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At least 937 records · Page 52Linked to original sources
PubMed2004

Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to Paraquat-induced photooxidative stress and to nitric oxide-induced cell death.

Ascorbate peroxidases (APX), localized in the cytosol, peroxisomes, mitochondria and chloroplasts of plant cells, catalyze the reduction of H(2)O(2) to water by using ascorbic acid (ASA) as specific electron donor. The chloroplastic isoenzymes of APX are involved in the water-water cycle, which contributes to the photophosphorylation coupled to the photosynthetic electron transport. In order to better clarify the contribution of thylakoidal APX (tAPX) to the reactive oxygen species (ROS) scavenging activity, as well as to the fine modulation of ROS for signaling, we produced Arabidopsis lines overexpressing tAPX. These lines show an increased resistance to treatment with the O(2)(-) generating herbicide Paraquat (Pq). However, when challenged with photoinhibitory treatments at high light or low temperature, or with iron (Fe) or copper (Cu) overload, the tAPX-overexpressing lines show no increased resistance with respect to controls, indicating that in such experimental conditions, tAPX overexpression does not reinforce plant defenses against the oxidative stresses tested. Interestingly, the nitric oxide (NO)-donor sodium nitroprusside (SNP) represses accumulation of tAPX transcript; SNP also partially inhibits tAPX enzymatic activity. After treatment with SNP, the tAPX-overexpressing lines show reduced symptoms of damage with respect to control plants treated with SNP. These transgenic lines confirm that H(2)O(2) acts in partnership with NO in causing cell death and highlight the important role of tAPX in the fine modulation of H(2)O(2) for signaling.

Arabidopsis↗
PubMed2006

Chronic lithium administration to FTDP-17 tau and GSK-3beta overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert.

Glycogen synthase kinase-3 (GSK-3) has been proposed as the main kinase able to aberrantly phosphorylate tau in Alzheimer's disease (AD) and related tauopathies, raising the possibility of designing novel therapeutic interventions for AD based on GSK-3 inhibition. Lithium, a widely used drug for affective disorders, inhibits GSK-3 at therapeutically relevant concentrations. Therefore, it was of great interest to test the possible protective effects of lithium in an AD animal model based on GSK-3 overexpression. We had previously generated a double transgenic model, overexpressing GSK-3beta in a conditional manner, using the Tet-off system and tau protein carrying a triple FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation. This transgenic line shows tau hyperphosphorylation in hippocampal neurones accompanied by neurofibrillary tangles (NFTs). We used this transgenic model to address two issues: first, whether chronic lithium treatment is able to prevent the formation of aberrant tau aggregates that result from the overexpression of FTDP-17 tau and GSK-3beta; second, whether lithium is able to change back already formed NFTs in aged animals. Our data suggest that progression of the tauopathy can be prevented by administration of lithium when the first signs of neuropathology appear. Furthermore, it is still possible to partially reverse tau pathology in advanced stages of the disease, although NFT-like structures cannot be changed. The same results were obtained after shut-down of GSK-3beta overexpression, supporting the possibility that GSK-3 inhibition is not sufficient to reverse NFT-like aggregates.

Age Factors↗
PubMed1994

Recombination between viral RNA and transgenic plant transcripts.

Transformed plants expressing the 3' two-thirds of the cowpea chlorotic mottle virus (CCMV) capsid gene were inoculated with a CCMV deletion mutant lacking the 3' one-third of the capsid gene. Although the deletion inoculum replicates in inoculated cells, systemic infections occur only if recombination restores a functional capsid gene. Four of 125 inoculated transgenic plants, representing three different transgenic lines, became systemically infected. Analysis of viral RNA confirmed that RNA recombination had united the transgenic messenger RNA and the challenging virus through aberrant homologous recombination.

Amino Acid Sequence↗
PubMed2005

Chitinase gene transformation through Agrobacteriumand its explanation in soybean in order to induce resistance to root rot caused by Rhizoctonia solani.

Chitinase gene (chi) of bean which has been cloned in recombinant binary plasmid vector, pBI121 with 35s promoter of Cauliflower mosaic virus (CaMV), were used for transformation of soybean using strain LBA4404 of Agrobacterium. The plasmid contained nptII gene that is a resistant gene to kanomycin as selector marker and Gus gene as reporter. Cotyledon explants of Williams and Clark cultivars were inoculated by Agrobacterium suspension with pBI121 and were cultured in regeneration medium. After complete regeneration of explants to seedling in B5 medium amended with kanomycin, polymerase chain reaction analysis were conducted to ensure conjugation of nptII, Gus, CHN genes in transformants seedling of soybean. Results showed that some lines of soybean contained Gus and CHN genes. More ever, chitinase activity in leaf extract of transformed soybean lines was significantly more than untransformed soybean, exception one sample. Bioassay of chitinase activity of transgenic lines on in vitro condition prevented mycelial growth of Rhizoctonia solani in comparison with untransformed control leaf extract.

