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At least 37 records · Page 2Linked to original sources

Behavior of the hobo transposable element with regard to TPE repeats in transgenic lines of Drosophila melanogaster.

The hobo transposable element of Drosophila melanogaster is known to induce a hybrid dysgenesis syndrome. Moreover it displays a polymorphism of a microsatellite in its coding region: TPE repeats. In European populations, surveys of the distribution of hobo elements with regard to TPE repeats revealed that the 5TPE element is distributed along a frequency gradient, and it is even more frequent than the 3TPE element in Western populations. This suggests that the invasive ability of the hobo elements could be related to the number of TPE repeats they contain. To test this hypothesis we monitored the evolution of 16 lines derived from five initial independent transgenic lines bearing the 3TPE element and/or the 5TPE element. Four lines bearing 5TPE elements and four bearing 3TPE elements were used as a noncompetitive genetic background to compare the evolution of the 5TPE element to that of the 3TPE element. Eight lines bearing both elements provided a competitive genetic context to study potential interactions between these two elements. We studied genetic and molecular aspects of the first 20 generations. At the molecular level, we showed that the 5TPE element is able to spread within the genome at least as efficiently as the 3TPE element. Surprisingly, at the genetic level we found that the 5TPE element is less active than the 3TPE element, and moreover may be able to regulate the activity of the 3TPE element. Our findings suggest that the invasive potential of the 5TPE element could be due not only to its intrinsic transposition capacity but also to a regulatory potential.

Animals↗

Nutritional properties of tubers of conventionally bred and transgenic lines of potato resistant to necrotic strain of Potato virus Y (PVYN).

The potential effect of genetic modification on nutritional properties of potatoes transformed to improve resistance to a necrotic strain of Potato virus Y was determined in a rat experiment. Autoclaved tubers from four transgenic lines were included to a diet in the amount of 40% and compared with the conventional cv. Irga. The experiment lasted 3 weeks and special attention was paid to nutritional properties of diets, caecal metabolism and serum indices. Genetic modification of potato had no negative effect on the chemical composition and nutritional properties of tubers, ecosystem of the caecum, activity of serum enzymes and non-specific defence mechanism of the rats. Obtained results indicate that transgenic potato with improved resistance to PVY(N): line R1F (truncated gene coding for PVY(N) polymerase in sense orientation), R2P (truncated gene coding for PVY(N) polymerase in antisense orientation), and NTR1.16 (non-translated regions of PVY(N) genome in sense orientation) are substantial and nutritional equivalence to the non-transgenic cultivar. Tubers of transgenic line NTR2.27 (non-translated regions of PVY(N) genome in antisense orientation) increased the bulk of caecal digesta and the production of SCFA as compared to tubers of the conventional cultivar and the other transgenic clones. Taking into account some deviations, it seems reasonable to undertake a long-term feeding study to confirm the nutritional properties of tubers of transgenic lines.

Animals↗

Analysis of the amplification and transcription of the C3-22 gene of Rhynchosciara americana (Diptera: Sciaridae) in transgenic lines of Drosophila melanogaster.

Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.

Animals↗

A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination.

We describe here a mouse line bearing a bovine keratin K5Cre recombinase transgene. These mice showed a dual pattern of Cre-mediated recombination, depending on the parent transmitting the transgene. In paternal transmission, recombination occurred specifically in the skin and stratified epithelia-as expected according to the expression of endogenous keratin K5. However, constitutive recombination between loxP sites transmitted by the sperm took place when the mother possessed the K5Cre transgene, even when the transgene was absent in the progeny. Cre expression in late-stage oocytes, with the Cre protein persisting into the developing embryo, leads to the constitutive recombination observed. Thus, this transgenic line allows for both tissue-specific and generalized recombination, depending on the breeding scheme.

Animals↗

A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines.

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, we have isolated and characterized the pax2.1-promoter using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5' region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1-expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, which do not normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires eng2 and eng3 as known targets for Pax2.1 regulation. We suggest that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo.

Animals↗

A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ.

A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.

Animals↗

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

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

Brassica↗

Tomato transgenic lines and Tetranychus urticae: changes in plant suitability and susceptibility.

