animal. Recent advances have been made in the understanding of rheumatoid arthritis, pulmonary inflammation, multiple sclerosis and inflammatory bowel disease through the use of transgenic animals in models of human inflammatory disease. PMID:14662397

Brodmerkel, Carrie M; Vaddi, Krishna

2003-12-01

241

A Built-In Strategy for Containment of Transgenic Plants: Creation of Selectively Terminable Transgenic Rice  

PubMed Central

Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation. PMID:18350155

Zhao, Te; Cheng, Jiaan; Tu, Juming; Ye, Gongyin; Shen, Zhicheng

2008-01-01

242

Identification of a Novel Transport-independent Function of PiT1/SLC20A1 in the Regulation of TNF-induced Apoptosis*  

PubMed Central

PiT1/SLC20A1 is a sodium-dependent Pi transporter expressed by most mammalian cells. Interestingly, PiT1 transcription has been shown to be up-regulated by the tumor necrosis factor ? (TNF), and we have now investigated the possible involvement of PiT1 in TNF-induced apoptosis. We show that PiT1-depleted cells are more sensitive to the proapoptotic activity of TNF (i.e. when the antiapoptotic NF?B pathway is inactivated). These observations were made in the human HeLa cancer cell line either transiently or stably depleted in PiT1 by RNA interference and in immortalized mouse embryonic fibroblasts isolated from PiT1 knock-out embryos. Depletion of the closely related family member PiT2 had no effect on TNF-induced apoptosis, showing that this effect was specific to PiT1. The increased sensitivity of PiT1-depleted cells was evident regardless of the presence or absence of extracellular Pi, suggesting that a defect in Pi uptake was not involved in the observed phenotype. Importantly, we show that the re-expression of a Pi uptake mutant of PiT1 in PiT1?/? mouse embryonic fibroblasts delays apoptosis as efficiently as the WT protein, showing that this function of PiT1 is unrelated to its transport activity. Caspase-8 is more activated in PiT1-depleted cells, and our data reveal that the sustained activation of the MAPK JNK is up-regulated in response to TNF. JNK activity is actually involved in PiT1-depleted cell death because specific JNK inhibitors delay apoptosis. PMID:20817733

Salaün, Christine; Leroy, Christine; Rousseau, Alice; Boitez, Valérie; Beck, Laurent; Friedlander, Gérard

2010-01-01

243

Effects of ?-synuclein overexpression in transgenic Caenorhabditis elegans strains.  

PubMed

The neural protein ?-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson's disease pathogenesis. We have generated ?-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human ?-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, ?-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing ?- synuclein showed significant reductions (p <0.05) in lifespan, motility and pharyngeal pumping, as compared to wildtype worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms displayed YFP-labelled inclusions on a diffuse CFP background. These findings implied that the ?-synuclein moieties of these fusion proteins still aggregated together in vivo, whereas CFP or YFP moieties alone did not. This in turn suggested that Foerster Resonanace Energy Transfer (FRET) between CFP and YFP labels in ?-synuclein aggregates could allow the extent of aggregation to be quantified. Accordingly, we also showed that net FRET signals increased 2- fold between L4 and adult SC+SV worms. PMID:23244416

Bodhicharla, Rakesh; Nagarajan, Archana; Winter, Jody; Adenle, Ademola; Nazir, Aamir; Brady, Declan; Vere, Kelly; Richens, Jo; O'Shea, Paul; Bell, David R; de Pomerai, David

2012-12-01

244

Effects of ?-Synuclein Overexpression in Transgenic Caenorhabditis elegans Strains  

PubMed Central

The neural protein ?-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson’s disease pathogenesis. We have generated ?-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human ?-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, ?-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing ?-synuclein showed significant reductions (p < 0.05) in lifespan, motility and pharyngeal pumping, as compared to wild-type worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms displayed YFP-labelled inclusions on a diffuse CFP background. These findings implied that the ?-synuclein moieties of these fusion proteins still aggregated together in vivo, whereas CFP or YFP moieties alone did not. This in turn suggested that Foerster Resonanace Energy Transfer (FRET) between CFP and YFP labels in ?-synuclein aggregates could allow the extent of aggregation to be quantified. Accordingly, we also showed that net FRET signals increased 2-fold between L4 and adult SC+SV worms. PMID:23244416

Bodhicharla, Rakesh; Nagarajan, Archana; Winter, Jody; Adenle, Ademola; Nazir, Aamir; Brady, Declan; Vere, Kelly; Richens, Jo; O’Shea, Paul; Bell, David R; de Pomerai, David

2012-01-01

245

Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV.  

PubMed Central

Transgenic mouse lines carrying several copies of the mouse apo A-IV gene were produced. Lipoprotein composition and function, and aortic lesion development were examined. Apo A-IV levels in the plasma of transgenic mice were elevated threefold compared with nontransgenic littermates on a chow diet, and sixfold in mice fed an atherogenic diet. Plasma concentrations of total cholesterol, HDL cholesterol, triglycerides, and free fatty acids were similar in transgenic and control mice fed a chow diet. However, with the atherogenic diet, male transgenic mice exhibited significantly higher levels of plasma triglycerides (P < 0.05), total cholesterol (P < 0.01), HDL cholesterol (P < 0.0001), and free fatty acids (P < 0.05), and lower levels of unesterified cholesterol (P < 0.05), than nontransgenic littermates. Expression of the apo A-IV transgene had a protective effect against the formation of diet-induced aortic lesions, with transgenics exhibiting lesion scores of approximately 30% those seen in control mice. HDL-sized lipoproteins isolated from transgenic mice fed the atherogenic diet promoted cholesterol efflux from cholesterol-loaded human monocytes more efficiently than comparable lipoproteins from nontransgenic counterparts. Plasma from transgenics also exhibited higher endogenous cholesterol esterification rates. Taken together, these results suggest that apo A-IV levels influence the metabolism and antiatherogenic properties of HDL. PMID:9109435

Cohen, R D; Castellani, L W; Qiao, J H; Van Lenten, B J; Lusis, A J; Reue, K

1997-01-01

246

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

NASA Technical Reports Server (NTRS)

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.

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

2000-01-01

247

T-DNA insertion alters the terpenoid content composition and bioactivity of transgenic Artemisia annua.  

PubMed

In this study, the interference of T-DNA insertion upon Agrobacterium-mediated transformation on the biochemical expression of the host genome is discussed. Plant extracts of transgenic Artemisia annua L. with or without an overexpressed famesyl pyrophosphate synthase gene have been investigated for their bioactivity and metabolic profile in comparison with wild type A. annua. The highest antimicrobial activity against Staphylococcus aureus, Bacillus subtilis and Candida albicans was observed in the T253 transgenic lines. Moreover, the crude extract from T253 showed higher antimalarial activity against the Plasmodium faciparum K1 strain than those of the others. The terpenoid constituents and antimicrobial properties of the plant samples were grouped by hierarchical clustering analysis. The clustering showed that squalene is a putative compound that might be involved in increasing the bioactivity of the transgenic line. In addition, T253 had a triterpene content that was about twice as great as that of the T253-2 line, which had a higher content of sesquiterpenes. However, both lines were transformed by the same FPS gene. These results suggested that the different bioactive properties observed in each transgenic line may be caused by variations in their terpenoid composition, which is affected by T-DNA insertion at different positions in the host plant. PMID:24689216

Karaket, Netiya; Wiyakrutta, Suthep; Lacaille-Dubois, Marie-Aleth; Supaibulwatana, Kanyaratt

2014-03-01

248

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

PubMed

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

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

2015-02-01

249

A 90-Day Dietary Toxicity Study of Genetically Modified Rice T1C-1 Expressing Cry1C Protein in Sprague Dawley Rats  

PubMed Central

In a 90-day study, Sprague Dawley rats were fed transgenic T1C-1 rice expressing Cry1C protein and were compared with rats fed non-transgenic parental rice Minghui 63 and rats fed a basal diet. No adverse effects on animal behavior or weight gain were observed during the study. Blood samples were collected and analyzed, and standard hematological and biochemical parameters were compared. A few of these parameters were found to be significantly different, but were within the normal reference intervals for rats of this breed and age, and were thus not considered to be treatment-related. Following sacrifice, a large number of organs were weighed, and macroscopic and histopathological examinations were performed with no changes reported. The aim of this study was to use a known animal model to determine the safety of the genetically modified (GM) rice T1C-1. The results showed no adverse or toxic effects due to T1C-1 rice when tested in this 90-day study. PMID:23300690

Tang, Xueming; Han, Fangting; Zhao, Kai; Xu, Yan; Wu, Xiao; Wang, Jinbin; Jiang, Lingxi; Shi, Wei

2012-01-01

250

Simultaneous down-regulation of caffeic/5-hydroxy ferulic acid-O-methyltransferase I and cinnamoyl-coenzyme A reductase in the progeny from a cross between tobacco lines homozygous for each transgene. Consequences for plant development and lignin synthesis.  

PubMed

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078

Pinçon, G; Chabannes, M; Lapierre, C; Pollet, B; Ruel, K; Joseleau, J P; Boudet, A M; Legrand, M

2001-05-01

251

Characterization of transgenic tobacco plants containing bacterial bphC gene and study of their phytoremediation ability.  

PubMed

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme. PMID:24933894

Viktorovtá, Jitka; Novakova, Martina; Trbolova, Ladislava; Vrchotova, Blanka; Lovecka, Petra; Mackova, Martina; Macek, Tomas

2014-01-01

252

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

PubMed Central

Background Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. Principal Findings By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein ? (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. Significance In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta. PMID:22363401

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

2012-01-01

253

[Phytate-phosphorus uptake and utilization by transgenic tobacco carrying Bacillus subtilis phytase gene].  

PubMed

Phytate is the major form of organic phosphorus in soil. Elevating the phyrase activity in transgenic plants may be an effective approach to promote their phytate-phosphorus utilization, but little is known about the applied conditions. In this study, several transgenic tobacco lines carrying Bacillus subtilis phytase gene were compared with wild-type tobacco, in terms of their ability in acquiring phosphorus from phytate in sterilized agar, sand and soil. In sterilized agar, transgenic tobacco plants were more efficient in phytate-phosphorus uptake and utilization, and their biomass and total phosphorus content were 3.6-10.7 and 2.2-4.6 fold of the wild-type's, respectively. In sand and soil systems, however, there were no significant differences in biomass and total phosphorus content between the trsansgenic and wild-type tobacco plants. These results indicated that Bacillus phytase transgene could only improve the phytate-phosphorus uptake by transgenic plants under sterilized condition, and its effectiveness might be limited under natural conditions because of microbial decomposition and mineral fixation. Therefore, further research is needed to understand the limiting factors on the functions of the transgene. PMID:16515194

Kong, Fanli; Lin, Wenliang; Yan, Xiaolong; Liao, Hong

2005-12-01

254

Tol2-mediated generation of a transgenic haplochromine cichlid, Astatotilapia burtoni.  