Chitinases↗
PubMed1996

Effects of hyperthyroidism on expression of a phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice.

Thyroid hormone (T3) responsiveness of the PEPCK promoter in vivo was examined in both PEPCK/bGH(460) and PEPCK/bGH(335) mouse lines. Transgenic and non-transgenic littermates were treated with methimazole or PTU for 6 or 4 weeks, respectively, then treated +/- T3 for 10 days. In PEPCK/bGH(460) and PEPCK/bGH(355) transgenic mice, the bGH mRNA was decreased by 65% and 46%, respectively, in hyperthyroid mice when compared to euthyroid controls. Endogenous PEPCK mRNA was decreased by 33% in hyperthyroid non-transgenic mice. The conclusion of this study is that chronic hyperthyroidism in mice inhibits PEPCK-directed expression of the transgene when either the -460/+73 or the -355/+73 promoter/regulatory elements are used.

Animals↗
PubMed2005

Endothelial cell-specific expression of Cre recombinase in transgenic mice.

Endothelial cells participate in angiogenesis, vascular homeostasis, thrombosis, inflammation and vascular wall remodeling. To study the function of genes in endothelial cells using Cre-loxP system, we generated Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by Tie2 promoter. Total six founder mice carrying the Tie2-Cre transgene were identified by genomic PCR and Southern blot. The integration efficiency is 11%. In order to test the excision activity and tissue distribution of the Cre recombinase, the Tie2-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4(Co/Co)) or the reporter line ROSA26. PCR of multiple tissue DNA from Tie2-Cre; Smad4(Co/+) mice revealed the Cre activity in all tissues containing endothelial cells. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre; ROSA26 double transgenic embryos by lacZ staining. Therefore, this mouse line may serve as a valuable tool for endothelial cell lineage analyses and conditional gene ablation in endothelial cells.

Animals↗
PubMed2003

Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior.

The neuropeptide galanin is expressed in the dorsal root ganglia (DRG) and spinal cord and is thought to be involved in the modulation of pain processing. However, its mechanisms of action are complex and poorly understood, as both facilitatory and inhibitory effects have been described. To understand further the role played by galanin in nociception, we have generated two transgenic lines that overexpress galanin in specific populations of primary afferent DRG neurons in either an inducible or constitutive manner. In the first line, a previously defined enhancer region from the galanin locus was used to target galanin to the DRG (Gal-OE). Transgene expression recapitulates the spatial endogenous galanin distribution pattern in DRG neurons and markedly overexpresses the peptide in the DRG after nerve injury but not in the uninjured state. In the second line, an enhancer region of the c-Ret gene was used to constitutively and ectopically target galanin overexpression to the DRG (Ret-OE). The expression of this second transgene does not alter significantly after nerve injury. Here, we report that intact Ret-OE, but not Gal-OE, animals have significantly elevated mechanical and thermal thresholds. After nerve damage, using a spared nerve-injury model, mechanical allodynia is attenuated markedly in both the Gal-OE and Ret-OE mice compared with WT controls. These results support an inhibitory role for galanin in the modulation of nociception both in intact animals and in neuropathic pain states.

Animals↗
PubMed2000

Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase.

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.

Arabidopsis↗
PubMed1993

Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives.

Transgenes in mice often exhibit different expression patterns in different transgenic lines. While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression. Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites. In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of a lacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome. Expression of lacZ was assayed by histochemical staining for Escherichia coli beta-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation. Several of the randomly integrated cell lines expressed lacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells. Targeted cell lines with lacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines. Targeted cell lines transcribing lacZ in the same orientation as HPRT transcription did not express high levels of lacZ in any differentiated cell type. Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter.

Animals↗
PubMed2002

Instability of CGG repeats in transgenic mice.

Dynamic mutation resulting in the expansion of CGG repeats in the untranslated region (UTR) of the first exon of the FMR1 gene in humans results in fragile X syndrome. Long stretches of CGG repeats that are known to be highly unstable in humans have so far failed to show similar intergenerational instability in transgenic mice. We generated transgenic lines that show a dramatic increase from 26 to >300 repeats in three generations. One of the salient features of our transgene is the inclusion of the origin of replication of simian virus-40 (SV40), which is known to exclude nucleosomes. Three founder mice in FVB/NJ background show expansion of CGG repeats present in the transgene, supporting a postzygotic mechanism for CGG expansion that is independent of a genomic imprinting effect. We discuss here the results of analyzing one of the lines established.

