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At least 433 records · Page 24Linked to original sources
PubMed2006

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance.

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.

Aluminum↗
PubMed2006

Transgenic tobacco plants overexpressing chitinases of fungal origin show enhanced resistance to biotic and abiotic stress agents.

Genes encoding defense-related proteins have been used to alter the resistance of plants to pathogens and other environmental challenges, but no single fungal gene overexpression has produced broad-spectrum stress resistance in transgenic lines. We have generated transgenic tobacco (Nicotiana tabacum) lines that overexpress the endochitinases CHIT33 and CHIT42 from the mycoparasitic fungus Trichoderma harzianum and have evaluated their tolerance to biotic and abiotic stress. Both CHIT33 and CHIT42, individually, conferred broad resistance to fungal and bacterial pathogens, salinity, and heavy metals. Such broad-range protective effects came off with no obvious detrimental effect on the growth of tobacco plants.

Chitinases↗
PubMed2002

Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids.

Cysteine proteinase inhibitors (cystatins) confer resistance to plant-parasitic nematodes when expressed in transgenic plants. The survival and growth of nymphs of the peach-potato aphid, Myzus persicae, were adversely affected when cystatins were added to artificial diets. When aphids were clip-caged onto transgenic plants expressing chicken egg white cystatin (CEWc) there was no adverse effect on aphid fitness. Field populations of aphids on transgenic Desiree potatoes, expressing CEWc or a modified version of oryzacystatin I, were not significantly different from populations on control Desiree plants. The effect of other nematode management options on aphid numbers was also studied. A conventionally bred cultivar, with partial nematode resistance, supported higher populations of aphids than the transgenic lines at the beginning of the sampling period. Peak aphid densities on the untreated control and untreated transgenic lines were 7 and 5.2 aphids per plant. Aldicarb, commonly used to control nematodes on potatoes, reduced the value to less than 0.2 aphids per plant. The results demonstrate that levels of expression in the plant tissue actually consumed are important in determining the risk of cystatins to nontarget invertebrates. The study also highlights the importance of including currently used management options in any assessment of the impact of transgenic plants on nontarget organisms.

Aldicarb↗
PubMed2003

Characterization of expression at the human XIST locus in somatic, embryonal carcinoma, and transgenic cell lines.

X inactivation requires XIST, a functional RNA that is expressed exclusively from, and localizes to, the inactive X in female somatic cells. In mouse, low-level unstable transcription of Xist is observed prior to the time of inactivation, and an antisense transcript, Tsix, is a critical regulator of early Xist expression. To examine the presence and impact of an antisense transcript in humans we have characterized the extent of sense and antisense transcription in human somatic, transgenic, and embryonal carcinoma (EC) cell lines. Downstream antisense expression at the human XIST locus was not detected in somatic cells, but was detected in the EC line N-Tera2D1 and in somatic cells with an ectopic XIST locus. Presence of the antisense did not disrupt the stability or localization of the sense transcript. We have also identified additional sense transcripts in EC and female somatic cells and demonstrate that the 5' flanking JPX/ENOX gene is expressed from both the active and the inactive X chromosome in somatic cell hybrids, delimiting the extent of inactive X-specific transcriptional control in somatic cells. These analyses reveal similarities to and differences from the murine Xist and Tsix transcripts and generate a complex picture of developmentally regulated transcription through the region.

Animals↗
PubMed2005

Cells of human aminopeptidase N (CD13) transgenic mice are infected by human coronavirus-229E in vitro, but not in vivo.

Aminopeptidase N, or CD13, is a receptor for serologically related coronaviruses of humans, pigs, and cats. A mouse line transgenic for the receptor of human coronavirus-229E (HCoV-229E) was created using human APN (hAPN) cDNA driven by a hAPN promoter. hAPN-transgenic mice expressed hAPN mRNA in the kidney, small intestine, liver, and lung. hAPN protein was specifically expressed on epithelial cells of the proximal convoluted renal tubules, bronchi, alveolar sacs, and intestinal villi. The hAPN expression pattern within transgenic mouse tissues matched that of mouse APN and was similar in mice heterozygous or homozygous for the transgene. Primary embryonic cells and bone marrow dendritic cells derived from hAPN-transgenic mice also expressed hAPN protein. Although hAPN-transgenic mice were resistant to HCoV-229E in vivo, primary embryonic cells and bone marrow dendritic cells were infected in vitro. hAPN-transgenic mice are valuable as a source of primary mouse cells expressing hAPN. This hAPN-transgenic line will also be used for crossbreeding experiments with other knockout, immune deficient, or transgenic mice to identify factors, in addition to hAPN, that are required for HCoV-229E infection.