PubMed

Cichlid fishes represent one of the most species-rich and rapid radiations of a vertebrate family. These ~2200 species, predominantly found in the East African Great Lakes, exhibit dramatic differences in anatomy, physiology, and behavior. However, the genetic bases for this radiation, and for the control of their divergent traits, are unknown. A flood of genomic and transcriptomic data promises to suggest mechanisms underlying the diversity, but transgenic technology will be needed to rigorously test the hypotheses generated. Here we demonstrate the successful use of the Tol2 transposon system to generate transgenic Astatotilapia burtoni, a haplochromine cichlid from Lake Tanganyika, carrying the GFP transgene under the control of the ubiquitous EF1? promoter. The transgene integrates into the genome, is successfully passed through the germline, and the widespread GFP expression pattern is stable across siblings and multiple generations. The stable inheritance and expression patterns indicate that the Tol2 system can be applied to generate A. burtoni transgenic lines. Transgenesis has proven to be a powerful technology for manipulating genes and cells in other model organisms and we anticipate that transgenic A. burtoni and other cichlids will be used to test the mechanisms underlying behavior and speciation. PMID:24204902

Juntti, Scott A; Hu, Caroline K; Fernald, Russell D

2013-01-01

255

Tol2-Mediated Generation of a Transgenic Haplochromine Cichlid, Astatotilapia burtoni  

PubMed Central

Cichlid fishes represent one of the most species-rich and rapid radiations of a vertebrate family. These ?2200 species, predominantly found in the East African Great Lakes, exhibit dramatic differences in anatomy, physiology, and behavior. However, the genetic bases for this radiation, and for the control of their divergent traits, are unknown. A flood of genomic and transcriptomic data promises to suggest mechanisms underlying the diversity, but transgenic technology will be needed to rigorously test the hypotheses generated. Here we demonstrate the successful use of the Tol2 transposon system to generate transgenic Astatotilapia burtoni, a haplochromine cichlid from Lake Tanganyika, carrying the GFP transgene under the control of the ubiquitous EF1? promoter. The transgene integrates into the genome, is successfully passed through the germline, and the widespread GFP expression pattern is stable across siblings and multiple generations. The stable inheritance and expression patterns indicate that the Tol2 system can be applied to generate A. burtoni transgenic lines. Transgenesis has proven to be a powerful technology for manipulating genes and cells in other model organisms and we anticipate that transgenic A. burtoni and other cichlids will be used to test the mechanisms underlying behavior and speciation. PMID:24204902

Fernald, Russell D.

2013-01-01

256

Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp.] plants.  

PubMed

Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia. The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination. This paper reports the development of transgenic pigeon pea [ Cajanus cajun (L.) Millsp.] expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus. A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciensstrain EHA105. Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation. The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction. Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization. The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein. The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein. The transgenic plants were fertile and the transgene expressed in the progeny. PMID:12789415

Satyavathi, V V; Prasad, V; Khandelwal, Abha; Shaila, M S; Sita, G Lakshmi

2003-03-01

257

Nitrogen 15 T1 measurements of semicrystalline nylon 6  

NASA Astrophysics Data System (ADS)

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.

Powell, Douglas G.; Mathias, Lon J.

1989-05-01

258

Two-year field analysis of reduced recalcitrance transgenic switchgrass.  

PubMed

Switchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility. PMID:24751162

Baxter, Holly L; Mazarei, Mitra; Labbe, Nicole; Kline, Lindsey M; Cheng, Qunkang; Windham, Mark T; Mann, David G J; Fu, Chunxiang; Ziebell, Angela; Sykes, Robert W; Rodriguez, Miguel; Davis, Mark F; Mielenz, Jonathan R; Dixon, Richard A; Wang, Zeng-Yu; Stewart, C Neal

2014-09-01

259

Transformation of pecan and regeneration of transgenic plants.  

PubMed

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis. PMID:24201878

McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E

1993-09-01

260

An ecological assessment of transgenic crops  

Microsoft Academic Search

Since the first commercial release of a transgenic crop in 1994, the land area planted to these crops has expanded to over 90 million ha worldwide, with approximately 8.5 million farmers in 21 countries cultivating transgenic crops. Public apprehension has mounted apace. Concerns include: (i) the potential for gene flow into wild plant populations or soil organisms; (ii) adverse effects

Janice E. Thies; Medha H. Devare

2007-01-01

261

Transgenic animals in inflammatory disease models  

Microsoft Academic Search

Inflammatory diseases affect a significant portion of the population worldwide and have been intensely studied for several decades. The advent of transgenic technology has allowed researchers to study individual gene contributions to the pathogenesis of these diseases. This has been done using standard inflammatory disease models in transgenic animals and by identifying novel models through the spontaneous generation of disease

Carrie M Brodmerkel; Krishna Vaddi

2003-01-01

262

Safety and Public Acceptance of Transgenic Products  

Microsoft Academic Search

Public acceptance of transgenic (genetically engineered, GE) prod- ucts is influenced by the perception of direct or indirect risks and benefits and the credibility of regulatory agencies that evaluate food and environmental safety. In North America acceptance of GE foods is holding steady, while knowledge about them remains low. Develop- ment of transgenic foods with improved nutritional properties or other

Patrick F. Byrne

2006-01-01

263

Plants rendered herbicide-susceptible by cauliflower mosaic virus–elicited suppression of a 35S promoter-regulated transgene  

Microsoft Academic Search

Crop plants genetically modified for herbicide tolerance were some of the first to be released into the environment. Frequently, the cauliflower mosaic virus (CaMV) 35S promoter is used to drive expression of the herbicide tolerance transgene. We analyzed the response to CaMV infection of a transgenic oilseed rape line containing the bialaphos tolerance gene (BAR) from Streptomyces hygroscopicus, regulated by

Maria M. Kreike; Simon N. Covey; Robert Pitcher; Anthony M. Page; Philip J. Dale; Nadia S. Al-Kaff

2000-01-01

264

Interleukin5 Transgenic Mice Show Enhanced Resistance to Primary Infections with Nippostrongylus brasiliensis but Not Primary Infections with Toxocara canis  

Microsoft Academic Search

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes, Nippostrongylus brasiliensis and Toxocara canis. Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all dis- played enhanced resistance to N. brasiliensis. Although the timing of final worm expulsion was similar in trans-

LINDSAY A. DENT; CHRISTINE M. DALY; GRAHAM MAYRHOFER; TRUDY ZIMMERMAN; ANN HALLETT; LEON P. BIGNOLD; JENETTE CREANEY; JIM C. PARSONS

1999-01-01

265

Endogenous immunoglobulin expression in mu transgenic mice.  

PubMed

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes. PMID:1902746

Iacomini, J; Yannoutsos, N; Bandyopadhay, S; Imanishi-Kari, T

1991-02-01

266

Recent patents on plant transgenic technology.  

PubMed

Plant transformation technology plays an ever-increasing role in improving crop plant traits and addressing research questions. In recent years, a number of important patents on plant transgenic technologies have been published.These patents represent significant new progresses, many representing novel approaches in the field of plant transgenic technology. This review highlights selected as well as the most significant advancements made mostly within the past 2 years in plant transgenic research. These advancements include the improvements of plant transformation systems, development of new plant selectable markers, engineering for plant stress tolerance, production of vaccines in transgenic plants, etc. Of these, the engineering for plant stress tolerance accounts for a high percentage of the transgenic field of inventions. Some results are drastic and unexpected, having a great potential in their applications towards a wider range of plant species. This review also tries to avoid terms and languages pertaining to the patent and legal documents to ease readings. PMID:20156132

Yin, Xiaoyan; Zhang, Zhanyuan J

2010-06-01

267

Field performance of transgenic sugarcane expressing isomaltulose synthase.  

PubMed

Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3?years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217?mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars. Sugarcane is vegetatively propagated from stem cuttings known as setts. Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field-sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. There was no apparent adverse effect of IM accumulation on vigour assessed by stalk height and diameter or other visual indicators including germination of setts and establishment of stools. There was some inconsistency in IM levels in juice, between samplings of the vegetatively propagated transgenic lines. Until the causes are resolved, it is prudent to selectively propagate from stalks with higher IM levels in the initial vegetative field generations. Pol/Brix ratio allowed rapid identification of lines with high IM levels, using common sugar industry instruments. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial-scale production of IM if higher activity can be engineered in appropriate developmental patterns. PMID:21895946

Basnayake, Shiromani W V; Morgan, Terrance C; Wu, Luguang; Birch, Robert G

2012-02-01

268

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.  

PubMed

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo. PMID:24560743

Varga, E; Nemes, C; Davis, R P; Ujhelly, O; Klincumhom, N; Polgar, Z; Muenthaisong, S; Pirity, M K; Dinnyes, A

2014-04-01

269

[Construction of transgenic tobacco expressing popW and analysis of its biological phenotype].  

PubMed

In a previous study, we cloned popW from Ralstonia solanacearum strain ZJ3721, coding PopW, a new harpin protein. The procaryotically expressed PopW can induce resistance to Tobacco mosaic virus (TMV), enhance growth and improve quality of tobacco, when sprayed onto tobacco leaves. Here, we constructed an expression vector pB- popW by cloning popW into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciens strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens. After screening on MS medium containing kanamycin, PCR and RT-PCR analysis, 21 T3 lines were identified as positive transgenic. Genomic intergration and expression of the transferred gene were determined by PCR and RT-PCR. And GUS staining analysis indicated that the protein expressed in transgenic tobacco was bioactive and exhibited different expression levels among lines. Disease bioassays showed that the transgenic tobacco had enhanced resistance to TMV with biocontrol efficiency up to 54.25%. Transgenic tobacco also exhibited enhanced plant growth, the root length of 15 d old seedlings was 1.7 times longer than that of wild type tobacco. 60 d after transplanting to pots, the height, fresh weight and dry weight of transgenic tobacco were 1.4, 1.7, 1.8 times larger than that of wild type tobacco, respectively. PMID:25195247

Wang, Cui; Liu, Hongxia; Cao, Jing; Wang, Chao; Guo, Jianhua

2014-04-01

270

Myocardial T1 mapping: modalities and clinical applications  

PubMed Central

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

Jellis, Christine L.

2014-01-01

271

Up-Regulation of 1-Deoxy-d-Xylulose-5-Phosphate Synthase Enhances Production of Essential Oils in Transgenic Spike Lavender1  

PubMed Central

Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared to controls (from 101.5% to 359.0% and from 12.2% to 74.1% yield increase compared to controls in leaves and flowers, respectively). T0 transgenic plants were grown for 2 years, self-pollinated, and the T1 seeds obtained. The inheritance of the DXS transgene was studied in the T1 generation. The increased essential oil phenotype observed in the transgenic T0 plants was maintained in the progeny that inherited the DXS transgene. Total chlorophyll and carotenoid content in DXS progenies that inherited the transgene depended on the analyzed plant, showing either no variation or a significant decrease in respect to their counterparts without the transgene. Transgenic plants had a visual phenotype similar to untransformed plants (controls) in terms of morphology, growth habit, flowering, and seed germination. Our results demonstrate that the MEP pathway contributes to essential oil production in spike lavender. They also demonstrate that the DXS enzyme plays a crucial role in monoterpene precursor biosynthesis and, thus, in essential oil production in spike lavender. In addition, our results provide a strategy to increase the essential oil production in spike lavender by metabolic engineering of the MEP pathway without apparent detrimental effects on plant development and fitness. PMID:16980564

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

2006-01-01

272

Transgenic RNA interference (RNAi)-derived field resistance to cassava brown streak disease.  

PubMed

Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (?CP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ?CP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ?CP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease. PMID:22845735

Ogwok, Emmanuel; Odipio, John; Halsey, Mark; Gaitán-Solís, Eliana; Bua, Anton; Taylor, Nigel J; Fauquet, Claude M; Alicai, Titus

2012-12-01

273

Enhanced polyhydroxybutyrate production in transgenic sugarcane.  