A critical aspect dealing with the use of transgenic plants is the global evaluation of their environmental impact. The polyphagous mite Tetranychus urticae can be considered a suitable species to investigate unpredictable and undesirable effects on phytophagous arthropods. Three tomato near isogenic lines, that is, the cv. Riogrande (RIG), the transgenic lines RC332 (containing the Gox gene and showing high glucose oxidase activity), and MS498 (containing the KTI3 gene and exhibiting a high trypsin inhibition) were used in laboratory and greenhouse trials. Trichomes and contents of C and N of the leaves, differences in development and oviposition of T. urticae and damage caused were evaluated for each line. The laboratory trials evidenced that (1) the intrinsic rate of increase of two strains of T. urticae (T from tomato, B from bindweed), reared on the lower surface of tomato leaflets, was significantly lower in RIG than in transgenic lines and doubling time ranged between 6.9 and 11.6 days in the first and between 3.9 and 5.3 days in the latter; (2) the glandular four-lobed trichomes were always higher in RIG than in other genotypes; (3) the N leaf content was from 1.3 to 1.9 fold lower and the C/N ratio from 1.3 to 1.9 fold higher in RIG than in other lines. The greenhouse experiment, that lasted over a month and was performed by inducing an initially equal infestation of strain T, evidenced: (1) no significant difference between plant lines in the final mite infestation (motile stages per plant), nevertheless an almost double number of spider mites was counted in RC332; (2) a significantly higher percentage of damaged leaves and a significant higher average damage index on RC332 than on RIG (79% and 2.3 in the former, and 62% and 2.1 in the latter, respectively), even if in both transgenics a higher level of the most severe damages and a shorter time to approach them were observed; (4) a comparable number of mites causing the same damage level in all genotypes and a strong linear relation between the first four levels of damage and mite infestation. Although in the laboratory studies both transgenic lines enhanced the T. urticae population increase, the glasshouse studies were not as conclusive and they only suggest the possibility of any real difference between the transgenic and non-transgenic genotypes.

Animals↗

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

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

Animals↗

[Obtaining stem borer-resistant homozygous transgenic lines of Minghui 81 harboring novel cry1Ac gene via particle bombardment].

A modified cry1Ac gene was generated by fusing with Lys-Asp-Glu-Lue (KDEL), an endoplasmic reticulum retention signal at the 3'-ends, with signal peptide coding sequence of Soybean kunitz trypsin inhibitor (SKTI) at the 5'-ends. Vector containing the modified cry1Ac gene coding region flanked by the corn ubiquitin 1 promoter and the nopaline synthase gene (nos) terminator with Hygromycin Phosphotransferase (hpt) gene as a plant selection marker was constructed. The modified cry1Ac gene in which toxic protein targeted to endoplasmic retention was successfully transferred into Minghui 81 (Oryza sativa L. subsp. indica), an elite restoring line of commercial CMS indica hybrid rice, through particle bombardment and obtained fertile transformants. Homozygous transgenic rice lines were obtained in the third generation exploiting self-seed set reproduction and HygromycinB selection. These transgenic lines were confirmed with polymerase chain reaction (PCR) amplification, Southern blotting and ELISA detection. Pest insect-resistant bioassay indicated that some of the homozygous cry1Ac-transgenic rice plants of T2 progeny showed high-level resistance against striped stem borer (Chilo suppressalis) at field trials.

Animals↗

Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Adipocytes↗

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate.

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange-red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3' region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20-25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Animals↗

A novel transgenic line of mice exhibiting autosomal recessive male-specific lethality and non-alcoholic fatty liver disease.

We have isolated a Meis1a transgenic mouse line exhibiting recessive male-specific lethality and non-alcoholic fatty liver disease (NAFLD), which coincides with pubescence and is androgen-dependent. The phenotype is due to disruption of an endogenous locus, since other Meis1a transgenic lines do not exhibit these phenotypes. Necropsy analysis revealed hepatic microvesicular steatosis in pubescent male homozygous mice, which is absent in transgenic females. The transgene insertion site was localized to chromosome 1 and further refined by cloning the flanking regions. Sequence analysis shows that the integration site disrupts a putative metallo-beta-lactamase gene with a 21.3 kb deletion encompassing exons 5-7.

Amino Acid Sequence↗

Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection.

The present report provides the first extensive characterization of the OT-I TCR transgenic line, which produces MHC class I-restricted, ovalbumin-specific, CD8+ T cells (OT-I cells). These cells are shown to be positively selected in vivo in H-2b C57BL/6 mice and in bm5 mice, which express the Kbm5 mutant molecule. In contrast, OT-I cells were not selected by mutant Kb molecules in bm1, bm3, bm8, bm10, bm11 or bm23 mice. Interestingly, however, when positive selection was examined in vitro in foetal thymic organ culture (FTOC), bm1 and bm8 were still poorly selective, but the bm3 haplotype now selected as efficiently as B6. The ability to select in vitro correlated with the capacity to present the ovalbumin (OVA) peptide to OT-I cells, as measured by induction of an OVA-specific proliferative response. These results suggest that a lower affinity TCR:MHC interaction may be necessary for positive selection in FTOC compared with selection in situ.

Animals↗

Human insulin gene in transgenic mouse lines.

