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Sample records for transgenic tobacco expressing

  1. Expression of chimeric HCV peptide in transgenic tobacco plants ...

    African Journals Online (AJOL)

    Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV. AK El Attar, AM Shamloul, AA Shalaby, BY Riad, A Saad, HM Mazyad, JM Keith ...

  2. Differential leaf resistance to insects of transgenic sweetgum (Liquidambar styraciflua) expressing tobacco anionic peroxidase.

    Science.gov (United States)

    Dowd, P F; Lagrimini, L M; Herms, D A

    1998-07-01

    Leaves of transgenic sweetgum (Liquidambar styraciflua) trees that expressed tobacco anionic peroxidase were compared with leaves of L. styraciflua trees that did not express the tobacco enzyme. Leaves of the transgenic trees were generally more resistant to feeding by caterpillars and beetles than wild-type leaves. However, as for past studies with transgenic tobacco and tomato expressing the tobacco anionic peroxidase, the degree of relative resistance depended on the size of insect used and the maturity of the leaf. Decreased growth of gypsy moth larvae appeared mainly due to decreased consumption, and not changes in the nutritional quality of the foliage. Transgenic leaves were more susceptible to feeding by the corn earworm, Helicoverpa zea. Thus, it appears the tobacco anionic peroxidase can contribute to insect resistance, but its effects are more predictable when it is expressed in plant species more closely related to the original gene source.

  3. Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects

    OpenAIRE

    Jube, Sandro; Borthakur, Dulal

    2007-01-01

    Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms, because it is easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. Many bacterial proteins involved in the synthesis of commercial products are currently engineered for production in tobacco. Bacterial enzymes synthesized in tobacco can enhance protection against abiotic stresses and diseases, and provide a system to test appli...

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  5. Transgenic tobacco plants expressing BoRS1 gene from Brassica ...

    Indian Academy of Sciences (India)

    Transgenic tobacco plants expressing BoRS1 gene from Brassica oleracea var. acephala show enhanced tolerance to water stress ... Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R & D Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200030, ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-15

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

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

    International Nuclear Information System (INIS)

    Dixit, Prachy; Mukherjee, Prasun K.; Sherkhane, Pramod D.; Kale, Sharad P.; Eapen, Susan

    2011-01-01

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

  8. Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

    Science.gov (United States)

    2011-01-01

    Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

  9. Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression

    Directory of Open Access Journals (Sweden)

    Fischer Lukas

    2009-04-01

    Full Text Available Abstract Background Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L. BY-2 cells with a gene encoding green fluorescent protein (GFP using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. Results The majority (~90% of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. Conclusion The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is

  10. Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene

    Science.gov (United States)

    Powell Abel, Patricia; Nelson, Richard S.; de, Barun; Hoffmann, Nancy; Rogers, Stephen G.; Fraley, Robert T.; Beachy, Roger N.

    1986-05-01

    A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

  11. Use of a simple semiquantitative method for an appraisal of green fluorescent protein (GFP) gene expression in transgenic tobacco plants

    Czech Academy of Sciences Publication Activity Database

    Hraška, M.; Rakouský, S.; Kocábek, Tomáš

    2005-01-01

    Roč. 49, - (2005), s. 313-316 ISSN 0006-3134 R&D Projects: GA MZe QE1123 Keywords : gene expression * transgenic tobacco Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 0.792, year: 2005

  12. Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene.

    Science.gov (United States)

    Jo, Jinki; Won, Sung-Hye; Son, Daeyoung; Lee, Byung-Hyun

    2004-09-01

    Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.

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

    Directory of Open Access Journals (Sweden)

    Liu Yong

    2011-01-01

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

  14. [Construction of transgenic tobacco expressing tomato GGPS2 gene and analysis of its low light tolerance].

    Science.gov (United States)

    Li, Cuiping; Dong, Weihua; Zhang, Xingguo

    2015-05-01

    To explore the influence of low light on the synthesis of carotenoids, chlorophyll and the adaptability of transgenic plants with tomato Solanum lycopersicon L. GGPS2 gene, we constructed a vector containing a GGPS2 gene with green fluorescent protein (GFP) as report gene under the control of a cauliflower mosaic virus 35S promoter and introduced it into tobacco Nicotiana tabacum L. cv. Wisconsin 38 by Agrobacterium tumefaciens-mediated transformation. PCR analysis of the DNA from kanamycin resistant tobacco indicated that the transgenic tobacco containing the nptII gene, SlaGGPS2 gene and without contamination of Agrobacterium. We also detected the root tip of kanamycin resistant tobacco showing characteristic fluorescence. The contents of carotenoid, chlorophyll and photosynthesis of transgenic tobacco increased in comparison with wild tobacco after low light treatment. In addition, leaf mass per unit area, total dry weight, ratio of root to shoot in transgenic tobacco were all higher than that of the wild tobacco, which proved that the transgenic tobacco could increase the accumulation of biomass and promote it transport to root. The transgenic tobacco with SlaGGPS2 gene can increase the contents of carotenoid, chlorophyll, enhance the photosynthetic rate, promote the biomass accumulation and its distribution to root. Hence, the transgenic tobacco with SlaGGPS2 gene had increased low light tolerance and the SlaGGPS2 gene maybe can be used in other crops.

  15. A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants.

    Science.gov (United States)

    Yang, I C; Iommarini, J P; Becker, D K; Hafner, G J; Dale, J L; Harding, R M

    2003-08-01

    Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

  16. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    Science.gov (United States)

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  17. [Effect of constitutive expression of ARGOS-LIKE gene on dimensions of cells and organs of transgenic tobacco plants].

    Science.gov (United States)

    Kuluev, B R; Khiazev, A V; Safiullina, M G; Cemeris, A V

    2013-05-01

    Transgenic tobacco plants that overexpress the ARGOS-LIKE (ARL) gene of Arabidopsis thaliana have been developed. The transgenic plants possessed increased dimensions of leaves and stem, whereas the magnitude of flowers was modified to a lesser degree. The increase in the organ dimensions was a result of stimulation of cell expansion; the cell quantity in the organ was even decreased. Ectopic expression of the ARL gene was promoted in order to increase in the level of mRNA of tobacco expansine NtEXPA5. It has been shown that the ARL gene of A. thaliana can be used to obtain transgenic plants with increased sizes of the leaves and stem.

  18. Transgenic tobacco plants constitutively expressing peanut BTF3 exhibit increased growth and tolerance to abiotic stresses.

    Science.gov (United States)

    Pruthvi, V; Rama, N; Parvathi, M S; Nataraja, K N

    2017-05-01

    Abiotic stresses limit crop growth and productivity worldwide. Cellular tolerance, an important abiotic stress adaptive trait, involves coordinated activities of multiple proteins linked to signalling cascades, transcriptional regulation and other diverse processes. Basal transcriptional machinery is considered to be critical for maintaining transcription under stressful conditions. From this context, discovery of novel basal transcription regulators from stress adapted crops like peanut would be useful for improving tolerance of sensitive plant types. In this study, we prospected a basal transcription factor, BTF3 from peanut (Arachis hypogaea L) and studied its relevance in stress acclimation by over expression in tobacco. AhBTF3 was induced under PEG-, NaCl-, and methyl viologen-induced stresses in peanut. The constitutive expression of AhBTF3 in tobacco increased plant growth under non stress condition. The transgenic plants exhibited superior phenotype compared to wild type under mannitol- and NaCl-induced stresses at seedling level. The enhanced cellular tolerance of transgenic plants was evidenced by higher cell membrane stability, reactive oxygen species (ROS) scavenging activity, seedling survival and vigour than wild type. The transgenic lines showed better in vitro regeneration capacity on growth media supplemented with NaCl than wild type. Superior phenotype of transgenic plants under osmotic and salinity stresses seems to be due to constitutive activation of genes of multiple pathways linked to growth and stress adaptation. The study demonstrated that AhBTF3 is a positive regulator of growth and stress acclimation and hence can be considered as a potential candidate gene for crop improvement towards stress adaptation. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

  19. Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

    DEFF Research Database (Denmark)

    Jach, G; Görnhardt, B; Mundy, J

    1995-01-01

    cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco....... Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack......cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II chitinase (CHI), a class-II beta-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1992-12-01

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

  1. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

    Science.gov (United States)

    Gutiérrez, Sonia P; Saberianfar, Reza; Kohalmi, Susanne E; Menassa, Rima

    2013-05-10

    Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels

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

    Directory of Open Access Journals (Sweden)

    Nidhi Thakur

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

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

    Science.gov (United States)

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

    2014-01-01

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

  4. [The creation of transgenic tobacco plants expressing fragments of the ARGOS and NtEXPA4 genes in antisense orientation].

    Science.gov (United States)

    Kuluev, B R; Kniazev, A V; Postrigan', B N; Chemeris, A V

    2014-01-01

    Transgenic tobacco plants expressing the fragments of the ARGOS and NtEXPA4 genes in antisense orientation have been created. Eleven lines of transgenic plants were investigated and five of them were characterized by a decrease in the sizes of the leaves and flowers as compared to control. Stalk sizes decreased when only the NtEXPA4 gene fragment was used. The organ size of the experimental plants decreased because of a reduction in the level of both cell division and cell expansion. Two lines of transgenic tobacco plants expressing the part of the ARGOS gene in antisense orientation were characterized by a reduction in the level of the NtEXPA1 and NtEXPA4 gene expression.

  5. Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

    Science.gov (United States)

    Agarwal, Parinita; Patel, Khantika; Agarwal, Pradeep K

    2018-04-01

    Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

  6. Characterization of mechanical properties of transgenic tobacco roots expressing a recombinant monoclonal antibody against tooth decay.

    Science.gov (United States)

    Hassan, Sally; Liu, Wei; Ma, Julian K-C; Thomas, Colin R; Keshavarz-Moore, Eli

    2008-07-01

    In this article, we describe a new approach that allows the determination of the magnitude of force required to break single plant roots. Roots were taken from transgenic tobacco plants, expressing a secreted monoclonal antibody. They were divided into four key developmental stages. A novel micromanipulation technique was used to pull to breakage, single tobacco roots in buffer in order to determine their breaking force. A characteristic uniform step-wise increase in the force up to a peak force for breakage was observed. The mean breaking force and mean work done were 101mN and 97microJ per root respectively. However, there was a significant increase in breaking force from the youngest white roots to the oldest, dark red-brown roots. We speculate that this was due to increasing lignin deposition with root stage of development (shown by phloroglucinol staining). No significant differences between fresh root mass, original root length, or mean root diameter for any of the root categories were found, displaying their uniformity, which would be beneficial for bioprocessing. In addition, no significant difference in antibody yield from the different root categories was found. These data show that it is possible to characterise the force requirements for root breakage and should assist in the optimisation of recombinant protein extraction from these roots. (c) 2008 Wiley Periodicals, Inc.

  7. Over-expression of SlJA2 decreased heat tolerance of transgenic tobacco plants via salicylic acid pathway.

    Science.gov (United States)

    Liu, Zhong-Ming; Yue, Meng-Meng; Yang, Dong-Yue; Zhu, Shao-Bo; Ma, Na-Na; Meng, Qing-Wei

    2017-04-01

    Over-expression of SlJA2 decreased the accumulation of SA, which resulted in significant physiological and gene expression changes in transgenic tobacco plants, leading to the decreased heat tolerance of transgenic tobacco. NAC family, the largest transcription factors in plants, responses to different environmental stimuli. Here, we isolated a typical NAC transcription factor (SlJA2) from tomato and got transgenic tobacco with SlJA2 over-expression. Expression of SlJA2 was induced by heat stress (42 °C), chilling stress (4 °C), drought stress, osmotic stress, abscisic acid, and salicylic acid. Over-expression of SlJA2 decreased the accumulation of salicylic acid by regulating expression of salicylic acid degradation gene under heat stress. Compared to WT plants, stomatal apertures and water loss increased in transgenic plants, and the damage of photosynthetic apparatus and chlorophyll breakdown were more serious in transgenic plants under heat stress. Meanwhile, more H 2 O 2 and O 2 ·- were accumulated transgenic plants and proline synthesis was restricted, which resulted in more serious oxidative damage compared to WT. qRT-PCR analysis showed that over-expression of SlJA2 could down-regulate genes involved in reactive oxygen species scavenging, proline biosynthesis, and response to heat stress. All the above results indicated that SlJA2 may be a negative regulator responded to plant's heat tolerance. Thus, this study provides new insight into roles of NAC family member in plant response to abiotic stress.

  8. [Morphological features of transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the Dahlia mosaic virus promoter].

    Science.gov (United States)

    Kuluev, B R; Kniazev, A V; Cheremis, A V; Vakhitov, V A

    2013-01-01

    Transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the 35S promoter and the promoter of dahlia mosaic virus were obtained. The transgenic plants were characterized by increase in the length of the leaves, flower sizes, stem height, and weight of seeds; at the same time, the degree of increase was greater in the case of use of the dahlia mosaic virus promoter as a regulator of transcription. Ectopic expression of the AINTEGUMENTA gene promoted prolongation of leaf growth, while sizes of epidermal cells of the leaves remained unchanged.

  9. [Estradiol inducible and flower-specific expression of ARGOS and ARGOS-LIKE genes in transgenic tobacco plants].

    Science.gov (United States)

    Kuluev, B R; Kniazev, A V; Nikonorov, Iu M; Cheremis, A V

    2014-08-01

    Transgenic tobacco plants expressing Arabidopsis thaliana ARGOS and ARGOS-LIKE genes under the control of the chalcone synthase promoter of Petunia hybrid L., as well as the estradiol inducible XVE system, have been obtained. The part of transgenic plants with flower-specific expression of the target genes was characterized by increased flower size, caused by an increase in cell size and quantity in the case of the ARGOS gene and by a stimulation of cell growth via stretching in the case of the ARGOS-LIKE gene. An enhanced expression level of the NtEXPA1, NtEXPA4 genes encoding expansins, NtEXGT gene encoding endo-xyloglucan transferase, and the AINTEGUMENTA-like gene was detected in the flowers of transgenic tobacco plants. In the case of inducible expression of ARGOS and ARGOS-LIKE genes, an increase in leaf, stem and flower size was revealed in several lines of transgenic plants as compared to control. Expression of the ARGOS gene also affected cell number and size in this case, while the ARGOS-LIKE gene mainly influenced cell size via stretching. Inducible expression of the ARGOS gene in flowers mainly provided an enhanced containment of AINTEGUMENTA-like mRNA, while ARGOS-LIKE gene expression resulted in the activation of NtEXPA1 and NtEXGT genes.

  10. [Induced expression of Serratia marcescens ribonuclease III gene in transgenic Nicotiana tabacum L. cv. SR1 tobacco plants].

    Science.gov (United States)

    Zhirnov, I V; Trifonova, E A; Romanova, A V; Filipenko, E A; Sapotsky, M V; Malinovsky, V I; Kochetov, A V; Shumny, V K

    2016-11-01

    Transgenic Nicotiana tabacum L. cv. SR1 plants, characterized by an increase in the level of dsRNA-specific hydrolytic activity after induction by wounding, were obtained. The Solanum lycopersicum anionic peroxidase gene promoter (new for plant genetic engineering) was for the first time used for the induced expression of the target Serratia marcescens RNase III gene. Upon infection with the tobacco mosaic virus (TMV), the transgenic plants of the obtained lines did not differ significantly from the control group in the level of TMV capsid protein accumulation. In general, no delay in the development of the infection symptoms was observed in transgenic plants as compared with the control group. The obtained transgenic plants represent a new model for the study of the biological role of endoribonucleases from the RNase III family, including in molecular mechanisms of resistance to pathogens.

  11. Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco.

    Science.gov (United States)

    Shukla, Pawan; Singh, Naveen Kumar; Kumar, Dilip; Vijayan, Sambasivam; Ahmed, Israr; Kirti, Pulugurtha Bharadwaja

    2014-06-01

    Usable male sterility systems have immense potential in developing hybrid varieties in crop plants, which can also be used as a biological safety containment to prevent horizontal transgene flow. Barnase-Barstar system developed earlier was the first approach to engineer male sterility in plants. In an analogous situation, we have evolved a system of inducing pollen abortion and male sterility in transgenic tobacco by expressing a plant gene coding for a protein with known developmental function in contrast to the Barnase-Barstar system, which deploys genes of prokaryotic origin, i.e., from Bacillus amyloliquefaciens. We have used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion in three transgenic lines. Furthermore, transcript analysis displayed the expression of cysteine protease in these male sterile lines and the expression of the protein was identified in western blot analysis using its polyclonal antibody raised in the rabbit system.

  12. Expression of the Native Cholera Toxin B Subunit Gene and Assembly as Functional Oligomers in Transgenic Tobacco Chloroplasts

    Science.gov (United States)

    Daniell, Henry; Lee, Seung-Bum; Panchal, Tanvi; Wiebe, Peter O.

    2012-01-01

    The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1% of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expresssion levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast-synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast- synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  14. Generation of Resistance to the Diphenyl Ether Herbicide, Oxyfluorfen, via Expression of the Bacillus subtilis Protoporphyrinogen Oxidase Gene in Transgenic Tobacco Plants.

    Science.gov (United States)

    Choi, K W; Han, O; Lee, H J; Yun, Y C; Moon, Y H; Kim, M; Kuk, Y I; Han, S U; Guh, J O

    1998-01-01

    In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.

  15. Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants.

    Science.gov (United States)

    Bao, Gegen; Zhuo, Chunliu; Qian, Chunmei; Xiao, Ting; Guo, Zhenfei; Lu, Shaoyun

    2016-01-01

    Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while L-ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co-expressing stylo 9-cis-epoxycarotenoid dioxygenase (SgNCED1) and yeast D-arabinono-1,4-lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild-type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co-expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought-responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold-responsive genes, but not in SgNCED1 plants. Co-expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold-responsive genes. Our result suggests that co-expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Expression of TaWRKY44, a wheat WRKY gene, in transgenic tobacco confers multiple abiotic stress tolerances

    Directory of Open Access Journals (Sweden)

    Xiatian eWang

    2015-08-01

    Full Text Available The WRKY transcription factors have been reported to be involved in various plant physiological and biochemical processes. In this study, we successfully assembled ten unigenes from expressed sequence tags (ESTs of wheat and designated them as TaWRKY44–TaWRKY53, respectively. Among these genes, a subgroup I gene, TaWRKY44, was found to be upregulated by treatments with PEG6000, NaCl, 4°C, abscisic acid (ABA, H2O2 and gibberellin (GA. The TaWRKY44-GFP fusion protein was localized to the nucleus of onion epidermal cells, and TaWRKY44 was able to bind to the core DNA sequences of TTGACC and TTAACC in yeast. The N-terminal of TaWRKY44 showed transcriptional activation activity. Expression of TaWRKY44 in tobacco plants conferred drought and salt tolerance and transgenic tobacco exhibited a higher survival rate, relative water content (RWC, soluble sugar, proline and superoxide dismutase (SOD content, as well as higher activities of catalase (CAT and peroxidase (POD, but less ion leakage (IL, lower contents of malondialdehyde (MDA, and H2O2. In addition, expression of TaWRKY44 also increased the seed germination rate in the transgenic lines under osmotic stress conditions while exhibiting a lower H2O2 content and higher SOD, CAT and POD activities. Expression of TaWRKY44 upregulated the expression of some reactive oxygen species (ROS-related genes and stress-responsive genes in tobacco under osmotic stresses. These data demonstrate that TaWRKY44 may act as a positive regulator in drought/salt/osmotic stress responses by either efficient ROS elimination through direct or indirect activation of the cellular antioxidant systems or activation of stress-associated gene expression.

  17. Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco.

    Science.gov (United States)

    Langen, Gregor; Imani, Jafargholi; Altincicek, Boran; Kieseritzky, Gernot; Kogel, Karl-Heinz; Vilcinskas, Andreas

    2006-05-01

    A cDNA encoding gallerimycin, a novel antifungal peptide from the greater wax moth Galleria mellonella, was isolated from a cDNA library of genes expressed during innate immune response in the caterpillars. Upon ectopic expression of gallerimycin in tobacco, using Agrobacterium tumefaciens as a vector, gallerimycin conferred resistance to the fungal pathogens Erysiphe cichoracearum and Sclerotinia minor. Quantification of gallerimycin mRNA in transgenic tobacco by real-time PCR confirmed transgenic expression under control of the inducible mannopine synthase promoter. Leaf sap and intercellular washing fluid from transgenic tobacco inhibited in vitro germination and growth of the fungal pathogens, demonstrating that gallerimycin is secreted into intercellular spaces. The feasibility of the use of gallerimycin to counteract fungal diseases in crop plants is discussed.

  18. The expression of analgesic-antitumor peptide (AGAP) from Chinese Buthus martensii Karsch in transgenic tobacco and tomato.

    Science.gov (United States)

    Lai, Linlin; Huang, Tingting; Wang, Yi; Liu, Yanfeng; Zhang, Jinghai; Song, Yongbo

    2009-05-01

    The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze-thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.

  19. Accumulation of nickel in transgenic tobacco

    Science.gov (United States)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TFtransgenic 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.

  20. Expression of chickpea CIPK25 enhances root growth and tolerance to dehydration and salt stress in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Mukesh Kumar Meena

    2015-09-01

    Full Text Available Calcium signaling plays an important role in adaptation and developmental processes in plants and animals. A class of calcium sensors, known as Calcineurin B-like (CBL proteins sense specific temporal changes in cytosolic Ca2+ concentration and regulate activities of a group of ser/thr protein kinases called CBL-interacting protein kinases (CIPKs. Although a number of CIPKs have been shown to play crucial roles in the regulation of stress signaling, no study on the function of CIPK25 or its orthologues has been reported so far. In the present study, an orthologue of Arabidopsis CIPK25 was cloned from chickpea (Cicer arietinum. CaCIPK25 gene expression in chickpea increased upon salt, dehydration, and different hormonal treatments. CaCIPK25 gene showed differential tissue-specific expression. 5'-upstream activation sequence (5'-UAS of the gene and its different truncated versions were fused to a reporter gene and studied in Arabidopsis to identify promoter regions directing its tissue-specific expression. Replacement of a conserved threonine residue with an aspartic acid at its catalytic site increased the kinase activity of CaCIPK25 by 2.5-fold. Transgenic tobacco plants overexpressing full-length and the high active versions of CaCIPK25 displayed a differential germination period and longer root length in comparison to the control plants. Expression of CaCIPK25 and its high active form differentially increased salt and water-deficit tolerance demonstrated by improved growth and reduced leaf chlorosis suggesting that the kinase activity of CaCIPK25 was required for these functions. Expressions of the abiotic stress marker genes were enhanced in the CaCIPK25-expressing tobacco plants. Our results suggested that CaCIPK25 functions in root development and abiotic stress tolerance.

  1. Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants.

    Science.gov (United States)

    Wang, Yuling; Zhang, Xinfu; Yang, Shaolan; Wang, Caihong; Lu, Guilong; Wang, Ran; Yang, Yingjie; Li, Dingli

    2017-12-30

    Lignin, a natural macromolecular compound, plays an important role in the texture and taste of fruit. Hard end is a physiological disorder of pear fruit, in which the level of lignification in fruit tissues is dramatically elevated. Cinnamyl alcohol dehydrogenase and expansin genes (PpCAD2 and PpEXP2, respectively) exhibit higher levels of expression in 'Whangkeumbae' (Pyrus pyrifolia) pear fruit exhibiting this physiological disorder, relative to control fruit without symptoms. These genes were isolated from pear fruit and subsequently expressed in tobacco (Nicotiana tabacum) to investigate their function. Histochemical staining for lignin revealed that the degree of lignification in leaf veins and stem tissues increased in plants transformed with sense constructs and decreased in plants transformed with antisense constructs of PpCAD2. The expression of native NtCADs was also inhibited in the antisense PpCAD2 transgenic tobacco. Sense and antisense PpCAD2 transgenic tobacco exhibited an 86.7% increase and a 60% decrease in CAD activity, respectively, accompanied by a complementary response in lignin content in root tissues. The basal portion of the stem in PpEXP2 transgenic tobacco was bent and highly lignified. Additionally, the level of cellulose also increased in the stem of PpEXP2 transgenic tobacco. Collectively, these results suggested that PpCAD2 and PpEXP2 genes play a significant role in lignin accumulation in transgenic tobacco plants, and it is inferred that these two genes may also participate in the increased lignification observed in hard end pear fruit. Copyright © 2017. Published by Elsevier B.V.

  2. Resistance to multiple viruses in transgenic tobacco expressing fused, tandem repeat, virus-derived double-stranded RNAs.

    Science.gov (United States)

    Chung, Bong Nam; Palukaitis, Peter

    2011-12-01

    Transgenic tobacco plants expressing fused, tandem, inverted-repeat, double-stranded RNAs derived either from the three viruses [potato virus Y (PVY), potato virus A (PVA), and potato leafroll virus (PLRV)] or the five viruses [PVY, PVA, PLRV as well as tobacco rattle virus (TRV), and potato mop-top virus (PMTV)] were generated in this study to examine whether resistance could be achieved against these three viruses or five viruses, respectively, in the same plant. The transgenic lines were engineered to produce 600- or 1000-bp inverted hairpin transcripts with an intron, in two orientations each, which were processed to silencing-inducing RNAs (siRNAs). Fewer lines were regenerated from the transformants with either 1000-bp inverted hairpin transcripts, or a sense-intron-antisense orientation versus antisense-intron-sense orientation. Resistances to PVA and two strains of PVY (-O and -N) were achieved in plants from most of lines examined, as well as resistance to co-infection by a mixture of PVY-O and PVA, applied to the plants by either rub inoculation or using aphids. This was regardless of the orientation of the inserted sequences for the 600-bp insert lines, but only for one orientation of the 1000-bp insert lines. The lines containing the 1000-bp inserts also showed resistance to infection by TRV inoculated by rub inoculation and PMTV inoculated by grafting. However, all the lines showed only low-to-moderate (15-43%) resistance to infection by PLRV transmitted by aphids. The resistances to the various viruses correlated with the levels of accumulation of siRNAs, indicating that the multiple resistances were achieved by RNA silencing.

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

    Science.gov (United States)

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

    2010-08-01

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

  4. Transgenic tobacco plants expressing BoRS1 gene from Brassica ...

    Indian Academy of Sciences (India)

    Unknown

    BoRS1 gene; dry matter accumulation; leaf chlorophyll content; relative water content; tobacco; water stress http://www.ias.ac.in/ ... techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the ... treme environments during long history of gene evolu- tion, which can be used ...

  5. Transgenic tobacco plants expressing BoRS1 gene from Brassica ...

    Indian Academy of Sciences (India)

    Unknown

    Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the. Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through ...

  6. Transgenic tobacco plants expressing BoRS1 gene from Brassica ...

    Indian Academy of Sciences (India)

    Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, the Brassica oleracea var. acephala BoRS1 gene was transferred into tobacco through ...

  7. Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.

    Science.gov (United States)

    Nahar, Noor; Rahman, Aminur; Nawani, Neelu N; Ghosh, Sibdas; Mandal, Abul

    2017-11-01

    We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200μM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100μM arsenate for 35days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28μg/g d wt.) than that found in the shoots of non-transgenic controls (40μg/g d wt.). However, the arsenic content in the roots of transgenic plants was significantly higher (2400μg/g d. wt.) than that (2100μg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Simultaneous Expression of PDH45 with EPSPS Gene Improves Salinity and Herbicide Tolerance in Transgenic Tobacco Plants.

    Science.gov (United States)

    Garg, Bharti; Gill, Sarvajeet S; Biswas, Dipul K; Sahoo, Ranjan K; Kunchge, Nandkumar S; Tuteja, Renu; Tuteja, Narendra

    2017-01-01

    To cope with the problem of salinity- and weed-induced crop losses, a multi-stress tolerant trait is need of the hour but a combinatorial view of such traits is not yet explored. The overexpression of PDH45 (pea DNA helicase 45) and EPSPS (5-enoylpruvyl shikimate-3-phosphate synthase) genes have been reported to impart salinity and herbicide tolerance. Further, the understanding of mechanism and pathways utilized by PDH45 and EPSPS for salinity and herbicide tolerance will help to improve the crops of economical importance. In the present study, we have performed a comparative analysis of salinity and herbicide tolerance to check the biochemical parameters and antioxidant status of tobacco transgenic plants. Collectively, the results showed that PDH45 overexpressing transgenic lines display efficient tolerance to salinity stress, while PDH45+EPSPS transgenics showed tolerance to both the salinity and herbicide as compared to the control [wild type (WT) and vector control (VC)] plants. The activities of the components of enzymatic antioxidant machinery were observed to be higher in the transgenic plants indicating the presence of an efficient antioxidant defense system which helps to cope with the stress-induced oxidative-damages. Photosynthetic parameters also showed significant increase in PDH45 and PDH45+EPSPS overexpressing transgenic plants in comparison to WT, VC and EPSPS transgenic plants under salinity stress. Furthermore, PDH45 and PDH45+EPSPS synergistically modulate the jasmonic acid and salicylic acid mediated signaling pathways for combating salinity stress. The findings of our study suggest that pyramiding of the PDH45 gene with EPSPS gene renders host plants tolerant to salinity and herbicide by enhancing the antioxidant machinery thus photosynthesis.

  9. Multiple Different Defense Mechanisms Are Activated in the Young Transgenic Tobacco Plants Which Express the Full Length Genome of the Tobacco Mosaic Virus, and Are Resistant against this Virus

    Science.gov (United States)

    Jada, Balaji; Soitamo, Arto J.; Siddiqui, Shahid Aslam; Murukesan, Gayatri; Aro, Eva-Mari; Salakoski, Tapio; Lehto, Kirsi

    2014-01-01

    Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter (Siddiqui et al., 2007. Mol Plant Microbe Interact, 20: 1489–1494). Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7–8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses (Potato viruses Y, A, and X) induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV

  10. Multiple different defense mechanisms are activated in the young transgenic tobacco plants which express the full length genome of the Tobacco mosaic virus, and are resistant against this virus.