PubMed

Polyhydroxybutyrate (PHB) is a bacterial polyester that has properties similar to some petrochemically produced plastics. Plant-based production has the potential to make this biorenewable plastic highly competitive with petrochemical-based plastics. We previously reported that transgenic sugarcane produced PHB at levels as high as 1.8% leaf dry weight without penalty to biomass accumulation, suggesting scope for improving PHB production in this species. In this study, we used different plant and viral promoters, in combination with multigene or single-gene constructs to increase PHB levels. Promoters tested included the maize and rice polyubiquitin promoters, the maize chlorophyll A/B-binding protein promoter and a Cavendish banana streak badnavirus promoter. At the seedling stage, the highest levels of polymer were produced in sugarcane plants when the Cavendish banana streak badnavirus promoter was used. However, in all cases, this promoter underwent silencing as the plants matured. The rice Ubi promoter enabled the production of PHB at levels similar to the maize Ubi promoter. The maize chlorophyll A/B-binding protein promoter enabled the production of PHB to levels as high as 4.8% of the leaf dry weight, which is approximately 2.5 times higher than previously reported levels in sugarcane. This is the first time that this promoter has been tested in sugarcane. The highest PHB-producing lines showed phenotypic differences to the wild-type parent, including reduced biomass and slight chlorosis. PMID:22369516

Petrasovits, Lars A; Zhao, Lihan; McQualter, Richard B; Snell, Kristi D; Somleva, Maria N; Patterson, Nii A; Nielsen, Lars K; Brumbley, Stevens M

2012-06-01

274

Evaluation of tolerance to Pierce's disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene.  

PubMed

SUMMARY Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs. Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogen Botrytis cinerea. To test this hypothesis, Vitis vinifera cvs. 'Thompson Seedless' and 'Chardonnay' were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressing pPGIP. Leaves of transgenic plants infected with B. cinerea had reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lower Xylella titres and better re-growth after pruning than the untransformed controls. PMID:20565637

Agüero, Cecilia B; Uratsu, Sandra L; Greve, Carl; Powell, Ann L T; Labavitch, John M; Meredith, Carole P; Dandekar, Abhaya M

2005-01-01

275

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

276

TRANSGENIC PAPAYA: A CASE FOR WORLDWIDE CONTROL OF PAPAYA RINGSPOT VIRUS  

Technology Transfer Automated Retrieval System (TEKTRAN)

Papaya ringspot virus (PRSV) was detected in the main papaya growing region of Hawaii in 1992. By 1994 Hawaii's papaya industry was facing devastating damage from PRSV. Efforts to develop resistant transgenic papaya were started in the mid 1980s. By 1991, a resistant line was identified, field tri...

277

Transgenic Manipulation of the Metabolism of Polyamines in Poplar Cells1  

E-print Network

, Rotorua, New Zealand (C.W.) The metabolism of polyamines (putrescine, spermidine, and spermine) has become in spermidine, and a small reduction in spermine as compared with NT cells. The conversion of labeled ornithine to a substantial reduction in putrescine and spermidine in both cell lines. The results show that: (a) Transgenic

278

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

279

Biosynthesis and cocoon-export of a recombinant globular protein in transgenic silkworms  

Microsoft Academic Search

A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the

Corinne Royer; Audrey Jalabert; Martine Da Rocha; Anne-Marie Grenier; Bernard Mauchamp; Pierre Couble; Gérard Chavancy

2005-01-01

280

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

PubMed

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

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

2015-01-01

281

Neuroprotective Effects of Creatine in a Transgenic Mouse Model of Huntington's Disease  

Microsoft Academic Search

Huntington's disease (HD) is a progressive neurodegenerative illness for which there is no effective therapy. We examined whether creatine, which may exert neuroprotective effects by increasing phosphocreatine levels or by stabilizing the mito- chondrial permeability transition, has beneficial effects in a transgenic mouse model of HD (line 6\\/2). Dietary creatine sup- plementation significantly improved survival, slowed the devel- opment of

Robert J. Ferrante; Ole A. Andreassen; Bruce G. Jenkins; Alpaslan Dedeoglu; Stefan Kuemmerle; James K. Kubilus; Rima Kaddurah-Daouk; Steven M. Hersch; M. Flint Beal

2000-01-01

282

Creatine therapy provides neuroprotection after onset of clinical symptoms in Huntington's disease transgenic mice  

Microsoft Academic Search

While there have been enormous strides in the understanding of Huntington's disease (HD) pathogenesis, treatment to slow or prevent disease progression remains elusive. We previ- ously reported that dietary creatine supplementation signifi- cantly improves the clinical and neuropathological phenotype in transgenic HD mice lines starting at weaning, before clinical symptoms appear. We now report that creatine administration started after onset

Alpaslan Dedeoglu; James K. Kubilus; Lichuan Yang; Kimberly L. Ferrante; Steven M. Hersch; M. Flint Beal; Robert J. Ferrante

2003-01-01

283

Nature Methods A genome-scale shRNA resource for transgenic RNAi in Drosophila  

E-print Network

. Dependence of shRNA-mediated knockdowns on mother age. Supplementary Table 1 Analysis of oogenesis phenotypes for viability. Supplementary Table 3 Transgenic long-hairpin RNAi lines analyzed for oogenesis phenotypes for germline expression and their abilities to generate oogenesis and maternal effect phenotypes. b. Luciferase

Perrimon, Norbert

284

Protection and conservation of Caricaceae germplasm with PRSV resistant transgenic papaya  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

285

Age-Dependent Penetrance of Disease in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis  

Microsoft Academic Search

The mutation gly93 ? ala of Cu,Zn superoxide dismutase (SOD) is found in patients with familial amyotrophic lateral sclerosis and causes motor neuron disease when expressed in transgenic mice. The progression of clinical and pathological disease was studied in a line of mice designated G1H. Clinical disease started at 91 ± 14 days of age with fine shaking of the

Arlene Y. Chiu; Ping Zhai; Mauro C. Dal Canto; Theresa M. Peters; Young W. Kwon; Susan M. Prattis; Mark E. Gurney

1995-01-01

286

Defective axonal transport in a transgenic mouse model of amyotrophic lateral sclerosis  

Microsoft Academic Search

AMYOTROPHIC lateral sclerosis (ALS) is a degenerative disease of motor neurons, characterized by depositions of neurofilaments in the perikarya and proximal axons. The pathogenesis of ALS remains poorly understood, but two lines of evidence suggest that neurofilament accumulation may play a causal role. First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration1á€-3 and, second, variant alleles of the

Jean-François Collard; Francine Côté; Jean-Pierre Julien

1995-01-01

287

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

EPA Science Inventory

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

288

Rapid characterization of transgenic and non-transgenic soybean oils by chemometric methods using NIR spectroscopy  

NASA Astrophysics Data System (ADS)

Near infrared (NIR) spectroscopy and multivariate classification were applied to discriminate soybean oil samples into non-transgenic and transgenic. Principal Component Analysis (PCA) was applied to extract relevant features from the spectral data and to remove the anomalous samples. The best results were obtained when with Support Vectors Machine-Discriminant Analysis (SVM-DA) and Partial Least Squares-Discriminant Analysis (PLS-DA) after mean centering plus multiplicative scatter correction. For SVM-DA the percentage of successful classification was 100% for the training group and 100% and 90% in validation group for non transgenic and transgenic soybean oil samples respectively. For PLS-DA the percentage of successful classification was 95% and 100% in training group for non transgenic and transgenic soybean oil samples respectively and 100% and 80% in validation group for non transgenic and transgenic respectively. The results demonstrate that NIR spectroscopy can provide a rapid, nondestructive and reliable method to distinguish non-transgenic and transgenic soybean oils.

Luna, Aderval S.; da Silva, Arnaldo P.; Pinho, Jéssica S. A.; Ferré, Joan; Boqué, Ricard

289

Geometric Mean Growth Discrete time: t, (t + 1), (t+2), ...  

E-print Network

, hence persist Recall: Temporal variation > 0 Geometric mean Arithmetic mean Arithmetic mean = i iip1 Geometric Mean Growth Discrete time: t, (t + 1), (t+2), ... Annual organisms: Non) .... (t ­ 1) ] Single quantity to characterize expected long-term growth Geometric mean growth rate ^ #12

Caraco, Thomas

290

Traffic Characteristics of the T1 NSFNET Backbone  

Microsoft Academic Search

This paper presents the results of a measurementstudy of the T1 NSFNET backbone. We first discussthe measurement environment and approach to datacollection. We then present measurements results for:long-term growth in traffic volume, including attributionto domains and protocols; trend in average packetsize on the network, both over long and medium termintervals; most popular sources, destinations, and sitepairs; traffic locality; international distribution

Kimberly C. Claffy; George C. Polyzos; Hans-werner Braun

1993-01-01

291

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.  

PubMed

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism. PMID:24101203

Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

2013-10-01

292

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice*  

PubMed Central

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism. PMID:24101203

Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

2013-01-01

293

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells  

Microsoft Academic Search

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus0nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg\\/l

G. A. Aguado-Santacruz; Q. Rascón-Cruz; J. Cabrera-Ponce; A. Martínez-Hernández; V. Olalde-Portugal; L. Herrera-Estrella

2002-01-01

294

Clinical Implications of Microsatellite Instability in T1 Colorectal Cancer  

PubMed Central

Purpose The estimation of regional lymph node metastasis (LNM) risk in T1 colorectal cancer is based on histologic examination and imaging of the primary tumor. High-frequency microsatellite instability (MSI-H) is likely to decrease the possibility of metastasis to either regional lymph nodes or distant organs in colorectal cancers. This study evaluated the clinical implications of MSI in T1 colorectal cancer with emphasis on the usefulness of MSI as a predictive factor for regional LNM. Materials and Methods A total of 133 patients who underwent radical resection for T1 colorectal cancer were included. Genomic DNA was extracted from normal and tumor tissues and amplified by polymerase chain reaction (PCR). Five microsatellite markers, BAT-25, BAT-26, D2S123, D5S346, and D17S250, were used. MSI and clinicopathological parameters were evaluated as potential predictors of LNM using univariate and multivariate analyses. Results Among 133 T1 colorectal cancer patients, MSI-H, low-frequency microsatellite instability (MSI-L), and microsatellite stable (MSS) colorectal cancers accounted for 7.5%, 6%, and 86.5%, respectively. MSI-H tumors showed a female predominance, a proximal location and more retrieved lymph nodes. Twenty-two patients (16.5%) had regional LNM. Lymphovascular invasion and depth of invasion were significantly associated with LNM. There was no LNM in 10 MSI-H patients; however, MSI status was not significantly correlated with LNM. Disease-free survival did not differ between patients with MSI-H and those with MSI-L/MSS. Conclusion MSI status could serve as a negative predictive factor in estimating LNM in T1 colorectal cancer, given that LNM was not detected in MSI-H patients. However, validation of our result in a different cohort is necessary. PMID:25510762

Kang, Jeonghyun; Lee, Hak Woo; Kim, Im-kyung; Kim, Nam Kyu; Sohn, Seung-Kook

2015-01-01

295

Molecular control of transgene escape from genetically modified plants  

Microsoft Academic Search

Potential risks of gene escape from transgenic crops through pollen and seed dispersal are being actively discussed and have slowed down full utilization of gene technology in crop improvement. To ban the transgene flow, barren zones and ‘terminator’ technology were developed as GMO risk management technologies in transgenic crops. Unfortunately, the technologies have not protected reliably the transgene migration to

Viktor Kuvshinov; Kimmo Koivu; Anne Kanerva; Eija Pehu

2001-01-01

296

Advancing environmental risk assessment for transgenic biofeedstock crops  

Microsoft Academic Search

Transgenic modification of plants is a key enabling technology for developing sustainable biofeedstocks for biofuels production. Regulatory decisions and the wider acceptance and development of transgenic biofeedstock crops are considered from the context of science-based risk assessment. The risk assessment paradigm for transgenic biofeedstock crops is fundamentally no different from that of current generation transgenic crops, except that the focus

Jeffrey D Wolt

2009-01-01

297

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

PubMed Central

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

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

2014-01-01

298

Lung specific expression of a human mutant p53 affects cell proliferation in transgenic mice  

Microsoft Academic Search

The human mutant p53(273H) has been shown in vitro to have both dominant-negative and gain-of-function properties, as well as to retain partial\\u000a DNA-binding and transcriptional activation functions. We have developed a line of transgenic mice in which the human mutant\\u000a p53(273H) is expressed in a lung specific manner (p53\\u000a +\\/+\\/TG). Crossing of the transgenic mice with p53 knockout mice led to

Wenrui Duan; Li Gao; David Jin; Gregory A. Otterson; Miguel A. Villalona-Calero

2008-01-01

299

Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function  

Microsoft Academic Search

Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function and dysfunction. We used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type–specific promoter elements. We performed an extensive functional characterization of the newly established VGAT-ChR2(H134R)-EYFP, ChAT-ChR2(H134R)-EYFP, Tph2-ChR2(H134R)-EYFP and Pvalb(H134R)-ChR2-EYFP BAC transgenic mouse lines and

Shengli Zhao; Jonathan T Ting; Hisham E Atallah; Li Qiu; Jie Tan; Bernd Gloss; George J Augustine; Karl Deisseroth; Minmin Luo; Ann M Graybiel; Guoping Feng

2011-01-01

300

Thirty years of transgenic research in plants.  