Transgenic mouse lines carrying the human insulin gene flanked by 4 kilobases (kb) in 5' and 5.5 kb in 3' were obtained. The presence of the human C-peptide in serum and urine, and of specific transcripts of the human gene in pancreas RNA indicate that the human DNA fragment contains the sequences necessary for correct phenotypic expression of the gene.

Animals↗

Competence of oat ( Avena sativa L.) shoot apical meristems for integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing hva1.

Three oat ( Avena sativa L.) cultivars have been successfully transformed using an efficient and reproducible in vitro culture system for differentiation of multiple shoots from shoot apical meristems. The transformation was performed using microprojectile bombardment with two plasmids (pBY520 and pAct1-D) containing linked ( hva1-bar) and non-linked ( gus) genes. The hva1 and bar genes cointegrated with a frequency of 100% as expected, and 61.6% of the transgenic plants carried all three genes. Molecular and biochemical analyses in R0, R1 and R2 progenies confirmed stable integration and expression of all transgenes. Localization of the GUS protein in R0 and R1 plants revealed that high-expression of gus occurred in vascular tissues and in the pollen grains of mature flowers. The constitutive expression of HVA1 protein was observed at all developmental stages of transgenic plants, and was particularly stronger during the early seedling stages. R2 progeny of five independent transgenic lines was tested in vitro for tolerance to osmotic (salt and mannitol) stresses. As compared to non-transgenic control plants, transgenic plants maintained a higher growth and showed significantly ( P < 0.05) increased tolerance to stress conditions. Less than 10% of transgenic plants showed symptoms of wilting or death of leaves and, when these symptoms present were delayed in transgenic plants as compared to 80% of non-transgenic plants, either wilted or died. These symptoms confirmed the increased in vitro tolerance in hva1-expressing transgenic plants to non-transgenic plants, providing strong evidence that the HVA1 protein may play an important role in the protection of oats against salinity and possible water-deficiency stress conditions.

Journal Article↗

Spatiotemporal expression of zebrafish keratin 18 during early embryogenesis and the establishment of a keratin 18:RFP transgenic line.

Zebrafish cytokeratin 18 (K18) is one of the type I keratin genes expressed the earliest after amputation of the zebrafish fin, but its spatiotemporal expression during early development is unclear. Whole-mount in situ hybridization revealed that k18 was a maternally inherited gene and that its expression is restricted to the single layer of enveloping cells on the surface of embryos during the gastrula stage. At later stages, K18 expression was detected in the epithelial cells, pronephric duct, digestive tract, dorsal aorta, and fins. By using microinjection, we generated the transgenic line Tg(k18(2.9):RFP), which carries an upstream 2.9-kb segment of k18 gene fused with a red fluorescent protein (RFP) reporter. The spatiotemporal distributions of red fluorescent signal of Tg(k18(2.9):RFP) line correlated well with endogenous k18 transcripts detected by whole-mount in situ hybridization, indicating that this line is capable of recapitulating endogenous k18 expression patterns. We noticed that the red fluorescence appeared strongly in the dorsal, pectoral, pelvic, anal, and caudal fins when transgenic fish became adults. Interestingly, we also found that when F1 female from the Tg(k18(2.9):RFP) line were mated with wild-type males, 100% (326/326) of F2 offspring expressed red fluorescence at the one-cell stage. In contrast, when F1 male from the Tg(k18(2.9):RFP) line were mated with wild-type females, only 49.8% (138/277) of F2 embryos exhibited red fluorescence. On the basis of these findings, we suggest that the transcript of zebrafish K18 is inherited as a maternal effect. We believe that Tg(k18(2.9):RFP) fish should be an excellent experimental animal for studying the zygotic regulatory mechanism of k18.

Animals↗

Neurocristopathy resembling neurofibromatosis type 1 in an NGF-SV40 transgenic line.

BACKGROUND: Animal models of carcinogenesis have been produced in transgenic mice by directing the expression of oncogenes such as SV40 T antigen and myc to different tissues by creating fusions with promoter/enhancer elements of various mammalian or viral genes. RESULTS: A transgenic mouse line was created in which SV40 T antigen is under the control of the mouse nerve growth factor (NGF) promoter. While the oncogene is expressed in a wide range of NGF producing tissues, it specifically causes the development of either neurofibromas or neurofibrosarcomas similar to those found in the human disease neurofibromatosis type 1 (NF1). These tumours are completely penetrant and appear after a mean latency of about 8 months. In contrast to the previously reported neurofibromatosis mouse model HTLV-1 tax, the tumours in these transgenic mice arise in Schwann cells rather than perineural fibroblasts and have a very restricted tissue distribution. In a cell line cloned from a neurofibroma from these mice, NGF was detected in the culture medium at levels similar to those produced by cultured primary Schwann cells. CONCLUSION: As all animal model for a heritable neurocristopathy resembling NF1, this mouse should allow study of the pathology and treatment of this disease.

Animals↗