Animals↗
PubMed2006

Long-range downstream enhancers are essential for Pax6 expression.

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.

Animals↗
PubMed2026

The transgenic Vip3A poplar plant confers high resistance against Hyphantria cunea Drury.

Poplar is severely damaged by Hyphantria cunea (fall webworm), which significantly reduces tree productivity. However, conventional pest management methods are largely ineffective against fall webworm infestation. In this study, we demonstrated that the Vip3A protein possesses high insecticidal activity against H. cunea by overexpressing a synthetic THI1-Vip3A gene in poplar plants. A dicot codon-optimized Vip3A gene, fused with the THI1 chloroplast signal peptide sequence, was chemically synthesized and introduced into the poplar cv. '741' genome via Agrobacterium-mediated transformation. PCR, RT-PCR, and ELISA analyses confirmed the integration and successful expression of the transgene at both the mRNA and protein levels. The Vip3A protein concentration in chloroplasts was approximately 4.8-fold higher than in the whole leaf extract, indicating that the Vip3A protein was successfully targeted to and accumulated within the chloroplasts by the THI1 signal peptide. Subsequently, four transgenic lines with high Vip3A expression were subjected to H. cunea infestation. Compared to wild-type plants, these four transgenic lines exhibited significantly higher resistance, resulting in pest mortality rates exceeding 95% and significantly reduced leaf damage. Together, these results indicate that Vip3A possesses high insecticidal activity against H. cunea. Therefore, transgenic THI1-Vip3A poplar plants can serve as valuable germplasm for breeding poplar cultivars with high resistance to H. cunea infestation.

Plants, Genetically Modified↗
PubMed2006

Molecular analysis, cytogenetics and fertility of introgression lines from transgenic wheat to Aegilops cylindrica host.

Natural hybridization and backcrossing between Aegilops cylindrica and Triticum aestivum can lead to introgression of wheat DNA into the wild species. Hybrids between Ae. cylindrica and wheat lines bearing herbicide resistance (bar), reporter (gus), fungal disease resistance (kp4), and increased insect tolerance (gna) transgenes were produced by pollination of emasculated Ae. cylindrica plants. F1 hybrids were backcrossed to Ae. cylindrica under open-pollination conditions, and first backcrosses were selfed using pollen bags. Female fertility of F1 ranged from 0.03 to 0.6%. Eighteen percent of the sown BC1s germinated and flowered. Chromosome numbers ranged from 30 to 84 and several of the plants bore wheat-specific sequence-characterized amplified regions (SCARs) and the bar gene. Self fertility in two BC1 plants was 0.16 and 5.21%, and the others were completely self-sterile. Among 19 BC1S1 individuals one plant was transgenic, had 43 chromosomes, contained the bar gene, and survived glufosinate treatments. The other BC1S1 plants had between 28 and 31 chromosomes, and several of them carried SCARs specific to wheat A and D genomes. Fertility of these plants was higher under open-pollination conditions than by selfing and did not necessarily correlate with even or euploid chromosome number. Some individuals having supernumerary wheat chromosomes recovered full fertility.

Breeding↗
PubMed2001

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation.

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.

Animals↗
PubMed2001

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants.

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED50 levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED50 level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED50 showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.

Alternaria↗
PubMed2005

Combinatorial microarray analysis revealing arabidopsis genes implicated in cytokinin responses through the His->Asp Phosphorelay circuitry.