PubMed

In 1983, the first transgenic tissues and plants were generated by means of disarmed Agrobacterium tumefaciens strains, in which the oncogenes had been replaced by antibiotic resistance markers. Hence, this Special Issue of The International Journal of Developmental Biology celebrates 30 years of transgenic research in plants! Eminent scientists working in the field of plant transformation or plant biotechnology have contributed to this publication and reviewed the state of the art of their particular subdomain or summarized the importance of transgenic research in the discovery of new mechanisms and the establishment of an entirely new field, such as epigenetics. PMID:24166426

Van Lijsebettens, Mieke; Angenon, Geert

2013-01-01

301

Expression of multiple proteins in transgenic plants  

DOEpatents

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

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

2002-01-01

302

Late Blight Resistance of RB Transgenic Potato Lines  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

303

[Triptolide inhibits cell proliferation by downregulating phosphorylation of estrogen reporters in 4T1 tumor-bearing mice].  

PubMed

In order to investigate the anti-proliferative effects of triptolide (TP) on 4T1 mice breast cancer cell line in vitro and in mouse model, as well as the possible mechanisms, we detected the effect of TP on cell proliferation by MTT assay or Crystal Violet Staining in our research. Flowcytometry combined with FITC-Annexin V/PI staining were used for detecting TP induced 4T1 cell apoptosis. The protein expression of ERalpha, p-ERalpha, ERbeta, p-ERbeta, ERK, p-ERK, p38, p-p38, SAPK/JNK, and p-SAPK/JNK was tested by western blotting. We also compare TP with chemotherapy drug doxorubicin in 4T1 tumor bearing BLAB/c mice model, the Xenogen bioluminescence imaging, H&E, and IHC result indicated that TP exhibits an anticancer proliferation activity. As a result, TP in 100, 10, 1, 0.1 micromol x L(-1), all inhibited the proliferation of 4T1 cells by MTT assay and Crystal Violet Staining. TP which concentrations is 10, 1, 0.1 micromol x L(-1) could induce the apoptosis of 4T1 cells and reduce the cell proliferation. TP in 200 microg x kg(-1) could inhibit the tumor growth in vivo. The anticancer proliferation of TP was involved in its effect on reducing expression of ERalpha, p-ERalpha, ERbeta, and p-ERbeta, but nothing to do with the activation of MAPK signaling pathway. PMID:24791503

Pan, Guo-Feng; Gao, Jian-Li; Zhang, Qi; Lv, Gui-Yuan; Chen, Su-Hong

2013-12-01

304

Human apolipoprotein AII is a pro-atherogenic molecule when it is expressed in transgenic mice at a level similar to that in humans: evidence of a potentially relevant species-specific interaction with diet  

Microsoft Academic Search

We report on the effect of human apolipoprotein (apo) A-II transgene expression on atherosclerosis suscep- tibility in two transgenic lines (25.3 and 11.1) whose plasma human apoA-II concentrations ( , 23 and 96 mg\\/dl, re- spectively) span the normal range in humans. After 9 months of an atherogenic diet, 25.3 and 11.1 transgenic mice developed aortic atherosclerotic lesions that were

Joan Carles Escolà-Gil; Àfrica Marzal-Casacuberta; Josep Julve-Gil; Brian Y. Ishida; Jordi Ordóñez-Llanos; Lawrence Chan; Francesc González-Sastre; Francisco Blanco-Vaca

305

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

PubMed

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

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

2014-04-01

306

Transgenic chickens expressing human urokinase-type plasminogen activator.  

PubMed

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders. PMID:23960123

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

2013-09-01

307

Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene.  

PubMed Central

A full-length cDNA coding for a mutant dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3), cloned from a mouse fibroblast cell line grown in high concentrations of methotrexate (MTX), was microinjected into mouse embryos to produce transgenic mice. The DHFR cDNA product is 270-fold more resistant to MTX than the wild-type enzyme. Seventeen transgenic mouse lines, identified by Southern blotting of tail or spleen DNA, carried between 1 and 400 copies of the foreign gene per cell. Eight lines have thus far been tested for resistance to MTX. Control mice were treated until death; MTX was withdrawn from transgenic mice when a cumulative MTX dose uniformly fatal for controls was reached. The major site of MTX toxicity was the gastrointestinal tract, with death of controls resulting from fluid and weight loss. Transgenic animals were relatively resistant to these symptoms and tolerated significantly more MTX than control animals. These results show that genes conferring resistance to chemotherapeutic agents can, after transfer into intact organisms, produce systemic drug resistance. PMID:3467329

Isola, L M; Gordon, J W

1986-01-01

308

Generation and characterization of a Tet-On (rtTA-M2) transgenic rat  

PubMed Central

Background The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter. Results The homozygous rat line (ROSA-rtTA-M2) generated by lentiviral vector injection, has a single integration site and was derived from the offspring of a genetic mosaic founder with multiple transgene integrations. The rtTA-M2 transgene integrated into an intron of a putative gene on chromosome 2 and does not appear to affect the tissue-specificity or expression of that gene. Fibroblasts from the ROSA-rtTA-M2 rats were transduced with a TetO7/CMV-EGFP lentivirus and exhibited doxycycline dose-dependent expression of the EGFP reporter transgene, in vitro. In addition, doxycycline-inducible EGFP expression was observed, in vivo, when the TetO7/CMV-EGFP lentivirus was injected into testis, kidney and muscle tissues of ROSA-rtTA-M2 rats. Conclusions This conditional expression rat model may have application for transgenic overexpression or knockdown studies of gene function in development, disease and gene therapy. PMID:20158911

2010-01-01

309

Cosuppression of the ? Subunits of ?-Conglycinin in Transgenic Soybean Seeds Induces the Formation of Endoplasmic Reticulum–Derived Protein Bodies  

PubMed Central

The expression of the ? and ?? subunits of ?-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in ?-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced ?-conglycinin levels, endoplasmic reticulum (ER)–derived vesicles were observed that resembled precursor accumulating–vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER–derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein ?-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories. PMID:11340189

Kinney, Anthony J.; Jung, Rudolf; Herman, Eliot M.

2001-01-01

310

AN APPROACH TO TRANSGENIC CROP MONITORING  

EPA Science Inventory

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

311

Trends in surgical management of T1 renal cell carcinoma.  

PubMed

Historically, open radical nephrectomy (ORN) represented the standard of care for localized renal cell carcinoma (RCC). While the incidence of T1 RCC is rising, treatment options are developing fast and the standard of care according to European and American guidelines has changed to partial nephrectomy (PN), or laparoscopic radical nephrectomy in patients not suitable for PN. To assess the implementation of guideline recommendations and to profile recent surgical and technical innovations, we reviewed the current literature. We observed that ORN still represents the most commonly used treatment in T1 RCC patients. Utilization of PN increased over time but implementation is still in progress. Whereas PN is frequently used in tertiary care centers, population-based studies suggest discrepancies in the diffusion of standard of care treatments. Alternative minimally invasive approaches for PN are available but their superiority is not yet proven. Further efforts in improving the training of urologic surgeons are required to continue the implementation of guideline recommendations. PMID:24414526

Schiffmann, Jonas; Bianchi, Marco; Sun, Maxine; Becker, Andreas

2014-02-01

312

T1? experiment as a noise spectrum analyzer  

NASA Astrophysics Data System (ADS)

We performed a T1? (spin-locking) experiment on a superconducting flux qubit, enabling us to resolve the environmental noise in the intermediate-frequency range. By driving the qubit along its state polarization, in the rotating frame, it is effectively spin-locked: the decohering effect of low-frequency noise is thereby dramatically reduced compared to Rabi oscillations. We measured the T1? relaxation rate in the rotating frame, under different driving amplitudes and flux biases. Relating this driven relaxation rate to the noise at the corresponding Rabi frequency, we extracted the noise power spectral densities of the energy-bias (flux) and tunnel-coupling terms of the qubit's Hamiltonian at frequencies ranging from 0.5 to 100 MHz. In the flux-noise spectrum, we observed features due to non-Gaussian noise, which can be modeled by a strong random-telegraph fluctuator, supporting observations in the decoherence of a spin-echo.

Yan, Fei; Gustavsson, Simon; Bylander, Jonas; Yoshihara, Fumiki; Nakamura, Yasunobu; Cory, David; Oliver, William

2012-02-01

313

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype.  

PubMed

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease. PMID:11063721

Pritchard, L; Sloane-Stanley, J A; Sharpe, J A; Aspinwall, R; Lu, W; Buckle, V; Strmecki, L; Walker, D; Ward, C J; Alpers, C E; Zhou, J; Wood, W G; Harris, P C

2000-11-01

314

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).  

PubMed

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. PMID:16802117

Richter, A; Jacobsen, H-J; de Kathen, A; de Lorenzo, G; Briviba, K; Hain, R; Ramsay, G; Kiesecker, H

2006-11-01

315

Biosafety of kanamycin-resistant transgenic plants  

Microsoft Academic Search

Kanamycin resistance is one of the most frequently used selection markers for obtaining transgenic plants. The introduction\\u000a of these transgenic plants into agricultural practice will cause the kanamycin resistance gene and the gene product to be\\u000a present on a large scale. The desirability of this situation is analysed. The nature, properties and applications of the antibiotic\\u000a kanamycin are briefly reviewed,

Jan-Peter Nap; Jacques Bijvoet; Willem J. Stiekema

1992-01-01

316

Genetic analysis of putative transgenic cattle  

E-print Network

the genomic incorporation of DNA that has been microinjected into the male pronuclei of single or two celled embryos. Mice, rabbits, sheep, and pigs have been successfully transfected with DNA. Transgenic cattle, however, have not yet been reported... whether microinjected DNA is incorporated into the genome of these putative transgenic cattle. Molecular methods were developed to detect the presence of as little as a single copy of the foreign DNA in the genomes of the animals as evidenced...

Fontenot, Gregory Kenneth

2012-06-07

317

Transgenic Wheat, Barley and Oats: Future Prospects  

NASA Astrophysics Data System (ADS)

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

Dunwell, Jim M.

318

An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice  

PubMed Central

Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agouti?basic) was expressed in transgenic mice under the control of the ?-actin promoter (BAPa?basic). Three independent lines of BAPa?basic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agouti?basic protein was functional in vivo. However, none of the BAPa?basic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agouti?basic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agouti?basic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPa?basic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein–protein interactions that contribute to effective antagonism of melanocortin receptors. PMID:10411918

Miltenberger, R. J.; Mynatt, R. L.; Bruce, B. D.; Wilkison, W. O.; Woychik, R. P.; Michaud, E. J.