3' Flanking Region↗
PubMed1994

Mosaic expression of an Hprt transgene integrated in a region of Y heterochromatin.

The sensitivity of small transgenes to position effects on their expression suggests that they could serve as indicators of the chromatin properties at their integration site. In particular, they might be expected to provide information on the functional properties of mammalian heterochromatin. We have produced a transgenic line that carries a mouse Hprt minigene on the Y chromosome. In situ hybridization localized the transgene to the heterochromatic portion of the Y. Analysis of transgene expression by isoelectric focusing indicated that the transgene is expressed in a mosaic pattern, and expressing cells have different levels of transgene activity. These findings can be explained as a position effect variegation induced by Y heterochromatin. However, two other transgenes, located at autosomal sites, also showed mosaic activity. If the mosaic transgene expression is attributed to the influence of the chromatin at the insertion site, the Y heterochromatin would appear less potent than some autosomal regions at inducing variegation. An alternative explanation consistent with our results is that the mosaic expression is a semi-autonomous characteristic of these transgene loci. Transgene-expressing and non-expressing cells differed in their ability to grow and be cloned in vitro, indicating that cellular differentiation affected the chromatin structure of the transgene locus on the Y. Karyotype analysis of male mice with the Y-linked transgene and from control male mice carrying the human HPRT transgene, or the mouse Pgk-1 gene at autosomal sites, indicated that the transgene-carrying Y is prone to non-disjunction, generating cells with two (or more) or no Y chromosomes in equal proportion. Further studies will determine if the propensity of this Y chromosome to mitotic errors is also observed in vivo.

Animals↗
PubMed1997

Live astrocytes visualized by green fluorescent protein in transgenic mice.

Green fluorescent protein (hGFP-S65T) was expressed in transgenic mice under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Tissues from two independent transgenic lines were characterized by Northern blot analysis and by confocal microscopy. The expression pattern in these two lines was identical in all tissues examined, and similar to that found previously with a lacZ transgene driven by the same promoter. Bright fluorescence was observed in the cell bodies and processes of unfixed or fixed astrocytes, using both whole mount and brain slice preparations, from multiple areas of the central nervous system. However, in contrast to GFAP-lacZ transgenics, retinal Müller cells expressed the GFP transgene in response to degeneration of neighboring photoreceptors. These data indicate that the 2.2-kb hGFAP promoter contains sufficient regulatory elements to direct expression in Müller cells, and that GFP is a suitable reporter gene for use in living preparations of the mammalian nervous system. Such mice should prove useful for studies of dynamic changes in astrocyte morphology during development, and in response to physiological and pathological conditions.

Animals↗
PubMed1993

Tissue-specific expression of the rat androgen-binding protein/sex hormone-binding globulin gene in transgenic mice.

The testicular Sertoli cell produces an extracellular androgen-binding protein (ABP) that binds testosterone and dihydrotestosterone with high affinity. The ABP gene also encodes plasma sex hormone-binding globulin (SHBG), which is produced by the liver of most species. Unlike the human, adult rats and mice do not express SHBG. A 5.5-kb rat genomic DNA fragment was found to contain the entire coding regions of ABP and 1.5 kb upstream of the transcription start site. To aid in identification of the promoter and enhancer regions of the ABP/SHBG gene, we developed transgenic mice that express the rat gene. The 5.5-kb DNA was microinjected into the pronuclei of fertilized mice ova, which were transferred to the reproductive tract of pseudopregnant females. Three of the offspring were identified as carriers of the rat gene by Southern hybridization and these founders were bred with normal mice to establish heterozygous transgenic lines. Northern blot analysis, RNA-PCR and sequencing of the PCR products from the adult transgenic mice revealed extremely high levels of the rat ABP mRNA in the testis, but no detectable rat ABP mRNA in liver, kidney or brain. Primer extension experiments showed that the correct transcript ion start site is utilized in the transgenes. These data demonstrate that the 5.5-kb genomic DNA fragment contains an element(s) capable of directing ABP gene expression in the testis. This enhancer should prove useful for the targeting of specific gene products to the mature Sertoli cell in transgenic animals.

Androgen-Binding Protein↗
PubMed2000

Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.