    Science.gov (United States)

    Jada, Balaji; Soitamo, Arto J; Siddiqui, Shahid Aslam; Murukesan, Gayatri; Aro, Eva-Mari; Salakoski, Tapio; Lehto, Kirsi

    2014-01-01

    Previously described transgenic tobacco lines express the full length infectious Tobacco mosaic virus (TMV) genome under the 35S promoter (Siddiqui et al., 2007. Mol Plant Microbe Interact, 20: 1489-1494). Through their young stages these plants exhibit strong resistance against both the endogenously expressed and exogenously inoculated TMV, but at the age of about 7-8 weeks they break into TMV infection, with typical severe virus symptoms. Infections with some other viruses (Potato viruses Y, A, and X) induce the breaking of the TMV resistance and lead to synergistic proliferation of both viruses. To deduce the gene functions related to this early resistance, we have performed microarray analysis of the transgenic plants during the early resistant stage, and after the resistance break, and also of TMV-infected wild type tobacco plants. Comparison of these transcriptomes to those of corresponding wild type healthy plants indicated that 1362, 1150 and 550 transcripts were up-regulated in the transgenic plants before and after the resistance break, and in the TMV-infected wild type tobacco plants, respectively, and 1422, 1200 and 480 transcripts were down-regulated in these plants, respectively. These transcriptome alterations were distinctly different between the three types of plants, and it appears that several different mechanisms, such as the enhanced expression of the defense, hormone signaling and protein degradation pathways contributed to the TMV-resistance in the young transgenic plants. In addition to these alterations, we also observed a distinct and unique gene expression alteration in these plants, which was the strong suppression of the translational machinery. This may also contribute to the resistance by slowing down the synthesis of viral proteins. Viral replication potential may also be suppressed, to some extent, by the reduction of the translation initiation and elongation factors eIF-3 and eEF1A and B, which are required for the TMV replication

  11. Cloning and functional analysis in transgenic tobacco of a tapetum ...

    African Journals Online (AJOL)

    Jane

    2010-10-11

    Oct 11, 2010 ... State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University,. Beijing ..... of GUS activity in the anther and pollen of transgenic tobacco T0 plants conferred by the constructs. ..... A chalcone synthase-like gene is highly expressed in the tapetum of.

  12. Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO2.

    Science.gov (United States)

    Occhialini, Alessandro; Lin, Myat T; Andralojc, P John; Hanson, Maureen R; Parry, Martin A J

    2016-01-01

    Introducing a carbon-concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO2 fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild-type and required supplementary CO2 . Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild-type growth rates, although still requiring elevated CO2 . We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non-native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen-use efficiency that may be achieved provided that adequate CO2 is available near the enzyme. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  13. Dehydrins Impart Protection against Oxidative Stress in Transgenic Tobacco Plants.

    Science.gov (United States)

    Halder, Tanmoy; Upadhyaya, Gouranga; Basak, Chandra; Das, Arup; Chakraborty, Chandrima; Ray, Sudipta

    2018-01-01

    Environmental stresses generate reactive oxygen species (ROS) which might be detrimental to the plants when produced in an uncontrolled way. However, the plants ameliorate such stresses by synthesizing antioxidants and enzymes responsible for the dismutation of ROS. Additionally, the dehydrins were also able to protect the inactivation of the enzyme lactate dehydrogenase against hydroxyl radicals (OH ⋅ ) generated during Fenton's reaction. SbDhn1 and SbDhn2 overexpressing transgenic tobacco plants were able to protect against oxidative damage. Transgenic tobacco lines showed better photosynthetic efficiency along with high chlorophyll content, soluble sugar and proline. However, the malonyl dialdehyde (MDA) content was significantly lower in transgenic lines. Experimental evidence demonstrates the protective effect of dehydrins on electron transport chain in isolated chloroplast upon methyl viologen (MV) treatment. The transgenic tobacco plants showed significantly lower superoxide radical generation () upon MV treatment. The accumulation of the H 2 O 2 was also lower in the transgenic plants. Furthermore, in the transgenic plants the expression of ROS scavenging enzymes was higher compared to non-transformed (NT) or vector transformed (VT) plants. Taken together these data, during oxidative stress dehydrins function by scavenging the () directly and also by rendering protection to the enzymes responsible for the dismutation of () thereby significantly reducing the amount of hydrogen peroxides formed. Increase in proline content along with other antioxidants might also play a significant role in stress amelioration. Dehydrins thus function co-operatively with other protective mechanisms under oxidative stress conditions rendering protection in stress environment.

  14. Dehydrins Impart Protection against Oxidative Stress in Transgenic Tobacco Plants

    Directory of Open Access Journals (Sweden)

    Tanmoy Halder

    2018-02-01

    Full Text Available Environmental stresses generate reactive oxygen species (ROS which might be detrimental to the plants when produced in an uncontrolled way. However, the plants ameliorate such stresses by synthesizing antioxidants and enzymes responsible for the dismutation of ROS. Additionally, the dehydrins were also able to protect the inactivation of the enzyme lactate dehydrogenase against hydroxyl radicals (OH⋅ generated during Fenton’s reaction. SbDhn1 and SbDhn2 overexpressing transgenic tobacco plants were able to protect against oxidative damage. Transgenic tobacco lines showed better photosynthetic efficiency along with high chlorophyll content, soluble sugar and proline. However, the malonyl dialdehyde (MDA content was significantly lower in transgenic lines. Experimental evidence demonstrates the protective effect of dehydrins on electron transport chain in isolated chloroplast upon methyl viologen (MV treatment. The transgenic tobacco plants showed significantly lower superoxide radical generation ( upon MV treatment. The accumulation of the H2O2 was also lower in the transgenic plants. Furthermore, in the transgenic plants the expression of ROS scavenging enzymes was higher compared to non-transformed (NT or vector transformed (VT plants. Taken together these data, during oxidative stress dehydrins function by scavenging the ( directly and also by rendering protection to the enzymes responsible for the dismutation of ( thereby significantly reducing the amount of hydrogen peroxides formed. Increase in proline content along with other antioxidants might also play a significant role in stress amelioration. Dehydrins thus function co-operatively with other protective mechanisms under oxidative stress conditions rendering protection in stress environment.

  15. Split-Cre complementation restores combination activity on transgene excision in hair roots of transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Mengling Wen

    Full Text Available 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.

  16. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco.

    Science.gov (United States)

    Kater, M M; Franken, J; van Aelst, A; Angenent, G C

    2000-08-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion.

  17. Stress-Inducible Expression of an F-box Gene TaFBA1 from Wheat Enhanced the Drought Tolerance in Transgenic Tobacco Plants without Impacting Growth and Development.

    Science.gov (United States)

    Kong, Xiangzhu; Zhou, Shumei; Yin, Suhong; Zhao, Zhongxian; Han, Yangyang; Wang, Wei

    2016-01-01

    E3 ligase plays an important role in the response to many environment stresses in plants. In our previous study, constitutive overexpression of an F-box protein gene TaFBA1 driven by 35S promoter improved the drought tolerance in transgenic tobacco plants, but the growth and development in transgenic plants was altered in normal conditions. In this study, we used stress-inducible promoter RD29A instead of 35S promoter, as a results, the stress-inducible transgenic tobacco plants exhibit a similar phenotype with wild type (WT) plants. However, the drought tolerance of the transgenic plants with stress-inducible expressed TaFBA1 was enhanced. The improved drought tolerance of transgenic plants was indicated by their higher seed germination rate and survival rate, greater biomass and photosynthesis than those of WT under water stress, which may be related to their greater water retention capability and osmotic adjustment. Moreover, the transgenic plants accumulated less reactive oxygen species, kept lower MDA content and membrane leakage under water stress, which may be related to their higher levels of antioxidant enzyme activity and upregulated gene expression of some antioxidant enzymes. These results suggest that stress induced expression of TaFBA1 confers drought tolerance via the improved water retention and antioxidative compete ability. Meanwhile, this stress-inducible expression strategy by RD29A promoter can minimize the unexpectable effects by 35S constitutive promoter on phenotypes of the transgenic plants.

  18. Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration.

    Science.gov (United States)

    Gayen, Srimonta; Mandal, Chandi Charan; Samanta, Milan Kumar; Dey, Avishek; Sen, Soumitra Kumar

    2016-04-01

    Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

  19. Ectopic Expression of the Coleus R2R3 MYB-Type Proanthocyanidin Regulator Gene SsMYB3 Alters the Flower Color in Transgenic Tobacco.

    Directory of Open Access Journals (Sweden)

    Qinlong Zhu

    Full Text Available Proanthocyanidins (PAs play an important role in plant disease defense and have beneficial effects on human health. We isolated and characterized a novel R2R3 MYB-type PA-regulator SsMYB3 from a well-known ornamental plant, coleus (Solenostemon scutellarioides, to study the molecular regulation of PAs and to engineer PAs biosynthesis. The expression level of SsMYB3 was correlated with condensed tannins contents in various coleus tissues and was induced by wounding and light. A complementation test in the Arabidopsis tt2 mutant showed that SsMYB3 could restore the PA-deficient seed coat phenotype and activated expression of the PA-specific gene ANR and two related genes, DFR and ANS. In yeast two-hybrid assays, SsMYB3 interacted with the Arabidopsis AtTT8 and AtTTG1 to reform the ternary transcriptional complex, and also interacted with two tobacco bHLH proteins (NtAn1a and NtJAF13-1 and a WD40 protein, NtAn11-1. Ectopic overexpression of SsMYB3 in transgenic tobacco led to almost-white flowers by greatly reducing anthocyanin levels and enhancing accumulation of condensed tannins. This overexpression of SsMYB3 upregulated the key PA genes (NtLAR and NtANR and late anthocyanin structural genes (NtDFR and NtANS, but downregulated the expression of the final anthocyanin gene NtUFGT. The formative SsMYB3-complex represses anthocyanin accumulation by directly suppressing the expression of the final anthocyanin structural gene NtUFGT, through competitive inhibition or destabilization of the endogenous NtAn2-complex formation. These results suggested that SsMYB3 may form a transcription activation complex to regulate PA biosynthesis in the Arabidopsis tt2 mutant and transgenic tobacco. Our findings suggest that SsMYB3 is involved in the regulation of PA biosynthesis in coleus and has the potential as a molecular tool for manipulating biosynthesis of PAs in fruits and other crops using metabolic engineering.

  20. Phytoextraction of Cadmium from Contaminated Soil Using Transgenic Tobacco Plants

    OpenAIRE

    , Hatice Daghan; , Andreas Schaeffer; , Rainer Fischer; , Ulrich Commandeur

    2008-01-01

    Phytoremediation, the use of plants to clean up toxic metals from contaminated soil or water, represents one of the most promising, effective and technically affordable solutions. Transgenic tobacco plants, constitutively expressing Metallothionein H (MTII) genes from Chinese hamster (Ch) and Saccharomyces cerevisiae (Sc), were designed for phytoextraction of cadmium (Cd) contaminated soil. The recombinant proteins were targeted to the vacuole, as conŞrmed by immunogold labelling. In this stu...

  1. Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene.

    Science.gov (United States)

    Badejo, Adebanjo A; Tanaka, Nobukazu; Esaka, Muneharu

    2008-01-01

    GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type.

  2. Lipid transfer proteins in coffee: isolation of Coffea orthologs, Coffea arabica homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants.

    Science.gov (United States)

    Cotta, Michelle G; Barros, Leila M G; de Almeida, Juliana D; de Lamotte, Fréderic; Barbosa, Eder A; Vieira, Natalia G; Alves, Gabriel S C; Vinecky, Felipe; Andrade, Alan C; Marraccini, Pierre

    2014-05-01

    The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5' deletions, were fused to the reporter gene β-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.

  3. Overexpression of CsANR increased flavan-3-ols and decreased anthocyanins in transgenic tobacco.

    Science.gov (United States)

    Kumar, Vinay; Yadav, Sudesh Kumar

    2013-06-01

    Anthocyanins and flavan-3-ols are distributed widely in plants and synthesized by a common biosynthetic pathway. Anthocyanin reductase (ANR) represents branching-point enzyme of this pathway converting anthocyanidins to flavan-3-ols. Since tea contains highest amount of flavonoids, a cDNA encoding anthocyanin reductase from tea (CsANR) was overexpressed in transgenic tobacco to check the influence on anthocyanin and flavan-3-ols. The transgenic tobacco was confirmed by genomic PCR and expression of transgene was analyzed through semiquantitative PCR. Interestingly flowers of transgenic tobacco were light pink/white in color instead of dark pink in wild tobacco, documenting the decrease in anthocyanins content. Upon measurement, flower anthocyanin content was found to be lesser. While flavan-3-ols (epicatechin and epigallocatechin) contents were increased in leaf tissue of transgenic lines. The expressions of other endogenous flavonoid biosynthetic pathway genes in different floral parts (sepal, petal, stamen, and carpel) of CsANR overexpressing tobacco as well as wild tobacco were analyzed. The transcript levels of PAL and CHI genes were downregulated, while transcript levels of F3H, FLS, CHS, ANR1, and ANR2 genes were upregulated in all floral parts of CsANR transgenic plants compared to wild tobacco. The expressions of DFR and ANS genes were also spatially modulated in different floral parts due to overexpression of CsANR. Thus, CsANR overexpression increased flavan-3-ols and decreased anthocyanin content by modulating the expressions of various flavonoid biosynthetic pathway genes in flower of tobacco. These changes might be responsible for the observed pollen tube in the pollens of CsANR overexpressing transgenic tobacco when they were still in the anther before pollination.

  4. Evaluation of the defense system in chloroplasts to photooxidative stress caused by paraquat using transgenic tobacco plants expressing catalase from Escherichia coli.

    Science.gov (United States)

    Miyagawa, Y; Tamoi, M; Shigeoka, S

    2000-03-01

    We evaluated the defense system in chloroplasts to photooxidative stress imposed by paraquat treatment under illumination in transgenic tobacco plants with increased tolerance to drought stress at a high light intensity produced by catalase from Escherichia coli targeted to chloroplasts [Shikanai et al. (1998) FEBS Lett. 428: 47]. At 24 h after the paraquat application, Chl was destroyed in the wild-type plants, but not in transgenic plants. Photosynthetic activities monitored by CO2 fixation and Chl fluorescence were much less affected by the paraquat treatment in transgenic lines. The activities of chloroplastic ascorbate peroxidase (APX) isoenzymes decreased in parallel with the depletion of ascorbate (AsA) in leaves in both lines. Paraquat treatment had no effect on the transcript level of chloroplastic APX isoenzymes, while it significantly lowered the level of their proteins. These data suggest that the depletion of AsA in chloroplasts under severe stress conditions inactivates and degrades chloroplastic APX isoenzymes.

  5. GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange Expressão do gene GUS controlado por promotor de citros em plantas transgênicas de tabaco e laranja 'Valência'

    Directory of Open Access Journals (Sweden)

    Fernando Alves de Azevedo

    2006-11-01

    Full Text Available The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL gene promoter (CsPP. Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.O objetivo deste trabalho foi realizar a transformação de plantas de tabaco e laranja 'Valência' com o gene GUS controlado pelo promotor do gene da fenilalanina amônia-liase (PAL de citros (CsPP. Foi realizada transformação genética por meio do co-cultivo de explantes de tabaco e laranja 'Valência' com Agrobacterium tumefaciens que continha o vetor binário CsPP-GUS/2201. Após a transformação e a regeneração, a detecção da atividade de GUS por ensaios histoquímicos revelou que o promotor CsPP, preferencialmente, mas não exclusivamente, confere expressão gênica em tecidos do xilema de tabaco. Expressão mais baixa de GUS também foi detectada na região de tecido de pecíolo, em plantas transgênicas (CsPP de tabaco e laranja 'Valência'.

  6. Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

    International Nuclear Information System (INIS)

    McCormac, A.; Whitelam, G.; Smith, H.

    1992-01-01

    A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162-170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants

  7. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    Science.gov (United States)

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  8. Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system

    Science.gov (United States)

    Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

    2012-01-01

    The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g−1 of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples. PMID:22428884

  9. Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

    Science.gov (United States)

    Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

    2012-01-01

    The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

  10. Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco.

    Science.gov (United States)

    da Silva, Leonardo F; Dias, Cristiano V; Cidade, Luciana C; Mendes, Juliano S; Pirovani, Carlos P; Alvim, Fátima C; Pereira, Gonçalo A G; Aragão, Francisco J L; Cascardo, Júlio C M; Costa, Marcio G C

    2011-07-01

    Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).

  11. Potential of MuS1 Transgenic Tobacco for Phytoremediation of the Urban Soils Contaminated with Cadmium

    Science.gov (United States)

    Kim, K. H.; Kim, Y. N.; Kim, S. H.

    2010-05-01

    Urban soils are prone to contamination by trace elements such as Cd, Cu, Pb and Zn. Phytoremediation is one of the attractive remediation methods for soils contaminated with trace elements due to its non-destructive and environmentally-friendly characteristic. Scientists have tried to find hyper-accumulator plants in nature or to develop transgenic plant through genetic engineering. This study was carried out to identify a potential of MuS1 transgenic tobacco for phytoremediation of the urban soils contaminated with Cd. MuS1 is known as a multiple stress related gene with several lines. The previous study using RT-PCR showed that the expression of MuS1 gene in tobacco plant induced tolerance to Cd stress. For this study, MuS1 transgenic tobacco and wild-type tobacco (control) were cultivated in a hydroponic system treated with Cd (0, 50, 100 and 200μM Cd) for 3 weeks. At harvest, both tobacco and nutrient solution were collected and were analyzed for Cd. Effect of Cd treatment on morphological change of the tobacco leaves was also observed by variable-pressure scanning electron microscopy (VP-SEM). The tolerance of MuS1 transgenic tobacco to Cd stress was better than that of wild-type tobacco at all Cd levels. Especially, wild-type tobacco showed chlorosis and withering with 200μM Cd treatment, whereas MuS1 transgenic tobacco gradually recovered from Cd damage. Wild-type tobacco accumulated more Cd (4.65mg per plant) than MuS1 transgenic tobacco (2.37mg per plant) with 200μM Cd treatment. Cd translocation rate from root to leaves was 81.8 % for wild-type tobacco compared to 37.1 % for MuS1 transgenic tobacco. Result of VP-SEM showed that the number of trichome in the leaves for wild-type tobacco increased in comparison with that for untreated samples after 3 weeks, while that for MuS1 transgenic tobacco was not changed by Cd treatment. Results showed that the mechanism of the recovery of the MuS1 tobacco plant was not by high level of Cd uptake and accumulation

  12. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

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

  13. Cloning and functional analysis in transgenic tobacco of a tapetum ...

    African Journals Online (AJOL)

    The 5'-flanking region of 1174 bp upstream of the translation start point (TSP) of a reported Arabidopsis anther-specific gene, Anther7 gene (ATA7), which putatively encodes a protein related to lipid transfer protein, was cloned and functionally analyzed in transgenic tobacco after been fused with β- glucuronidase (GUS) ...

  14. Promoter Sequences for Defining Transgene Expression

    Science.gov (United States)

    Jones, Huw D.; Sparks, Caroline A.

    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.

  15. Positron emission tomography : measurement of transgene expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vaalburg, W

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

  16. Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production.

    Science.gov (United States)

    Nookaraju, Akula; Pandey, Shashank K; Fujino, Takeshi; Kim, Ju Young; Suh, Mi Chung; Joshi, Chandrashekhar P

    2014-07-01

    We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes. The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the "food versus fuel" issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and leafy cotyledon2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80% increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.

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

    Science.gov (United States)

    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

    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.

  18. Photosynthesis and protective mechanisms during ageing in transgenic tobacco leaves with over-expressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content

    Czech Academy of Sciences Publication Activity Database

    Mýtinová, Zuzana; Haisel, Daniel; Wilhelmová, Naděžda

    2006-01-01

    Roč. 44, č. 4 (2006), s. 599-605 ISSN 0300-3604 R&D Projects: GA ČR GA522/03/0312 Institutional research plan: CEZ:AV0Z50380511 Keywords : senescence * cytokinins * carotenoids * chlorophyll * xanthophylls cycle * photosynthetic efficiency * chlorophyll fluorescence * tobacco Subject RIV: CE - Biochemistry Impact factor: 0.782, year: 2006

  19. The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89

    Directory of Open Access Journals (Sweden)

    Yu Liu

    Full Text Available BACKGROUND: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1 gene was isolated from apple peel (var. white winterpearmain, and transformed into tobacco (Nicotiana tabacum NC89. The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT. Our studies focused on the transgenic tobacco phenotype. RESULTS: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn in transgenic plants. Reactive oxygen species (ROS such as hydrogen peroxide (H2O2 and superoxide radicals (O2._ had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage. CONCLUSIONS: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS played an important role in the premature aging of transgenic plants.

  20. Tobacco OPBP1 Enhances Salt Tolerance and Disease Resistance of Transgenic Rice

    Directory of Open Access Journals (Sweden)

    Xujun Chen

    2008-12-01

    Full Text Available Osmotin promoter binding protein 1 (OPBP1, an AP2/ERF transcription factor of tobacco, has been demonstrated to function in disease resistance and salt tolerance in tobacco. To increase stress tolerant capability of rice, we generated rice plants with an OPBP1 overexpressing construct. Salinity shock treatment with 250 mM NaCl indicated that most of the OPBP1 transgenic plants can survive, whereas the control seedlings cannot. Similar recovery was found by using the seedlings grown in 200 mM NaCl for two weeks. The OPBP1 transgenic and control plants were also studied for oxidative stress tolerance by treatment with paraquat, showing the transgenic lines were damaged less in comparison with the control plants. Further, the OPBP1 overexpression lines exhibited enhanced resistance to infections of Magnaporthe oryzae and Rhizoctonia solani pathogens. Gene expressing analysis showed increase in mRNA accumulation of several stress related genes. These results suggest that expression of OPBP1 gene increase the detoxification capability of rice.

  1. Expression of multiple proteins in transgenic plants

    Science.gov (United States)

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  3. Protection of the photosynthetic apparatus from extreme dehydration and oxidative stress in seedlings of transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Concepción Almoguera

    Full Text Available A genetic program that in sunflower seeds is activated by Heat Shock transcription Factor A9 (HaHSFA9 has been analyzed in transgenic tobacco seedlings. The ectopic overexpression of the HSFA9 program protected photosynthetic membranes, which resisted extreme dehydration and oxidative stress conditions. In contrast, heat acclimation of seedlings induced thermotolerance but not resistance to the harsh stress conditions employed. The HSFA9 program was found to include the expression of plastidial small Heat Shock Proteins that accumulate only at lower abundance in heat-stressed vegetative organs. Photosystem II (PSII maximum quantum yield was higher for transgenic seedlings than for non-transgenic seedlings, after either stress treatment. Furthermore, protection of both PSII and Photosystem I (PSI membrane protein complexes was observed in the transgenic seedlings, leading to their survival after the stress treatments. It was also shown that the plastidial D1 protein, a labile component of the PSII reaction center, and the PSI core protein PsaB were shielded from oxidative damage and degradation. We infer that natural expression of the HSFA9 program during embryogenesis may protect seed pro-plastids from developmental desiccation.

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

    Science.gov (United States)

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

    2014-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Shiyi Zhou

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

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

    African Journals Online (AJOL)

    Study on the characteristics of integration and expression is the basis of genetic stability of foreign genes in transgenic trees. To obtain insight into the relationship of transgene copy number and expression level, we screened 22 transgenic birch lines. Southern blot analysis of the transgenic birch plants indicated that the ...

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... Study on the characteristics of integration and expression is the basis of genetic stability of foreign genes in transgenic trees. To obtain insight into the relationship of transgene copy number and expression level, we screened 22 transgenic birch lines. Southern blot analysis of the transgenic birch.

  8. RNAi-mediated mortality of the whitefly through transgenic expression of double-stranded RNA homologous to acetylcholinesterase and ecdysone receptor in tobacco plants

    Science.gov (United States)

    The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses affecting plants worldwide. Using RNA interference (RNAi) to downregulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus dise...

  9. Colocalization of barley lectin and sporamin in vacuoles of transgenic tobacco plants

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, M.R.; Borkhsenious, O.N.; Raikhel, N.V. (Michigan State Univ., East Lansing (United States)); Matsuoka, K.; Nakamura, K. (Nagoya Univ. (Japan))

    1993-02-01

    Various targeting motifs have been identified for plant proteins delivered to the vacuole. For barley (Hordeum vulgare) lectin, a typical Gramineae lectin and defense-related protein, the vacuolar information is contained in a carboxyl-terminal propeptide. In contrast, the vacuolar targeting information of sporamin, a storage protein from the tuberous roots of the sweet potato (Ipomoea batatas), is encoded in an amino-terminal propeptide. Both proteins were expressed simultaneously in transgenic tobacco plants to enable analysis of their posttranslational processing and subcellular localization by pulse-chase labeling and electron-microscopic immunocytochemical methods. The pulse-chase experiments demonstrated that processing and delivery to the vacuole are not impaired by the simultaneous expression of barley lectin and sporamin. Both proteins were targeted quantitatively to the vacuole, indication that the carboxyl-terminal and amino-terminal propeptided are equally recognized by the vacuolar protein-sorting machinery. Double-labeling experiments showed that barley lectin and sporamin accumulate in the same vacuole of transgenic tobacco (Nicotiana tabacum) leaf and root cells. 35 refs., 5 figs., 3 tabs.

  10. Protective effect of oral administration of transgenic tobacco seeds against verocytotoxic Escherichia coli strain in piglets.

    Science.gov (United States)

    Rossi, Luciana; Dell'Orto, Vittorio; Vagni, Simona; Sala, Vittorio; Reggi, Serena; Baldi, Antonella

    2014-03-01

    The use of transgenic plants as delivery system for antigenic proteins is attractive for its simplicity and increases likelihood for local immune response at sites of infection. The aim of this study was to evaluate the protective effect of oral administration of tobacco seeds, expressing the FedA, the major protein of the F18 adhesive fimbriae, and B subunit of verocytotoxin, against verocytotoxin-producing E. coli (VTEC) strain in piglets. Forty-three early weaned piglets, were randomly divided into 4 experimental groups: 3 test groups and a control. Treatment groups orally received a bolus, with different dose of tobacco seeds on 0, 1, 2, 14 days post primary administration. After challenge, with 1*10(10) CFU of O138 Escherichia coli strain, piglets showed clinical scores significantly higher in the control group compared to orally immunized groups (P administration of recombinant tobacco seeds expressing antigenic proteins against VTEC strains can induce a protective effect against challenger strain in piglets.

  11. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants

    OpenAIRE

    Ma, J.K.-C.; Drossard, J.; Lewis, D.; Altmann, F.; Boyle, J.; Christou, P.; Cole, T.; Dale, P.; van Dolleweerd, C.J.; Isitt, V.; Katinger, D.; Lobedan, M.; Mertens, H.; Paul, M.J.; Rademacher, T.

    2015-01-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehens...

  12. PDH45 overexpressing transgenic tobacco and rice plants provide salinity stress tolerance via less sodium accumulation.

    Science.gov (United States)

    Nath, Manoj; Garg, Bharti; Sahoo, Ranjan Kumar; Tuteja, Narendra

    2015-01-01

    Salinity stress negatively affects the crop productivity worldwide, including that of rice. Coping with these losses is a major concern for all countries. The pea DNA helicase, PDH45 is a unique member of helicase family involved in the salinity stress tolerance. However, the exact mechanism of the PDH45 in salinity stress tolerance is yet to be established. Therefore, the present study was conducted to investigate the mechanism of PDH45-mediated salinity stress tolerance in transgenic tobacco and rice lines along with wild type (WT) plants using CoroNa Green dye based sodium localization in root and shoot sections. The results showed that under salinity stress root and shoot of PDH45 overexpressing transgenic tobacco and rice accumulated less sodium (Na(+)) as compared to their respective WT. The present study also reports salinity tolerant (FL478) and salinity susceptible (Pusa-44) varieties of rice accumulated lowest and highest Na(+) level, respectively. All the varieties and transgenic lines of rice accumulate differential Na(+) ions in root and shoot. However, roots accumulate high Na(+) as compared to the shoots in both tobacco and rice transgenic lines suggesting that the Na(+) transport in shoot is somehow inhibited. It is proposed that the PDH45 is probably involved in the deposition of apoplastic hydrophobic barriers and consequently inhibit Na(+) transport to shoot and therefore confers salinity stress tolerance to PDH45 overexpressing transgenic lines. This study concludes that tobacco (dicot) and rice (monocot) transgenic plants probably share common salinity tolerance mechanism mediated by PDH45 gene.

  13. A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    Zhibin Liu

    2013-12-01

    Full Text Available Ascorbate peroxidase (APX plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP fusion protein. Quantitative polymerase chain reaction (qPCR analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT, the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H2O2 content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS-scavenging capacity under short-term NaCl stress conditions.

  14. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

    Directory of Open Access Journals (Sweden)

    Burgos Lorenzo

    2010-07-01

    Full Text Available Abstract Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII transgene as well as of an internal control (β-actin, used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot

  15. RNAi-mediated resistance to SMV and BYMV in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Lo Thi Mai Thu

    2016-09-01

    Full Text Available Soybean mosaic virus (SMV and bean yellow mosaic virus (BYMV are two typical types of viruses that cause mosaic in soybean plants. Multiple viral infections at the same site can lead to 66% to 80% yield reduction. We have aimed to improve SMV and BYMV resistance in Vietnamese soybeans using gene transfer techniques under the mechanism of RNAi. In this study, we present newly generated transgenic tobacco plants carrying RNAi [CPi (SMV-BYMV] resistance to the two types of viruses; 73.08% of transgenic tobacco lines proved to be fully resistant to SMV and BYMV. In addition, the number of virus copies in transgenic tobacco plants was reduced on average by more than 51% compared to the control plants (wild type. This promising result shows the potential of transerring the CPi (SMV-BYMV structure in soybean to increase resistance of soybean to SMV and BYMV and advance the aims of antiviral soybean breeding in Vietnam.

  16. Identification of the CAD gene from Eucalyptus urophylla GLU4 and its functional analysis in transgenic tobacco.

    Science.gov (United States)

    Chen, B W; Xiao, Y F; Li, J J; Liu, H L; Qin, Z H; Gai, Y; Jiang, X N

    2016-12-02

    Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin biosynthesis. The genus Eucalyptus belongs to the family Myrtaceae, which is the main cultivated species in China. Eucalyptus urophylla GLU4 (GLU4) is widely grown in Guangxi. It is preferred for pulping because of its excellent cellulose content and fiber length. Based on GLU4 and CAD gene expression, a Eucalyptus variety low in lignin content should be obtained using transgenic technology, which could reduce the cost of pulp and improve the pulping rate, and have favorable prospects for application. However, the role and function of CAD in GLU4 is still unclear. In the present study, EuCAD was cloned from GLU4 and identified using bioinformatic tools. Subsequently, in order to evaluate its impact on lignin synthesis, a full-length EuCAD RNAi vector was constructed, and transgenic tobacco was obtained via Agrobacterium-mediated transformation. A significant decrease in CAD expression and lignin content in transgenic tobacco demonstrated a key role for EuCAD in lignin biosynthesis and established a regulatory role for RNAi. In our study, the direct molecular basis of EuCAD expression was determined, and the potential regulatory effects of this RNAi vector on lignin biosynthesis in E. urophylla GLU4 were demonstrated. Our results provide a theoretical basis for the study of lignin biosynthesis in Eucalyptus.