1999-01-01

319

Myc-Induced Liver Tumors in Transgenic Zebrafish Can Regress in tp53 Null Mutation  

PubMed Central

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53-/- mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53-/- mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53. PMID:25612309

Sun, Lili; Nguyen, Anh Tuan; Spitsbergen, Jan M.; Gong, Zhiyuan

2015-01-01

320

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens.  

PubMed

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96 h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-?B, tumor necrosis factor (TNF)-?, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24 h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases. PMID:21642002

Pan, Chieh-Yu; Peng, Kuan-Chieh; Lin, Cheng-Hui; Chen, Jyh-Yih

2011-08-01

321

Transgene flow: Facts, speculations and possible countermeasures.  

PubMed

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

Ryffel, Gerhart U

2014-10-01

322

Cellular, Molecular and Functional Characterisation of YAC Transgenic Mouse Models of Friedreich Ataxia  

PubMed Central

Background Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats). Methodology/Principal Findings We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R. Conclusions/Significance Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy. PMID:25198290

Anjomani Virmouni, Sara; Sandi, Chiranjeevi; Al-Mahdawi, Sahar; Pook, Mark A.

2014-01-01

323

Age and lesion-induced increases of GDNF transgene expression in brain following intracerebral injections of DNA nanoparticles.  

PubMed

In previous studies that used compacted DNA nanoparticles (DNP) to transfect cells in the brain, we observed higher transgene expression in the denervated striatum when compared to transgene expression in the intact striatum. We also observed that long-term transgene expression occurred in astrocytes as well as neurons. Based on these findings, we hypothesized that the higher transgene expression observed in the denervated striatum may be a function of increased gliosis. Several aging studies have also reported an increase of gliosis as a function of normal aging. In this study we used DNPs that encoded for human glial cell line-derived neurotrophic factor (hGDNF) and either a non-specific human polyubiquitin C (UbC) or an astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The DNPs were injected intracerebrally into the denervated or intact striatum of young, middle-aged or aged rats, and glial cell line-derived neurotrophic factor (GDNF) transgene expression was subsequently quantified in brain tissue samples. The results of our studies confirmed our earlier finding that transgene expression was higher in the denervated striatum when compared to intact striatum for DNPs incorporating either promoter. In addition, we observed significantly higher transgene expression in the denervated striatum of old rats when compared to young rats following injections of both types of DNPs. Stereological analysis of GFAP(+) cells in the striatum confirmed an increase of GFAP(+) cells in the denervated striatum when compared to the intact striatum and also an age-related increase; importantly, increases in GFAP(+) cells closely matched the increases in GDNF transgene levels. Thus neurodegeneration and aging may lay a foundation that is actually beneficial for this particular type of gene therapy while other gene therapy techniques that target neurons are actually targeting cells that are decreasing as the disease progresses. PMID:25453772

Yurek, D M; Hasselrot, U; Cass, W A; Sesenoglu-Laird, O; Padegimas, L; Cooper, M J

2015-01-22

324

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

PubMed Central

Background Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. Methodology/Principal Findings Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. Conclusions/Significance Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops. PMID:24595215

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

2014-01-01

325

Activation of the T1 neuronal circuit is necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila melanogaster.  

PubMed

The molecular and cellular events mediating complex behaviors in animals are largely unknown. Elucidating the circuits underlying behaviors in simple model systems may shed light on how these circuits function. In drosophila, courtship behavior provides a tractable model for studying the underlying basis of innate behavior. The male-specific pheromone 11-cis-vaccenyl acetate (cVA) modulates courtship behavior and is detected by T1 neurons, located on the antenna of male and female flies. The T1 neurons express the odorant receptor Or67d and are exquisitely tuned to cVA pheromone. However, cVA-induced changes in mating behavior have also been reported upon manipulation of olfactory neurons expressing odorant receptor Or65a. These findings raise the issue of whether multiple olfactory-driven circuits underlie cVA-induced behavioral responses and what role these circuits play in behavior. Here, we engineered flies in which the Or67d circuit is specifically activated in the absence of cVA to determine the role of this circuit in behavior. We created transgenic flies that express a dominant-active, pheromone-independent variant of the extracellular pheromone receptor, LUSH. We found that, similar to the behaviors elicited by cVA, engineered male flies have dramatically reduced courtship, whereas engineered females showed enhanced courtship. cVA exposure did not enhance the dominant LUSH-triggered effects on behavior in the engineered flies. Finally, we show the effects of both cVA and dominant LUSH on courtship are reversed by genetically removing Or67d. These findings demonstrate that the T1/Or67d circuit is necessary and sufficient to mediate sexually dimorphic courtship behaviors. PMID:20164344

Ronderos, David S; Smith, Dean P

2010-02-17

326

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

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

2014-01-01

327

T1 and susceptibility contrast at high fields  

NASA Astrophysics Data System (ADS)

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.

Neelavalli, Jaladhar

328

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii.  

PubMed

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2(t) and T2, and was considered as part of the same allele at the Glu-D (t) 1locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2(t) and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an omega-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of omega-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F(2)grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii ( Glu-D (t) 1 and Gli-D (t) 1) segregated as alleles of the Glu-D1 and Gli-D1loci of bread wheat. A new locus encoding the omega-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively. PMID:12582724

Gianibelli, M. C.; Lagudah, E. S.; Wrigley, C. W.; MacRitchie, F.

2002-02-01

329

Transgenic technologies to induce sterility  

PubMed Central

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes. PMID:19917077

Catteruccia, Flaminia; Crisanti, Andrea; Wimmer, Ernst A

2009-01-01

330

A Primer for Using Transgenic Insecticidal Cotton in Developing Countries  

PubMed Central

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

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

2009-01-01

331

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

PubMed

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

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

2011-01-01

332

Analysis of chiral amino acids in conventional and transgenic maize.  

PubMed

In this work, a new chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) method is proposed to identify and quantify D- and L-amino acids in three lines of transgenic maize and their corresponding nontransgenic parental lines grown under identical conditions. The optimized procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC-LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 80 mM SDS, and 20 mM beta-CD at pH 10.0. The D- and L-forms of Arg, Ser, Ala, Glu, and Asp, corresponding to the majority amino acids usually found in maize, are separated in less than 25 min with efficiencies up to 890,000 plates/m and high sensitivity (i.e., LODs as low as 160 nM were obtained for D-Arg for a signal-to-noise ratio of three), allowing the detection of 1% D-Arg in the presence of 99% of its opposite enantiomer. Using this method, different D-amino acids are detected in all investigated maize samples providing the reproducible quantification of the D-enantiomeric excess (% d-aa) for each amino acid calculated as % D-aa = 100D-aa/(D-aa + L-aa). Thus, significant differences were observed among the % d-aa values for the different conventional varieties (Aristis, Tietar, and PR33P66 maize) as could be expected from their natural variability. More interestingly, comparing each conventional maize with its corresponding transgenic line, very similar % D-aa values were obtained for one of the studied maize couples (Tietar vs Tietar-Bt) what could be presented as a new proof of their substantial equivalence. However, significant differences in the % d-aa values were observed for the other lines of maize studied. It is concluded that enantioselective procedures can open new perspectives in the study of transgenic organisms in order to corroborate (or not) the equivalence with their conventional counterparts. PMID:17523597

Herrero, Miguel; Ibáñez, Elena; Martín-Alvarez, Pedro J; Cifuentes, Alejandro

2007-07-01

333

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

PubMed

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens. PMID:18184061

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

2008-02-01

334

Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates.  

PubMed Central

BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 8 PMID:10551903

Charreau, B.; Ménoret, S.; Tesson, L.; Azimzadeh, A.; Audet, M.; Wolf, P.; Marquet, R.; Verbakel, C.; Ijzermans, J.; Cowan, P.; Pearse, M.; d'Apice, A.; Soulillou, J. P.; Anegon, I.

1999-01-01

335

[The use of transgenic animals in biomedical research in Germany. Part 1: Status Report 2001-2003].  

PubMed

While the German Federal Government has set itself the goal to make an active contribution to reducing animal experiments, the use of transgenic animals in biomedical research continuously increases every year. It is against this background that the study at hand aimed at providing an overview over the goals and the contents of research projects performed in Germany, in the course of which transgenic animals were produced or used in experimental procedures. Specifically, it was envisaged to spell out those specific areas of research, for which transgenic animals mainly were being used. Subsequently it was evaluated whether the research goals revealed might also be pursued with non animal test methods. In a literature survey, a total of 577 scientific publications relevant for the purposes of the study were collected. This material enables conclusions on those scientific areas, in which transgenic animals are used, applying to fundamental research, but not on their use in routine procedures in applied research or for the maintenance of transgenic breeds, since such purposes do not tend to be the subject of publications in scientific journals. According to the topics covered by the publications, main areas of biomedical research with transgenic animals can be found in the fields of neurobiology, immunology, cardiology, embryology and oncology. However their use can be discerned in all other areas of fundamental biomedical research as well. In accordance with the official German laboratory animal statistics, the vast majority of transgenic animals used were mice, followed by rats and pigs. Additionally, singular research projects with fish, rabbits and chicken were recorded. (In the official German laboratory animals statistics, very small numbers of transgenic hamsters, sheep and amphibians were also recorded in the past years.) A high percentage of the rats were used in cardiovascular research, whereas transgenic pigs as a rule were produced and bred as organ donors in xenotransplantation research. The majority of research projects either dealt with the experimental use of already established transgenic animal lines, or they described that transgenic animals specifically were produced for the purpose of the respective research project. Mostly, transgenesis was initiated by inserting the foreign gene into the germ cell genome. In some research projects, it was reported that the transgenic material was inserted into normally bred animals some time after parturition. PMID:16344907

Sauer, Ursula G; Kolar, Roman; Rusche, Brigitte

2005-01-01

336

14 N quadrupole resonance and 1 H T 1 dispersion in the explosive RDX  

NASA Astrophysics Data System (ADS)

The explosive hexahydro-1,3,5-trinitro-s-triazine (CH 2-N-NO 2) 3, commonly known as RDX, has been studied by 14N NQR and 1H NMR. NQR frequencies and relaxation times for the three ?+ and ?- lines of the ring 14N nuclei have been measured over the temperature range 230-330 K. The 1H NMR T1 dispersion has been measured for magnetic fields corresponding to the 1H NMR frequency range of 0-5.4 MHz. The results have been interpreted as due to hindered rotation of the NO 2 group about the N-NO 2 bond with an activation energy close to 92 kJ mol -1. Three dips in the 1H NMR dispersion near 120, 390 and 510 kHz are assigned to the ?0, ?- and ?+ transitions of the 14NO 2 group. The temperature dependence of the inverse line-width parameters T2? of the three ?+ and ?- ring nitrogen transitions between 230 and 320 K can be explained by a distribution in the torsional oscillational amplitudes of the NO 2 group about the N-NO 2 bond at crystal defects whose values are consistent with the latter being mainly edge dislocations or impurities in the samples studied. Above 310 K, the 14N line widths are dominated by the rapid decrease in the spin-spin relaxation time T2 due to hindered rotation of the NO 2 group. A consequence of this is that above this temperature, the 1H T1 values at the quadrupole dips are dominated by the spin mixing time between the 1H Zeeman levels and the combined 1H and 14N spin-spin levels.