In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep histidine kinase (AHK)-->histidine-containing phosphotransmitter (AHP)-->response regulator (ARR) phosphorelay signaling circuitry, which is initiated by the cytokinin receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-->Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we used a combinatorial microarray strategy by employing not only wild-type plants, but also certain transgenic lines in which the cytokinin-mediated His-->Asp phosphorelay signaling circuitry has been genetically manipulated. These transgenic lines employed were ARR21-overexpressing and ARR22-overexpressing plants, each of which exhibits a characteristic phenotype with regard to the cytokinin-mediated His-->Asp phosphorelay. The results of extensive microarray analyses with these plants allowed us systematically to identify a certain number of genes that were up-regulated at the level of transcription in response to cytokinin directly or indirectly. Among them, some representatives were examined further in wild-type plants to support the idea that certain genes encoding transcription factors are rapidly and specifically induced at the level of transcription by cytokinin in a manner similar to that of the type-A ARR genes, which are the hallmarks of the His-->Asp phosphorelay signaling circuitry. Several interesting transcription factors were thus identified as being cytokinin responsive, including those belonging to the AP2/EREBP family, MYB family, GATA family or bHLH family. Including these, the presented list of cytokinin-up-regulated genes (214) will provide us with valuable bases for understanding the His-->Asp phosphorelay in A. thaliana.

Arabidopsis↗
PubMedDate not supplied

Effective resistance to potyvirus infection conferred by expression of antisense RNA in transgenic plants.

Approximately 660 nt including the carboxy-terminal portion of the bean yellow mosaic potyvirus (BYMV) coat protein gene, complete 3' noncoding sequence and a short poly(A) tract were introduced to produce antisense RNA in transgenic Nicotiana benthamiana. Original (R0) transformants were selfed, and homozygous second generation (R2) populations challenged with infected sap, purified virus, or viral RNA. One transgenic line of 10 examined was extremely resistant to infection by mechanical inoculation of 100 micrograms/ml BYMV or 50 micrograms/ml BYMV RNA (the highest concentrations tested); no virus could be detected in inoculated leaves of this line. Nine other lines were systemically infected when inoculated with BYMV or BYMV RNA, with initial symptoms indistinguishable from those in nontransgenic plants. One of these lines subsequently developed reduced symptoms and then symptomless, virus-free leaves (complete recovery from BYMV infection), while other lines produced leaves with reduced symptoms and reduced virus titer compared to the controls (partial recovery). No transgenic lines had resistance to infection, nor recovery from symptom expression, following inoculation with pepper mottle or turnip mosaic potyviruses. Antisense RNA from 3' regulatory regions can confer multiple degrees of resistance to potyviruses, including extreme resistanc to infection, presumably by interference in virus replication.

Base Sequence↗
PubMed1999

Lymphoid specificity of a copy number-related expression of the H2-Kb transgene.

Transcriptional regulation of the MHC class I genes leading to their developmental and tissue specific expression is still poorly understood in spite of the recovery of a large variety of cis-controlling sequences and trans-acting factors pertaining to the 5' enhancer and the downstream regulatory element. Here we produced a series transgenic lines of mice with a genomic subclone of the H2-Kb gene consisting of 367 bp of the 5' upstream region, the coding region and 1.5 kb of the 3' downstream region and carrying all hitherto known regulatory sequences. The comparison of nine transgenic lines carrying the same H2-Kb transgene made it possible to ask whether the cis-information present in the transgene was sufficient for the tissue- and developmental-specific expression and its copy number dependence. We found the proper developmental onset of expression of the transgene at day 13 p.c. and correct tissue specific mRNA levels in adult mice. While in lymphoid tissues and in lung the number of transgene copies still correlated with RNA levels, the copy number dependence was completely lost in liver, kidney and embryonic tissues. Comparison with previously published H2-Kb transgenes indicates that the H2-Kb locus-controlling region is composed of more than one element.

Animals↗
PubMed1995

The cyclic AMP response element directs tyrosine hydroxylase expression in catecholaminergic central and peripheral nervous system cell lines from transgenic mice.