  17. A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco.

    Science.gov (United States)

    Velten, Jeff; Cakir, Cahid; Cazzonelli, Christopher I

    2010-03-29

    In part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation. A spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production. Messenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

  18. The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.

    Science.gov (United States)

    Li, Le; Miao, Weiguo; Liu, Wenbo; Zhang, Shujian

    2017-01-01

    Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum-a synonym of X. campestris pv. malvacearum (Smith 1901-1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1-45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

  19. The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Le Li

    Full Text Available Harpins, encoded by hrp (hypersensitive response and pathogenicity genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR. HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum-a synonym of X. campestris pv. malvacearum (Smith 1901-1978. A putative signal peptide (1-MNSLNTQIGANSSFL-15 of hpaXm was predicted in the nitroxyl-terminal (N-terminalby SignalP (SignalP 3.0 server. Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.. Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1-45 bp in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall.

  20. Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco.

    Science.gov (United States)

    Osakabe, Yuriko; Osakabe, Keishi; Chiang, Vincent L

    2009-01-01

    We characterized promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clalpha, from Pinus taeda. Histochemical- and quantitative assays of GUS expression in the vascular tissue were performed using transgenic tobacco plants expressing promoter-GUS reporters. Deletion analysis of the Pta4Clalpha promoter showed that the region -524 to -252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLalpha was also detected in the xylem cells under bending stress. DNA-protein complexes were detected in the reactions of the Pta4CLalpha promoter fragments with the nuclear proteins of xylem of P. taeda. The AC elements in the Pta4CLalpha promoter appeared to have individual roles during xylem development that are activated in a coordinated manner in response to stress in transgenic tobacco.

  1. Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress.

    Science.gov (United States)

    Hacham, Yael; Matityahu, Ifat; Amir, Rachel

    2017-07-01

    Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions. © 2017 Scandinavian Plant Physiology Society.

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

    Science.gov (United States)

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

  3. An Alcohol Dehydrogenase Gene from Synechocystis sp. Confers Salt Tolerance in Transgenic Tobacco

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    So Young Yi

    2017-11-01

    Full Text Available Synechocystis salt-responsive gene 1 (sysr1 was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1–2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

  4. Transgenic Nicotiana tabacum plants expressing a fungal copper transporter gene show enhanced acquisition of copper.

    Science.gov (United States)

    Singh, Sudhir; Korripally, Premsagar; Vancheeswaran, Ramachandran; Eapen, Susan

    2011-10-01

    The diets of two-thirds of the world's population are deficient in one or more essential elements and one of the approaches to enhance the levels of mineral elements in food crops is by developing plants with ability to accumulate them in edible parts. Besides conventional methods, transgenic technology can be used for enhancing metal acquisition in plants. Copper is an essential element, which is often deficient in human diet. With the objective of developing plants with improved copper acquisition, a high-affinity copper transporter gene (tcu-1) was cloned from fungus Neurospora crassa and introduced into a model plant (Nicotiana tabacum). Integration of the transgene was confirmed by Southern blot hybridization. Transgenic tobacco plants (T(0) and T(1)) expressing tcu-1, when grown in hydroponic medium spiked with different concentrations of copper, showed higher acquisition of copper (up to 3.1 times) compared with control plants. Transgenic plants grown in soil spiked with copper could also take up more copper compared with wild-type plants. Supplementation of other divalent cations such as Cd(2+) and Zn(2+) did not alter uptake of Cu by transgenic plants. The present study has shown that expression of a heterologous copper transporter in tobacco could enhance acquisition of copper.

  5. Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth

    Czech Academy of Sciences Publication Activity Database

    Schnablová, Renáta; Synková, Helena; Vičánková, Anna; Burketová, Lenka; Eder, Josef; Cvikrová, Milena

    2006-01-01

    Roč. 44, - (2006), s. 526-534 ISSN 0981-9428 R&D Projects: GA ČR GA206/03/0310 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinins * ipt * transgenic tobacco * in vitro cultivation Subject RIV: EF - Botanics Impact factor: 1.847, year: 2006

  6. Response to mild water stress in transgenic Pssu-ipt tobacco

    Czech Academy of Sciences Publication Activity Database

    Synková, Helena; Valcke, R.

    2001-01-01

    Roč. 112, - (2001), s. 513-523 ISSN 0031-9317 R&D Projects: GA ČR GA206/01/1061 Institutional research plan: CEZ:AV0Z5038910 Keywords : mild water * transgenic tobacco Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.760, year: 2001

  7. The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing

    NARCIS (Netherlands)

    Mlynarova, L.; Hricova, A.; Loonen, A.; Nap, J.P.H.

    2003-01-01

    We present isogenic transgenic tobacco lines that carry at a given chromosomal position a -glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site-specific recombination

  8. Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus.

    Directory of Open Access Journals (Sweden)

    Guixia Hao

    Full Text Available Citrus Huanglongbing (HLB associated with 'Candidatus Liberibacter asiaticus' (Las and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri

  9. Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

    Science.gov (United States)

    Keller, H; Pamboukdjian, N; Ponchet, M; Poupet, A; Delon, R; Verrier, J L; Roby, D; Ricci, P

    1999-02-01

    The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

  10. Overexpression of monoubiquitin improves photosynthesis in transgenic tobacco plants following high temperature stress.

    Science.gov (United States)

    Tian, Fengxia; Gong, Jiangfeng; Zhang, Jin; Feng, Yanan; Wang, Guokun; Guo, Qifang; Wang, Wei

    2014-09-01

    The ubiquitin/26S proteasome system (Ub/26S) is implicated in abiotic stress responses in plants. In this paper, transgenic tobacco plants overexpressing Ta-Ub2 from wheat were used to study the functions of Ub in the improvement of photosynthesis under high temperature (45°C) stress. We observed higher levels of Ub conjugates in transgenic plants under high temperature stress conditions compared to wild type (WT) as a result of the constitutive overexpression of Ta-Ub2, suggesting increased protein degradation by the 26S proteasome system under high temperature stress. Overexpressing Ub increased the photosynthetic rate (Pn) of transgenic tobacco plants, consistent with the improved ATPase activity in the thylakoid membrane and enhanced efficiency of PSII photochemistry. The higher D1 protein levels following high temperature stress in transgenic plants than WT were also observed. These findings imply that Ub may be involved in tolerance of photosynthesis to high temperature stress in plants. Compared with WT, the transgenic plants showed lower protein carbonylation and malondialdehyde (MDA) levels, less reactive oxygen species (ROS) accumulation, but higher antioxidant enzyme activity under high temperature stress. These findings suggest that the improved antioxidant capacity of transgenic plants may be one of the most important mechanisms underlying Ub-regulated high temperature tolerance. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  12. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

    Science.gov (United States)

    Aharoni, A; De Vos, C H; Wein, M; Sun, Z; Greco, R; Kroon, A; Mol, J N; O'Connell, A P

    2001-11-01

    Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

  13. A CBL-Interacting Protein Kinase TaCIPK2 Confers Drought Tolerance in Transgenic Tobacco Plants through Regulating the Stomatal Movement.

    Science.gov (United States)

    Wang, Yan; Sun, Tao; Li, Tingting; Wang, Meng; Yang, Guangxiao; He, Guangyuan

    2016-01-01

    In plants, the CBL-CIPK signaling pathways play key roles in the response to abiotic stresses. However, functional studies of CIPKs in the important staple crop wheat are very rare. In this study, we identified a CIPK gene from wheat, designated TaCIPK2. Expression analysis results showed that TaCIPK2 could be up-regulated in wheat leaves by polyethylene glycol, abscisic acid and H2O2 treatments. Subcellular localization analyses revealed that TaCIPK2 was present in whole wheat epidermal cells. A yeast two-hybrid assay indicated that TaCIPK2 interacted with TaCBL1, 2, 3 and 4 in vitro. Transgenic tobacco plants over-expressing TaCIPK2 exhibited increased drought tolerance, indicated by a larger proportion of green cotyledons and higher survival rates under the osmotic and drought stress conditions compared with control plants. Additionally, physiological index analyses revealed that the transgenic tobacco plants had lower water loss rates and ion leakage, accumulated less malondialdehyde and H2O2, and had higher catalase and superoxide dismutase activities than the control plants. The transgenic plants also exhibited faster stomatal closure following exposure to osmotic stress conditions. The seed germination rates and stomatal aperture of TaCIPK2-overexpressing tobacco plants decreased after exogenous abscisic acid treatment was applied, implying that the transgenic tobacco plants were more sensitive to exogenous abscisic acid than the control plants. Our results indicate that TaCIPK2 plays a positive regulatory role in drought stress responses in transgenic tobacco plants.

  14. Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

    DEFF Research Database (Denmark)

    Ghiasi, Seyed Mojtaba; Salmanian, A H; Chinikar, S

    2011-01-01

    glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed...... the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies...... in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition...

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

    Science.gov (United States)

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

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  16. Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Farooqahmed S Kittur

    Full Text Available Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P (20 U/ml provides 2-fold better cytoprotection (44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M (21%. The cytoprotective effect of the asialo-rhuEPO(P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

  17. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Amit Kumar Chaturvedi

    Full Text Available Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13 showed significantly enhanced salt (NaCl, osmotic (PEG and metals (Zn++, Cu++ and Cd++ tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl, osmotic (PEG and metals (Zn++, Cu++ and Cd++ stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2-; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

  18. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco.

    Science.gov (United States)

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

    2014-01-01

    Heavy metals are common pollutants of the coastal saline area and Salicornia brachiata an extreme halophyte is frequently exposed to various abiotic stresses including heavy metals. The SbMT-2 gene was cloned and transformed to tobacco for the functional validation. Transgenic tobacco lines (L2, L4, L6 and L13) showed significantly enhanced salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) tolerance compared to WT plants. Transgenic lines did not show any morphological variation and had enhanced growth parameters viz. shoot length, root length, fresh weight and dry weight. High seed germination percentage, chlorophyll content, relative water content, electrolytic leakage and membrane stability index confirmed that transgenic lines performed better under salt (NaCl), osmotic (PEG) and metals (Zn++, Cu++ and Cd++) stress conditions compared to WT plants. Proline, H2O2 and lipid peroxidation (MDA) analyses suggested the role of SbMT-2 in cellular homeostasis and H2O2 detoxification. Furthermore in vivo localization of H2O2 and O2-; and elevated expression of key antioxidant enzyme encoding genes, SOD, POD and APX evident the possible role of SbMT-2 in ROS scavenging/detoxification mechanism. Transgenic lines showed accumulation of Cu++ and Cd++ in root while Zn++ in stem under stress condition. Under control (unstressed) condition, Zn++ was accumulated more in root but accumulation of Zn++ in stem under stress condition suggested that SbMT-2 may involve in the selective translocation of Zn++ from root to stem. This observation was further supported by the up-regulation of zinc transporter encoding genes NtZIP1 and NtHMA-A under metal ion stress condition. The study suggested that SbMT-2 modulates ROS scavenging and is a potential candidate to be used for phytoremediation and imparting stress tolerance.

  19. Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

    Directory of Open Access Journals (Sweden)

    Baschieri Selene

    2007-02-01

    Full Text Available Abstract Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa and a truncated form (p25, 25 kDa. Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

  20. The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants.

    Science.gov (United States)

    Spena, A; Prinsen, E; Fladung, M; Schulze, S C; Van Onckelen, H

    1991-06-01

    The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[14C]lysine when fed with [14C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterized by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.

  1. Transgenic tobacco plants overexpressing a grass PpEXP1 gene exhibit enhanced tolerance to heat stress.

    Directory of Open Access Journals (Sweden)

    Qian Xu

    Full Text Available Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars.

  2. Calcium electrotransfer for termination of transgene expression in muscle

    DEFF Research Database (Denmark)

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

    2011-01-01

    Gene electrotransfer is expanding in clinical use, thus we have searched for an emergency procedure to stop transgene expression in case of serious adverse events. Calcium is cytotoxic at high intracellular levels, so we tested effects of calcium electrotransfer on transgene expression in muscle....... A clinical grade calcium solution (20 µl, 168 mM) was injected into transfected mouse or rat tibialis cranialis muscle. Ca(2+) uptake was quantified using calcium 45 ((45)Ca), and voltage and time between injection and pulsation were varied. Extinction of transgene expression was investigated by using both...... voltage pulses of 1000 V/cm. Using these parameters, in vivo imaging showed that transgene expression significantly decreased 4 hr after Ca(2+) electrotransfer and was eliminated within 24 hr. Similarly, serum erythropoietin was reduced by 46% at 4 hr and to control levels at 2 days. Histological analyses...

  3. Comparison of nutritional value of transgenic peanut expressing bar and rcg3 genes with non-transgenic counterparts

    International Nuclear Information System (INIS)

    Robab, U.E.; )

    2014-01-01

    The transgenic peanut plants expressing bar and rcg3 genes were subjected to assessment of any change in nutritional value of the crop at various locations. The protein and fat contents of transgenic lines were compared with the non-transgenic parent varieties. Protein content in the transgenic lines was higher as compared to that in non-transgenic counterparts and differences among locations for fat and protein content were significant. No differences among fatty acids were recorded for genes, events and locations. Irrespective of small differences, all the values were in range described for this crop and transgenic lines appeared to be substantially equivalent to non-transgenic parent varieties. (author)

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

    Science.gov (United States)

    Huber, Reinhard C; Remuge, Liliana; Carlisle, Ailsa; Lillico, Simon; Sandøe, Peter; Sørensen, Dorte B; Whitelaw, C Bruce A; Olsson, I Anna S

    2012-08-01

    Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology--animal welfare--has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly 4 months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs. The present study may furthermore serve as starting point for researchers aiming at a systematic characterization of welfare relevant effects in the line of transgenic pigs they are working with.

  5. The SbASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Jha, Bhavanath; Lal, Sanjay; Tiwari, Vivekanand; Yadav, Sweta Kumari; Agarwal, Pradeep K

    2012-12-01

    Salinity severely affects plant growth and development. Plants evolved various mechanisms to cope up stress both at molecular and cellular levels. Halophytes have developed better mechanism to alleviate the salt stress than glycophytes, and therefore, it is advantageous to study the role of different genes from halophytes. Salicornia brachiata is an extreme halophyte, which grows luxuriantly in the salty marshes in the coastal areas. Earlier, we have isolated SbASR-1 (abscisic acid stress ripening-1) gene from S. brachiata using cDNA subtractive hybridisation library. ASR-1 genes are abscisic acid (ABA) responsive, whose expression level increases under abiotic stresses, injury, during fruit ripening and in pollen grains. The SbASR-1 transcript showed up-regulation under salt stress conditions. The SbASR-1 protein contains 202 amino acids of 21.01-kDa molecular mass and has 79 amino acid long signatures of ABA/WDS gene family. It has a maximum identity (73 %) with Solanum chilense ASR-1 protein. The SbASR-1 has a large number of disorder-promoting amino acids, which make it an intrinsically disordered protein. The SbASR-1 gene was over-expressed under CaMV 35S promoter in tobacco plant to study its physiological functions under salt stress. T(0) transgenic tobacco seeds showed better germination and seedling growth as compared to wild type (Wt) in a salt stress condition. In the leaf tissues of transgenic lines, Na(+) and proline contents were significantly lower, as compared to Wt plant, under salt treatment, suggesting that transgenic plants are better adapted to salt stress.

  6. Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis.

    Science.gov (United States)

    Li, Li-Ya; Cai, Qiu-Yi; Yu, Dian-Si; Guo, Chang-Hong

    2011-08-01

    The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.

  7. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    International Nuclear Information System (INIS)

    Tamot, B.K.; Pauls, K.P.; Glick, R.

    2003-01-01

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  8. Overexpression of a R2R3 MYB gene MdSIMYB1 increases tolerance to multiple stresses in transgenic tobacco and apples.

    Science.gov (United States)

    Wang, Rong-Kai; Cao, Zhong-Hui; Hao, Yu-Jin

    2014-01-01

    MYB transcription factors (TFs) involve in plant abiotic stress tolerance and response in various plant species. In this study, rapid amplification of cDNA ends (RACE) was conducted to isolate the R2R3-MYB TF gene MdSIMYB1 from apples (Malus × domestica). The gene transcripts were abundant in the leaves, flowers and fruits, compared to other organs, and were induced by abiotic stresses and plant hormones. We observed the subcellular localization of an MdSIMYB1-GFP fusion protein in the nucleus. Furthermore, the MdSIMYB1 gene was introduced into the tobacco genome and ectopically expressed in transgenic lines. The results indicate that MdSIMYB1 transgenic tobacco seed germination is insensitive to abscisic acid and NaCl treatment. Additionally, it was found that the ectopic expression of MdSIMYB1 enhanced the tolerance of plants to high salinity, drought and cold tolerance by upregulating the stress-responsive genes NtDREB1A, NtERD10B and NtERD10C. Meanwhile, the transgenic tobacco exhibited robust root growth because of the enhanced expression of the auxin-responsive genes NtIAA4.2, NtIAA4.1 and NtIAA2.5 under stress conditions, which is conducive to stress tolerance. Finally, transgenic apple lines were obtained and tested. Transgenic apple lines that were overexpressing MdSIMYB1 exhibited a higher tolerance to abiotic stress than the wild-type control, but suppression of MdSIMYB1 resulted in lower tolerance. Our results indicate that MdSIMYB1 may be utilized as a target gene for enhancing stress tolerance in important crops. © 2013 Scandinavian Plant Physiology Society.

  9. The stability of transgene expression and effect of DNA methylation ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... transformation system (Zhan et al., 2003). Stable expression of foreign gene is important for commercial use of genetic transformation in long-lived tree species as well as for ecological risk-assessment studies. However, analysis of the instable/stable transgene expression in tree is more problematic than in ...

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

    Science.gov (United States)

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

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

  11. Destabilizing domains mediate reversible transgene expression in the brain.

    Directory of Open Access Journals (Sweden)

    Khalid Tai

    Full Text Available Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD of the Escherichia coli dihydrofolate reductase (DHFR that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinson's disease and should be further investigated.

  12. Heat shock transcription factors involved in seed desiccation tolerance and longevity retard vegetative senescence in transgenic tobacco.

    Science.gov (United States)

    Almoguera, Concepción; Personat, José-María; Prieto-Dapena, Pilar; Jordano, Juan

    2015-08-01

    Transcription factors normally expressed in sunflower seeds delayed vegetative senescence induced by severe stress in transgenic tobacco. This revealed a novel connection between seed heat shock factors, desiccation tolerance and vegetative longevity. HaHSFA9 and HaHSFA4a coactivate a genetic program that, in sunflower (Helianthus annuus L.), contributes to seed longevity and desiccation tolerance. We have shown that overexpression of HaHSFA9 in transgenic tobacco seedlings resulted in tolerance to drastic dehydration and oxidative stress. Overexpression of HaHSFA9 alone was linked to a remarkable protection of the photosynthetic apparatus. In addition, the combined overexpression of HaHSFA9 and HaHSFA4a enhanced all these stress-resistance phenotypes. Here, we find that HaHSFA9 confers protection against damage induced by different stress conditions that accelerate vegetative senescence during different stages of development. Seedlings and plants that overexpress HaHSFA9 survived lethal treatments of dark-induced senescence. HaHSFA9 overexpression induced resistance to effects of culture under darkness for several weeks. Only some homoiochlorophyllous resurrection plants are able to withstand this experimental severe stress condition. The combined overexpression of HaHSFA9 and HaHSFA4a did not result in further slowing of dark-induced seedling senescence. However, combined expression of the two transcription factors caused improved recovery of the photosynthetic organs of seedlings after lethal dark treatments. At later stages of vegetative development, HaHSFA9 delayed the appearance of senescence symptoms in leaves of plants grown under normal illumination. This delay was observed under either control or stress treatments. Thus, HaHSFA9 delayed both natural and stress-induced leaf senesce. These novel observations connect transcription factors involved in desiccation tolerance with leaf longevity.

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

    Directory of Open Access Journals (Sweden)

    Déborah Ley

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

  14. Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

    Science.gov (United States)

    Nesler, Andrea; DalCorso, Giovanni; Fasani, Elisa; Manara, Anna; Di Sansebastiano, Gian Pietro; Argese, Emanuele; Furini, Antonella

    2017-03-25

    Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Fangmeng Duan

    2017-11-01

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

  16. Identification of abnormal gene expression in bovine transgenic somatic cell nuclear transfer embryos

    OpenAIRE

    Cho, Jongki; Kang, Sungkeun; Lee, Byeong Chun

    2014-01-01

    This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogene...

  17. In trans silencing properties of a tobacco transgene locus depend on its epigenetic state

    Czech Academy of Sciences Publication Activity Database

    Fojtová, Miloslava; Van Houdt, H.; Depicker, A.; Kovařík, Aleš

    2004-01-01

    Roč. 26, č. 3 (2004), s. 147 ISSN 0137-5881. [Congress of the Federation of European Societies of Plant Biology /14./. 23.08.2004-27.08.2004, Cracow] Keywords : plant transgenes * gene expression * silencing Subject RIV: BO - Biophysics

  18. Transgenic expression of nonclassically secreted FGF suppresses kidney repair.

    Directory of Open Access Journals (Sweden)

    Aleksandr Kirov

    Full Text Available FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC, which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.

  19. Comparison of Membrane Targeting Strategies for the Accumulation of the Human Immunodeficiency Virus p24 Protein in Transgenic Tobacco

    Directory of Open Access Journals (Sweden)

    Jean-Marc Neuhaus

    2013-06-01

    Full Text Available Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal or a tail-anchored fusion (where the p24 sequence is cytosolic resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants.

  20. A wheat WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available WRKY transcription factors are reported to be involved in defense regulation, stress response and plant growth and development. However, the precise role of WRKY transcription factors in abiotic stress tolerance is not completely understood, especially in crops. In this study, we identified and cloned 10 WRKY genes from genome of wheat (Triticum aestivum L.. TaWRKY10, a gene induced by multiple stresses, was selected for further investigation. TaWRKY10 was upregulated by treatment with polyethylene glycol, NaCl, cold and H2O2. Result of Southern blot indicates that the wheat genome contains three copies of TaWRKY10. The TaWRKY10 protein is localized in the nucleus and functions as a transcriptional activator. Overexpression of TaWRKY10 in tobacco (Nicotiana tabacum L. resulted in enhanced drought and salt stress tolerance, mainly demonstrated by the transgenic plants exhibiting of increased germination rate, root length, survival rate, and relative water content under these stress conditions. Further investigation showed that transgenic plants also retained higher proline and soluble sugar contents, and lower reactive oxygen species and malonaldehyde contents. Moreover, overexpression of the TaWRKY10 regulated the expression of a series of stress related genes. Taken together, our results indicate that TaWRKY10 functions as a positive factor under drought and salt stresses by regulating the osmotic balance, ROS scavenging and transcription of stress related genes.

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

    Science.gov (United States)

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

    2003-03-28

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

  2. Calcium electrotransfer for termination of transgene expression in muscle

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  3. MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers drought and salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Zhang, Zhiqiang; Wang, Yafang; Chang, Leqin; Zhang, Tong; An, Jie; Liu, Yushi; Cao, Yuman; Zhao, Xia; Sha, Xuyang; Hu, Tianming; Yang, Peizhi

    2016-02-01

    The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized from alfalfa and validated for its function of tolerance toward drought and salt stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase (ZEP) plays important roles in plant response to various environment stresses due to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the expression characteristics and the biological functions of ZEP in alfalfa (Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned, characterized and overexpressed in Nicotiana tabacum. The open reading frame of MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide. Amino acid sequence alignment indicated that deduced MsZEP protein was highly homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP was grouped into a branch with other legume plants. Real-time quantitative PCR revealed that MsZEP gene expression was clearly tissue-specific, and the expression levels were higher in green tissues (leaves and stems) than in roots. MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment, while the expression levels in roots showed different trends. Besides, the results showed that nodules could up-regulate the MsZEP expression under non-stressful conditions and in the earlier stage of different abiotic stress. Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to drought and salt stress by affecting various physiological pathways, ABA levels and stress-responsive genes expression. Taken together, these results suggested that the MsZEP gene may be involved in alfalfa responses to different abiotic stresses and nodules, and could enhance drought and salt tolerance of transgenic tobacco by heterologous expression.

  4. Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

    Science.gov (United States)

    Imran, Muhammad; Asad, Shaheen; Barboza, Andre Luiz; Galeano, Esteban; Carrer, Helaine; Mukhtar, Zahid

    2017-04-01

    Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4 - EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T 1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4 - EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

  5. The production of viral vectors designed to express large and difficult to express transgenes within neurons.

    Science.gov (United States)

    Holehonnur, Roopashri; Lella, Srihari K; Ho, Anthony; Luong, Jonathan A; Ploski, Jonathan E

    2015-02-24

    Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons. We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains. We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express

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

    Directory of Open Access Journals (Sweden)

    Kanoria Shaveta

    2012-11-01

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

  7. HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

    Directory of Open Access Journals (Sweden)

    Lehto Kirsi

    2011-04-01

    Full Text Available Abstract Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs. These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter the helper component-proteinase (HC-Pro derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent. Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1 were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S

  8. Evaluation of deoxynivalenol production in dsRNA Carrying and Cured Fusarium graminearum isolates by AYT1 expressing transformed tobacco

    Directory of Open Access Journals (Sweden)

    Mohammad Hasan shahhosseiny

    2015-12-01

    Full Text Available Introduction: Fusarium head blight (FHB, is the most destructive disease of wheat, producing the mycotoxin deoxynivalenol, a protein synthesis inhibitor, which is harmful to humans and livestock. dsRNAmycoviruses-infected-isolates of Fusariumgraminearum, showed changes in morphological and pathogenicity phenotypes including reduced virulence towards wheat and decreased production of trichothecene mycotoxin (deoxynivalenol: DON. Materials and methods: Previous studies indicated that over expression of yeast acetyl transferase gene (ScAYT1 encoding a 3-O trichothecene acetyl transferase that converts deoxynivalenol to a less toxic acetylated form, leads to suppression of the deoxynivalenol sensitivity in pdr5 yeast mutants. To identify whether ScAYT1 over-expression in transgenic tobacco plants can deal with mycotoxin (deoxynivalenol in fungal extract and studying the effect of dsRNA contamination on detoxification and resistance level, we have treated T1 AYT1 transgenic tobacco seedlings with complete extraction of normal F. graminearum isolate carrying dsRNA metabolites. First, we introduced AYT1into the model tobacco plants through Agrobacterium-mediated transformation in an attempt to detoxify deoxynivalenol. Results: In vitro tests with extraction of dsRNA carrying and cured isolates of F. graminearum and 10 ppm of deoxynivalenol indicated variable resistance levels in transgenic plants. Discussion and conclusion: The results of this study indicate that the transgene expression AYT1 and Fusarium infection to dsRNA can induce tolerance to deoxynivalenol, followed by increased resistance to Fusarium head blight disease of wheat.

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

    Science.gov (United States)

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

    2010-01-01

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

  10. Inheritance and effectiveness of two transgenes determining PVY resistance in progeny from crossing independently transformed tobacco lines.

    Science.gov (United States)

    Czubacka, Anna; Sacco, Ermanno; Olszak-Przybyś, Hanna; Doroszewska, Teresa

    2017-05-01

    Genetic transformation of plants allows us to obtain improved genotypes enriched with the desired traits. However, if transgenic lines were to be used in breeding programs the stability of inserted transgenes is essential. In the present study, we followed the inheritance of transgenes in hybrids originated from crossing two transgenic tobacco lines resistant to Potato virus Y (PVY): MN 944 LMV with the transgene containing Lettuce mosaic virus coat protein gene (LMV CP) and AC Gayed ROKY2 with PVY replicase gene (ROKY2). Progeny populations generated by successive self-pollination were analyzed with respect to the transgene segregation ratio and resistance to Potato virus Y in tests carried out under greenhouse conditions. The presence of the virus in inoculated plants was detected by DAS-ELISA method. The results demonstrated the Mendelian fashion of inheritance of transgenes which were segregated independently and stably. As a result, we obtained T 4 generation of hybrid with both transgenes stacked and which was highly resistant to PVY.

  11. Expression of GLUT-1 in oral squamous cell carcinoma in tobacco and non-tobacco users.

    Science.gov (United States)

    Azad, Neha; Kumari Maurya, Malti; Kar, Meenakshi; Goel, Madhu Mati; Singh, Ajay Kumar; Sagar, Mala; Mehrotra, Divya; Kumar, Vijay

    2016-01-01

    GLUTs are a family of proteins that mediate glucose transport through the membrane, expressed in head and neck squamous cell carcinoma. GLUT-1 positivity in malignant cells indicates increased proliferative activity, energy requirements, aggressive behaviour and poor radiation response. To observe the expression of GLUT-1 protein in oral squamous cell carcinoma in tobacco and non-tobacco users and to correlate the expression with histopathological grading and pathological staging. 50 cases (25 tobacco and 25 non-tobacco) of oral squamous cell carcinoma, selected during period of August 2014 to July 2015. Histopathological grading, TNM and staging were done. Immunohistochemical staining was performed using standard protocol for paraffin embedded sections. Analysis was performed on SPSS software (Windows version 17.0). Significant association of GLUT-1 expression was found with history of tobacco (p GLUT-1 expression in stage II, stage III and stage IV was found as compared to stage I. GLUT-1 immunoexpression also shows progressive switch from membranous to cytoplasmic to combined location correlating with histopathologic grade and pTNM stage. GLUT-1 expression correlates significantly with histological grade and pTNM staging of oral squamous cell carcinoma. It also significantly correlates with tobacco addiction. Thus, GLUT-1 expression may serve as a biomarker for patients of oral squamous cell carcinoma.