Smith, John A. S.; Blanz, Martin; Rayner, Timothy J.; Rowe, Michael D.; Bedford, Simon; Althoefer, Kaspar

2011-12-01

337

14N quadrupole resonance and 1H T1 dispersion in the explosive RDX.  

PubMed

The explosive hexahydro-1,3,5-trinitro-s-triazine (CH2-N-NO2)3, commonly known as RDX, has been studied by 14N NQR and 1H NMR. NQR frequencies and relaxation times for the three ?+ and ?- lines of the ring 14N nuclei have been measured over the temperature range 230-330 K. The 1H NMR T1 dispersion has been measured for magnetic fields corresponding to the 1H NMR frequency range of 0-5.4 M Hz. The results have been interpreted as due to hindered rotation of the NO2 group about the N-NO2 bond with an activation energy close to 92 kJ mol(-1). Three dips in the 1H NMR dispersion near 120, 390 and 510 kHz are assigned to the ?0, ?- and ?+ transitions of the 14NO2 group. The temperature dependence of the inverse line-width parameters T2? of the three ?+ and ?- ring nitrogen transitions between 230 and 320 K can be explained by a distribution in the torsional oscillational amplitudes of the NO2 group about the N-NO2 bond at crystal defects whose values are consistent with the latter being mainly edge dislocations or impurities in the samples studied. Above 310 K, the 14N line widths are dominated by the rapid decrease in the spin-spin relaxation time T2 due to hindered rotation of the NO2 group. A consequence of this is that above this temperature, the 1H T1 values at the quadrupole dips are dominated by the spin mixing time between the 1H Zeeman levels and the combined 1H and 14N spin-spin levels. PMID:21978662

Smith, John A S; Blanz, Martin; Rayner, Timothy J; Rowe, Michael D; Bedford, Simon; Althoefer, Kaspar

2011-12-01

338

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

NASA Technical Reports Server (NTRS)

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

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

2000-01-01

339

Compositional equivalency of RNAi-mediated virus-resistant transgenic soybean and its nontransgenic counterpart.  

PubMed

RNA silencing or RNA interference (RNAi), which is triggered by double-stranded RNA (dsRNA), is an evolutionarily conserved process that is active in a wide variety of eukaryotic organisms. Engineering plants with hairpin construct in which the viral gene is arranged in inverted repeats (IR) renders plants resistant to plant virus infection. However, there is no report on whether biologically important changes occurred by the insertion of IR, which confer transgenic plants virus resistance. In the present study, the compositions of virus-resistant transgenic soybean seeds developed by insertion of three short IRs, each containing the specific, highly conserved sequences derived from one virus, were compared with those of nontransgenic counterparts by applying the principle of substantial equivalence to determine whether significant undesirable biological changes occurred by IR insertion. The results revealed that the nutrient components as well as antinutrient contents of these virus-resistant soybean lines are substantially equivalent to those of the nontransgenic counterparts, and the majority of the measured amounts of nutritional components and antinutrient contents are well within the range of values reported for other commercial soybean lines. The results imply that no biologically important changes occurred by the insertion of IRs in the RNAi-mediated virus-resistant transgenic soybeans. The results can serve as baseline information for developing RNAi-mediated transgenic soybean cultivars or other crops with broader spectrum virus resistance. PMID:24754373

Zhang, Xiuchun; Zhao, Pingjuan; Wu, Kunxin; Zhang, Yuliang; Peng, Ming; Liu, Zhixin

2014-05-14

340

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

NASA Astrophysics Data System (ADS)

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

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

2013-11-01

341

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

NASA Technical Reports Server (NTRS)

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

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

2003-01-01

342

Rederivation of transgenic mice from iPS cells derived from frozen tissue  

PubMed Central

In mice, induced pluripotent stem (iPS) cells with embryonic stem (ES)-like characteristics have been derived by ectopic expression of four transcription factors in somatic cells: Sox2, Oct3/4, Klf4 and/or c-Myc. To date, iPS cells have only be made from freshly harvested tissues and cells. However, if iPS cells could be derived from frozen tissues and cells, then cryopreservation of tissues such as mouse tails could conceivably become a reliable alternative to the more traditional formats, like germplasm and ES cells, for the archiving of genetically altered mouse lines. To test this hypothesis, we sought to demonstrate that a live transgenic mouse line could be recovered from transgenic iPS cells derived from cryopreserved mouse tissues. Tails and tail-derived fibroblasts from a DsRED transgenic mouse were cryopreserved in the presence of 5% dimethylsulfoxide (DMSO) in liquid nitrogen for 1 week and 1 month, respectively. Afterward, tissues and cells were thawed and underwent nuclear reprogramming by molecular transfection to derive iPS cells which generated germline confirmed transgenic mice. Our results demonstrate for the first time that iPS cells can be efficiently derived from frozen-stored-thawed tail tissue and fibroblasts and used to re-establish a transgenic mouse line. Therefore, this study provides conclusive evidence that, as a practical matter, frozen tails and fibroblasts can be used as an effective and reliable alternative to frozen germplasm and ES cells for the storage, maintenance, and distribution of genetically-altered mutant mice. PMID:20397045

Lee, Angus Yiu-Fai

2010-01-01

343

J =0 ,T =1 pairing-interaction selection rules  

NASA Astrophysics Data System (ADS)

Wave functions arising from a pairing Hamiltonian E (0 ), i.e., one in which the interaction is only between J =0+,T =1 pairs, lead to magnetic dipole and Gamow-Teller (GT) transition rates that are much larger than those from an interaction E (Jmax) in which a proton and a neutron couple to J =2 j . With realistic interactions the results are between the two extremes. In the course of this study we found that certain M 1 and GT matrix elements vanish with E (0 ) . These are connected to seniority and reduced isospin selection rules. We also relate our results to the single j scissors mode.

Harper, Matthew; Zamick, Larry

2015-01-01

344

Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells.  

PubMed

There are growing numbers of recombinant proteins that have been expressed in milk. Thus one can consider the placement of any gene of interest under the control of the regulatory elements of a milk protein gene in a dairy farm animal. Among the transgene introducing techniques, only nuclear transfer (NT) allows 100 % efficiency and bypasses the mosaicism associated with counterpart techniques. In this study, in an attempt to produce a transgenic goat carrying the human coagulation factor IX (hFIX) transgene, goat fetal fibroblasts were electroporated with a linearized marker-free construct in which the transgene was juxtaposed to ?-casein promoter designed to secret the recombinant protein in goat milk. Two different lines of transfected cells were used as donors for NT to enucleated oocytes. Two transgenic goats were liveborn. DNA sequencing of the corresponding transgene locus confirmed authenticity of the cloning procedure and the complementary experiments on the whey demonstrated expression of human factor IX in the milk of transgenic goats. In conclusion, our study has provided the groundwork for a prosperous and promising approach for large-scale production and therapeutic application of hFIX expressed in transgenic goats. PMID:22869287

Amiri Yekta, Amir; Dalman, Azam; Eftekhari-Yazdi, Poopak; Sanati, Mohammad Hossein; Shahverdi, Abdol Hossein; Fakheri, Rahman; Vazirinasab, Hamed; Daneshzadeh, Mohammad Taghi; Vojgani, Mahdi; Zomorodipour, Alireza; Fatemi, Nayeralsadat; Vahabi, Zeinab; Mirshahvaladi, Shahab; Ataei, Fariba; Bahraminejad, Elmira; Masoudi, Najmehsadat; Rezazadeh Valojerdi, Mojtaba; Gourabi, Hamid

2013-02-01

345

Transgenic models of prion disease.  

PubMed

There is growing concern that bovine spongiform encephalopathy (BSE) may have passed from cattle to humans, resulting in approximately 70 cases of an atypical, variant CJD (vCJD) in teenagers and young adults. We report here that transgenic (Tg) mice expressing full-length bovine (Bo) PrP serially propagate BSE prions and that there is no species barrier for transmission from cattle to Tg(BoPrP) mice. Surprisingly, these same mice were also highly susceptible to vCJD and natural sheep scrapie. The incubation times (approximately 250 d), neuropathology, and PrP(Sc) isoforms in Tg(BoPrP) mice inoculated with vCJD and BSE brain extracts were indistinguishable and differed dramatically from those seen in these mice injected with natural scrapie. In efforts to identify PrP sequences required for prion formation, we found that a redacted prion protein of only 106 amino acids (PrP106) containing two large deletions supported prion propagation. In Tg(PrP106) mice, an artificial transmission barrier for the passage of full-length mouse prions was diminished by the coexpression of full-length wt MoPrP(C), suggesting that wt MoPrP acts in trans to accelerate the replication of "miniprions" containing PrP(Sc)106. Following a single passage (approximately 300 d) in Tg(PrP106) mice, the miniprions efficiently transmitted disease to Tg(PrP106) mice after only approximately 66 days. Our findings with Tg(BoPrP) mice provide compelling evidence that prions from cattle with BSE have infected humans and caused fatal neurodegeneration, the unique features of miniprions offer new insights into the mechanism of prion replication, and the trans-acting effects of full-length PrP coexpression suggest a new approach to the development of even more efficient animal models for prion diseases. PMID:11214913

Scott, M R; Supattapone, S; Nguyen, H O; DeArmond, S J; Prusiner, S B

2000-01-01

346

Relationships between LDH-A, Lactate and Metastases in 4T1 Breast Tumors  

PubMed Central

Purpose To investigate the relationship between LDH-A expression, lactate concentration, cell metabolism and metastases in murine 4T1 breast tumors. Experimental Design Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using shRNA technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging-MRSI) and metastases was assessed. Results LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose utilization and glycolysis and increase in oxygen consumption, ROS and cellular ATP levels, compared to control (NC) cells cultured in 25 mM glucose. In vivo studies showed lower lactate levels in KD9, KD5, KD317 tumors than in NC or 4T1 wild-type tumors (p<0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased. Conclusions We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a pre-clinical setting and provides a non-invasive imaging strategy to monitor LDH-A targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease. PMID:23833310

Rizwan, Asif; Serganova, Inna; Khanin, Raya; Karabeber, Hazem; Ni, Xiaohui; Thakur, Sunitha; Zakian, Kristen L.; Blasberg, Ronald; Koutcher, Jason A.

2013-01-01

347

Chimeric elk/mouse prion proteins in transgenic mice  

PubMed Central

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions. PMID:23100369

Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L.; DeArmond, Stephen J.

2013-01-01

348

Design Consideration on the ACTS T1-VSAT  

NASA Technical Reports Server (NTRS)

The Advanced Communications Technology Satellite (ACTS) was developed by NASA as an experimental platform to demonstrate to industry the high risk technologies need for the next generation of communication satellites. The ACTS contains many state-of-the-art technologies including time division multiple accessing, adaptive error correction coding, multiple electronically steerable hopping beam antennas, spectrally efficient SMSK modulation, demand assignment multiple access protocol and on-board baseband circuit switching. An integral part of this testbed is the ACTS T1-very small aperture terminal (VSAT). The requirements imposed on the T1-VSAT demanded numerous design tradeoffs; balancing risk, cost, and implementation. Some of these considerations include: the method used for determining link quality, the development of a low cost and reliable Ka-band high power amplifier, the integration and testing environment for use in development and production and the low cost signal processing solution to high rate burst data. This paper discusses the tradeoffs considered in the selected implementation and the results obtained over the past two years of operation. Recommendations for improvements are also included.

Lilley, Richard D.; Robinson, Dennis D.