Enhancer elements regulating the neuronal gene, tyrosine hydroxylase (TH), were identified in TH-expressing peripheral nervous system PATH and central nervous system CATH cell lines. Mutational analysis in which rat TH 5'-flanking sequences directed chloramphenicol acetyltransferase (CAT) reporter gene expression demonstrated that mutating the cyclic AMP response element (CRE) at -45 base pair reduced expression by 80-90%. A CRE linked to an enhancerless TH promoter fully supported expression. Cotransfection of a dominant-negative CREB protein reduced expression 50-60%, suggesting that the CRE is bound by CREB or a CREB dimerization partner. Although mutating the AP1/dyad (AD) element at -205 base pair only modestly reduced CAT levels, AD minimal enhancer constructs gave 45-80% of wild type expression when positioned at -91 or -95. However, in its native context at -205, the AD could not support expression. In contrast, a CRE, moved from its normal position at -45 to -206, gave full activity. These results indicate that the CRE is critical for TH transcription in central nervous system CATH and peripheral nervous system PATH cells, whereas the AD is less important and its enhancer activity is context-and/or position-dependent. These results represent the first attempts to map regulatory elements directing TH expression in central nervous system cell lines.

Adrenal Gland Neoplasms↗
PubMed2005

Efficient recombination in pancreatic islets by a tamoxifen-inducible Cre-recombinase.

We generated pdx1(PB)CreERtrade mark transgenic mice in which a pancreatic endocrine-specific enhancer (pdx1(PB)) drives expression of a tamoxifen (TM)-inducible Cre recombinase/estrogen receptor fusion protein. We previously showed that this enhancer directs expression to immature endocrine cells as well as postnatal islets. This transgene provides spatial and temporal control of gene inactivation in pancreatic islets. Three transgenic lines were generated and crossed with R26R mice to assess recombination efficiency. TM-dependent lacZ expression was observed in islets from all three lines. One line was chosen for further study based on its strong islet-specific recombination in embryos and adults. In this line, a dose-dependent increase in recombination efficiency was observed in endocrine cells. Our data suggest that this transgenic line will be a valuable tool to inactivate genes in pancreatic endocrine cells during development or in the adult. The dose-dependent nature of recombination suggests a potential use for this line in the generation of genetic mosaic animals.

Animals↗
PubMed1992

Tissue-specific and hormonally regulated expression of the puromycin N-acetyltransferase-encoding gene under control of the rabbit uteroglobin promoter in transgenic mice.

Transgenic mice bearing two fragments of the rabbit uteroglobin 5'-flanking region fused to the new reporter gene (pac) encoding puromycin N-acetyltransferase (PAC) showed a different pattern of expression. Transgenic lines (C0.4) harboring a 404-bp fragment (-396/+8) had a uterus-specific expression slightly inducible by estrogen, lacking detectable expression in other tissues where the uteroglobin-encoding gene is naturally expressed in rabbit. Transgenic lines (C3.2) bearing a longer fragment of 3.2-kb (-3254/+8) showed hormonally regulated expression in the uterus and the male genital tract, and detectable expression in the lung. In addition, the nonstimulated uterine expression of the transgene was higher in C0.4 lines than in C3.2 lines. It could be concluded that all sequences required for uterus-specific expression should be present within the 404-bp fragment, and that other upstream (-396) sequences are responsible for expression in the lung and male genital tract, as well as for a possible down modulation of expression in the uterus.

Acetyltransferases↗
PubMed2000

Responses of transgenic mouse lines p53(+/-) and Tg.AC to agents tested in conventional carcinogenicity bioassays.

The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.

Administration, Oral↗
PubMed2004

Utility of the FLP-FRT recombination system for genetic manipulation of rice.

To develop an FLP-FRT recombination system- (derived from 2 mu plasmid of Saccharomyces cerevisiae) based marker gene removal application for rice, we introduced the gene for FLP recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome. FLP activity was monitored in callus and regenerated plants by an assay based on the deletion of the FRT-flanked DNA fragment, leading to the activation of the beta-glucuronidase gene. FLP activity was detected both in the callus and leaves of some of the transgenic lines. Based on our comparison of the recombination efficiency of the FLP-FRT system expressed in the transgenic lines with that of the widely used Cre-lox system (derived from bacteriophage P1), we suggest that the FLP-FRT system is a useful tool for the genetic manipulation of rice.

DNA Nucleotidyltransferases↗