  12. Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco

    Science.gov (United States)

    Deb, Debasish; Shrestha, Ankita; Maiti, Indu B.; Dey, Nrisingha

    2018-01-01

    Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach. PMID:29556246

  13. Expressive freedom and tobacco advertising: a Canadian perspective.

    Science.gov (United States)

    Manfredi, Christopher P

    2002-03-01

    In 1989, Canada enacted the Tobacco Products Control Act (TPCA), which prohibited tobacco advertising, required health warnings on tobacco packaging, and restricted promotional activities. Canada's tobacco companies challenged the TPCA's constitutionality, arguing that it infringed on freedom of expression. Although it seemed likely that the Canadian Supreme Court would uphold the legislation, in 1995 the court declared the impugned provisions to be unconstitutional. The decision is testimony to the constraining force of liberalism on tobacco regulation, but it is also evidence of the power of political will. While the Canadian government could have used the decision to justify withdrawing from further confrontations with powerful commercial interests, it chose instead to enact new tobacco control legislation in 1997.

  14. Human HLA-Ev (147) Expression in Transgenic Animals.

    Science.gov (United States)

    Matsuura, R; Maeda, A; Sakai, R; Eguchi, H; Lo, P-C; Hasuwa, H; Ikawa, M; Nakahata, K; Zenitani, M; Yamamichi, T; Umeda, S; Deguchi, K; Okuyama, H; Miyagawa, S

    2016-05-01

    In our previous study, we reported on the development of substituting S147C for HLA-E as a useful gene tool for xenotransplantation. In this study we exchanged the codon of HLA-Ev (147), checked its function, and established a line of transgenic mice. A new construct, a codon exchanging human HLA-Ev (147) + IRES + human beta 2-microgloblin, was established. The construct was subcloned into pCXN2 (the chick beta-actin promoter and cytomegalovirus enhancer) vector. Natural killer cell- and macrophage-mediated cytotoxicities were performed using the established the pig endothelial cell (PEC) line with the new gene. Transgenic mice with it were next produced using a micro-injection method. The expression of the molecule on PECs was confirmed by the transfection of the plasmid. The established molecules on PECs functioned well in regulating natural killer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity. We have also successfully generated several lines of transgenic mice with this plasmid. The expression of HLA-Ev (147) in each mouse organ was confirmed by assessing the mRNA. The chick beta-actin promoter and cytomegalovirus enhancer resulted in a relatively broad expression of the gene in each organ, and a strong expression in the cases of the heart and lung. A synthetic HLA-Ev (147) gene with a codon usage optimized to a mammalian system represents a critical factor in the development of transgenic animals for xenotransplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology.

    Science.gov (United States)

    Agrawal, Aditi; Rajamani, Vijayalakshmi; Reddy, Vanga Siva; Mukherjee, Sunil Kumar; Bhatnagar, Raj K

    2015-10-01

    The success of Bt transgenics in controlling predation of crops has been tempered by sporadic emergence of resistance in targeted insect larvae. Such emerging threats have prompted the search for novel insecticidal molecules that are specific and could be expressed through plants. We have resorted to small RNA-based technology for an investigative search and focused our attention to an insect-specific miRNA that interferes with the insect molting process resulting in the death of the larvae. In this study, we report the designing of a vector that produces artificial microRNA (amiR), namely amiR-24, which targets the chitinase gene of Helicoverpa armigera. This vector was used as transgene in tobacco. Northern blot and real-time analysis revealed the high level expression of amiR-24 in transgenic tobacco plants. Larvae feeding on the transgenic plants ceased to molt further and eventually died. Our results demonstrate that transgenic tobacco plants can express amiR-24 insectice specific to H. armigera.

  16. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    Science.gov (United States)

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

    2011-01-01

    Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

  17. Optical modulation of transgene expression in retinal pigment epithelium

    Science.gov (United States)

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

    2013-03-01

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

  18. A cold-induced myo-inositol transporter-like gene confers tolerance to multiple abiotic stresses in transgenic tobacco plants.

    Science.gov (United States)

    Sambe, Mame Abdou Nahr; He, Xueying; Tu, Qinghua; Guo, Zhenfei

    2015-03-01

    A full length cDNA encoding a myo-inositol transporter-like protein, named as MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a species with greater cold tolerance than alfalfa (M. sativa subsp. sativa). MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2-4 h after exogenous myo-inositol treatment, 24-96 h with cold, and 96 h by salinity. Given that myo-inositol accumulates higher in falcata after 24 h of cold treatment, myo-inositol is proposed to be involved in cold-induced expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in the roots of plants grown on myo-inositol containing medium, suggesting that transgenic plants had higher myo-inositol transport activity than the wild-type. Transgenic plants survived better to freezing temperature, and had lower ion leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm ) after chilling treatment. In addition, greater plant fresh weight was observed in transgenic plants as compared with the wild-type when plants were grown under drought or salinity stress. The results suggest that MfINT-like mediated transport of myo-inositol is associated with plant tolerance to abiotic stresses. © 2014 Scandinavian Plant Physiology Society.

  19. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    Science.gov (United States)

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. © 2015 Society for Experimental Biology, Association of

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

    Science.gov (United States)

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

    2016-10-01

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

  1. Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield.

    Science.gov (United States)

    Han, Qiang; Wang, Zhenzhen; He, Yunxin; Xiong, Yehui; Lv, Shun; Li, Shupeng; Zhang, Zhigang; Qiu, Dewen; Zeng, Hongmei

    2017-08-30

    RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3 , a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA- HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing ds HaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera . Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.

  2. Heterologous expression of gentian MYB1R transcription factors suppresses anthocyanin pigmentation in tobacco flowers.

    Science.gov (United States)

    Nakatsuka, Takashi; Yamada, Eri; Saito, Misa; Fujita, Kohei; Nishihara, Masahiro

    2013-12-01

    Single-repeat MYB transcription factors, GtMYB1R1 and GtMYB1R9 , were isolated from gentian. Overexpression of these genes reduced anthocyanin accumulation in tobacco flowers, demonstrating their applicability to modification of flower color. RNA interference (RNAi) has recently been used to successfully modify flower color intensity in several plant species. In most floricultural plants, this technique requires prior isolation of target flavonoid biosynthetic genes from the same or closely related species. To overcome this limitation, we developed a simple and efficient method for reducing floral anthocyanin accumulation based on genetic engineering using novel transcription factor genes isolated from Japanese gentians. We identified two single-repeat MYB genes--GtMYB1R and GtMYB1R9--predominantly expressed in gentian petals. Transgenic tobacco plants expressing these genes were produced, and their flowers were analyzed for flavonoid components and expression of flavonoid biosynthetic genes. Transgenic tobacco plants expressing GtMYB1R1 or GtMYB1R9 exhibited significant reductions in floral anthocyanin accumulation, resulting in white-flowered phenotypes. Expression levels of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) genes were preferentially suppressed in these transgenic tobacco flowers. A yeast two-hybrid assay demonstrated that both GtMYB1R1 and GtMYB1R9 proteins interacted with the GtbHLH1 protein, previously identified as an anthocyanin biosynthesis regulator in gentian flowers. In addition, a transient expression assay indicated that activation of the gentian GtDFR promoter by the GtMYB3-GtbHLH1 complex was partly canceled by addition of GtMYB1R1 or GtMYB1R9. These results suggest that GtMYB1R1 and GtMYB1R9 act as antagonistic transcription factors of anthocyanin biosynthesis in gentian flowers. These genes should consequently be useful for manipulating anthocyanin accumulation via genetic engineering in

  3. Overexpression of a specific soybean GmGSTU4 isoenzyme improves diphenyl ether and chloroacetanilide herbicide tolerance of transgenic tobacco plants.

    Science.gov (United States)

    Benekos, Kostantinos; Kissoudis, Christos; Nianiou-Obeidat, Irini; Labrou, Nikolaos; Madesis, Panagiotis; Kalamaki, Mary; Makris, Antonis; Tsaftaris, Athanasios

    2010-10-01

    Plant glutathione transferases (GSTs) superfamily consists of multifunctional enzymes and forms a major part of the plants herbicide detoxification enzyme network. The tau class GST isoenzyme GmGSTU4 from soybean, exhibits catalytic activity towards the diphenyl ether herbicide fluorodifen and is active as glutathione-dependent peroxidase (GPOX). Transgenic tobacco plants of Basmas cultivar were generated via Agrobacterium transformation. The aim was to evaluate in planta, GmGSTU4's role in detoxifying the diphenyl ether herbicides fluorodifen and oxyfluorfen and the chloroacetanilides alachlor and metolachlor. Transgenic tobacco plants were verified by PCR and Southern blot hybridization and expression of GmGSTU4 was determined by RT-PCR. Leaf extracts from transgenic plants showed moderate increase in GST activity towards CDNB and a significant increase towards fluorodifen and alachlor, and at the same time an increased GPOX activity towards cumene hydroperoxide. GmGSTU4 overexpressing plants when treated with 200 μM fluorodifen or oxyfluorfen exhibited reduced relative electrolyte leakage compared to wild type plants. Moreover all GmGSTU4 overexpressing lines exhibited significantly increased tolerance towards alachlor when grown in vitro at 7.5 mg/L alachlor compared to wild type plants. No significant increased tolerance was observed to metolachlor. These results confirm the contribution of this particular GmGSTU4 isoenzyme from soybean in the detoxification of fluorodifen and alachlor, and provide the basis towards the development of transgenic plants with improved phytoremediation capabilities for future use in environmental cleanup of herbicides. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.

    Science.gov (United States)

    Neubauer, Katja; Hühns, Maja; Hausmann, Tina; Klemke, Friederike; Lockau, Wolfgang; Kahmann, Uwe; Pistorius, Elfriede K; Kragl, Udo; Broer, Inge

    2012-03-31

    A chimeric cyanophycin synthetase gene composed of the cphATe coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

    OpenAIRE

    Rossi, Luciana; Di Giancamillo, Alessia; Reggi, Serena; Domeneghini, Cinzia; Baldi, Antonella; Sala, Vittorio; Dell'Orto, Vittorio; Coddens, Annelies; Cox, Eric; Fogher, Corrado

    2013-01-01

    Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and ...

  6. Mutations in the pqe-1 Gene Enhance Transgene Expression in Caenorhabditis elegans

    Science.gov (United States)

    Yamada, Koji; Tsuchiya, Jun-ichi; Iino, Yuichi

    2012-01-01

    Although various genetic tools have been developed and used as transgenes, the expression of the transgenes often is hampered by negative regulators. Disrupting such negative regulators of gene expression is potentially a way to overcome the common problem of low expression of transgenes. To find such regulators whose mutations enhance transgene expression in Caenorhabditis elegans, we took advantage of a newly developed reporter transgene, lin-11pAΔ::venus. This transgene induces expression of a fluorescent protein, Venus, in specific neurons including AIZ, where the expression was stochastic. The frequency of reporter expression in AIZ seemed to be correlated with the strength of transgene expression. By using this system, in which a moderate increase of expression was converted to all-or-none expression states, we describe here a forward genetic screen for mutations that enhance the expression of transgenes. Through the screen, we found that mutations in the pqe-1 gene, which encodes a Q/P-rich nuclear protein with an exonuclease domain, increase the chance of reporter expression in AIZ. The fluorescence intensity in RIC, in which all lin-11pAΔ::venus animals show reporter expression, was increased in pqe-1 mutants, suggesting that pqe-1 reduces the expression level of the transgene. Expression of transgenes with other promoters, 3′UTR, or reporter genes was also enhanced by the pqe-1 mutation, suggesting that the effect was not specific to a particular type of transgenes, whereas the effect did not seem to extend to endogenous genes. We propose that pqe-1 mutants can be used to increase the expression of various useful transgenes. PMID:22870397

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

    Science.gov (United States)

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

    2015-01-01

    Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression. PMID:25926732

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Expression of chimeric HCV peptide in transgenic tobacco plants ...

    African Journals Online (AJOL)

    associated and sporadic non-A non-B hepatitis affecting more than 180 million worldwide. Vaccine development for HCV has been difficult and there is no vaccine or effective therapy against this virus. In this paper, we describe the development of an ...

  10. Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture

    Science.gov (United States)

    K?í?ová, Kate?ina; Depicker, Ann; Kova?ík, Ale?

    2013-01-01

    In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided. PMID:23770973

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

    Science.gov (United States)

    Schwartz, Robert J.

    1999-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Susan K Morton

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

  13. Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells.

    Science.gov (United States)

    Sato, Yasushi; Whetten, Ross W

    2006-11-01

    We previously showed that eight laccase genes (Lac 1-Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147-155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO(4) in the reaction mixtures increased in the range from 1 to 100 microM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.

  14. Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

    Science.gov (United States)

    Ryan, Annette C; Hewitt, C Nicholas; Possell, Malcolm; Vickers, Claudia E; Purnell, Anna; Mullineaux, Philip M; Davies, William J; Dodd, Ian C

    2014-01-01

    Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  15. ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco.

    Science.gov (United States)

    Sun, Liping; Liu, Yang; Kong, Xiangpei; Zhang, Dan; Pan, Jiaowen; Zhou, Yan; Wang, Li; Li, Dequan; Yang, Xinghong

    2012-08-01

    Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H₂O₂. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant. The overexpression of ZmHSP16.9 enhanced tolerance to heat and oxidative stress in transgenic tobacco.

  16. Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

    Science.gov (United States)

    Obembe, Olawole O.; Jacobsen, Evert; Timmers, Jaap; Gilbert, Harry; Blake, Anthony W.; Knox, J. Paul; Visser, Richard G. F.

    2007-01-01

    We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth. PMID:17622484

  17. Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model.

    Science.gov (United States)

    Rossi, Luciana; Di Giancamillo, Alessia; Reggi, Serena; Domeneghini, Cinzia; Baldi, Antonella; Sala, Vittorio; Dell'Orto, Vittorio; Coddens, Annelies; Cox, Eric; Fogher, Corrado

    2013-01-01

    Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

  18. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    Science.gov (United States)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

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

    Directory of Open Access Journals (Sweden)

    Quanmei Yan

    2014-01-01

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

  20. Regulated expression of transgenes in embryonic stem cell-derived neural cells.

    Science.gov (United States)

    Lorberbaum, David S; Gottlieb, David

    2011-02-01

    Discovery and characterization of gene promoters, enhancers and repressor binding elements is an important research area in neuroscience. Here, the suitability of embryonic stem cells and their neural derivatives as a model system for this research is investigated. Three neural transgenic constructs (from the Mnx1, Fabp7, and tuba1a genes) that have been validated in transgenic mice were inserted into embryonic stem cells as stable transgenes. These transgenic embryonic stem cells were differentiated into neural cultures and the pattern of transgene expression across a series of inducing conditions determined. The pattern of expression matched that predicted from transgenic mouse experiments for each of the three transgenes. The results show that embryonic stem cells and their neural derivatives comprise a promising model for investigating the mechanisms that control cell- and temporal-specific neural gene transcription. Copyright © 2010 Wiley-Liss, Inc.

  1. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na(+) loading in xylem and confers salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Yadav, Narendra Singh; Shukla, Pushp Sheel; Jha, Anupama; Agarwal, Pradeep K; Jha, Bhavanath

    2012-10-11

    Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na(+)/H(+) antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K(+)/Na(+) ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na(+) content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na(+) content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na(+) loading to xylem from root and leaf tissues. Transgenic lines also showed increased K(+) and Ca(2+) content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na(+) efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na(+) content in different organs and also affect the other transporters activity indirectly. These

  2. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

    Science.gov (United States)

    2012-01-01

    Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other transporters activity indirectly

  3. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Yadav Narendra

    2012-10-01

    Full Text Available Abstract Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1 gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC, chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other

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

    Science.gov (United States)

    Hakamata, Yoji; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

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

  5. Contribution of Epigenetic Modifications to the Decline in Transgene Expression from Plasmid DNA in Mouse Liver

    Directory of Open Access Journals (Sweden)

    Lei Zang

    2015-08-01

    Full Text Available Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression.

  6. A ThDREB gene from Tamarix hispida improved the salt and drought tolerance of transgenic tobacco and T. hispida.

    Science.gov (United States)

    Yang, Guiyan; Yu, Lili; Zhang, Kaimin; Zhao, Yulin; Guo, Yucong; Gao, Caiqiu

    2017-04-01

    Dehydration-responsive element-binding (DREB) transcription factors are important abiotic stress tolerance related genes, and some reports on the roles of DREB have primarily addressed herbal plants. To explore the abiotic stress tolerance role of DREB (ThDREB) from Tamarix hispida, a ThDREB gene with a complete ORF of 783 bp that encodes a 28.74 kDa protein with 260 amino acids, was isolated and functionally annotated. ThDREB expression was highly induced by NaCl, PEG, NaHCO 3 and CdCl 2 treatments, and the highest expression level (369.2-fold of control) was found for the roots that were under NaCl stress for 6 h. The tobacco plants that were transformed by ThDREB were conferred with higher germination rates, fresh weights and root lengths than the wild type (WT) tobacco plants under NaCl and mannitol treatments. The total chlorophyll content (tcc), superoxide dismutase (SOD) and peroxidase (POD) activities were also higher in the transgenic lines in comparison with the WT, and the malondialdehyde (MDA) and H 2 O 2 content, electrolyte leakage (EL) rate and ROS as tracked by staining were generated to a lesser degree in ThDREB transgenic plants than in the WT under NaCl and mannitol stress. Furthermore, the transient overexpression analysis of ThDREB in T. hispida also improved plant salt and drought tolerance in comparison with the empty vector-transformed lines. Our results indicated that ThDREB expression could effectively improve tolerance to salt and drought stress by enhancing the antioxidase activity that keeps the ROS at a low accumulation level and makes them easy to scavenge. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. [The establishment and identification of GPx-1(P198L) gene systemic expression transgenic mice].

    Science.gov (United States)

    Wang, Su-qin; Zhu, Yan-he; Lin, Lin; Gao, Deng-feng; Niu, Xiao-lin

    2015-01-01

    To generate systemic expression human cellular glutathione peroxidase-1 (GPx-1) (198Leu) transgenic mice model in order to investigate the functional variants in GPx-1 gene in oxidative stress-related diseases. After linearization with BamnH I and Acc I, the transgenic construct GPx-1 (198Leu) was microinjected into the zygotes of C57BL/6J mice to generate transgenic mice, whose genotype was detected by PCR with specific primers. The GPx-1 gene expression profile was determined by Western blotting. 13 transgenic founder mice were successfully generated. Western blotting result showed that the protein expression level of 4 transgenic mice in hearts were higher than that of wild type mice. Human GPx-1PSL transgenic mice was successfully established. This kind of animal model is of significance for making further researches on oxidative stress-related diseases.

  8. A short synthetic MAR positively affects transgene expression in rice and Arabidopsis

    NARCIS (Netherlands)

    Geest, van der A.H.M.; Welter, M.E.; Woosley, A.T.; Pareddy, D.R.; Pavelko, S.E.; Skokut, M.; Ainley, W.M.

    2004-01-01

    Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number

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

    Directory of Open Access Journals (Sweden)

    Douglas Strathdee

    2006-12-01

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

  10. Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

    Directory of Open Access Journals (Sweden)

    Yun Seong-Jo

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Inhibition of cell proliferation, cell expansion and differentiation by the Arabidopsis SUPERMAN gene in transgenic tobacco plants.

    Science.gov (United States)

    Bereterbide, A; Hernould, M; Castera, S; Mouras, A

    2001-11-01

    Plant development depends upon the control of growth, organization and differentiation of cells derived from shoot and root meristems. Among the genes involved in flower organ determination, the cadastral gene SUPERMAN controls the boundary between whorls 3 and 4 and the growth of the adaxial outer ovule integument by down-regulating cell divisions. To determine the precise function of this gene we overexpressed ectopically the Arabidopsis thaliana (L.) Heynh. SUPERMAN gene in tobacco (Nicotiana tabacum L.). The transgenic plants exhibited a dwarf phenotype. Histologically and cytologically detailed analyses showed that dwarfism is correlated with a reduction in cell number, which is in agreement with the SUPERMAN function in Arabidopsis. Furthermore, a reduction in cell expansion and an impairment of cell differentiation were observed in tobacco organs. These traits were observed in differentiated vegetative and floral organs but not in meristem structures. A potential effect of the SUPERMAN transcription factor in the control of gibberellin biosynthesis is discussed.

  13. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  14. Enhancement of α-linolenic acid content in transgenic tobacco seeds by targeting a plastidial ω-3 fatty acid desaturase (fad7) gene of Sesamum indicum to ER.

    Science.gov (United States)

    Bhunia, Rupam Kumar; Chakraborty, Anirban; Kaur, Ranjeet; Maiti, Mrinal K; Sen, Soumitra Kumar

    2016-01-01

    Expression of sesame plastidial FAD7 desaturase modified with the endoplasmic reticulum targeting and retention signals, enhances the α-linolenic acid accumulation in seeds of Nicotiana tabacum. In plants, plastidial ω-3 fatty acid desaturase-7 (FAD7) catalyzes the formation of C16 and C18 trienoic fatty acids using organellar glycerolipids and participate in the membrane lipid formation. The plastidial ω-3 desaturases (FAD7) share high sequence homology with the microsomal ω-3 desaturases (FAD3) at the amino acid level except the N-terminal organelle transit peptide. In the present study, the predicted N-terminal plastidial signal peptide of fad7 gene was replaced by the endoplasmic reticulum signal peptide and an endoplasmic reticulum retention signal was placed at the C-terminal. The expression of the modified sesame ω-3 desaturase increases the α-linolenic acid content in the range of 4.78-6.77 % in the seeds of transgenic tobacco plants with concomitant decrease in linoleic acid content. The results suggested the potential of the engineered plastidial ω-3 desaturase from sesame to influence the profile of α-linolenic acid in tobacco plant by shifting the carbon flux from linoleic acid, and thus it can be used in suitable genetic engineering strategy to increase the α-linolenic acid content in sesame and other vegetable oils.

  15. Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions

    OpenAIRE

    Lee, Young Pyo; Baek, Kwang-Hyun; Lee, Haeng-Soon; Kwak, Sang-Soo; Bang, Jae-Woog; Kwon, Suk-Yoon

    2010-01-01

    Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal gr...

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

    Science.gov (United States)

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

    2016-06-14

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

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

    Directory of Open Access Journals (Sweden)

    Dismuke Adria D

    2009-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Kohn Aimee

    2009-01-01

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

  19. A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco

    NARCIS (Netherlands)

    Mlynarova, L.; Nap, J.P.H.

    2003-01-01

    To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox

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

    OpenAIRE

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

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human ?-interferon and ?-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian ...

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

    Directory of Open Access Journals (Sweden)

    Gonzalez-Ruiz Gloriene

    2011-08-01

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

  2. Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

    Science.gov (United States)

    Ghosh, Gourab; Ganguly, Shreeparna; Purohit, Arnab; Chaudhuri, Rituparna Kundu; Das, Sampa; Chakraborti, Dipankar

    2017-07-01

    Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T 0 transformants were selected on the basis of PCR and protein expression profile. T 1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T 1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T 2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T 3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T 0 , T 1, and T 2 ). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

  3. From gene to harvest: insights into upstream process development for the GMP production of a monoclonal antibody in transgenic tobacco plants.

    Science.gov (United States)

    Sack, Markus; Rademacher, Thomas; Spiegel, Holger; Boes, Alexander; Hellwig, Stephan; Drossard, Juergen; Stoger, Eva; Fischer, Rainer

    2015-10-01

    The EU Sixth Framework Programme Integrated Project 'Pharma-Planta' developed an approved manufacturing process for recombinant plant-made pharmaceutical proteins (PMPs) using the human HIV-neutralizing monoclonal antibody 2G12 as a case study. In contrast to the well-established Chinese hamster ovary platform, which has been used for the production of therapeutic antibodies for nearly 30 years, only draft regulations were initially available covering the production of recombinant proteins in transgenic tobacco plants. Whereas recombinant proteins produced in animal cells are secreted into the culture medium during fermentation in bioreactors, intact plants grown under nonsterile conditions in a glasshouse environment provide various 'plant-specific' regulatory and technical challenges for the development of a process suitable for the acquisition of a manufacturing licence for clinical phase I trials. During upstream process development, several generic steps were addressed (e.g. plant transformation and screening, seed bank generation, genetic stability, host plant uniformity) as well as product-specific aspects (e.g. product quantity). This report summarizes the efforts undertaken to analyse and define the procedures for the GMP/GACP-compliant upstream production of 2G12 in transgenic tobacco plants from gene to harvest, including the design of expression constructs, plant transformation, the generation of production lines, master and working seed banks and the detailed investigation of cultivation and harvesting parameters and their impact on biomass, product yield and intra/interbatch variability. The resulting procedures were successfully translated into a prototypic manufacturing process that has been approved by the German competent authority. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Transgenic plants expressing GLK1 and CCA1 having increased nitrogen assimilation capacity

    Science.gov (United States)

    Coruzzi, Gloria [New York, NY; Gutierrez, Rodrigo A [Santiago, CL; Nero, Damion C [Woodside, NY

    2012-04-10

    Provided herein are compositions and methods for producing transgenic plants. In specific embodiments, transgenic plants comprise a construct comprising a polynucleotide encoding CCA1, GLK1 or bZIP1, operably linked to a plant-specific promote, wherein the CCA1, GLK1 or bZIP1 is ectopically overexpressed in the transgenic plants, and wherein the promoter is optionally a constitutive or inducible promoter. In other embodiments, transgenic plants in which express a lower level of CCA1, GLK1 or bZIP1 are provided. Also provided herein are commercial products (e.g., pulp, paper, paper products, or lumber) derived from the transgenic plants (e.g., transgenic trees) produced using the methods provided herein.

  5. Preservation and faithful expression of transgene via artificial seeds in alfalfa.

    Directory of Open Access Journals (Sweden)

    Wenting Liu

    Full Text Available Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12-15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species.

  6. Insect-resistance and high-yield transgenic tobacco obtained by ...

    African Journals Online (AJOL)

    The modified synthesized VHb gene and insectidal gene (GFMcryIA) were transferred to tobacco plants by Agrobacterium-mediated transformation. The bivalent genes were inserted successfully into the tobacco genome and detected by PCR amplification. Southern blot and Western blot analyses showed that VHb gene ...

  7. Insect-resistance and high-yield transgenic tobacco obtained by ...

    African Journals Online (AJOL)

    hope&shola

    2010-10-04

    Oct 4, 2010 ... The modified synthesized VHb gene and insectidal gene (GFMcryIA) were transferred to tobacco plants by Agrobacterium-mediated transformation. The bivalent genes were inserted successfully into the tobacco genome and detected by PCR amplification. Southern blot and Western blot analyses showed.

  8. Oviduct-Specific Expression of Human Neutrophil Defensin 4 in Lentivirally Generated Transgenic Chickens

    Science.gov (United States)

    Liu, Tongxin; Wu, Hanyu; Cao, Dainan; Li, Qingyuan; Zhang, Yaqiong; Li, Ning; Hu, Xiaoxiang

    2015-01-01

    The expression of oviduct-specific recombinant proteins in transgenic chickens is a promising technology for the production of therapeutic biologics in eggs. In this study, we constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. After the antimicrobial activity of the recombinant HNP4 protein was tested at the cellular level, a 2.8-kb ovalbumin promoter was used to drive HNP4 expression specifically in oviduct tissues. From 669 injected eggs, 218 chickens were successfully hatched. Ten G0 roosters, with semens identified as positive for the transgene, were mated with wild-type hens to generate G1 chickens. From 1,274 total offspring, fifteen G1 transgenic chickens were positive for the transgene, which was confirmed by PCR and Southern blotting. The results of the Southern blotting and genome walking indicated that a single copy of the HNP4 gene was integrated into chromosomes 1, 2, 3, 4, 6 and 24 of the chickens. As expected, HNP4 expression was restricted to the oviduct tissues, and the levels of both transcriptional and translational HNP4 expression varied greatly in transgenic chickens with different transgene insertion sites. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml. These results indicated that the production of transgenic chickens that expressed HNP4 protein in egg whites was successful. PMID:26020529

  9. Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants.

    Science.gov (United States)

    Ko, Moonkyung; Cho, Jung Hyun; Seo, Hyo-Hyoun; Lee, Hyun-Hwa; Kang, Ha-Young; Nguyen, Thai Son; Soh, Hyun Cheol; Kim, Young Soon; Kim, Jeong-Il

    2016-08-01

    Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling

  10. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    Science.gov (United States)

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (ptransgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

  11. Transgene expression in microalgae – from tools to applications

    Directory of Open Access Journals (Sweden)

    Lior eDoron

    2016-04-01

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

  12. Stability of transgene expression, field performance and recombination breeding of transformed barley lines

    DEFF Research Database (Denmark)

    Horvath, H.; Jensen, L.G.; Wong, O.T.

    2001-01-01

    Stable inheritance of the transgene, consistent expression and competitive agronomic properties of transgenic crops are important parameters for successful use of the latter. These properties have been analyzed with 18 homozygous transgenic barley lines of the cultivar Golden Promise. The lines...... in homozygous transgenic T-3 plants, and these remained constant over a 3-year period. In micro-malting experiments, the heat-stable enzyme reached levels of up to 1.4 mug.mg(-1) protein and survived kiln drying at levels of 70-100%. In the field trials of 1997 and 1998 the transgenic lines had a reduced 1000......, and ari-e. Two improvements were achieved: (1) F-3 lines homozygous for the expression of heat-stable (1,3-1,4)-beta -glucanase were found among lines that were homozygous for each of the four morphological phenotypes; (2) improved 1000-grainweights and yields with respect to those of the original...

  13. Improving expression of reporter transgene in stem cell by construction of different lentiviral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Tae, Seong Ho; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of); Le, Uyenchi N.; Padmanabhan, Parasuraman [Singapore Bio-Imaging Imaging Consortium, Singapore (Singapore)

    2007-07-01

    For stem cell trafficking applications, it is imperative to express transgenes at desired and stable levels. In recent years, lentivirus-mediated gene transfer was shown to be an efficient method to stably introduce genetic modifications in target cells, even if these are in proliferative or nonproliferative states. Moreover, transgene expression levels can be controlled by using different promoters. The present study was designed to compare the potency of various promoters regulating expression of imaging reporter genes in embryonic H9c2 cardiomyoblasts derived from rat heart. Lentiviral vector was produced by the transient transfection of plasmids carrying required genes and those encoding for virus coating proteins into 293T cells. Harvested viral constructs were incubated with Hela and H9c2 cells, respectively. Transgene expressions were detected by several imaging modalities and evaluated by enzymatic assays. Results - We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. Here we show that lentivirus-mediated gene transfer allows efficient and stable transgene expression in embryonic cardiomyoblasts in vitro and that transgene expression levels can be varied by using different well-characterized gene promoters. In vivo trials about gene expression will probably further determine the potential of long-term trafficking stem cells using lentivirus.