1996-01-01

349

Generation of a Focused Poly(amino ether) Library: Polymer-mediated Transgene Delivery and Gold-Nanorod based Theranostic Systems  

PubMed Central

A focused library of twenty-one cationic poly(amino ethers) was synthesized following ring-opening polymerization of two diglycidyl ethers by different oligoamines. The polymers were screened in parallel for plasmid DNA (pDNA) delivery, and transgene expression efficacies of individual polymers were compared to those of 25 kDa polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Seven lead polymers that demonstrated higher transgene expression than PEI in pancreatic and prostate cancer cells lines were identified from the screen. All seven lead polymers showed highest transgene expression at a polymer:pDNA weight ratio of 5:1 in the MIA PaCa-2 pancreatic cancer cell line. Among the conditions studied, transgene expression efficacy correlated with minimal polymer cytotoxicity but not polyplex sizes. In addition, this study indicated that methylene spacing between amine centers in the monomers, amine content, and molecular weight of the polymers are all significant factors and should be considered when designing polymers for transgene delivery. A lead effective polymer was employed for coating gold nanorods, leading to theranostic nanoassemblies that possess combined transgene delivery and optical imaging capabilities, leading to potential theranostic systems. PMID:23382773

Vu, Lucas; Ramos, James; Potta, Thrimoorthy; Rege, Kaushal

2012-01-01

350

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.  

PubMed

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis. PMID:15761663

Taniguchi, T; Kurita, M; Ohmiya, Y; Kondo, T

2005-03-01

351

Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.  

PubMed Central

Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism. Images PMID:8670892

Flavell, D M; Wells, T; Wells, S E; Carmignac, D F; Thomas, G B; Robinson, I C

1996-01-01

352

Structure function relationships of transgenic starches with engineered phosphate substitution and starch branching.  

PubMed

Potato tuber starch was genetically engineered in the plant by the simultaneous antisense suppression of the starch branching enzyme (SBE) I and II isoforms. Starch prepared from 12 independent lines and three control lines were characterised with respect to structural and physical properties. The lengths of the amylopectin unit chains, the concentrations of amylose and monoesterified phosphate were significantly increased in the transgenically engineered starches. Size exclusion chromatography with refractive index detection (SEC-RI) indicated a minor decrease in apparent molecular size of the amylose and the less branched amylopectin fractions. Differential scanning calorimetry (DSC) revealed significantly higher peak temperatures for gelatinisation and retrogradation of the genetically engineered starches whereas the enthalpies of gelatinisation were lower. Aqueous gels prepared from the transgenic starches showed increased gel elasticity and viscosity. Principle component analysis (PCA) of the data set discriminated the control lines from the transgenic lines and revealed a high correlation between phosphate concentration and amylopectin unit chain length. The PCA also indicated that the rheological characteristics were primarily influenced by the amylose concentration. The phosphate and the amylopectin unit chain lengths had influenced primarily the pasting and rheological properties of the starch gels. PMID:16024070

Blennow, Andreas; Wischmann, Bente; Houborg, Karen; Ahmt, Tina; Jørgensen, Kirsten; Engelsen, Søren Balling; Bandsholm, Ole; Poulsen, Peter

2005-08-01

353

Comparison of DNA walking methods for isolation of transgene-flanking regions in GM potato.  

PubMed

An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis. PMID:21225379

Cullen, Danny; Harwood, Wendy; Smedley, Mark; Davies, Howard; Taylor, Mark

2011-09-01

354

Transgenic study of parallelism in plant morphological evolution  

PubMed Central

Developmental constraint is indicated when one finds that similar genetic mechanisms are responsible for independent origins of the same derived phenotype. We studied three independent origins of rosette flowering within the mustard family and attempted to evaluate the extent to which the same mechanisms were involved in each transition from the ancestral phenotype, inflorescence flowering. We used transformation to move a candidate gene, LFY, and its cis-regulatory sequences from rosette-flowering species into an inflorescence-flowering recipient, Arabidopsis thaliana, in place of its endogenous LFY gene. The transgenic phenotypes of experimental and control lines (containing an A. thaliana LFY transgene) and the expression driven by the cis-regulatory sequences show that changes at the LFY locus might have contributed to the evolution of rosette flowering in two of the three lineages. In the third case, changes upstream of LFY are implicated. Our data suggest that changes in a single developmental regulatory program were involved in multiple origins of the same derived trait but that the specific genetic changes were different in each case. PMID:15096600

Yoon, Ho-Sung; Baum, David A.

2004-01-01

355

Membrane cofactor protein (MCP; CD46) expression in transgenic mice  

PubMed Central

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an ? 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction. PMID:11422193

Kemper, C; Leung, M; Stephensen, C B; Pinkert, C A; Liszewski, M K; Cattaneo, R; Atkinson, J P

2001-01-01

356

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.  

PubMed

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize. PMID:12354967

Gordon-Kamm, W. J.; Spencer, T. M.; Mangano, M. L.; Adams, T. R.; Daines, R. J.; Start, W. G.; O'Brien, J. V.; Chambers, S. A.; Adams, W. R.; Willetts, N. G.; Rice, T. B.; Mackey, C. J.; Krueger, R. W.; Kausch, A. P.; Lemaux, P. G.

1990-07-01

357

Metabolic syndrome without obesity: Hepatic overexpression of 11?-hydroxysteroid dehydrogenase type 1 in transgenic mice  

PubMed Central

In obese humans and rodents there is increased expression of the key glucocorticoid (GC) regenerating enzyme, 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1), in adipose tissue. This increased expression appears to be of pathogenic importance because transgenic mice overexpressing 11?-HSD1 selectively in adipose tissue exhibit a full metabolic syndrome with visceral obesity, dyslipidemia, insulin-resistant diabetes, and hypertension. In this model, while systemic plasma GC levels are unaltered, GC delivery to the liver via the portal vein is increased. 11?-HSD1 is most highly expressed in liver where inhibition or deficiency of its activity improves glucose and lipid homeostasis. To determine the potential contribution of elevated intrahepatic GCs alone toward development of insulin-resistant syndromes we generated transgenic mice expressing increased 11?-HSD1 activity selectively in the liver under transcriptional control of hepatic regulatory sequences derived from the human apoE gene (apoE-HSD1). Transgenic lines with 2- and 5-fold-elevated 11?-HSD1 activity exhibited mild insulin resistance without altered fat depot mass. ApoE-HSD1 transgenic mice exhibited fatty liver and dyslipidemia with increased hepatic lipid synthesis/flux associated with elevated hepatic LXR? and PPAR? mRNA levels as well as impaired hepatic lipid clearance. Further, apoE-HSD1 transgenic mice have a marked, transgene-dose-associated hypertension paralleled by incrementally increased liver angiotensinogen expression. These data suggest that elevated hepatic expression of 11?-HSD1 may relate to the pathogenesis of specific fatty liver, insulin-resistant, and hypertensive syndromes without obesity in humans as may occur in, for example, myotonic dystrophy, and possibly, the metabolically obese, normal-weight individual. PMID:15118095

Paterson, Janice M.; Morton, Nicholas M.; Fievet, Catherine; Kenyon, Christopher J.; Holmes, Megan C.; Staels, Bart; Seckl, Jonathan R.; Mullins, John J.

2004-01-01

358

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

PubMed

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

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

2014-01-01

359

Quantitative analysis of lentiviral transgene expression in mice over seven generations.  

PubMed

Lentiviral transgenesis is now recognized as an extremely efficient and cost-effective method to produce transgenic animals. Transgenes delivered by lentiviral vectors exhibited inheritable expression in many species including those which are refractory to genetic modification such as non-human primates. However, epigenetic modification was frequently observed in lentiviral integrants, and transgene expression found to be inversely correlated with methylation density. Recent data showed that about one-third lentiviral integrants exhibited hypermethylation and low expression, but did not demonstrate whether those integrants with high expression could remain constant expression and hypomethylated during long term germline transmission. In this study, using lentiviral eGFP transgenic mice as the experimental animals, lentiviral eGFP expression levels and its integrant numbers in genome were quantitatively analyzed by fluorescent quantitative polymerase-chain reaction (FQ-PCR), using the house-keeping gene ribosomal protein S18 (Rps18) and the single copy gene fatty acid binding protein of the intestine (Fabpi) as the internal controls respectively. The methylation densities of the integrants were quantitatively analyzed by bisulfite sequencing. We found that the lentiviral integrants with high expression exhibited a relative constant expression level per integrant over at least seven generations. Besides, the individuals containing these integrants exhibited eGFP expression levels which were positively and almost linearly correlated with the integrant numbers in their genomes, suggesting that no remarkable position effect on transgene expression of the integrants analyzed was observed. In addition, over seven generations the methylation density of these integrants did not increase, but rather decreased remarkably, indicating that these high expressing integrants were not subjected to de novo methylation during at least seven generations of germline transmission. Taken together, these data suggested that transgenic lines with long term stable expression and no position effect can be established by lentiviral transgenesis. PMID:20091347

Wang, Yong; Song, Yong-tao; Liu, Qin; Liu, Cang'e; Wang, Lu-lu; Liu, Yu; Zhou, Xiao-yang; Wu, Jun; Wei, Hong

2010-10-01

360

Toxins for Transgenic Resistance to Hemipteran Pests  

PubMed Central

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

Chougule, Nanasaheb P.; Bonning, Bryony C.

2012-01-01

361

Toxins for transgenic resistance to hemipteran pests.  

PubMed

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

Chougule, Nanasaheb P; Bonning, Bryony C

2012-06-01

362

Editorial Manager(tm) for Transgenic Research Manuscript Draft  

E-print Network

: Original research papers Section/Category: Keywords: BDNF; neurotrophic factor; YAC; transgene; expression (BDNF) is an attractive component for the treatment of various neurodegenerative diseases pharmacological induction of endogenous BDNF synthesis in brain. A transgenic mouse model has been established

363

Lectin cDNA and transgenic plants derived therefrom  

DOEpatents

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

Raikhel, Natasha V. (Okemos, MI)

2000-10-03

364

Role of nutrient-sensing taste 1 receptor (T1R) family members in gastrointestinal chemosensing.  

PubMed

Luminal nutrient sensing by G-protein-coupled receptors (GPCR) expressed on the apical domain of enteroendocrine cells activates intracellular pathways leading to secretion of gut hormones that control vital physiological processes such as digestion, absorption, food intake and glucose homeostasis. The taste 1 receptor (T1R) family of GPCR consists of three members: T1R1; T1R2; T1R3. Expression of T1R1, T1R2 and T1R3 at mRNA and protein levels has been demonstrated in the intestinal tissue of various species. It has been shown that T1R2-T1R3, in association with G-protein gustducin, is expressed in intestinal K and L endocrine cells, where it acts as the intestinal glucose (sweet) sensor. A number of studies have demonstrated that activation of T1R2-T1R3 by natural sugars and artificial sweeteners leads to secretion of glucagon-like peptides 1&2 (GLP-1 and GLP-2) and glucose dependent insulinotropic peptide (GIP). GLP-1 and GIP enhance insulin secretion; GLP-2 increases intestinal growth and glucose absorption. T1R1-T1R3 combination co-expressed on the apical domain of cholecystokinin (CCK) expressing cells is a luminal sensor for a number of L-amino acids; with amino acid-activation of the receptor eliciting CCK secretion. This article focuses on the role of the gut-expressed T1R1, T1R2 and T1R3 in intestinal sweet and L-amino acid sensing. The impact of exploiting T1R2-T1R3 as a nutritional target for enhancing intestinal glucose absorption and gut structural maturity in young animals is also highlighted. PMID:24382171

Shirazi-Beechey, Soraya P; Daly, Kristian; Al-Rammahi, Miran; Moran, Andrew W; Bravo, David

2014-06-01

365

Phytoremediation of Mercury and Organomercurials in Chloroplast Transgenic Plants: Enhanced Root Uptake, Translocation to Shoots, and Volatilization  