  14. Improving expression of reporter transgene in stem cell by construction of different lentiviral vectors

    International Nuclear Information System (INIS)

    Tae, Seong Ho; Min, Jung Joon; Le, Uyenchi N.; Padmanabhan, Parasuraman

    2007-01-01

    For stem cell trafficking applications, it is imperative to express transgenes at desired and stable levels. In recent years, lentivirus-mediated gene transfer was shown to be an efficient method to stably introduce genetic modifications in target cells, even if these are in proliferative or nonproliferative states. Moreover, transgene expression levels can be controlled by using different promoters. The present study was designed to compare the potency of various promoters regulating expression of imaging reporter genes in embryonic H9c2 cardiomyoblasts derived from rat heart. Lentiviral vector was produced by the transient transfection of plasmids carrying required genes and those encoding for virus coating proteins into 293T cells. Harvested viral constructs were incubated with Hela and H9c2 cells, respectively. Transgene expressions were detected by several imaging modalities and evaluated by enzymatic assays. Results - We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. We observed that the level of stable transgene expression in lentivirus-transduced myoblasts could be modulated over several orders of magnitude, with the Ubiquitin (Ub) promoter exhibiting the highest activity, intermediate expression was observed with the CAG promoter, whereas expression observed with the CMV promoter was very weak. Here we show that lentivirus-mediated gene transfer allows efficient and stable transgene expression in embryonic cardiomyoblasts in vitro and that transgene expression levels can be varied by using different well-characterized gene promoters. In vivo trials about gene expression will probably further determine the potential of long-term trafficking stem cells using lentivirus

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

    Science.gov (United States)

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

    2012-01-01

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

  16. From transgene expression to public acceptance of transgenic plants: a matter of predictability

    NARCIS (Netherlands)

    Nap, J.P.H.; Mlynárová, L.; Stiekema, W.J.

    1996-01-01

    A good strategy for acceptable legislation of transgenic plants can be thought to be composed of several stacked levels of decision-making. These levels range from global to individual to cellular to nuclear and beyond. Any decision will depend on decisions made on the level below. Various examples

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Clare C McArthur

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2013-01-01

    The fruit fly Drosophila melanogaster represents a key model organism for analyzing how neuronal circuits regulate behavior. The mushroom body in the central brain is a particularly prominent brain region that has been intensely studied in several insect species and been implicated in a variety of behaviors, e.g., associative learning, locomotor activity, and sleep. Drosophila melanogaster offers the advantage that transgenes can be easily expressed in neuronal subpopulations, e.g., in intrinsic mushroom body neurons (Kenyon cells). A number of transgenes has been described and engineered to visualize the anatomy of neurons, to monitor physiological parameters of neuronal activity, and to manipulate neuronal function artificially. To target the expression of these transgenes selectively to specific neurons several sophisticated bi- or even multipartite transcription systems have been invented. However, the number of transgenes that can be combined in the genome of an individual fly is limited in practice. To facilitate the analysis of the mushroom body we provide a compilation of transgenic fruit flies that express transgenes under direct control of the Kenyon-cell specific promoter, mb247. The transgenes expressed are fluorescence reporters to analyze neuroanatomical aspects of the mushroom body, proteins to restrict ectopic gene expression to mushroom bodies, or fluorescent sensors to monitor physiological parameters of neuronal activity of Kenyon cells. Some of the transgenic animals compiled here have been published already, whereas others are novel and characterized here for the first time. Overall, the collection of transgenic flies expressing sensor and reporter genes in Kenyon cells facilitates combinations with binary transcription systems and might, ultimately, advance the physiological analysis of mushroom body function. PMID:24065891

  1. Spatio Temporal Expression Pattern of an Insecticidal Gene (cry2A in Transgenic Cotton Lines

    Directory of Open Access Journals (Sweden)

    Allah BAKHSH

    2012-11-01

    Full Text Available The production of transgenic plants with stable, high-level transgene expression is important for the success of crop improvement programs based on genetic engineering. The present study was conducted to evaluate genomic integration and spatio temporal expression of an insecticidal gene (cry2A in pre-existing transgenic lines of cotton. Genomic integration of cry2A was evaluated using various molecular approaches. The expression levels of cry2A were determined at vegetative and reproductive stages of cotton at regular intervals. These lines showed a stable integration of insecticidal gene in advance lines of transgenic cotton whereas gene expression was found variable with at various growth stages as well as in different plant parts throughout the season. The leaves of transgenic cotton were found to have maximum expression of cry2A gene followed by squares, bolls, anthers and petals. The protein level in fruiting part was less as compared to other parts showing inconsistency in gene expression. It was concluded that for culturing of transgenic crops, strategies should be developed to ensure the foreign genes expression efficient, consistent and in a predictable manner.

  2. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression.

    Science.gov (United States)

    López-Paz, Cristina; Liu, Dianyi; Geng, Sa; Umen, James G

    2017-12-01

    Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data-mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL35a and RPL23, and ferredoxin, FDX1, whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A-RBCS2 (AR) hybrid promoter/terminator sequences. The RPL23 flanking sequences were further tested using the zeocin resistance gene sh-ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin-resistant transformants and higher levels of resistance than AR- or PSAD-based vectors. Chlamydomonas RPL23 sequences also enabled transgene expression in Volvox carteri. Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  3. Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

    OpenAIRE

    Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.; Nettelbeck, Dirk M.

    2010-01-01

    Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show t...

  4. Expression of the murine wild-type tyrosinase gene in transgenic rabbits.

    Science.gov (United States)

    Aigner, B; Besenfelder, U; Seregi, J; Frenyo, L V; Sahin-Toth, T; Brem, G

    1996-11-01

    The tyrosinase gene is known to be essential for melanization and has been shown to rescue pigmentation in albino mice. Previously we have described the strict copy-number-dependent expression of a murine wild-type tyrosinase gene construct over several generations in transgenic mice. In this study, we analysed the same gene construct as a marker gene for the transmission and expression of transgenes in rabbits. Using an albino hybrid strain, we produced transgenic rabbits expressing the murine tyrosinase gene. Strict correlation between integration and expression of the transgene and stable germline transmission of the integrated gene construct according to the Mendelian pattern of inheritance was observed. Thus, breeding control was facilitated by simple phenotypic examination of the transgenic animals. In contrast to mice transgenic for the same gene construct, tyrosinase-transgenic rabbits showed a greater variety in hue, intensity and extent of coat pigmentation, which is caused by the diversity in the loci affecting the melanization. Benefits and limitations of tyrosinase as a marker gene for the detection of homozygous individuals in the albino hybrid strain used are discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X. [Lawrence Livermore National Laboratory, CA (United States); Anderson, G. [Univ. of California, Davis, CA (United States)

    1994-12-31

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

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

    Science.gov (United States)

    Weber, Ricardo Luís Mayer; Wiebke-Strohm, Beatriz; Bredemeier, Christian; Margis-Pinheiro, Márcia; de Brito, Giovani Greigh; Rechenmacher, Ciliana; Bertagnolli, Paulo Fernando; de Sá, Maria Eugênia Lisei; Campos, Magnólia de Araújo; de Amorim, Regina Maria Santos; Beneventi, Magda Aparecida; Margis, Rogério; Grossi-de-Sa, Maria Fátima; Bodanese-Zanettini, Maria Helena

    2014-12-10

    Drought 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. Two 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. This 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.

  7. Expression profiling of microRNAs in optineurin (E50K) mutant transgenic mice.

    Science.gov (United States)

    Gao, Lin; Jiang, B O; Lei, Dawei; Zhou, Xinrong; Yuan, Huiping

    2016-02-01

    An E50K substitution in the transcription factor optineurin (OPTN) induces primary open-angle glaucoma (POAG). To explore the potential role of microRNAs (miRNAs) in E50K OPTN-induced POAG, miRNA expression profiling was performed on retinal samples from OPTN (E50K) transgenic and wild-type mice. The retinas were collected from 30 transgenic and 30 wild-type mice, and miRNA expression was evaluated using a genome-wide miRNA microarray. miRNAs that were differentially expressed in retinal samples from OPTN (E50K) transgenic mice were identified and validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additional gene ontology and signaling pathway analyses were performed using bioinformatics tools. A total of 48 miRNAs exhibited increased or decreased expression in the retinas from OPTN (E50K) transgenic mice when compared with the expression in the retinas from wild-type mice. A total of 5 miRNAs with increased expression in OPTN (E50K) transgenic mice could be grouped into one cluster as they belong to the miR-8 family and may act as regulators in the development of POAG in OPTN (E50K) transgenic mice. RT-qPCR results confirmed significantly increased expression of miR-141 in the retinas of OPTN (E50K) transgenic mice as compared to wild-type mice. In conclusion, these results show that certain miRNAs are differentially expressed in the retinas of OPTN (E50K) transgenic mice and may play roles in the pathogenesis of POAG induced by OPTN (E50K).

  8. Correlation between transgen expression and plasmid DNA loss in mouse liver.

    Science.gov (United States)

    Togashi, Ryohei; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2013-01-01

    Transgene expression from plasmid DNA is dependent on the expression efficiency per plasmid and the amount of intranuclear plasmid. In the present study, intranuclear dispositions of two types of plasmid DNAs (i.e. the pCpGfree and pLIVE plasmids) that maintain transgene expression in mouse liver were analyzed. In addition, the relationship between transgene expression and plasmid stability in the nucleus was examined. First, the pCpGfree and pLIVE plasmid DNAs, bearing the mouse secreted alkaline phosphatase (Seap) gene, were administered into mouse liver by the hydrodynamics-based method. Next, various Seap-plasmid DNAs containing different promoters, upstream and downstream sequences, and backbones were injected into mice, and both SEAP expression and plasmid DNA amounts were monitored for 28 days. At the 14- and 28-day time points, the amount of the pCpGfree plasmid DNA was one order of magnitude less than that of the pLIVE plasmid. Meanwhile, the expression efficiency per plasmid was one order of magnitude more efficient for the pCpGfree plasmid DNA. Moreover, the administration of various Seap-plasmid DNAs revealed that negative correlations exist between plasmid stability and SEAP expression level. The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter.

    Science.gov (United States)

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

    2008-01-01

    Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.

  10. Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions.

    Science.gov (United States)

    Lee, Young Pyo; Baek, Kwang-Hyun; Lee, Haeng-Soon; Kwak, Sang-Soo; Bang, Jae-Woog; Kwon, Suk-Yoon

    2010-05-01

    Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal growth and seed development. Furthermore, the transgenic seeds displayed increased CuZnSOD and APX enzymatic activities during seed development and maintained antioxidant enzymatic activity after two years of dried storage at room temperature. The two-year stored non-transgenic seeds (aged NT seeds) had higher levels of ion leakage than the two-year stored transgenic seeds (aged CA seeds), indicating membrane damage caused by ROS was more severe in the aged NT seeds than the aged CA seeds. The aged CA seeds decreased germination rates as compared to newly harvested transgenic and non-transgenic seeds. The aged CA seeds, however, significantly increased germination rates under various abiotic stress conditions as compared to aged NT seeds. These data strongly suggest that simultaneous over-expression of the CuZnSOD and APX genes in plastids improves seed longevity and germination under various environmental stress conditions by attenuating the effects of oxidative stress produced by elongated storage conditions and harsh environmental stresses.

  11. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.; Ku, Maurice S.

    2011-12-10

    Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in

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

    Directory of Open Access Journals (Sweden)

    Uzoma eIhemere

    2012-09-01

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

  13. Pronounced Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Sucrose Synthase May Reveal a Novel Sugar Signaling Pathway

    Science.gov (United States)

    Nguyen, Quynh Anh; Luan, Sheng; Wi, Seung G.; Bae, Hanhong; Lee, Dae-Seok; Bae, Hyeun-Jong

    2016-01-01

    Soluble sugars not only serve as nutrients, but also act as signals for plant growth and development, but how sugar signals are perceived and translated into physiological responses in plants remains unclear. We manipulated sugar levels in transgenic plants by overexpressing sucrose synthase (SuSy), which is a key enzyme believed to have reversible sucrose synthesis and sucrose degradation functions. The ectopically expressed SuSy protein exhibited sucrose-degrading activity, which may change the flux of sucrose demand from photosynthetic to non-photosynthetic cells, and trigger an unknown sucrose signaling pathway that lead to increased sucrose content in the transgenic plants. An experiment on the transition from heterotrophic to autotrophic growth demonstrated the existence of a novel sucrose signaling pathway, which stimulated photosynthesis, and enhanced photosynthetic synthesis of sucrose, which was the direct cause or the sucrose increase. In addition, a light/dark time treatment experiment, using different day length ranges for photosynthesis/respiration showed the carbohydrate pattern within a 24-h day and consolidated the role of sucrose signaling pathway as a way to maintain sucrose demand, and indicated the relationships between increased sucrose and upregulation of genes controlling development of the shoot apical meristem (SAM). As a result, transgenic plants featured a higher biomass and a shorter time required to switch to reproduction compared to those of control plants, indicating altered phylotaxis and more rapid advancement of developmental stages in the transgenic plants. PMID:26793204

  14. PITT: pronuclear injection-based targeted transgenesis, a reliable transgene expression method in mice.

    Science.gov (United States)

    Ohtsuka, Masato; Miura, Hiromi; Sato, Masahiro; Kimura, Minoru; Inoko, Hidetoshi; Gurumurthy, Channabasavaiah B

    2012-01-01

    Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

  15. Transgene expression in cowpea ( Vigna unguiculata (L.) Walp ...

    African Journals Online (AJOL)

    Pollen transformation shows potential as a fast and easy means of obtaining transformed plants carrying desirable transgenes. Agrobacterium tumefaciens has been suggested as the best natural plant genetic engineering system. Laboratory and screenhouse studies were undertaken to investigate the possibility of ...

  16. The stability of transgene expression and effect of DNA methylation ...

    African Journals Online (AJOL)

    In this paper, we selected transgenic birch (Betula platyphylla Suk) plants, which included nonsilencing plants, transcriptional silence plants including TP96, TP74, TP73 and the post-transcriptional silence ones (TP67 and TP72). The transcription of the bgt gene in different tissues and organs were significantly different.

  17. Transgene expression of lilies grown in the greenhouse and outdoors

    Science.gov (United States)

    Lilium longiflorum cv. Nellie White plants were transformed with either the bar-uidA fusion gene or the npt II and uidA genes and grown for two seasons in the greenhouse and outdoors in containers. All transgenes were under control of the CaMV 35S promoter. During the first year there was no differ...

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

    Indian Academy of Sciences (India)

    cent of certain human autoimmune disorders. Recent evi- dence has demonstrated that constitutive ..... showed traits of prematurity with a distinct tail and a yolk extension elongating away from the yolk (figure 2A, ... RNAs were isolated from three tails of 3 mpf (month post- fertilization) F0 transgenic zebrafish g1, g2 and g7, ...

  19. Tobacco

    Science.gov (United States)

    ... Second-hand smoke is the smoke that fills restaurants, offices or other enclosed spaces when people burn ... as smuggling, illicit manufacturing and counterfeiting. The tobacco industry and others often argue that high tobacco product ...

  20. Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

    NARCIS (Netherlands)

    Haring, Michel A.; Teeuwen-de Vroomen, Marianne J.; Nijkamp, H. John J.; Hille, Jacques

    1991-01-01

    In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of

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

    Science.gov (United States)

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

    2013-02-01

    As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli β-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes. © 2012 Royal Entomological Society.

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

    Science.gov (United States)

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

    2013-01-01

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

  3. Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

    International Nuclear Information System (INIS)

    Hufbauer, M.; Lazic, D.; Akguel, B.; Brandsma, J.L.; Pfister, H.; Weissenborn, S.J.

    2010-01-01

    Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

  4. The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco

    Czech Academy of Sciences Publication Activity Database

    Fojtová, Miloslava; Bleys, A.; Bedřichová, Jana; Van Houdt, H.; Křížová, Kateřina; Depicker, A.; Kovařík, Aleš

    2006-01-01

    Roč. 34, č. 8 (2006), s. 2280-2293 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GA521/04/0775; GA ČR(CZ) GD204/05/H505 Institutional research plan: CEZ:AV0Z50040507 Keywords : transgenes * epigenetic silencing * DNA methylation Subject RIV: BO - Biophysics Impact factor: 6.317, year: 2006

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Gloria Volohonsky

    2017-01-01

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

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

    Science.gov (United States)

    Hartley, Katharine O; Nutt, Stephen L; Amaya, Enrique

    2002-02-05

    The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.

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

    Science.gov (United States)

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

    2013-02-01

    We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans. © 2012 Royal Entomological Society.

  9. Foamy virus vectors expressing anti-HIV transgenes efficiently block HIV-1 replication.

    Science.gov (United States)

    Taylor, Jason A; Vojtech, Lucia; Bahner, Ingrid; Kohn, Donald B; Laer, Dorothee Von; Russell, David W; Richard, Robert E

    2008-01-01

    Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEMx174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy.

  10. Transgenic labeling of parvalbumin-expressing neurons with tdTomato

    Science.gov (United States)

    Kaiser, Tobias; Ting, Jonathan T.; Monteiro, Patrícia; Feng, Guoping

    2015-01-01

    Summary Parvalbumin (PVALB)-expressing fast-spiking interneurons subserve important roles in many brain regions by modulating circuit function and dysfunction of these neurons is strongly implicated in neuropsychiatric disorders including schizophrenia and autism. To facilitate the study of PVALB neuron function we need to be able to identify PVALB neurons in vivo. We have generated a bacterial artificial chromosome (BAC) transgenic mouse line expressing the red fluorophore tdTomato under the control of endogenous regulatory elements of the Pvalb gene locus (JAX # 027395). We show that the tdTomato transgene is faithfully expressed relative to endogenous PVALB expression throughout the brain. Furthermore, targeted patch clamp recordings confirm that the labeled populations in neocortex, striatum, and hippocampus are fast-spiking interneurons based on intrinsic properties. This new transgenic mouse line provides a useful tool to study PVALB neuron function in the normal brain as well as in mouse models of psychiatric disease. PMID:26318335

  11. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.

  12. Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function.

    Science.gov (United States)

    Daniell, Henry; Ruiz, Gricel; Denes, Bela; Sandberg, Laurence; Langridge, William

    2009-04-03

    Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function. Expression of the synthetic Insulin like Growth Factor 1 gene (IGF-1s, 60% AT) was observed in transformed E. coli. However, no native IGF-1 gene (IGF-1n, 41% AT) product was detected in the western blots in E. coli. Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR. Southern blot analysis confirmed that the transgenic lines were homoplasmic. The transgenic plant lines had IGF-1s expression levels of 11.3% of total soluble protein (TSP). The IGF-1n plants contained 9.5% TSP as IGF-1n, suggesting that the chloroplast translation machinery is more flexible than E. coli in codon preference and usage. The expression of IGF-1 was increased up to 32% TSP under continuous illumination by the chloroplast light regulatory elements. IgG-Sepharose affinity column chromatographic separation of Z domain containing chloroplast derived IGF-1 protein, single and two dimensional electrophoresis methods and mass spectrometer analysis confirmed the identity of human IGF-1 in transgenic chloroplasts. Two spots analyzed from 2-D focusing/phoresis acrylamide gel showed the correct amino acid sequence of human IGF-1 and the S. aureus Z-tag. Cell proliferation assays in human HU-3 cells demonstrated the biological activity of chloroplast derived IGF-1 even in the presence of the S. aureus Z tag. This study demonstrates that the human Insulin like Growth Factor-1 expressed in transgenic chloroplasts is identical to the native protein and is fully functional. The ability to use plant

  13. Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function

    Directory of Open Access Journals (Sweden)

    Sandberg Laurence

    2009-04-01

    Full Text Available Abstract Background Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function. Results Expression of the synthetic Insulin like Growth Factor 1 gene (IGF-1s, 60% AT was observed in transformed E. coli. However, no native IGF-1 gene (IGF-1n, 41% AT product was detected in the western blots in E. coli. Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR. Southern blot analysis confirmed that the transgenic lines were homoplasmic. The transgenic plant lines had IGF-1s expression levels of 11.3% of total soluble protein (TSP. The IGF-1n plants contained 9.5% TSP as IGF-1n, suggesting that the chloroplast translation machinery is more flexible than E. coli in codon preference and usage. The expression of IGF-1 was increased up to 32% TSP under continuous illumination by the chloroplast light regulatory elements. IgG-Sepharose affinity column chromatographic separation of Z domain containing chloroplast derived IGF-1 protein, single and two dimensional electrophoresis methods and mass spectrometer analysis confirmed the identity of human IGF-1 in transgenic chloroplasts. Two spots analyzed from 2-D focusing/phoresis acrylamide gel showed the correct amino acid sequence of human IGF-1 and the S. aureus Z-tag. Cell proliferation assays in human HU-3 cells demonstrated the biological activity of chloroplast derived IGF-1 even in the presence of the S. aureus Z tag. Conclusion This study demonstrates that the human Insulin like Growth Factor-1 expressed in transgenic chloroplasts is identical to the native

  14. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

    Science.gov (United States)

    Cho, Kwang-Soo; Han, Eun-Heui; Kwak, Sang-Soo; Cho, Ji-Hong; Im, Ju-Seong; Hong, Su-Young; Sohn, Hwang-Bae; Kim, Yun-Hee; Lee, Shin-Woo

    2016-01-01

    Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  15. Transgenic potato plants expressing cry3A gene confer resistance to Colorado potato beetle.

    Science.gov (United States)

    Mi, Xiaoxiao; Ji, Xiangzhuo; Yang, Jiangwei; Liang, Lina; Si, Huaijun; Wu, Jiahe; Zhang, Ning; Wang, Di

    2015-07-01

    The Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a fatal pest, which is a quarantine pest in China. The CPB has now invaded the Xinjiang Uygur Autonomous Region and is constantly spreading eastward in China. In this study, we developed transgenic potato plants expressing cry3A gene. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the cry3A gene expressed in leaves, stems and roots of the transgenic plants under the control of CaMV 35S promoter, while they expressed only in leaves and stems under the control of potato leaf and stem-specific promoter ST-LS1. The mortality of the larvae was higher (28% and 36%) on the transgenic plant line 35S1 on the 3rd and 4th days, and on ST3 (48%) on the 5th day after inoculation with instar larvae. Insect biomass accumulation on the foliage of the transgenic plant lines 35S1, 35S2 and ST3 was significantly lower (0.42%, 0.43% and 0.42%). Foliage consumption was lowest on transgenic lines 35S8 and ST2 among all plant foliage (7.47 mg/larvae/day and 12.46 mg/larvae/day). The different transgenic plant foliages had varied inhibition to larval growth. The survivors on the transgenic lines obviously were smaller than their original size and extremely weak. The transgenic potato plants with CPB resistance could be used to develop germplasms or varieties for controlling CPB damage and halting its spread in China. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  16. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin.

    Science.gov (United States)

    Han, Yuepeng; Vimolmangkang, Sornkanok; Soria-Guerra, Ruth Elena; Korban, Schuyler S

    2012-04-01

    Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated.

  17. Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

    Science.gov (United States)

    Marino, Nalini; Illingworth, Sam; Kodialbail, Prithvi; Patel, Ashvin; Calderon, Hugo; Lear, Rochelle; Fisher, Kerry D; Champion, Brian R; Brown, Alice C N

    2017-01-01

    Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

  18. Development of a versatile oncolytic virus platform for local intra-tumoural expression of therapeutic transgenes.

    Directory of Open Access Journals (Sweden)

    Nalini Marino

    Full Text Available Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.

  19. An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

    Science.gov (United States)

    Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

    2013-01-01

    On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

  20. An intergenic region shared by At4g35985 and At4g35987 in Arabidopsis thaliana is a tissue specific and stress inducible bidirectional promoter analyzed in transgenic arabidopsis and tobacco plants.

    Directory of Open Access Journals (Sweden)

    Joydeep Banerjee

    Full Text Available On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985 are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85 showed stronger expression (about 3.5 fold compared to the At4g35987 promoter (P87. The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.

  1. The Q System: A Repressible Binary System for Transgene Expression, Lineage Tracing and Mosaic Analysis

    OpenAIRE

    Potter, Christopher J.; Tasic, Bosiljka; Russler, Emilie V.; Liang, Liang; Luo, Liqun

    2010-01-01

    We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This ‘Q system’ offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications including: 1) intersectiona...

  2. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

    Science.gov (United States)

    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

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

    Directory of Open Access Journals (Sweden)

    Sebastian R Schreglmann

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

  4. Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing

    Czech Academy of Sciences Publication Activity Database

    Crhák Khaitová, Lucie; Fojtová, M.; Křížová, Kateřina; Lunerová Bedřichová, Jana; Fulneček, Jaroslav; Depicker, A.; Kovařík, Aleš

    2011-01-01

    Roč. 6, č. 5 (2011), s. 650-660 ISSN 1559-2294 R&D Projects: GA ČR(CZ) GD204/09/H002; GA MŠk(CZ) LC06004 Grant - others:GA ČR(CZ) GPP501/11/P667 Program:GP Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : transcriptional gene silencing * transgene epialleles * DNA methylation Subject RIV: BO - Biophysics Impact factor: 4.318, year: 2011

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

    Science.gov (United States)

    Ellett, Felix; Pase, Luke; Hayman, John W; Andrianopoulos, Alex; Lieschke, Graham J

    2011-01-27

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

  6. Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland.

    Science.gov (United States)

    Baldassarre, Hernan; Hockley, Duncan K; Olaniyan, Benjamen; Brochu, Eric; Zhao, Xin; Mustafa, Arif; Bordignon, Vilceu

    2008-10-01

    The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P milk secretion observed in these transgenic goats.

  7. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    Science.gov (United States)

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. Tobacco expressing pap1 increases the responses to par and uv-a by enhancing soluble sugars and flavonoids and elevating plant protections

    International Nuclear Information System (INIS)

    Sompornpailin, K.; Kanthang, S.

    2015-01-01

    Five lines of transgenic tobacco over-expressing Production of Anthocyanin Pigment 1 (PAP1) cDNA were analysis of metabolic response against the radiation and their protection of the plant under tissue culture condition. PAP1 transgenic and wild type (WT) plants were treated with the radiations of photosynthetically activate radiation (PAR) or PAR combined with UV-A. All lines of transgenic significantly increased in amounts of p-coumaric acid, naringenin apigenin more than WT under both treatments. Additional UV-A radiating to plant rose up kaempferol content in WT plant (1.5 times) and in PAP1 transgenics (1.8 times). These transgenic plants treated under both conditions had also increased anthocyanin substances (pelargonidin) with significant value after compared to WT. Content of total soluble sugar (TSS) was related to the content of total flavonoids in transgenic. PAR combined with UV-A had a lower induction of the electrolyte leakage percentage and malondialdehyde (MDA) level in the transgenic leaf tissue compared to WT tissue. The metabolic substance levels were considered on its protection of plant cells. In transgenic tissue, the enhancement of apigenin level strongly diminished the increase level of electrolyte leakage while the levels of TSS, p-coumaric acid and naringinin less affected. Moreover, the increase levels of kaempferol and pelargonidin associated with the decrease level of MDA, while the TSS level reversely responded. The PAP1 transgenic increased response of light by adaptation of their metabolites (TSS, p-coumaric acid and flavonoids) consequently enhance parameter indicating protections of the cell. (author)

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

    DEFF Research Database (Denmark)

    Chen, Yaling; Sun, Xiao-Feng; Zhou, Xin

    2017-01-01

    Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice....... Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges...... content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated...

  10. Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin βE subunit

    International Nuclear Information System (INIS)

    Hashimoto, Osamu; Ushiro, Yuuki; Sekiyama, Kazunari; Yamaguchi, Osamu; Yoshioka, Kazuki; Mutoh, Ken-Ichiro; Hasegawa, Yoshihisa

    2006-01-01

    Activins, TGF-β superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin β subunit genes, βC and βE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin βE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells

  11. LiPS-A3S, a human genomic site for robust expression of inserted transgenes

    Directory of Open Access Journals (Sweden)

    Andriana G Kotini

    2016-01-01

    Full Text Available Transgenesis of human pluripotent stem cells (hPSCs can enable and empower a variety of studies in stem cell research, including lineage tracing and functional genetics studies. While in recent years much progress has been made in the development of tools for gene targeting, little attention has been given to the identification of sites in the human genome where transgenes can be inserted and reliably expressed. In order to find human genomic sites capable of supporting long-term and high-level transgene expression in hPSCs, we performed a lentiviral screen in human induced pluripotent stem cells (iPSCs. We isolated 40 iPSC clones each harboring a single vector copy and characterized the level of transgene expression afforded by each unique integration site. We selected one clone, LiPS-A3 with an integration site in chromosome 15 maintaining robust expression without silencing and demonstrate that different transgenes can be inserted therein rapidly and efficiently through recombinase-mediated cassette exchange (RMCE. The LiPS-A3 line can greatly facilitate the insertion of reporter and other genes in hPSCs. Targeting transgenes in the LiPS-A3S genomic locus can find broad applications in stem cell research and possibly cell and gene therapy.

  12. Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1

    Science.gov (United States)

    Ihemere, Uzoma E.; Narayanan, Narayanan N.; Sayre, Richard T.

    2012-01-01

    We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants. PMID:22993514

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

    Science.gov (United States)

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

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50......% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression...

  15. EXPRESSION OF CHITINASE GENE IN TRANSGENIC RAPE PLANTS

    Directory of Open Access Journals (Sweden)

    Lu Longdou

    2005-08-01

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

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

    International Nuclear Information System (INIS)

    Ritchie, K.A.

    1986-01-01

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

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

    Directory of Open Access Journals (Sweden)

    David A. Dunn

    2012-01-01

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

  18. Nuclear expression of a mitochondrial DNA gene: mitochondrial targeting of allotopically expressed mutant ATP6 in transgenic mice.

    Science.gov (United States)

    Dunn, David A; Pinkert, Carl A

    2012-01-01

    Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE) represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W) mt-Atp6 transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses; P 0.05). This study illustrates a mouse model capable of circumventing in vivo mitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

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

    Science.gov (United States)

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

    2000-06-01

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

  20. Generation of highly productive polyclonal and monoclonal tobacco suspension lines from a heterogeneous transgenic BY-2 culture through flow cytometric sorting

    OpenAIRE

    Kirchhoff, Janina

    2012-01-01

    Over the last 20 years, plant cells gained increasing interest as host systems for the production of human and animal pharmaceuticals. Transgenic tobacco suspension cultures are an especially promising production host because of their beneficial growth, low maintenance requirement and the possibility of contained cultivation in bioreactors under controlled conditions. The excellent product quality of plant-produced proteins was demonstrated for a multitude of proteins, but the heterogeneous a...

  1. Expression of recombinant human lysozyme in egg whites of transgenic hens.

    Directory of Open Access Journals (Sweden)

    Dainan Cao

    Full Text Available Chicken egg lysozyme (cLY is an enzyme with 129 amino acid (AA residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY at 57.66 ± 4.10 μg/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 μg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.

  2. Expression of recombinant human lysozyme in egg whites of transgenic hens.

    Science.gov (United States)

    Cao, Dainan; Wu, Hanyu; Li, Qingyuan; Sun, Yingmin; Liu, Tongxin; Fei, Jing; Zhao, Yaofeng; Wu, Sen; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Chicken egg lysozyme (cLY) is an enzyme with 129 amino acid (AA) residue enzyme. This enzyme is present not only in chicken egg white but also in mucosal secretions such as saliva and tears. The antibacterial properties of egg white can be attributed to the presence of lysozyme, which is used as an anti-cancer drug and for the treatment of human immunodeficiency virus (HIV) infection. In this study, we constructed a lentiviral vector containing a synthetic cLY signal peptide and a 447 bp synthetic human lysozyme (hLY) cDNA sequence driven by an oviduct-specific ovalbumin promoter, and microinjected into the subgerminal cavity of stage X chick embryos to generate transgenic chicken. The transgene inserted in the chicken chromosomes directs the synthesis and secretion of hLY which has three times higher specific activity than cLY. Three G1 transgenic chickens were identified, the only female of which expressed recombinant human lysozyme (rhLY) at 57.66 ± 4.10 μg/ml in the egg white and the G2 transgenic hens of the G1 transgenic cock A011 expressed rhLY at 48.72 ± 1.54 μg/ml. This experiment demonstrated that transgenic hens with stable oviduct-specific expression of recombinant human lysozyme proteins can be created by microinjection of lentiviral vectors. The results of this research could be contribute to the technological development using transgenic hens as a cost-effective alternative to other mammalian systems, such as cow, sheep and goats, for the production of therapeutic proteins and other applications.

  3. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Directory of Open Access Journals (Sweden)

    Bin Yang

    Full Text Available BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. CONCLUSIONS/SIGNIFICANCE: Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  4. Characterization of bioactive recombinant human lysozyme expressed in milk of cloned transgenic cattle.

    Science.gov (United States)

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-03-16

    There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences. Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.

  5. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L. increases seed protein content and weight without augmenting nitrogen supplying.

    Directory of Open Access Journals (Sweden)

    Xiao-Qiang Zhao

    Full Text Available Heavy nitrogen (N application to gain higher yield of wheat (Triticum aestivum L. resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed, respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  6. BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

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    Yuki Muranishi

    2012-01-01

    Full Text Available Dickkopf (DKK family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.

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

    Directory of Open Access Journals (Sweden)

    Pan Liao

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

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

    Science.gov (United States)

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

    2015-01-01

    Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls. PMID:26202070

  9. A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants.

    Science.gov (United States)

    Anderson, J M; Palukaitis, P; Zaitlin, M

    1992-01-01

    Nicotiana tabacum cv. Turkish Samsun NN plants were transformed with a modified and truncated replicase gene encoded by RNA-2 of cucumber mosaic virus strain Fny. The replicase gene had been modified by deleting a 94-base-pair region spanning nucleotides 1857-1950; the deletion also caused a shift in the open reading frame, resulting in a truncated translation product approximately 75% as large as the full-length protein. Upon transformation via Agrobacterium tumefaciens, transgenic plants were obtained that were resistant to virus disease when challenged with either cucumber mosaic virus virions or RNA at concentrations up to 500 micrograms/ml or 50 micrograms/ml, respectively, the highest concentrations tested. This resistance was absolute, as neither symptoms nor virus could be detected in uninoculated leaves, even after prolonged incubation (120 days after inoculation). These data suggest, therefore, that such a "replicase-mediated" resistance strategy may be applicable to other plant and animal viruses. Images PMID:1528890

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

    Science.gov (United States)

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

    2014-04-01

    A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well-known bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Intracerebral transplants of primary muscle cells: a potential 'platform' for transgene expression in the brain

    Science.gov (United States)

    Jiao, S.; Schultz, E.; Wolff, J. A.

    1992-01-01

    After the transplantation of rat primary muscle cells into the caudate or cortex of recipient rats, the muscle cells were able to persist for at least 6 months. Muscle cells transfected with expression plasmids prior to transplantation were able to express reporter genes in the brains for at least 2 months. These results suggest that muscle cells might be a useful 'platform' for transgene expression in the brain.

  12. Tetracycline-regulated transgene expression in hippocampal neurones following transfection with adenoviral vectors.

    Science.gov (United States)

    Harding, T C; Geddes, B J; Noel, J D; Murphy, D; Uney, J B

    1997-12-01

    A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)-responsive On/Off regulatory elements into type-5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet-On and AdTet-Off systems. Following transfection with the AdTet-On system no EGFP-fluorescent cells could be detected until doxycycline was added. The AdTet-Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.

  13. Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1

    Science.gov (United States)

    Liu, Chunxin; Bolund, Lars; Vajta, Gábor; Dou, Hongwei; Yang, Wenxian; Xu, Ying; Luan, Jing; Wang, Jun; Yang, Huanming; Staunstrup, Nicklas Heine; Du, Yutao

    2013-01-01

    Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders. PMID:24146819

  14. Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

    Science.gov (United States)

    Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

  15. Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids.

    Science.gov (United States)

    Mi, Xiaoxiao; Liu, Xue; Yan, Haolu; Liang, Lina; Zhou, Xiangyan; Yang, Jiangwei; Si, Huaijun; Zhang, Ning

    2017-01-01

    Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0±1.43 (ST2) and 36.6±0.99 (35S3) aphids per plant, which corresponds to 24.9-53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

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

    Science.gov (United States)

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

    2015-02-01

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

  17. Phenotypic characterization of transgenic Japanese medaka (Oryzias latipes) that express a red fluorescent protein in hepatocytes.

    Science.gov (United States)

    Van Wettere, Arnaud J; Law, J Mac; Hinton, David E; Kullman, Seth W

    2014-01-01

    Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.

  18. Fitness aspects of transgenic Aedes fluviatilis mosquitoes expressing a Plasmodium-blocking molecule.

    Science.gov (United States)

    Santos, Maíra N; Nogueira, Paula M; Dias, Fernando B S; Valle, Denise; Moreira, Luciano A

    2010-12-01

    Vector-born diseases cause millions of deaths every year globally. Alternatives for the control of diseases such as malaria and dengue fever are urgently needed and the use of transgenic mosquitoes that block parasite/virus is a sound strategy to be used within control programs. However, prior to use transgenic mosquitoes as control tools, it is important to study their fitness since different biological aspects might influence their ability to disseminate and compete with wild populations. We previously reported the construction of four transgenic Aedes fluviatilis mosquito lines expressing a Plasmodium- blocking molecule (mutated bee venom phospholipase A(2)-mPLA(2)). Presently we studied two aspects of their fitness: body size, that has been used as a fitness-related status, and the expression of major enzymes classes involved in the metabolism of xenobiotics, including insecticides. Body size analysis (recorded by geometric wing morphometrics) indicated that both male and female mosquitoes were larger than the non-transgenic counterparts, suggesting that this characteristic might have an impact on their overall fitness. By contrast, no significant difference in the activity of enzymes related to metabolic insecticide resistance was detected in transgenic mosquitoes. The implication on fitness advantage of these features, towards the implementation of this strategy, is further discussed.

  19. Expression of cholera toxin B–proinsulin fusion protein in lettuce and tobacco chloroplasts – oral administration protects against development of insulitis in non-obese diabetic mice

    Science.gov (United States)

    Ruhlman, Tracey; Ahangari, Raheleh; Devine, Andrew; Samsam, Mohtahsem; Daniell, Henry

    2008-01-01

    Summary Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit–human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB–green fluorescent protein (CTB-GFP) or interferon–GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing β-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few β-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing β-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T1 progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases. PMID:17490448

  20. Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic mice.

    Science.gov (United States)

    Ruhlman, Tracey; Ahangari, Raheleh; Devine, Andrew; Samsam, Mohtahsem; Daniell, Henry

    2007-07-01

    Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit-human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB-green fluorescent protein (CTB-GFP) or interferon-GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing beta-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few beta-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing beta-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T(1) progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Liu Zhihui

    2008-08-01

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

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

    DEFF Research Database (Denmark)

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

    1987-01-01

    The complete soybean leghemoglobin lbc(3) gene was transferred into the legume Lotus corniculatus using an Agrobacterium rhizogenes vector system. Organ-specific expression of the soybean gene was observed in root nodules formed on regenerated transgenic plants after infection with Rhizobium loti...

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

    Science.gov (United States)

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

    2012-01-01

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

  5. Use of lentiviral vectors to deliver and express bicistronic transgenes in developing chicken embryos.

    Science.gov (United States)

    Semple-Rowland, Susan L; Berry, Jonathan

    2014-04-01

    The abilities of lentiviral vectors to carry large transgenes (∼8kb) and to efficiently infect and integrate these genes into the genomes of both dividing and non-dividing cells make them ideal candidates for transport of genetic material into cells and tissues. Given the properties of these vectors, it is somewhat surprising that they have seen only limited use in studies of developing tissues and in particular of the developing nervous system. Over the past several years, we have taken advantage of the large capacity of these vectors to explore the expression characteristics of several dual promoter and 2A peptide bicistronic transgenes in developing chick neural retina, with the goal of identifying transgene designs that reliably express multiple proteins in infected cells. Here we summarize the activities of several of these transgenes in neural retina and provide detailed methodologies for packaging lentivirus and delivering the virus into the developing neural tubes of chicken embryos in ovo, procedures that have been optimized over the course of several years of use in our laboratory. Conditions to hatch injected embryos are also discussed. The chicken-specific techniques will be of highest interest to investigators using avian embryos, development and packaging of lentiviral vectors that reliably express multiple proteins in infected cells should be of interest to all investigators whose experiments demand manipulation and expression of multiple proteins in developing cells and tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

    International Nuclear Information System (INIS)

    Keasler, Victor V.; Lerat, Herve; Madden, Charles R.; Finegold, Milton J.; McGarvey, Michael J.; Mohammed, Essam M.A.; Forbes, Stuart J.; Lemon, Stanley M.; Hadsell, Darryl L.; Grona, Shala J.; Hollinger, F. Blaine; Slagle, Betty L.

    2006-01-01

    Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis

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

    Directory of Open Access Journals (Sweden)

    Han Jigang

    2012-03-01

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

  8. Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

    Science.gov (United States)

    Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

    2006-10-01

    Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

  9. Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk.

    Science.gov (United States)

    Baldassarre, Hernan; Hockley, Duncan K; Doré, Monique; Brochu, Eric; Hakier, Bernard; Zhao, Xin; Bordignon, Vilceu

    2008-02-01

    The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1-5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.

  10. Tobacco

    Science.gov (United States)

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  11. Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice

    Science.gov (United States)

    Feng, Mingxiao; Field, Kevin; Chatzistamou, Ioulia; Shim, Minsub

    2016-01-01

    Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated β-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders. PMID:27750221

  12. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture...... by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used...

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

    Directory of Open Access Journals (Sweden)

    Shidlovsky Konstantin

    2010-11-01

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

  14. Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells

    OpenAIRE

    Cerisoli, Francesco; Cassinelli, Letizia; Lamorte, Giuseppe; Citterio, Stefania; Bertolotti, Francesca; Magli, Maria Cristina; Ottolenghi, Sergio

    2009-01-01

    The expression of Kit in multiple types of stem cells suggests that common transcriptional programs might regulate this gene in different stem cells. In this work, the authors used mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements. This study provides the basis for the elucidation of DNA sequences regulating a stem cell gene in multiple types of stem cells.

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

    OpenAIRE

    Uzoma eIhemere; Narayanan eNarayanan; Richard eSayre

    2012-01-01

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

  16. Characterization of Bioactive Recombinant Human Lysozyme Expressed in Milk of Cloned Transgenic Cattle

    OpenAIRE

    Yang, Bin; Wang, Jianwu; Tang, Bo; Liu, Yufang; Guo, Chengdong; Yang, Penghua; Yu, Tian; Li, Rong; Zhao, Jianmin; Zhang, Lei; Dai, Yunping; Li, Ning

    2011-01-01

    BACKGROUND: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study,...

  17. The constitutive expression of a two transgene construct enhances the abiotic stress tolerance of chrysanthemum.

    Science.gov (United States)

    Song, Aiping; An, Juan; Guan, Zhiyong; Jiang, Jiafu; Chen, Fadi; Lou, Wanghuai; Fang, Weimin; Liu, Zhaolei; Chen, Sumei

    2014-07-01

    Various abiotic stresses downgrade the quality and productivity of chrysanthemum. A construct carrying both CcSOS1 (from Chrysanthemum crassum) and CdICE1 (from Chrysanthemum dichrum) was constitutively expressed in the chrysanthemum variety 'Jinba'. The transgenic plants were superior to the wild type (WT) ones with respect to their sensitivity to low temperature, drought and salinity, as measured by visible damage and plant survival. Salinity stressed transgenic plants accumulated more proline, and their level of superoxide dismutase and peroxidase activity was higher than in WT plants. At the physiological level, they suffered less loss of viable leaf area, maintained a lower leaf electrolyte conductivity and retained more chlorophyll (a+b). The ratio between the K(+) and Na(+) content was higher in the root, stem and median leaves of salinity stressed transgenic plants than in those of WT plants. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth......Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...

  19. Expression and Purification of Recombinant Mouse Interleukin-4 and -6 from Transgenic Rice Seeds.

    Science.gov (United States)

    Fujiwara, Yoshihiro; Yang, Lijun; Takaiwa, Fumio; Sekikawa, Kenji

    2016-04-01

    Transgenic rice seed can be utilized as a bioreactor to produce high-value recombinant proteins. Mouse interleukin 4 (mIL-4) and mIL-6 were specifically expressed as secretory proteins in rice endosperm by ligating the N-terminal glutelin B-1 (GluB-1) signal peptide and the C-terminal KDEL endoplasmic reticulum retention signal under control of the endosperm-specific GluB-1 promoter. In the transgenic rice seed, mIL-4 and mIL-6 accumulated in levels up to 0.43 mg/g grain and 0.16 mg/g grain, respectively. The reducing agents and detergents required for extraction from the transgenic rice seeds differed between the two proteins, indicating differences in their intracellular localization within the endosperm cell. Purified mIL-4 and mIL-6 exhibited high activity and very low endotoxin contamination.

  20. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...... germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth...

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

    Indian Academy of Sciences (India)

    GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences. Functional GFP and BAFF proteins were expressed separately and confirmed in HeLa cells.

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

    Science.gov (United States)

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

    2007-10-01

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

  3. Transgenic mice with increased astrocyte expression of CCL2 show altered behavioral effects of alcohol.

    Science.gov (United States)

    Bray, Jennifer G; Roberts, Amanda J; Gruol, Donna L

    2017-06-23

    Emerging research provides strong evidence that activation of CNS glial cells occurs in neurological diseases and brain injury and results in elevated production of neuroimmune factors. These factors can contribute to pathophysiological processes that lead to altered CNS function. Recently, studies have also shown that both acute and chronic alcohol consumption can produce activation of CNS glial cells and the production of neuroimmune factors, particularly the chemokine ligand 2 (CCL2). The consequences of alcohol-induced increases in CCL2 levels in the CNS have yet to be fully elucidated. Our studies focus on the hypothesis that increased levels of CCL2 in the CNS produce neuroadaptive changes that modify the actions of alcohol on the CNS. We utilized behavioral testing in transgenic mice that express elevated levels of CCL2 to test this hypothesis. The increased level of CCL2 in the transgenic mice involves increased astrocyte expression. Transgenic mice and their non-transgenic littermate controls were subjected to one of two alcohol exposure paradigms, a two-bottle choice alcohol drinking procedure that does not produce alcohol dependence or a chronic intermittent alcohol procedure that produces alcohol dependence. Several behavioral tests were carried out including the Barnes maze, Y-maze, cued and contextual conditioned fear test, light-dark transfer, and forced swim test. Comparisons between alcohol naïve, non-dependent, and alcohol-dependent CCL2 transgenic and non-transgenic mice show that elevated levels of CCL2 in the CNS interact with alcohol in tests for alcohol drinking, spatial learning, and associative learning. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  4. Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants.

    Science.gov (United States)

    Choi, Hae-Woon; Yu, Xiao-Hong; Lemaux, Peggy G; Cho, Myeong-Je

    2009-08-01

    To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T(8) plant, having uidA (or gus) driven by the barley endosperm-specific B(1)-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T(4) plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F(1) progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F(2) seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F(2) plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F(3) and F(4) generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.

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

    DEFF Research Database (Denmark)

    Ambartsumian, N; Klingelhöfer, Jörg; Grigorian, M

    1998-01-01

    and constitutive 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) gene promoter. In transgenic animals the expression of the transgene RNA was detected in all organs, but only some of the organs showed elevated levels of the protein. Expression of the S100A4(Mts1) protein was downregulated in the organs...... that normally do not express the gene in the wild-type animal. The transgene RNA is detected in the polysomes indicating that it could be translated into the S100A4(Mts1) protein. The specificity of the S100A4(Mts1) protein expression is determined by a complex mechanism including regulation of translation and...

  6. Stress-inducible GmGSTU4 shapes transgenic tobacco plants metabolome towards increased salinity tolerance

    NARCIS (Netherlands)

    Kissoudis, Christos; Kalloniati, Chrissanthi; Flemetakis, Emmanouil; Madesis, Panagiotis; Labrou, Nikolaos E.; Tsaftaris, Athanasios; Nianiou-Obeidat, Irini

    2015-01-01

    The involvement of glutathione transferases (GSTs) in plant’s tolerance to abiotic stresses has been extensively studied; however, the metabolic changes occurring in the plants with altered GSTs expression have not been studied in detail. We have previously demonstrated that GmGSTU4

  7. Transcription activator-like effector hybrids for conditional control and rewiring of chromosomal transgene expression.

    Science.gov (United States)

    Li, Yi; Moore, Richard; Guinn, Michael; Bleris, Leonidas

    2012-01-01

    The ability to conditionally rewire pathways in human cells holds great therapeutic potential. Transcription activator-like effectors (TALEs) are a class of naturally occurring specific DNA binding proteins that can be used to introduce targeted genome modifications or control gene expression. Here we present TALE hybrids engineered to respond to endogenous signals and capable of controlling transgenes by applying a predetermined and tunable action at the single-cell level. Specifically, we first demonstrate that combinations of TALEs can be used to modulate the expression of stably integrated genes in kidney cells. We then introduce a general purpose two-hybrid approach that can be customized to regulate the function of any TALE either using effector molecules or a heterodimerization reaction. Finally, we demonstrate the successful interface of TALEs to specific endogenous signals, namely hypoxia signaling and microRNAs, essentially closing the loop between cellular information and chromosomal transgene expression.

  8. Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice

    International Nuclear Information System (INIS)

    Selden, R.F.; Wagner, T.E.; Blethen, S.; Yun, J.S.; Rowe, M.E.; Goodman, H.M.

    1988-01-01

    The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, the authors have expressed a mouse methallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties

  9. Increased adenovirus Type 5 mediated transgene expression due to RhoB down-regulation.

    Directory of Open Access Journals (Sweden)

    Dragomira Majhen

    Full Text Available Adenovirus type 5 (Ad5 is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

  10. High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus dragline silk protein.

    Directory of Open Access Journals (Sweden)

    Yoshihiko Kuwana

    Full Text Available Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol% native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

  11. Does pea lectin expressed transgenically in oilseed rape (Brassica napus) influence honey bee (Apis mellifera) larvae?

    Science.gov (United States)

    Lehrman, Anna

    2007-01-01

    The European honey bee (Apis mellifera) is important both for pollination and for honey production. Pollen is the major protein source for bees, which exposes them directly to changes in pollen quality e.g. through genetic engineering. In order to create a worst case scenario regarding pea lectin (PSL) expressed transgenically in oilseed rape anthers and pollen, the maximum amount of dried pollen that could be mixed in an artificial diet without negatively affecting larval performance (1.5% w/w) was fed to bee larvae. Pollen from two transgenic plant lines expressing PSL up to 1.2% of total soluble protein and pollen from one non-transgenic line was added to the same diet and used as a pollen control. When these three pollen diets and the control diet (without added pollen) were compared, no negative effect from the pollen of the transgenic plants could be detected on larval mortality, weight, or development time. An increased weight and a reduced developmental time were recorded for larvae on all diets containing pollen when compared to the diet without pollen.

  12. The ZmRCP-1 promoter of maize provides root tip specific expression of transgenes in plantain.

    Science.gov (United States)

    Onyango, Stephen O; Roderick, Hugh; Tripathi, Jaindra N; Collins, Richard; Atkinson, Howard J; Oduor, Richard O; Tripathi, Leena

    2016-12-01

    Bananas and plantains (Musa spp.) provide 25 % of the food energy requirements for more than 100 million people in Africa. Plant parasitic nematodes cause severe losses to the crop due to lack of control options. The sterile nature of Musa spp. hampers conventional breeding but makes the crop suitable for genetic engineering. A constitutively expressed synthetic peptide in transgenic plantain has provided resistance against nematodes. Previous work with the peptide in potato plants indicates that targeting expression to the root tip improves the efficacy of the defence mechanism. However, a promoter that will provide root tip specific expression of transgenes in a monocot plant, such as plantain, is not currently available. Here, we report the cloning and evaluation of the maize root cap-specific protein-1 (ZmRCP-1) promoter for root tip targeted expression of transgenes that provide a defence against plant parasitic nematodes in transgenic plantain. Our findings indicate that the maize ZmRCP-1 promoter delivers expression of β-glucuronidase (gusA) gene in roots but not in leaves of transgenic plantains. In mature old roots, expression of gusA gene driven by ZmRCP-1 becomes limited to the root cap. Invasion by the nematode Radopholus similis does not modify Root Cap-specific Protein-1 promoter activity. Root cap-specific protein-1 promoter from maize can provide targeted expression of transgene for nematode resistance in transgenic plantain.

  13. Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units

    International Nuclear Information System (INIS)

    Kuegler, S.; Lingor, P.; Schoell, U.; Zolotukhin, S.; Baehr, M.

    2003-01-01

    Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology

  14. RNA interference is responsible for reduction of transgene expression after Sleeping Beauty transposase mediated somatic integration.

    Directory of Open Access Journals (Sweden)

    Christina Rauschhuber

    Full Text Available BACKGROUND: Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. PRINCIPAL FINDINGS: To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. CONCLUSION: In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system.

  15. Restoration of stamen development and production of functional pollen in an alloplasmic CMS tobacco line by ectopic expression of the Arabidopsis thaliana SUPERMAN gene.

    Science.gov (United States)

    Bereterbide, Agnès; Hernould, Michel; Farbos, Isabelle; Glimelius, Kristina; Mouras, Armand

    2002-03-01

    The alloplasmic male-sterile tobacco line Nta(rep)S, combining the nucleus of Nicotiana tabacum with the cytoplasm of Nicotiana repanda, exhibits cadastral-type anomalies due to a fusion of several stamens with the pistil. These anomalies share similarities with Arabidopsis superman mutants. SUPERMAN (SUP) is a cadastral gene controlling the boundary between whorls 3 (androecium) and 4 (gynoecium). Thus we hypothesized that the expression of the tobacco SUP orthologue might be impaired in the alloplasmic Nta(rep)S line, and that the deficiency could be complemented by the Arabidopsis SUP gene. Here we show that the ectopic expression of SUP in the alloplasmic male-sterile tobacco line Nta(rep)S significantly increases the frequency of flowers possessing free stamens, inducing the recovery of a proper structure for whorls 3 and 4. Furthermore, flowers of transgenic plants show a significant improvement of the morphology of stamens, and more particularly of the anthers, which are able to produce few but functional pollen. The data show that ectopic expression of Arabidopsis SUP reactivates the regulatory cascade of anther development. The plausible causes of the developmental defects of anthers in the alloplasmic male-sterile tobacco line are discussed in relation to the model of regulation of the Arabidopsis SUP gene.

  16. Characterization of a Maize Wip1 Promoter in Transgenic Plants

    Directory of Open Access Journals (Sweden)

    Shengxue Zhang

    2013-12-01

    Full Text Available 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.

  17. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E.; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...... transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian...

  18. A Double-Switch Cell Fusion-Inducible Transgene Expression System for Neural Stem Cell-Based Antiglioma Gene Therapy

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available Recent progress in neural stem cell- (NSC- based tumor-targeted gene therapy showed that NSC vectors expressing an artificially engineered viral fusogenic protein, VSV-G H162R, could cause tumor cell death specifically under acidic tumor microenvironment by syncytia formation; however, the killing efficiency still had much room to improve. In the view that coexpression of another antitumoral gene with VSV-G can augment the bystander effect, a synthetic regulatory system that triggers transgene expression in a cell fusion-inducible manner has been proposed. Here we have developed a double-switch cell fusion-inducible transgene expression system (DoFIT to drive transgene expression upon VSV-G-mediated NSC-glioma cell fusion. In this binary system, transgene expression is coregulated by a glioma-specific promoter and targeting sequences of a microRNA (miR that is highly expressed in NSCs but lowly expressed in glioma cells. Thus, transgene expression is “switched off” by the miR in NSC vectors, but after cell fusion with glioma cells, the miR is diluted and loses its suppressive effect. Meanwhile, in the syncytia, transgene expression is “switched on” by the glioma-specific promoter. Our in vitro and in vivo experimental data show that DoFIT successfully abolishes luciferase reporter gene expression in NSC vectors but activates it specifically after VSV-G-mediated NSC-glioma cell fusion.

  19. A BAX inhibitor-1 gene in Capsicum annuum is induced under various abiotic stresses and endows multi-tolerance in transgenic tobacco.

    Science.gov (United States)

    Isbat, Mohammad; Zeba, Naheed; Kim, Seong Ryong; Hong, Choo Bong

    2009-10-15

    Programmed cell death (PCD) is a highly conserved cellular suicide process important in developmental processes and elimination of damaged cells upon environmental stresses. Among the important regulators of PCD, much interest has been centered on BCL2-associated x protein (BAX) as the pro-PCD factor. On the other hand, BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor that balances out the activity of BAX in the developmental processes and responses to environment. A cDNA clone coding a BI-1 gene was isolated from a cDNA library of heat-stressed hot pepper (Capsicum annuum) and named as CaBI-1. This gene contains an open reading frame (ORF) of 248 amino acids encoding a BI-1 protein. Genomic DNA-blot analysis for CaBI-1 suggested one or two loci in the C. annuum genome. Transcription of CaBI-1 was induced in response to high or low temperatures, drought, high salinity, flooding and heavy metal stresses, and ABA. We introduced the ORF of CaBI-1 under the control of the CaMV 35S promoter (P(35S)) into tobacco (Nicotiana tabacum cv. Wisconsin 38) genome by Agrobacterium-mediated transformation. The P(35S):CaBI-1 transgenic plants displayed markedly improved tolerance to high temperature, water deficit, and high salinity in comparison to the control plants. The results indicate that CaBI-1 is a BI-1 gene of which expression induced under various abiotic stresses and endows tolerance to several types of environmental stresses.

  20. Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression

    OpenAIRE

    Jacobs, Andreas; Dubrovin, Michael; Hewett, Jeff; Sena-Esteves, Miguel; Tan, Cui-Wen; Slack, Mark; Sadelain, Michele; Breakefield, Xandra O; Tjuvajev, Juri G

    1999-01-01

    Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk...

  1. Isolation and expression analysis of a tobacco AINTEGUMENTA ortholog (NtANTL).

    Science.gov (United States)

    Rieu, Ivo; Bots, Marc; Mariani, Celestina; Weterings, Koen A P

    2005-05-01

    The Arabidopsis AINTEGUMENTA (ANT) protein is essential for proper ovule development, but functions in cell proliferation and organ growth throughout the plant. Here we report the isolation of a full-length cDNA clone from tobacco (Nicotiana tabacum L.) that encodes a protein with high similarity to ANT and is preferentially expressed in the pistil. In situ hybridization analysis on the tobacco ovary shows that the expression pattern of the corresponding gene is different from that of ANT in Arabidopsis.

  2. The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

    Science.gov (United States)

    Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)

    2001-01-01

    To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

  3. Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance.

    Science.gov (United States)

    Kim, Yun-Hee; Kim, Myoung Duck; Choi, Young Im; Park, Sung-Chul; Yun, Dae-Jin; Noh, Eun Woon; Lee, Haeng-Soon; Kwak, Sang-Soo

    2011-04-01

    Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

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

    Science.gov (United States)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2009-06-30

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

  6. Polyketide synthesis in tobacco plants transformed with a Plumbago zeylanica type III hexaketide synthase.

    Science.gov (United States)

    Jadhav, Supriya; Phapale, Prasad; Thulasiram, Hirekodathakallu V; Bhargava, Sujata

    2014-02-01

    A type III polyketide synthase from Plumbago zeylanica (PzPKS) was cloned and expressed in tobacco plants to study whether the transgenic tobacco plants expressing PzPKS synthesize the pharmacologically important polyketide, plumbagin. High resolution mass spectrometry based metabolite profiling of two transgenic events and wild type tobacco plants was carried out to investigate changes in polyketides, including plumbagin. Ten polyketides, which included six pyrones and four naphthalene derivatives, were identified in PzPKS transgenic plants. While one pyrone, styryl-2-pyranone, was detected in both, wild type and transgenic tobacco plants, three pyrones were expressed only in the leaves of transgenic tobacco plants. The transgenic tobacco plants did not accumulate plumbagin, but showed accumulation of isoshinanolone in the roots, which is postulated to be the reduction product of plumbagin. In addition, leaves of transgenic tobacco plants accumulated 3-methyl-1,8-naphthalenediol, a postulated precursor of plumbagin. The results indicated the requirement of additional Plumbago-specific components in the biosynthetic pathway of this polyketide. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Functional expression of transgenic α1sDHPR channels in adult mammalian skeletal muscle fibres

    Science.gov (United States)

    DiFranco, Marino; Tran, Philip; Quiñonez, Marbella; Vergara, Julio L

    2011-01-01

    Abstract We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Q and SR Ca2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of them works as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the

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

    Directory of Open Access Journals (Sweden)

    Ryan C Smith

    Full Text Available 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.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.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.