PubMed Central

Transgenic tobacco plants engineered with bacterial merA and merB genes via the chloroplast genome were investigated to study the uptake, translocation of different forms of mercury (Hg) from roots to shoots, and their volatilization. Untransformed plants, regardless of the form of Hg supplied, reached a saturation point at 200 µM of phenylmercuric acetate (PMA) or HgCl2, accumulating Hg concentrations up to 500 µg g?1 with significant reduction in growth. In contrast, chloroplast transgenic lines continued to grow well with Hg concentrations in root tissues up to 2000 µg g?1. Chloroplast transgenic lines accumulated both the organic and inorganic Hg forms to levels surpassing the concentrations found in the soil. The organic-Hg form was absorbed and translocated more efficiently than the inorganic-Hg form in transgenic lines, whereas no such difference was observed in untransformed plants. Chloroplast-transgenic lines showed about 100-fold increase in the efficiency of Hg accumulation in shoots compared to untransformed plants. This is the first report of such high levels of Hg accumulation in green leaves or tissues. Transgenic plants attained a maximum rate of elemental-Hg volatilization in two days when supplied with PMA and in three days when supplied with inorganic-Hg, attaining complete volatilization within a week. The combined expression of merAB via the chloroplast genome enhanced conversion of Hg2+ into Hg,0 conferred tolerance by rapid volatilization and increased uptake of different forms of mercury, surpassing the concentrations found in the soil. These investigations provide novel insights for improvement of plant tolerance and detoxification of mercury. PMID:18200876

Hussein, Hussein S.; Ruiz, Oscar N.; Terry, Norman; Daniell, Henry

2008-01-01

366

Transgenes are Dispensable for the RNA Degradation Step of Cosuppression  

Microsoft Academic Search

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

Jean-Christophe Palauqui; Her Ve Vaucheret

1998-01-01

367

Fate of transgenes in the forest tree genome  

Microsoft Academic Search

During the last two decades, genetic engineering (GE) has been progressing at a steady pace in the forest trees. Transgenic\\u000a trees carrying a variety of different transgenes have been produced, and are undergoing confined field trials in the world.\\u000a However, there are questions regarding stability of transgene expression, and transgene escape that need to be addressed in\\u000a the long-lived forest

M. Raj Ahuja

2011-01-01

368

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola  

Microsoft Academic Search

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant\\u000a macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene

Ri-He Peng; Quan-Hong Yao; Ai-Sheng Xiong; Zong-Ming Cheng; Yi Li

2006-01-01

369

Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum  

Microsoft Academic Search

Genes encoding pathogenesis-related (PR-) proteins isolated from a cDNA library of Fusarium gramine- arum-infected wheat spikes of scab-resistant cultivar 'Sumai-3' were transformed into susceptible spring wheat, 'Bobwhite' using a biolistic transformation protocol, with the goal of enhancing levels of resist- ance against scab. Twenty-four putative transgenic lines expressing either a single PR-protein gene or combinations thereof were regenerated. Transgene expression

Ajith Anand; Tian Zhou; Harold N. Trick; Bikram S. Gill; William W. Bockus; Subbaratnam Muthukrishnan

2003-01-01

370

Maize transgenes containing zein promoters are regulated by opaque2  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

371

Regulating Transgenic Crops: A Comparative Analysis of Different Regulatory Processes  

Microsoft Academic Search

Transgenic crops have the potential to benefit both developed and developing countries. To ensure safe crops to humans and the environment, a strong, but not stifling, regulatory system needs to be established and properly implemented. This paper explores some essential components of a strong regulatory structure for transgenic crops. First, five different regulatory systems for transgenic crops – the United

Gregory Jaffe

2004-01-01

372

IDENTIFICATION OF ESCAPED TRANSGENIC CREEPING BENTGRASS IN OREGON  

EPA Science Inventory

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

373

Drosophila transgenes Spectrum estimation from X-ray scattering  

E-print Network

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

McCullagh, Peter

374

DEVELOPMENT AND CHARACTERIZATION OF A TRANSGENIC MOUSE MODEL FOR POLIOMYELITIS  

E-print Network

DEVELOPMENT AND CHARACTERIZATION OF A TRANSGENIC MOUSE MODEL FOR POLIOMYELITIS Ruibao Ren Submitted of a transgenic mouse model for poliomyelitis Ruibao Ren In this work, transgenic mice containing a human and histopathologically resembles human poliomyelitis. This study demonstrates that the absence of PVR is the determinant

Racaniello, Vincent

375

Endogenous allergen upregulation: Transgenic vs. traditionally bred crops  

Microsoft Academic Search

The safety assessment for transgenic food crops currently includes an evaluation of the endogenous allergy potential (via serum IgE screening) when the non-transgenic counterpart is a commonly allergenic food. The value of this analysis in the safety assessment of transgenic crops, especially with reference to recent requests to quantify individual allergen concentrations in raw commodities, is examined. We conclude that

Rod A. Herman; Gregory S. Ladics

2011-01-01

376

Single-copy insertion of transgenes in Caenorhabditis elegans  

Microsoft Academic Search

At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization

Christian Frøkjær-Jensen; M Wayne Davis; Christopher E Hopkins; Blake J Newman; Jason M Thummel; Søren-Peter Olesen; Morten Grunnet; Erik M Jorgensen

2008-01-01

377

Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices.  

PubMed

Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi.   The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea. PMID:22922179

Hassan, Fathi; Noorian, Mojgan Sharifi; Jacobsen, Hans-Jörg

2012-01-01

378

Overexpression of RoDELLA impacts the height, branching, and flowering behaviour of Pelargonium × domesticum transgenic plants.  

PubMed

KEY MESSAGE : We reported the cloning of a rose DELLA gene. We obtained transgenic Pelargonium lines overexpressing this gene which presented several phenotypes in plant growth, root growth, flowering time and number of inflorescences. Control of development is an important issue for production of ornamental plant. The plant growth regulator, gibberellins (GAs), plays a pivotal role in regulating plant growth and development. DELLA proteins are nuclear negative regulator of GA signalling. Our objective was to study the role of GA in the plant architecture and in the blooming of ornamentals. We cloned a rose DELLA homologous gene, RoDELLA, and studied its function by genetic transformation of pelargonium. Several transgenic pelargonium (Pelargonium × domesticum 'Autum Haze') lines were produced that ectopically expressed RoDELLA under the control of the 35S promoter. These transgenic plants exhibited a range of phenotypes which could be related to the reduction in GA response. Most of transgenic plants showed reduced growth associated to an increase of the node and branch number. Moreover, overexpression of RoDELLA blocked or delayed flowering in transgenic pelargonium and exhibited defects in the root formation. We demonstrated that pelargonium could be used to validate ornamental gene as the rose DELLA gene. RoDELLA overexpression modified many aspects of plant developmental pathways, as the plant growth, the transition of vegetative to floral stage and the ability of rooting. PMID:22898902

Hamama, L; Naouar, A; Gala, R; Voisine, L; Pierre, S; Jeauffre, J; Cesbron, D; Leplat, F; Foucher, F; Dorion, N; Hibrand-Saint Oyant, L

2012-11-01

379

Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish.  

PubMed

Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provides valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development. PMID:17335798

Davison, Jon M; Akitake, Courtney M; Goll, Mary G; Rhee, Jerry M; Gosse, Nathan; Baier, Herwig; Halpern, Marnie E; Leach, Steven D; Parsons, Michael J

2007-04-15

380

Novel behavioural characteristics of the APP(Swe)/PS1?E9 transgenic mouse model of Alzheimer's disease.  

PubMed

In order to better understand animal models of Alzheimer's disease, novel phenotyping strategies have been established for transgenic mouse models. In line with this, the current study characterised male APPxPS1 transgenic mice on mixed C57BL/6JxC3H/HeJ background for the first time for social recognition memory, sensorimotor gating, and spatial memory using the cheeseboard test as an alternative to the Morris water maze. Furthermore, locomotion, anxiety, and fear conditioning were evaluated in transgenic and wild type-like animals. APPxPS1 males displayed task-dependent hyperlocomotion and anxiety behaviours and exhibited social recognition memory impairments compared to wild type-like littermates. Spatial learning and memory, fear conditioning, and sensorimotor gating were unaffected in APPxPS1 transgenic mice. In conclusion, this study describes for the first time social recognition memory deficits in male APPxPS1 mice and suggests that spatial learning and memory deficits reported in earlier studies are dependent on the sex and genetic background of the APPxPS1 mouse line used. Furthermore, particular test conditions of anxiety and spatial memory paradigms appear to impact on the behavioural response of this transgenic mouse model for Alzheimer's disease. PMID:23419740

Cheng, David; Logge, Warren; Low, Jac Kee; Garner, Brett; Karl, Tim

2013-05-15

381

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

PubMed Central

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

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

2012-01-01

382

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

NASA Astrophysics Data System (ADS)

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

Murakami, Takashi; Kobayashi, Eiji

2005-04-01

383

MAIN-BELT COMET P/2012 T1 (PANSTARRS)  

SciTech Connect

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.

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 [Institute for Astronomy, University of Hawaii, 2680 Woodlawn Drive, Honolulu, HI 96822 (United States); Novakovic, Bojan [Department of Astronomy, Faculty of Mathematics, University of Belgrade, Studentski trg 16, 11000 Belgrade (Serbia); Fitzsimmons, Alan [Astrophysics Research Centre, Queens University Belfast, Belfast BT7 1NN (United Kingdom); Moskovitz, Nicholas A. [Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States); Sheppard, Scott S., E-mail: hsieh@ifa.hawaii.edu [Department of Terrestrial Magnetism, Carnegie Institution of Washington, 5241 Broad Branch Road, NW, Washington, DC 20015 (United States); and others

2013-07-01

384

Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1  

SciTech Connect

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

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

1995-06-01

385

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

PubMed Central

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

Huber, M C; Krüger, G; Bonifer, C

1996-01-01

386

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

Microsoft Academic Search

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 1T0, 2T0, and 3T0 showed the presence of

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

2006-01-01

387

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids  

Microsoft Academic Search

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line

V. A. Hilder; K. S. Powell; A. M. R. Gatehouse; J. A. Gatehouse; L. N. Gatehouse; Y. Shi; W. D. O. Hamilton; A. Merryweather; C. A. Newell; J. C. Timans; W. J. Peumans; E. van Damme; D. Boulter

1995-01-01

388

Expression of functional recombinant human growth hormone in transgenic soybean seeds  

Microsoft Academic Search

We produced human growth hormone (hGH), a protein that stimulates growth and cell reproduction, in genetically engineered\\u000a soybean [Glycine max (L.) Merrill] seeds. Utilising the alpha prime (??) subunit of ?-conglycinin tissue-specific promoter from soybean and the\\u000a ?-Coixin signal peptide from Coix lacryma-jobi, we obtained transgenic soybean lines that expressed the mature form of hGH in their seeds. Expression levels

Nicolau B. Cunha; André M. Murad; Thaís M. Cipriano; Ana Cláudia G. Araújo; Francisco J. L. Aragão; Adilson Leite; Giovanni R. Vianna; Timothy R. McPhee; Gustavo H. M. F. Souza; Michael J. Waters; Elíbio L. Rech

389

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

390

Comparative Genomic Analysis of Transgenic Poplar Dwarf Mutant Reveals Numerous Differentially Expressed Genes Involved in Energy Flow  

PubMed Central

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

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

2014-01-01

391

Overexpression of -tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a  

E-print Network

in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea

Govindjee "Gov"

392

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

Technology Transfer Automated Retrieval System (TEKTRAN)

In order to study the effects of independently increasing the levels of high-molecular-weight glutenin subunits Dx5 and Dy10, we added copie