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

    Science.gov (United States)

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

    2013-01-01

    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. 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. 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.

  10. A bovine oxytocin transgene in mice: expression in the female reproductive organs and regulation during pregnancy, parturition and lactation.

    Science.gov (United States)

    Ho, M Y; Murphy, D

    1997-12-31

    Transgene bovine oxytocin 3.5 (bOT3.5) consists of the bovine oxytocin structural gene flanked by 0.6 kbp of upstream and 1.9 kbp of downstream sequences. We have examined the expression of bOT3.5 in the female reproductive organs, and we show tissue-specific and physiological regulation dependent on the stage of pregnancy and lactation. In the ovary, no transgene expression could be detected during the estrus cycle, or during pregnancy. However, high levels of transgene RNA were found at day 1 of lactation. Expression dropped 10-fold by day 2 of lactation, and was undetectable thereafter. Interestingly, the expression of bOT3.5 in the mouse ovary at the beginning of lactation mimics that of the endogenous OT gene in the bovine ovary. Expression of the bOT3.5 transgene correlates with a parturition defect that results in considerable maternal mortality.

  11. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

    Science.gov (United States)

    Katter, Katharina; Geurts, Aron M; Hoffmann, Orsolya; Mátés, Lajos; Landa, Vladimir; Hiripi, László; Moreno, Carol; Lazar, Jozef; Bashir, Sanum; Zidek, Vaclav; Popova, Elena; Jerchow, Boris; Becker, Katja; Devaraj, Anantharam; Walter, Ingrid; Grzybowksi, Michael; Corbett, Molly; Filho, Artur Rangel; Hodges, Matthew R; Bader, Michael; Ivics, Zoltán; Jacob, Howard J; Pravenec, Michal; Bosze, Zsuzsanna; Rülicke, Thomas; Izsvák, Zsuzsanna

    2013-03-01

    Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

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

    Directory of Open Access Journals (Sweden)

    Moens Ugo

    2007-11-01

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

  13. Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials.

    Science.gov (United States)

    Wu, Jing-Jing; Liu, Yu-Wen; Sun, Meng-Xiang

    2011-07-01

    Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2-6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20 ng/ml to 20 μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.

  14. Expression of stabilized β-catenin in differentiated neurons of transgenic mice does not result in tumor formation

    International Nuclear Information System (INIS)

    Kratz, John E; Stearns, Duncan; Huso, David L; Slunt, Hilda H; Price, Donald L; Borchelt, David R; Eberhart, Charles G

    2002-01-01

    Medulloblastomas, embryonal tumors arising in the cerebellum, commonly contain mutations that activate Wnt signaling. To determine whether increased Wnt signaling in the adult CNS is sufficient to induce tumor formation, we created transgenic mice expressing either wild-type or activated β-catenin in the brain. Wild-type and mutant human β-catenin transgenes were expressed under the control of a murine PrP promoter fragment that drives high level postnatal expression in the CNS. Mutant β-catenin was stabilized by a serine to phenylalanine alteration in codon 37. Expression of the mutant transgene resulted in an approximately two-fold increase in β-catenin protein levels in the cortex and cerebellum of adult animals. Immunohistochemical analysis revealed nuclear β-catenin in hippocampal, cortical and cerebellar neurons of transgenic animals but not in non-transgenic controls. Tail kinking was observed in some transgenic animals, but no CNS malformations or tumors were detected. No tumors or morphologic alterations were detected in the brains of transgenic mice expressing stabilized β-catenin, suggesting that postnatal Wnt signaling in differentiated neurons may not be sufficient to induce CNS tumorigenesis

  15. Transgenic rice plants expressing synthetic cry2AX1 gene exhibits resistance to rice leaffolder (Cnaphalocrosis medinalis).

    Science.gov (United States)

    Manikandan, R; Balakrishnan, N; Sudhakar, D; Udayasuriyan, V

    2016-06-01

    Bacillus thuringiensis is a major source of insecticidal genes imparting insect resistance in transgenic plants. Level of expression of transgenes in transgenic plants is important to achieve desirable level of resistance against target insects. In order to achieve desirable level of expression, rice chloroplast transit peptide sequence was fused with synthetic cry2AX1 gene to target its protein in chloroplasts. Sixteen PCR positive lines of rice were generated by Agrobacterium mediated transformation using immature embryos. Southern blot hybridization analysis of T 0 transgenic plants confirmed the integration of cry2AX1 gene in two to five locations of rice genome and ELISA demonstrated its expression. Concentration of Cry2AX1 in transgenic rice events ranged 5.0-120 ng/g of fresh leaf tissue. Insect bioassay of T 0 transgenic rice plants against neonate larvae of rice leaffolder showed larval mortality ranging between 20 and 80 % in comparison to control plant. Stable inheritance and expression of cry2AX1 gene was demonstrated in T 1 progenies through Southern and ELISA. In T 1 progenies, the highest concentration of Cry2AX1 and mortality of rice leaffolder larvae were recorded as 150 ng/g of fresh leaf tissue and 80 %, respectively. The Cry2AX1 expression even at a very low concentration (120-150 ng/g) in transgenic rice plants was found effective against rice leaffolder larvae.

  16. New Wistar Kyoto and spontaneously hypertensive rat transgenic models with ubiquitous expression of green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Ana Isabel Garcia Diaz

    2016-04-01

    Full Text Available The Wistar Kyoto (WKY rat and the spontaneously hypertensive (SHR rat inbred strains are well-established models for human crescentic glomerulonephritis (CRGN and metabolic syndrome, respectively. Novel transgenic (Tg strains add research opportunities and increase scientific value to well-established rat models. We have created two novel Tg strains using Sleeping Beauty transposon germline transgenesis, ubiquitously expressing green fluorescent protein (GFP under the rat elongation factor 1 alpha (EF1a promoter on the WKY and SHR genetic backgrounds. The Sleeping Beauty system functioned with high transgenesis efficiency; 75% of new rats born after embryo microinjections were transgene positive. By ligation-mediated PCR, we located the genome integration sites, confirming no exonic disruption and defining a single or low copy number of the transgenes in the new WKY-GFP and SHR-GFP Tg lines. We report GFP-bright expression in embryos, tissues and organs in both lines and show preliminary in vitro and in vivo imaging data that demonstrate the utility of the new GFP-expressing lines for adoptive transfer, transplantation and fate mapping studies of CRGN, metabolic syndrome and other traits for which these strains have been extensively studied over the past four decades.

  17. Strategies for long-term expression of transgenes in the respiratory epithelium.

    Science.gov (United States)

    Gill, Deborah R; Bazzani, Reto P; Hyde, Stephen C

    2010-08-01

    Lung gene therapy is being developed to treat acute and chronic airway diseases, and many viral and non-viral gene transfer vectors have been evaluated in the airway epithelium lining the nose and lung. Stem cells have not been clearly defined in the airways and, currently, it is only possible to target progenitor cells to proliferate and repair the epithelium after inducing epithelial damage. However, the majority of airway epithelial cells are slowly dividing or terminally differentiated, thus necessitating repeated administration of gene transfer vectors for life-long transgene expression. Many improvements to adeno-associated virus and lentivirus vectors have led to increased airway transduction efficiencies, although achieving consistent repeated administration remains problematic because of immune system activation. Non-viral vectors appear to be less efficient, but can be successfully re-administered. The modification of plasmid sequences also offers maximum flexibility in increasing and extending the duration of transgene expression in the airways. This review describes recent developments in achieving persistent transgene expression in the airways by specifically targeting the cells of the respiratory epithelium lining the nose and lung.

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  20. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Science.gov (United States)

    Lo, Mandy Y M; Rival-Gervier, Sylvie; Pasceri, Peter; Ellis, James

    2012-01-01

    Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES) cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  1. Transgenic mice can express mutant human coagulation factor IX with higher level of clotting activity.

    Science.gov (United States)

    Yan, Jing-Bin; Wang, Shu; Huang, Wen-Ying; Xiao, Yan-Ping; Ren, Zhao-Rui; Huang, Shu-Zheng; Zeng, Yi-Tao

    2006-10-01

    To improve the available values of transgenic animals, we produced a mutant human coagulation factor IX minigene (including cDNA and intron I) with arginine at 338 changed to alanine (R338A-hFIX) by using a direct mutation technique. The R338A-hFIX minigene was then cloned into a plasmid carrying the goat beta-casein promoter to get a mammary gland-specific expression vector. The clotting activity in the supernatant of the transfected HC-11 cells increased to approximately three times more than that of wild-type hFIX. Nine transgenic mice (three females and six males) were produced, and the copy number of the foreign gene was very different, ranging from 1 to 43 in different lines. ELISA, Western blot, and clotting assay experiments showed that the transgenic mice could express R338A-hFIX, showing higher average levels of clotting activity than wild-type hFIX in the milk (103.76% vs. 49.95%). The highest concentration and clotting activity of hFIX reached 26 mug/mL and 1287% in one founder (F(0)-7), which was over 10 times higher than that in human plasma. Furthermore, RT-PCR, APTT assay, and histological analysis indicated that hFIX was expressed specifically in the mammary gland without affecting the intrinsic coagulation pathway and physiologic performance of the local tissue.

  2. Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog.

    Science.gov (United States)

    Fan, H; Oro, A E; Scott, M P; Khavari, P A

    1997-07-01

    Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC), has recently been implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. We used retroviral transduction of normal human keratinocytes to constitutively express SHH. SHH-expressing cells demonstrated increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia.

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

    Directory of Open Access Journals (Sweden)

    Tony Nolan

    2011-02-01

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

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

    Science.gov (United States)

    Nolan, Tony; Petris, Elisa; Müller, Hans-Michael; Cronin, Ann; Catteruccia, Flaminia; Crisanti, Andrea

    2011-02-04

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

  5. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Jacob Barbara A

    2009-02-01

    Full Text Available Abstract Background HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

  6. Development and Evaluation of Transgenic Nude Mice Expressing Ubiquitous Green Fluorescent Protein.

    Science.gov (United States)

    Iyer, Srikanth; Arindkar, Shailendra; Mishra, Alaknanda; Manglani, Kapil; Kumar, Jerald Mahesh; Majumdar, Subeer S; Upadhyay, Pramod; Nagarajan, Perumal

    2015-08-01

    Researchers had developed and characterized transgenic green/red fluorescent protein (GFP/RFP) nude mouse with ubiquitous RFP or GFP expression, but none has evaluated the level of immune cells and expression levels of GFP in this model. The nude GFP mice were evaluated by imaging, hematological indices, and flow cytometry to compare the proportion of immune T cells. Quantitative real-time PCR (qRT-PCR) was done for evaluating the relative expression of GFP transcripts in few organs of the nude GFP mice. The hematological and immune cells of nude GFP were within the range of nude mice. However, the gene expression levels were relatively less in various tissues compared with B6 GFP mice. These findings suggest that nude GFP is an ideal model resembling normal nude mice; however, GFP expression in various tissues by fluorescence should be considered, as the expression of GFP differs in various organs.

  7. Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes.

    Science.gov (United States)

    Cheng, Xin; Gao, Xiang-Cui; Wang, Jun-Ping; Yang, Xin-Ying; Wang, Yan; Li, Bao-Sheng; Kang, Fu-Biao; Li, Hai-Jun; Nan, Yue-Min; Sun, Dian-Xing

    2014-12-28

    To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons. The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA. The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES

  8. Expression of a transgene encoding mutant p193/CUL7 preserves cardiac function and limits infarct expansion after myocardial infarction

    NARCIS (Netherlands)

    Hassink, R. J.; Nakajima, H.; Nakajima, H. O.; Doevendans, P. A.; Field, L. J.

    2009-01-01

    Background: Transgenic mice expressing the dominant interfering p193 protein in cardiomyocytes (MHC-1152stop mice) exhibit an induction of cell cycle activity and altered remodelling after experimental myocardial infarction (MI). Objective: To determine whether the altered remodelling results in

  9. Induction of Immunity to a Breast Cancer Associated Mucin in Transgenic Mice Expressing the Human Antigen - A Preclinical Study

    National Research Council Canada - National Science Library

    Cohen, Edward

    1997-01-01

    .... Two approach 5 are being evaluated to augment the immunity to PEM. The studies are being carried out in transgenic mice that express human mucin as "self," mimicking as closely as possible the disease in humans...

  10. Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

    NARCIS (Netherlands)

    Mlynárová, Ľudmila; Loonen, Annelies; Heldens, Jos; Jansen, Ritsert C.; Keizer, Paul; Stiekema, Willem J.; Nap, Jan-Peter

    1994-01-01

    Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in

  11. Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth.

    Science.gov (United States)

    Merali, Zara; Mayer, Melinda J; Parker, Mary L; Michael, Anthony J; Smith, Andrew C; Waldron, Keith W

    2012-06-01

    Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications. Copyright © Physiologia Plantarum 2012.

  12. Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

    Directory of Open Access Journals (Sweden)

    Yu-Guo Yuan

    2014-01-01

    Full Text Available To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC containing a hybrid goat β-lactoglobulin (BLG promoter/cytomegalovirus (CMV enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT. Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2% from the non-transfected line and five recipients delivered six kids (1.8% from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P<0.05. Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

  13. A non-specific effect associated with conditional transgene expression based on Cre-loxP strategy in mice.

    Directory of Open Access Journals (Sweden)

    Linghua Qiu

    Full Text Available Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

  14. Assessment of long-term transgene expression in barley: Ds-mediated delivery of bar results in robust, stable, and heritable expression

    Science.gov (United States)

    The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes are frequently subject to silencing. Studies of the charact...

  15. Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice

    Directory of Open Access Journals (Sweden)

    Postina Rolf

    2009-02-01

    Full Text Available Abstract Background In a transgenic mouse model of Alzheimer disease (AD, cleavage of the amyloid precursor protein (APP by the α-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology. Results To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the α-secretase ADAM10, or a dominant-negative mutant (dn of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant. Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and β-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice. Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down syndrome patients. Conclusion In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of

  16. 3' self-inactivating long terminal repeat inserts for the modulation of transgene expression from lentiviral vectors.

    Science.gov (United States)

    Manic, Gwenola; Maurin-Marlin, Aurélie; Galluzzi, Lorenzo; Subra, Frédéric; Mouscadet, Jean-François; Bury-Moné, Stéphanie

    2012-04-01

    Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-15

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

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

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene....... Level and duration of transgene expression after gene electrotransfer to skin is essential and here we present data from two independent quantitative studies. Using in vivo bioimaging of a far-red fluorescent molecule, Katushka, allowing for continuous monitoring of local gene expression, compared...

  20. Enhanced motivation to alcohol in transgenic mice expressing human α-synuclein.

    Science.gov (United States)

    Rotermund, Carola; Reolon, Gustavo K; Leixner, Sarah; Boden, Cindy; Bilbao, Ainhoa; Kahle, Philipp J

    2017-11-01

    α-Synuclein (αSYN) is the neuropathological hallmark protein of Parkinson's disease (PD) and related neurodegenerative disorders. Moreover, the gene encoding αSYN (SNCA) is a major genetic contributor to PD. Interestingly, independent genome-wide association studies also identified SNCA as the most important candidate gene for alcoholism. Furthermore, single-nucleotide-polymorphisms have been associated with alcohol-craving behavior and alcohol-craving patients showed augmented αSYN expression in blood. To investigate the effect of αSYN on the addictive properties of chronic alcohol use, we examined consumption, motivation, and seeking responses induced by environmental stimuli and relapse behavior in transgenic mice expressing the human mutant [A30P]αSYN throughout the brain. The primary reinforcing effects of alcohol under operant self-administration conditions were increased, while consumption and the alcohol deprivation effect were not altered in the transgenic mice. The same mice were subjected to immunohistochemical measurements of immediate-early gene inductions in brain regions involved in addiction-related behaviors. Acute ethanol injection enhanced immunostaining for the phosphorylated form of cAMP response element binding protein in both amygdala and nucleus accumbens of αSYN transgenic mice, while in wild-type mice no effect was visible. However, at the same time, levels of cFos remain unchanged in both genotypes. These results provide experimental confirmation of SNCA as a candidate gene for alcoholism in addition to its known link to PD. © 2017 International Society for Neurochemistry.

  1. A multiplexed miRNA and transgene expression platform for simultaneous repression and expression of protein coding sequences.

    Science.gov (United States)

    Seyhan, Attila A

    2016-01-01

    Knockdown of single or multiple gene targets by RNA interference (RNAi) is necessary to overcome escape mutants or isoform redundancy. It is also necessary to use multiple RNAi reagents to knockdown multiple targets. It is also desirable to express a transgene or positive regulatory elements and inhibit a target gene in a coordinated fashion. This study reports a flexible multiplexed RNAi and transgene platform using endogenous intronic primary microRNAs (pri-miRNAs) as a scaffold located in the green fluorescent protein (GFP) as a model for any functional transgene. The multiplexed intronic miRNA - GFP transgene platform was designed to co-express multiple small RNAs within the polycistronic cluster from a Pol II promoter at more moderate levels to reduce potential vector toxicity. The native intronic miRNAs are co-transcribed with a precursor GFP mRNA as a single transcript and presumably cleaved out of the precursor-(pre) mRNA by the RNA splicing machinery, spliceosome. The spliced intron with miRNA hairpins will be further processed into mature miRNAs or small interfering RNAs (siRNAs) capable of triggering RNAi effects, while the ligated exons become a mature messenger RNA for the translation of the functional GFP protein. Data show that this approach led to robust RNAi-mediated silencing of multiple Renilla Luciferase (R-Luc)-tagged target genes and coordinated expression of functional GFP from a single transcript in transiently transfected HeLa cells. The results demonstrated that this design facilitates the coordinated expression of all mature miRNAs either as individual miRNAs or as multiple miRNAs and the associated protein. The data suggest that, it is possible to simultaneously deliver multiple negative (miRNA or shRNA) and positive (transgene) regulatory elements. Because many cellular processes require simultaneous repression and activation of downstream pathways, this approach offers a platform technology to achieve that dual manipulation efficiently

  2. Expression of recombinant interferon α-2a in tobacco chloroplasts ...

    African Journals Online (AJOL)

    Chloroplast transformation was accomplished upon bombardment of fully expanded 4 to 6 weeks-old tobacco leaves using helium gun. Green shoots regenerated from single antibiotic resistant cells were subjected to further rounds of selection and regeneration to develop homoplasmic clones. The molecular analysis of ...

  3. High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors

    Directory of Open Access Journals (Sweden)

    Lindbo John A

    2007-08-01

    Full Text Available Abstract Background Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use. Results We have constructed a Cauliflower mosaic virus (CaMV 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI. The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date. Conclusion These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.

  4. Transgenic Carrot Expressing Fusion Protein Comprising M. tuberculosis Antigens Induces Immune Response in Mice

    Directory of Open Access Journals (Sweden)

    Natalia V. Permyakova

    2015-01-01

    Full Text Available Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L. genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

  5. Transgenic overexpression of BAFF regulates the expression of immune-related genes in zebrafish, Danio rerio.

    Science.gov (United States)

    Zhang, Li; Liu, Chao; Zhou, Xin; Xie, Ying; Su, Libo; Geng, Qi; Liu, Binghui; Liu, Shufeng

    2016-12-01

    The B-cell activating factor (BAFF) is a member of tumour necrosis factor (TNF) superfamily that specifically regulates B lymphocyte proliferation and survival. Excess BAFF leads to overproduction of antibodies for secretion, anti-dsDNA antibodies and a lupus-like syndrome in mice. To investigate whether transgenic overexpression of the zebrafish BAFF leads to immunoglobulin changes and/or early maturing of the immune system, a Tol2-GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences. Functional GFP and BAFF proteins were expressed separately and confirmed in HeLa cells. The relative expression of immune-related genes (IgLC-1, IgLC-2, IgLC-3, IgD, IgM and IL-4), early lymphoid markers (Ikaros, Rag-1 and TCRAC), and the protooncogene Bcl-2 were evaluated by quantitative polymerase chain reaction (PCR) in F0 founder of transgenic zebrafish juveniles and adults. Ectopic expression of BAFF in adults was confirmed using Western blots and was shown to upregulate IgLC-1, IgLC-2, IgD, IgM, IgZ/T, Ikaros, Rag-1, TCRAC, IL-4 and Bcl-2 expression in juveniles on day 21 and IgLC-1, IgLC-2, IgD, IgM,IgZ/T, Rag-1, TCRAC and Bcl-2 expression in zebrafish three months postfertilization. The relative titers of specific IgM against Edwardsiella tarda WED were assessed using modified enzyme-linked immunosorbent assay (ELISA) with the whole body homogenate of zebrafish and demonstrated a significant increase in BAFF-transgenic group. Therefore, our findings provided novel insight into further exploration of modulating adaptive immunity and studying autoimmune diseases caused by regulating BAFF.

  6. Transgenic expression of BRCA1 disturbs hematopoietic stem and progenitor cells quiescence and function

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Lin; Shi, Guiying; Zhang, Xu; Dong, Wei; Zhang, Lianfeng, E-mail: zhanglf@cnilas.org

    2013-10-15

    The balance between quiescence and proliferation of HSCs is an important regulator of hematopoiesis. Loss of quiescence frequently results in HSCs exhaustion, which underscores the importance of tight regulation of proliferation in these cells. Studies have indicated that cyclin-dependent kinases are involved in the regulation of quiescence in HSCs. BRCA1 plays an important role in the repair of DNA double-stranded breaks, cell cycle, apoptosis and transcription. BRCA1 is expressed in the bone marrow. However, the function of BRCA1 in HSCs is unknown. In our study, we generated BRCA1 transgenic mice to investigate the effects of BRCA1 on the mechanisms of quiescence and differentiation in HSCs. The results demonstrate that over-expression of BRCA1 in the bone marrow impairs the development of B lymphocytes. Furthermore, BRCA1 induced an increase in the number of LSKs, LT-HSCs, ST-HSCs and MPPs. A competitive transplantation assay found that BRCA1 transgenic mice failed to reconstitute hematopoiesis. Moreover, BRCA1 regulates the expression of p21{sup waf1}/cip1 and p57{sup kip2}, which results in a loss of quiescence in LSKs. Together, over-expression of BRCA1 in bone marrow disrupted the quiescent of LSKs, induced excessive accumulation of LSKs, and disrupted differentiation of the HSCs, which acts through the down-regulated of p21{sup waf1}/cip1 and p57{sup kip2}. - Highlights: • Over-expression of BRCA1 results in impaired B lymphocyte development. • BRCA1 transgenic mice disrupted the quiescent of LSKs, induced excessive accumulation of LSKs. • BRCA1 impairs the function of HSCs through the down-regulated of p21{sup waf1/cip1} and p57{sup kip2}.

  7. Transgenic expression of BRCA1 disturbs hematopoietic stem and progenitor cells quiescence and function

    International Nuclear Information System (INIS)

    Bai, Lin; Shi, Guiying; Zhang, Xu; Dong, Wei; Zhang, Lianfeng

    2013-01-01

    The balance between quiescence and proliferation of HSCs is an important regulator of hematopoiesis. Loss of quiescence frequently results in HSCs exhaustion, which underscores the importance of tight regulation of proliferation in these cells. Studies have indicated that cyclin-dependent kinases are involved in the regulation of quiescence in HSCs. BRCA1 plays an important role in the repair of DNA double-stranded breaks, cell cycle, apoptosis and transcription. BRCA1 is expressed in the bone marrow. However, the function of BRCA1 in HSCs is unknown. In our study, we generated BRCA1 transgenic mice to investigate the effects of BRCA1 on the mechanisms of quiescence and differentiation in HSCs. The results demonstrate that over-expression of BRCA1 in the bone marrow impairs the development of B lymphocytes. Furthermore, BRCA1 induced an increase in the number of LSKs, LT-HSCs, ST-HSCs and MPPs. A competitive transplantation assay found that BRCA1 transgenic mice failed to reconstitute hematopoiesis. Moreover, BRCA1 regulates the expression of p21 waf1 /cip1 and p57 kip2 , which results in a loss of quiescence in LSKs. Together, over-expression of BRCA1 in bone marrow disrupted the quiescent of LSKs, induced excessive accumulation of LSKs, and disrupted differentiation of the HSCs, which acts through the down-regulated of p21 waf1 /cip1 and p57 kip2 . - Highlights: • Over-expression of BRCA1 results in impaired B lymphocyte development. • BRCA1 transgenic mice disrupted the quiescent of LSKs, induced excessive accumulation of LSKs. • BRCA1 impairs the function of HSCs through the down-regulated of p21 waf1/cip1 and p57 kip2

  8. Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance.

    Science.gov (United States)

    McGraw, D W; Fukuda, N; James, P F; Forbes, S L; Woo, A L; Lingrel, J B; Witte, D P; Matthay, M A; Liggett, S B

    2001-10-01

    Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.

  9. The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.

    Science.gov (United States)

    Potter, Christopher J; Tasic, Bosiljka; Russler, Emilie V; Liang, Liang; Luo, Liqun

    2010-04-30

    We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This "Q system" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional "logic gates" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis. 2010 Elsevier Inc. All rights reserved.

  10. Expression and Immunogenicity of the Mycobacterial Ag85B/ESAT-6 Antigens Produced in Transgenic Plants by Elastin-Like Peptide Fusion Strategy

    Directory of Open Access Journals (Sweden)

    Doreen Manuela Floss

    2010-01-01

    Full Text Available This study explored a novel system combining plant-based production and the elastin-like peptide (ELP fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

  11. Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12 receptor

    Science.gov (United States)

    Zhang, Y.; Ye, J.; Hu, L.; Zhang, S.; Zhang, S.H.; Li, Y.; Kunapuli, S.P.; Ding, Z.

    2012-01-01

    Summary Background In our previous in vitro study we reported a constitutively active chimeric P2Y12 receptor (cP2Y12) and found AR-C78511 is a potent inverse agonist at this receptor. The role of this cP2Y12 receptor in platelet activation and thrombosis is not clear. Objectives To investigate the physiological implications of the constitutively active P2Y12 receptor in platelet activation, thrombus formulation and evaluate the antiplatelet activity of AR-C78511 as an inverse agonist. Methods and Results We generated transgenic mice conditionally and platelet-specifically expressing cP2Y12. High expression of cP2Y12 receptor in platelets increased platelet reactivity as evidenced by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice displayed shortened bleeding time, more rapid and stable thrombus formation in mesenteric artery injured with FeCl3. The constitutive activity of cP2Y12 in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR-C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited superior antiplatelet effect over AR-C69931MX in transgenic mice. Conclusions These findings further emphasize the importance of P2Y12 in platelet activation, hemostasis and thrombosis, as well as the prothrombotic role of constitutive activity of P2Y12. Our data also validates the in vivo inverse agonist activity of AR-C78511 and confirms its superior antiplatelet activity over neutral antagonist. PMID:22906019

  12. Biotic and abiotic stress tolerance in transgenic tomatoes by constitutive expression of S-adenosylmethionine decarboxylase gene.

    Science.gov (United States)

    Hazarika, Pranjal; Rajam, Manchikatla Venkat

    2011-04-01

    Recent findings have implicated the role of polyamines (putrescine, spermidine and spermine) in stress tolerance. Therefore, the present work was carried out with the goal of generating transgenic tomato plants with human S-adenosylmethionine decarboxylase (samdc) gene, a key gene involved in biosynthesis of polyamines, viz. spermidine and spermine and evaluating the transgenic plants for tolerance to both biotic and abiotic stresses. Several putative transgenic tomato plants with normal phenotype were obtained, and the transgene integration and expression was validated by PCR, Southern blot analysis and RT-PCR analysis, respectively. The transgenic plants exhibited high levels of polyamines as compared to the untransformed control plants. They also showed increased resistance against two important fungal pathogens of tomato, the wilt causing Fusarium oxysporum and the early blight causing Alternaria solani and tolerance to multiple abiotic stresses such as salinity, drought, cold and high temperature. These results suggest that engineering polyamine accumulation can confer tolerance to both biotic and abiotic stresses in plants.

  13. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

    Science.gov (United States)

    Guo, Ying-Hui; Yu, Yue-Ping; Wang, Dong; Wu, Chang-Ai; Yang, Guo-Dong; Huang, Jin-Guang; Zheng, Cheng-Chao

    2009-01-01

    * Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

  14. Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations.

    Science.gov (United States)

    Padilla, Stephanie L; Reef, Daniel; Zeltser, Lori M

    2012-03-01

    Melanocortin signaling plays a central role in the regulation of phenotypes related to body weight and energy homeostasis. To specifically target and study the function of proopiomelanocortin (POMC) neurons, Pomc promoter elements have been utilized to generate reporter and Cre recombinase transgenic reagents. Across gestation, we find that Pomc is dynamically expressed in many sites in the developing mouse forebrain, midbrain, hindbrain, spinal cord, and retina. Although Pomc expression in most embryonic brain regions is transient, it is sufficient to direct Cre-mediated recombination of floxed alleles. We visualize the populations affected by this transgene by crossing Pomc-Cre mice to ROSA reporter strains and identify 62 sites of recombination throughout the adult brain, including several nuclei implicated in energy homeostasis regulation. To compare the relationship between acute Pomc promoter activity and Pomc-Cre-mediated recombination at the single cell level, we crossed Pomc-enhanced green fluorescent protein (eGFP) and Pomc-Cre;ROSA-tdTomato lines. We detect the highest concentration of Pomc-eGFP+ cells in the arcuate nucleus of the hypothalamus and dentate gyrus but also observe smaller populations of labeled cells in the nucleus of the solitary tract, periventricular zone of the third ventricle, and cerebellum. Consistent with the dynamic nature of Pomc expression in the embryo, the vast majority of neurons marked with the tdTomato reporter do not express eGFP in the adult. Thus, recombination in off-target sites could contribute to physiological phenotypes using Pomc-Cre transgenics. For example, we find that approximately 83% of the cells in the arcuate nucleus of the hypothalamus immunoreactive for leptin-induced phosphorylated signal transducer and activator of transcription 3 are marked with Pomc-Cre;ROSA-tdTomato; only 13% of these are eGFP+ POMC neurons.

  15. Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

    Directory of Open Access Journals (Sweden)

    Maria Arantzazu Sanchez-Fernandez

    Full Text Available BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK, a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

  16. Expression of thymosin alpha1 concatemer in transgenic tomato (Solanum lycopersicum) fruits.

    Science.gov (United States)

    Chen, Yuhui; Wang, Aoxue; Zhao, Lingxia; Shen, Guoan; Cui, Lijie; Tang, Kexuan

    2009-04-01

    Talpha1 (thymosin alpha1), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Talpha1 is one of two thymosin proteins that have potential future clinical applications. In order to express Talpha1 protein in plants, we designed and synthesized the Talpha1 gene according to the plant codon usage bias and created a novel 4 x Talpha1 concatemer (four copies of the Talpha1 gene arranged end-to-end in tandem, designated 4 x Talpha1). Subsequently, a plant binary expression vector, PG-pRD12-4 x Talpha1, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x Talpha1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Talpha1 protein reached a maximum of 6.098 microg/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4xTalpha1 protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x Talpha1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Talpha1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Talpha1 in plants, and also it will provide the basis for further development of the plant system to produce Talpha1.

  17. Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment

    DEFF Research Database (Denmark)

    Roos, Anna-Karin; Eriksson, Fredrik; Timmons, James A

    2009-01-01

    and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co....... METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA......-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. CONCLUSIONS/SIGNIFICANCE: This study provides important insights to how DNA delivery by intradermal...

  18. Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

    Science.gov (United States)

    Daniels, Richard W; Rossano, Adam J; Macleod, Gregory T; Ganetzky, Barry

    2014-01-01

    Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

  19. Cytokeratin 19 promoter directs the expression of Cre recombinase in various epithelia of transgenic mice.

    Science.gov (United States)

    Zhao, Gui-Feng; Zhao, Shuang; Liu, Jia-Jie; Wu, Ji-Cheng; He, Hao-Yu; Ding, Xiao-Qing; Yu, Xue-Wen; Huang, Ke-Qiang; Li, Zhi-Jie; Zheng, Hua-Chuan

    2017-03-14

    Cytokeratin 19 (K19) is expressed in various differentiated cells, including gastric, intestinal and bronchial epithelial cells, and liver duct cells. Here, we generated a transgenic mouse line, K19-Cre, in which the expression of Cre recombinase was controlled by the promoter of K19. To test the tissue distribution and excision activity of Cre recombinase, K19-Cre transgenic mice were bred with Rosa26 reporter strain and a mouse strain that carries PTEN conditional alleles (PTENLoxp/Loxp). At mRNA level, Cre was strongly expressed in the stomach, lung and intestine, while in stomach, lung, and liver at protein level. The immunoreactivity to Cre was strongly observed the cytoplasm of gastric, bronchial and intestinal epithelial cells. Cre activity was detectable in gastric, bronchial and intestinal epithelial cells, according to LacZ staining. In K19-Cre/PTEN Loxp/Loxp mice, PTEN was abrogated in stomach, intestine, lung, liver and breast, the former two of which were verified by in situ PCR. There appeared breast cancer with PTEN loss. These data suggest that K19 promoter may be a useful tool to study the pathophysiological functions of cytokeratin 19-positive cells, especially gastrointestinal epithelial cells. Cell specificity of neoplasia is not completely attributable to the cell-specific expression of oncogenes and cell-specific loss of tumor suppressor genes.

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

    Science.gov (United States)

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

    2006-08-01

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

  1. Expression and thermotolerance of calreticulin during pollen development in tobacco

    Czech Academy of Sciences Publication Activity Database

    Hrubá, Petra; Honys, David; Tupý, Jaroslav

    2005-01-01

    Roč. 18, č. 3 (2005), s. 143-148 ISSN 0934-0882 R&D Projects: GA ČR GP522/02/D075; GA MŠk LZ1K03018; GA AV ČR KJB6038409 Institutional research plan: CEZ:AV0Z50380511 Keywords : tobacco * pollen development * thermotolerance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.278, year: 2005

  2. HIV-1 transgene expression in rats induces differential expression of tumor necrosis factor alpha and zinc transporters in the liver and the lung

    Directory of Open Access Journals (Sweden)

    Guidot David M

    2011-10-01

    Full Text Available Abstract Background Highly effective antiviral treatment can suppress HIV-1 infection, but the chronic effects of HIV-1-related viral proteins, including gp120 and Tat, on organs such as the lungs can be damaging. HIV-1 transgenic rodent models are useful for studying the systemic effects of these proteins independently of viral infection. We have previously shown that HIV-1 transgene expression (and therefore, HIV-1-related protein expression in rats decreases alveolar macrophage zinc levels and phagocytic capacity by unknown mechanisms. We hypothesized that HIV-1 transgene expression induces chronic inflammation and zinc sequestration within the liver and thereby decreases zinc bioavailability in the lung. We examined the expression of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα, the zinc storage protein, metallothionein (MT1, and the zinc exporter, ZNT1 in the livers and the lungs of wild type and HIV-1 transgenic rats ± dietary zinc supplementation. In addition, we measured zinc levels, the zinc importing protein ZIP1, and the phagocytic capacity in the alveolar macrophages. Results HIV-1 transgene expression increased the liver-specific expression of TNFα, suggesting a chronic inflammatory response within the liver in response to HIV-1-related protein expression. In parallel, HIV-1 transgene expression significantly increased MT1 and ZNT1 expression in the liver as compared to the lung, a pattern that is consistent with zinc sequestration in the liver as occurs during systemic inflammation. Further, HIV-1 transgene expression decreased intracellular zinc levels and increased expression of ZIP1 in the alveolar macrophages, a pattern consistent with zinc deficiency, and decreased their bacterial phagocytic capacity. Interestingly, dietary zinc supplementation in HIV-1 transgenic rats decreased gene expression of TNFα, MT1, and ZNT1 in the liver while simultaneously increasing their expression in the lung. In parallel

  3. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

    Directory of Open Access Journals (Sweden)

    Guidot David M

    2008-04-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 infection and the consequent acquired immunodeficiency syndrome (AIDS has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef. Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1 and Transforming Growth Factor-β1 (TGFβ1, three factors associated with muscle catabolism. Results Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss, the oxidized form of the anti-oxidant cysteine (Cys, and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold and TGFβ1 (~5-fold and ~3-fold in heart and

  4. [Expression of plant antimicrobial peptide pro-SmAMP2 gene increases resistance of transgenic potato plants to Alternaria and Fusarium pathogens].

    Science.gov (United States)

    Vetchinkina, E M; Komakhina, V V; Vysotskii, D A; Zaitsev, D V; Smirnov, A N; Babakov, A V; Komakhin, R A

    2016-09-01

    The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.

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

    Science.gov (United States)

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

    2014-06-01

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

  6. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  7. Skeletal Phenotype of Transgenic Mice Expressing the Beta1 Integrin Cytoplasmic Tail In Osteoblasts

    Science.gov (United States)

    Globus, R. K.; vanderMeulen, M. C. H.; Damsky, D.; Kim, J.-B.; Amblard, D.; Amblard, D.; Nishimura, Y.; Almeida, E.; Iwaniec, U. T.; Wronski, T. J.; hide

    2002-01-01

    To define the physiologic role of beta1 integrin in bone formation and mechanical loading, transgenic mice were generated by expressing the cytoplasmic tall and transmembrane domain of Beta1 integrin under the control of the osteocalcin promoter. In cultured cells, this truncated fragment of Beta1 can act as a dominant negative. Previously, the matrix of calvariae was shown to be abnormal in transgenic (TG) compared to wildtype (WT) mice. In this study, we analyzed appendicular bone in TG and WT, male and female mice at 14, 35, 63, 90 and 365 days old (n=8-12/gp). To assess beta1 integrin function in mechanical loading, a pilot study using hindlimb unloading by tail suspension was performed. 35d old TG and WT females were hindlimb unloaded for 4 wks (n=3-5). Body mass, bone mineral content, histomorphometric (distal femur) and biomechanical parameters were analyzed. Statistical significance (P less than.05) was defined by ANOVA using the Tukey-Kramer post-hoc test. We confirmed transgene expression by immunoprecipitating then immunoblotting bone lysates using an antibody against the beta1 tail. Body masses of TG mice at 63, 90 and 365d old were greater (16-25%) than WT. Some TG female mice at 365d appeared obese; mean abdominal fat mass was 415% greater in TG than WT mice. Tibiae were longer (5-7%) in TG than WT mice at 63 and 90d. Tibial mineral mass of 35d males was 7% lower in TG than WT mice, but at 63d was 21% higher. The % osteoblast surface in 35d TG mice was 20% higher than WT, and at 63d was 17% lower, while % osteoclast surface did not differ. In 365d mice, cancellous bone volume (125%) and endocortical mineral apposition rate (40%) were greater in TG than WT males but not females. In WT mice, hindlimb unloading caused a reduction in mineral mass of tibiae (-20%) and lumbar vertebrae (-22%) relative to normally loaded controls. Surprisingly, hindlimb unloading also caused a relative reduction (-13%) in humerus mass. The effects of hindlimb unloading on

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

    Science.gov (United States)

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

    2012-01-01

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

  9. Transgenically enhanced expression of indole-3-acetic Acid confers hypervirulence to plant pathogens.

    Science.gov (United States)

    Cohen, Barry A; Amsellem, Ziva; Maor, Rudy; Sharon, Amir; Gressel, Jonathan

    2002-06-01

    ABSTRACT Fusarium oxysporum and F. arthrosporioides, pathogenic on Orobanche aegyptiaca, were transformed with two genes of the indole-3-acetamide (IAM) pathway leading to indole-3-acetic acid (IAA) to attempt to enhance virulence. Transgenic F. oxysporum lines containing both the tryptophan-2-monooxyngenase (iaaM) and indole-3-acetamide hydrolase (iaaH) genes produced significantly more IAA than the wild type. IAM accumulated in culture extracts of F. oxysporum containing iaaM alone. F. arthrosporioides containing only iaaM accumulated IAM and an unidentified indole. Some transformants of F. oxysporum expressing only the iaaM gene also produced more IAA than the wild type. Sub-threshold levels (that barely infect Orobanche) of transgenic F. oxysporum expressing both genes and of F. arthrosporioides expressing iaaM were more effective in suppressing the number and size of Orobanche shoots than the wild type on tomato plants grown in soil mixed with Orobanche seed. Stimulating an auxin imbalance enhanced pathogen virulence by affecting the host in a manner similar to low doses of auxin herbicides such as 2,4-dichlorophenoxy acetic acid.

  10. Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

    NARCIS (Netherlands)

    Olawole, O.; Jacobsen, E.; Timmers, J.F.P.; Gilbert, H.J.; Blake, W.; Knox, J.P.; Visser, R.G.F.; Vincken, J.P.

    2007-01-01

    We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase

  11. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  12. Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase.

    Science.gov (United States)

    Baldassarre, H; Schirm, M; Deslauriers, J; Turcotte, C; Bordignon, V

    2009-08-01

    The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of alpha-lactalbumin was determined using the IDRing assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 +/- 0.97 vs. 2.24 +/- 0.25 mg/ml, P 0.05). We concluded that a decreased/delayed expression of the alpha-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.

  13. Differential Analysis of Protein Expression in RNA-Binding-Protein Transgenic and Parental Rice Seeds Cultivated under Salt Stress

    OpenAIRE

    Nakamura, Rika; Nakamura, Ryosuke; Adachi, Reiko; Hachisuka, Akiko; Yamada, Akiyo; Ozeki, Yoshihiro; Teshima, Reiko

    2014-01-01

    Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in pr...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state...... of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates...... the role of Hes1 in a number of different research areas including organ specification, development and regeneration....

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene...... is a suitable time frame for vaccinations and is applicable, for example, in gene therapy for wound healing or treatment of cancer.......In development of novel vaccines, attention is drawn to DNA vaccinations. They are heat stable and can be easily produced. Gene electrotransfer is a simple and nonviral means of transferring DNA to cells and tissues and is attracting increasing interest. One very interesting perspective with gene...... electrotransfer is that choice of tissue can determine the duration of transgene expression. With gene electrotransfer to muscle, long-term expression, that is beyond 1 year, can be obtained, whereas gene electrotransfer to skin gives short-term expression, which is desirable in, for example, DNA vaccinations...

  16. Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops.

    Science.gov (United States)

    Bhunia, Rupam Kumar; Chakraborty, Anirban; Kaur, Ranjeet; Gayatri, T; Bhattacharyya, Jagannath; Basu, Asitava; Maiti, Mrinal K; Sen, Soumitra Kumar

    2014-11-01

    The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.

  17. Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

    International Nuclear Information System (INIS)

    Tuteja, N.

    2005-01-01

    Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

  18. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    International Nuclear Information System (INIS)

    Tomioka, Yukiko; Morimatsu, Masami; Nishijima, Ken-ichi; Usui, Tatsufumi; Yamamoto, Sayo; Suyama, Haruka; Ozaki, Kinuyo; Ito, Toshihiro

    2014-01-01

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation

  19. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Tomioka, Yukiko, E-mail: ytomi@muses.tottori-u.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Morimatsu, Masami, E-mail: mmorimat@vetmed.hokudai.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Nishijima, Ken-ichi, E-mail: nishijma@nubio.nagoya-u.ac.jp [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Nagoya 464-8603 (Japan); Usui, Tatsufumi, E-mail: usutatsu@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Yamamoto, Sayo, E-mail: ysayo@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Suyama, Haruka, E-mail: sharuka@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ozaki, Kinuyo, E-mail: k-ozaki@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ito, Toshihiro, E-mail: toshiito@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); and others

    2014-07-18

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

  20. Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer.

    Science.gov (United States)

    Yuan, Yu-Guo; An, Liyou; Yu, Baoli; Song, Shaozheng; Zhou, Feng; Zhang, Liqing; Gu, Yinyin; Yu, Minghui; Cheng, Yong

    2014-01-01

    To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β -lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P milk at 0.1-0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

  1. A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

    Science.gov (United States)

    Vaughan, Ashley M; Mikolajczak, Sebastian A; Camargo, Nelly; Lakshmanan, Viswanathan; Kennedy, Mark; Lindner, Scott E; Miller, Jessica L; Hume, Jen C C; Kappe, Stefan H I

    2012-12-01

    Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Transgenic expression of Dspp partially rescued the long bone defects of Dmp1-null mice.

    Science.gov (United States)

    Jani, Priyam H; Gibson, Monica P; Liu, Chao; Zhang, Hua; Wang, Xiaofang; Lu, Yongbo; Qin, Chunlin

    2016-01-01

    Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1(-/-)) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1(-/-) mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as "Dmp1(-/-);Dspp-Tg mice"). We characterized the long bones of the Dmp1(-/-);Dspp-Tg mice at different ages and compared them with those from Dmp1(-/-) and Dmp1(+/-) (normal control) mice. Our analyses showed that the long bones of Dmp1(-/-);Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1(-/-) mice. The long bones of Dmp1(-/-);Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1(-/-) mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1(-/-) mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that DSPP and DMP1 may function

  3. CCL2-ethanol interactions and hippocampal synaptic protein expression in a transgenic mouse model

    Directory of Open Access Journals (Sweden)

    Donna eGruol

    2014-04-01

    Full Text Available Chronic exposure to ethanol produces a number of detrimental effects on behavior. Neuroadaptive changes in brain structure or function underlie these behavioral changes and may be transient or persistent in nature. Central to the functional changes are alterations in the biology of neuronal and glial cells of the brain. Recent data show that ethanol induces glial cells of the brain to produce elevated levels of neuroimmune factors including CCL2, a key innate immune chemokine. Depending on the conditions of ethanol exposure, the upregulated levels of CCL2 can be transient or persistent and outlast the period of ethanol exposure. Importantly, results indicate that the upregulated levels of CCL2 may lead to CCL2-ethanol interactions that mediate or regulate the effects of ethanol on the brain. Glial cells are in close association with neurons and regulate many neuronal functions. Therefore, effects of ethanol on glial cells may underlie some of the effects of ethanol on neurons. To investigate this possibility, we are studying the effects of chronic ethanol on hippocampal synaptic function in a transgenic mouse model that expresses elevated levels of CCL2 in the brain through enhanced glial expression, a situation know to occur in alcoholics. Both CCL2 and ethanol have been reported to alter synaptic function in the hippocampus. In the current study, we determined if interactions are evident between CCL2 and ethanol at level of hippocampal synaptic proteins. Two ethanol exposure paradigms were used; the first involved ethanol exposure by drinking and the second involved ethanol exposure in a paradigm that combines drinking plus ethanol vapor. The first paradigm does not produce dependence on ethanol, whereas the second paradigm is commonly used to produce ethanol dependence. Results show modest effects of both ethanol exposure paradigms on the level of synaptic proteins in the hippocampus of CCL2 transgenic mice compared with their non-transgenic

  4. Expression of β-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants.

    Science.gov (United States)

    Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

    2016-03-01

    Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the aetiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β-glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript were confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography, time of flight mass spectrometer data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (per g DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to five-fold in BGL1 transgenic flowers. This study opens the possibility of increasing artemisinin content by manipulating trichomes' density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Expression of Beta-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants

    Science.gov (United States)

    Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

    2015-01-01

    Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript was confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography (HPLC, MS-TOF) data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (g-1DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to 5-fold in BGL1 transgenic flowers. The present study opens the possibility of increasing artemisinin content by manipulating trichomes density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. PMID:26360801

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

    Directory of Open Access Journals (Sweden)

    Ma Hao

    2011-10-01

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

  7. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Palm Kaia

    2009-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Results In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP. The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. Conclusion Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.

  8. Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

    Science.gov (United States)

    Katoh, Akiko; Fujihara, Hiroaki; Ohbuchi, Toyoaki; Onaka, Tatsushi; Young, W. Scott; Dayanithi, Govindan; Yamasaki, Yuka; Kawata, Mitsuhiro; Suzuki, Hitoshi; Otsubo, Hiroki; Suzuki, Hideaki; Murphy, David; Ueta, Yoichi

    2010-01-01

    We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the OXT-eCFP fusion gene was expressed in the supraoptic (SON) and the paraventricular nuclei (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence (ME) and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT-immunofluorescence, but not with AVP-immunofluorescence in the SON and the PVN. Although the expression levels of the OXT-eCFP fusion gene in the SON and the PVN showed a wide range of variation in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the OXT gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared to wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration, OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rat is a valuable new tool to identify OXT-producing neurones and their terminals. PMID:20026620

  9. Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids.

    Science.gov (United States)

    Chaveroux, Cédric; Bruhat, Alain; Carraro, Valérie; Jousse, Céline; Averous, Julien; Maurin, Anne-Catherine; Parry, Laurent; Mesclon, Florent; Muranishi, Yuki; Cordelier, Pierre; Meulle, Aline; Baril, Patrick; Do Thi, Anh; Ravassard, Philippe; Mallet, Jacques; Fafournoux, Pierre

    2016-07-01

    Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.

  10. Molecular Analyses of Transgenic Plants.

    Science.gov (United States)

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

    2016-01-01

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

  11. Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos.

    Science.gov (United States)

    Ariffin, Norazrin; Abdullah, Ruslan; Rashdan Muad, Mohd; Lourdes, Juanita; Emran, Nurul Ain; Ismail, Mohd Razi; Ismail, Ismanizan; Fadzil, Mohd Fazli Mohd; Ling, Kong Lih; Siddiqui, Yasmeen; Amir, Anna Aryani; Berahim, Zulkarami; Husni Omar, Mohd

    2011-09-01

    Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Lysozyme transgenic goats' milk positively impacts intestinal cytokine expression and morphology.

    Science.gov (United States)

    Cooper, Caitlin A; Brundige, Dottie R; Reh, Wade A; Maga, Elizabeth A; Murray, James D

    2011-12-01

    In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.

  13. Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

    NARCIS (Netherlands)

    Meade-White, K.; Race, B.; Trifilo, M.; Bossers, A.; Favara, C.; Lacasse, R.; Miller, M.; Williams, E.; Oldstone, M.; Race, R.; Chesebro, B.

    2007-01-01

    Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer

  14. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  15. Focal glomerulosclerosis in proviral and c-fms transgenic mice links Vpr expression to HIV-associated nephropathy

    International Nuclear Information System (INIS)

    Dickie, Peter; Roberts, Amanda; Uwiera, Richard; Witmer, Jennifer; Sharma, Kirti; Kopp, Jeffrey B.

    2004-01-01

    Clinical and morphologic features of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN), such as proteinuria, sclerosing glomerulopathy, tubular degeneration, and interstitial disease, have been modeled in mice bearing an HIV proviral transgene rendered noninfectious through a deletion in gag/pol. Exploring the genetic basis of HIVAN, HIV transgenic mice bearing mutations in either or both of the accessory genes nef and vpr were created. Proteinuria and focal glomerulosclerosis (FGS) only developed in mice with an intact vpr gene. Transgenic mice bearing a simplified proviral DNA (encoding only Tat and Vpr) developed renal disease characterized by FGS in which Vpr protein was localized to glomerular and tubular epithelia by immunohistochemistry. The dual transgenic progeny of HIV[Tat/Vpr] mice bred to HIV[ΔVpr] proviral transgenic mice displayed a more severe nephropathy with no apparent increase in Vpr expression, implying that multiple viral genes contribute to HIVAN. However, the unique contribution of macrophage-specific Vpr expression in the development of glomerular disease was underscored by the induction of FGS in multiple murine lines bearing a c-fms/vpr transgene

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

    Science.gov (United States)

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

    2010-12-01

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

  17. Expression of Plant Sweet Protein Brazzein in the Milk of Transgenic Mice

    Science.gov (United States)

    Yan, Sen; Song, Hong; Pang, Daxin; Zou, Qingjian; Li, Li; Yan, Quanmei; Fan, Nana; Zhao, Xiangjie; Yu, Hao; Li, Zhanjun; Wang, Haijun; Gao, Fei; Ouyang, Hongsheng; Lai, Liangxue

    2013-01-01

    Sugar, the most popular sweetener, is essential in daily food. However, excessive sugar intake has been associated with several lifestyle-related diseases. Finding healthier and more economical alternatives to sugars and artificial sweeteners has received increasing attention to fulfill the growing demand. Brazzein, which comes from the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is a protein that is 2,000 times sweeter than sucrose by weight. Here we report the production of transgenic mice that carry the optimized brazzein gene driven by the goat Beta-casein promoter, which specifically directs gene expression in the mammary glands. Using western blot analysis and immunohistochemistry, we confirmed that brazzein could be efficiently expressed in mammalian milk, while retaining its sweetness. This study presents the possibility of producing plant protein–sweetened milk from large animals such as cattle and goats. PMID:24155905

  18. Expression of plant sweet protein brazzein in the milk of transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sen Yan

    Full Text Available Sugar, the most popular sweetener, is essential in daily food. However, excessive sugar intake has been associated with several lifestyle-related diseases. Finding healthier and more economical alternatives to sugars and artificial sweeteners has received increasing attention to fulfill the growing demand. Brazzein, which comes from the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is a protein that is 2,000 times sweeter than sucrose by weight. Here we report the production of transgenic mice that carry the optimized brazzein gene driven by the goat Beta-casein promoter, which specifically directs gene expression in the mammary glands. Using western blot analysis and immunohistochemistry, we confirmed that brazzein could be efficiently expressed in mammalian milk, while retaining its sweetness. This study presents the possibility of producing plant protein-sweetened milk from large animals such as cattle and goats.

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

    Science.gov (United States)

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

    2006-11-01

    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.

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

    Science.gov (United States)

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

    2016-11-20

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

  1. Suppression of GPI-induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands.

    Science.gov (United States)

    Hirota, Tomoya; Tsuboi, Hiroto; Takahashi, Hiroyuki; Asashima, Hiromitsu; Ohta, Masaru; Wakasa, Yuhya; Matsumoto, Isao; Takaiwa, Fumio; Sumida, Takayuki

    2017-01-01

    To investigate the effects and mechanisms of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI 325-339 ) in mice model of GPI induced arthritis (GIA). We generated transgenic rice expressing APL12 which was analog peptide of hGPI 325-339 . The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined IL-17 production from splenocytes and inguinal lymph node (iLN) and mesenteric lymph nodes (mLN) cells and analyzed the expression levels of functional molecules from splenocytes and iLN cells. Prophylactic treatment of GIA mice with APL12 transgenic rice seeds (APL12-TG) significantly improved the severity of arthritis, histopathological arthritis scores, and decreased titers of serum anti-GPI antibodies, BAFF mRNA in iLN cells, IL-17 production in splenocytes and iLN cells compared with non-transgenic rice-treated mice. APL12-TG-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4 + CD25 + cells in the spleen. APL12-TG improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on regulatory T cells in spleen.

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

    Science.gov (United States)

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

    2013-12-01

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

  3. Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Procházková, Dagmar; Wilhelmová, Naděžda

    2009-01-01

    Roč. 53, č. 4 (2009), s. 691-696 ISSN 0006-3134 R&D Projects: GA ČR GP522/05/P558 Institutional research plan: CEZ:AV0Z50380511 Keywords : oxidative stress * reactive oxygen species * transgenic plants Subject RIV: ED - Physiology Impact factor: 1.656, year: 2009

  4. Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression

    Science.gov (United States)

    Mueller, Christian; Chulay, Jeffrey D.; Trapnell, Bruce C.; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A.; McElvaney, Noel G.; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N.; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R.; Ye, Guo-jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P.; Vonderheide, Robert H.; Hulme, Maigan A.; Brusko, Todd M.; Wilson, James M.; Flotte, Terence R.

    2013-01-01

    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy. PMID:24231351

  5. Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression.

    Science.gov (United States)

    Mueller, Christian; Chulay, Jeffrey D; Trapnell, Bruce C; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A; McElvaney, Noel G; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R; Ye, Guo-Jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P; Vonderheide, Robert H; Hulme, Maigan A; Brusko, Todd M; Wilson, James M; Flotte, Terence R

    2013-12-01

    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

  6. Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

    Science.gov (United States)

    Song, Hongman; Bush, Ronald A.; Vijayasarathy, Camasamudram; Fariss, Robert N.; Kjellstrom, Sten; Sieving, Paul A.

    2014-01-01

    Purpose. Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1. Methods. Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy. Function was evaluated by electroretinography. Potential protein partners of CA RAC1 were identified by co-immunoprecipitation of retinal extracts. Results. Constitutively active RAC1 expression in differentiating rods disrupted outer retinal lamination as early as postnatal day (P)6, and many photoreceptor cell nuclei were displaced apically into the presumptive subretinal space. These photoreceptors did not develop normal inner and outer segments and had abnormal placement of synaptic elements. Some photoreceptor nuclei were also mislocalized into the inner nuclear layer. Extensive photoreceptor degeneration was subsequently observed in the adult animal. Constitutively active RAC1 formed a complex with the polarity protein PAR6 and with microtubule motor dynein in mouse retina. The normal localization of the PAR6 complex was disrupted in CA RAC1-expressing rod photoreceptors. Conclusions. Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. PAR6 and dynein are two potential downstream effectors that may be involved in CA RAC1-mediated defective mouse photoreceptor morphogenesis. PMID:24651551

  7. Broader expression of the mouse platelet factor 4-cre transgene beyond the megakaryocyte lineage.

    Science.gov (United States)

    Pertuy, F; Aguilar, A; Strassel, C; Eckly, A; Freund, J-N; Duluc, I; Gachet, C; Lanza, F; Léon, C

    2015-01-01

    Transgenic mice expressing cre recombinase under the control of the platelet factor 4 (Pf4) promoter, in the context of a 100-kb bacterial artificial chromosome, have become a valuable tool with which to study genetic modifications in the platelet lineage. However, the specificity of cre expression has recently been questioned, and the time of its onset during megakaryopoiesis remains unknown. To characterize the expression of this transgene, we used double-fluorescent cre reporter mice. In the bone marrow, Pf4-cre-mediated recombination had occurred in all CD42-positive megakaryocytes as early as stage I of maturation, and in rare CD42-negative cells. In circulating blood, all platelets had recombined, along with only a minor fraction of CD45-positive cells. However, we found that all tissues contained recombined cells of monocyte/macrophage origin. When recombined, these cells might potentially modify the function of the tissues under particular conditions, especially inflammatory conditions, which further increase recombination in immune cells. Unexpectedly, a subset of epithelial cells from the distal colon showed signs of recombination resulting from endogenous Pf4-cre expression. This is probably the basis of the unexplained colon tumors developed by Apc(flox/flox) ;Pf4-cre mice, generated in a separate study on the role of Apc in platelet formation. Altogether, our results indicate early recombination with full penetrance in megakaryopoiesis, and confirm the value of Pf4-cre mice for the genetic engineering of megakaryocytes and platelets. However, care must be taken when investigating the role of platelets in processes outside hemostasis, especially when immune cells might be involved. © 2014 International Society on Thrombosis and Haemostasis.

  8. Over-expression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice

    Directory of Open Access Journals (Sweden)

    Ai-Ling Liu

    2013-01-01

    Full Text Available Heat shock proteins play an important role in plant stresstolerance and are mainly regulated by heat shock transcriptionfactors (Hsfs. In this study, we generated transgenic riceover-expressing OsHsfA7 and carried out morphologicalobservation and stress tolerance assays. Transgenic plantsexhibited less, shorter lateral roots and root hair. Under salttreatment, over-expressing OsHsfA7 rice showed alleviativeappearance of damage symptoms and higher survival rate, leafelectrical conductivity and malondialdehyde content of transgenicplants were lower than those of wild type plants. Meanwhile,transgenic rice seedlings restored normal growth but wild typeplants could not be rescued after drought and re-wateringtreatment. These findings indicate that over-expression ofOsHsfA7 gene can increase tolerance to salt and drought stressesin rice seedlings. [BMB Reports 2013; 46(1: 31-36

  9. Transient expression of P-type ATPases in tobacco epidermal cells

    DEFF Research Database (Denmark)

    Pedas, Lisbeth Rosager; Palmgren, Michael Broberg; Lopez Marques, Rosa Laura

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellula...

  10. UDP-galactose transporter gene hUGT1 expression in tobacco plants leads to hyper-galactosylated cell wall components.

    Science.gov (United States)

    Abedi, Tayebeh; Khalil, Mohamed Farouk Mohamed; Asai, Toshihiko; Ishihara, Nami; Kitamura, Kenji; Ishida, Nobuhiro; Tanaka, Nobukazu

    2016-05-01

    We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1-transgenic plants) displayed morphological, architectural, and physiological alterations, such as enhanced growth, increased accumulation of chlorophyll and lignin, and a gibberellin-responsive phenotype. In the present study, we demonstrated that hUGT1 expression altered the monosaccharide composition of cell wall matrix polysaccharides, such as pectic and hemicellulosic polysaccharides, which are biosynthesized in the Golgi lumen. An analysis of the monosaccharide composition of the cell wall matrix polysaccharides revealed that the ratio of galactose to total monosaccharides was significantly elevated in the hemicellulose II and pectin fractions of hUGT1-transgenic plants compared with that of control plants. A