We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed XFPs) were selectively expressed in neurons. All four XFPs labeled neurons in their entirety, including axons, nerve terminals, dendrites, and dendritic spines. Remarkably, each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene). For example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglion cells or only layer 5 cortical pyramids were labeled in others. In some lines, intense labeling of small neuronal subsets provided a Golgi-like vital stain. In double transgenic mice expressing two different XFPs, it was possible to differentially label 3 neuronal subsets in a single animal.

Animals↗
PubMed1998

The human pH aldolase A promoter directs widespread but muscle-predominant expression in transgenic mice.

In order to identify regulatory elements that direct widespread in vivo expression of a linked gene, we have examined one of the human aldolase A alternative promoters, the ubiquitous pH promoter, which is active in most foetal and adult tissues. We have used the pH promoter region to drive expression of an heterologous CAT reporter gene in transgenic mice. We show that a short 820 bp pH promoter fragment is able to confer a ubiquitous and reproducible activity pattern on the CAT reporter gene in most of the transgenic lines analysed, with a particularly high level of expression in adult skeletal muscle. Activity of this transgene was detected from early embryonic stages. Therefore, this pH promoter region appears to be a powerful tool to direct ubiquitous and early expression of a transgene in vivo. Deletion analysis revealed that: (i) the region between -651 and -369 bp relative to the pH promoter transcription start site includes DNA elements capable of overriding effects of the surrounding chromatin at the integration site, (ii) the region between -285 and -211 bp is involved in pH promoter tissue-specific expression pattern in skeletal muscle and/or nervous tissues, (iii) the region located between -211 and -108 bp is necessary for its ubiquitous and muscle-predominant activity and (iv) the most proximal region downstream from -108 bp is still sufficient to confer an activity in brain and lung.

Animals↗
PubMed2003

CHRK1, a chitinase-related receptor-like kinase, plays a role in plant development and cytokinin homeostasis in tobacco.

CHRK1 encodes a receptor-like kinase that contains a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. In this study, we showed that CHRK1 is mainly expressed in the shoot apex region including leaf primordia and young leaves, and germinating seedlings and vascular tissues, based on GUS activity of transgenic tobacco plants carrying the CHRK1 promoter-GUS fusion gene. Transgenic tobacco plants in which CHRK1 expression was suppressed exhibited pleiotrophic developmental abnormality, including formation of proliferating shooty calli from emerging seedlings and severely altered seedling development. At the cellular level, ectopic cell proliferation, reduced cell specificity, and aberrant chloroplast development were observed. The transgenic lines contained 3-fold higher level of cytokinin than the wild-type plants. Consistently, the transgenic seedlings exhibited a typical cytokinin response in the absence of hormone, such as deetiolation under the dark. Based on these results, we propose that CHRK1 is involved in a developmental signaling pathway regulating cell proliferation/differentiation and the endogenous cytokinin levels in tobacco.

Cell Division↗
PubMed2000

The Mom1AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64.

The Mom1 (Modifier of Min-1) region of distal chromosome 4 was identified during a screen for polymorphic modifiers of intestinal tumorigenesis in ApcMin/+ mice. Here, we demonstrate that the Mom1AKR allele consists of two genetic components. These include the secretory phospholipase Pla2g2a, whose candidacy as a Mom1 resistance modifier has now been tested with several transgenic lines. A second region, distal to Pla2g2a, has also been identified using fine structure recombinants. Pla2g2aAKR transgenic mice demonstrate a modest resistance to tumorigenesis in the small intestine and a very robust resistance in the large intestine. Moreover, the tumor resistance in the colon of Pla2g2aAKR animals is dosage-dependent, a finding that is consistent with our observation that Pla2g2a is expressed in goblet cells. By contrast, mice carrying the distal Mom1 modifier demonstrate a modest tumor resistance that is confined to the small intestine. Thus, the phenotypes of these two modifier loci are complementary, both in their quantitative and regional effects. The additive effects and tight linkage of these modifiers may have been necessary for the initial identification of the Mom1 region.

Adenomatous Polyposis Coli↗
PubMed2004

Reduction of cell size induced by enod40 in Arabidopsis thaliana.

An extensive analysis of organ and cell size was performed in three different Arabidopsis lines transformed with the early nodulin gene enod40 under control of the CaMV35S promoter. All three transgenic lines presented a significant decrease in the mean size of both epidermal internode and leaf mesophyll cells. Flow cytometric and image analysis of enod40-transfected protoplasts prepared from wild-type Arabidopsis cell suspensions showed that transient expression of the gene resulted in reduced forward light scattering (a factor correlated with particle size) and cell size. The direct administration of ENOD40 peptide to fresh protoplasts also resulted in reduced forward scattering with respect to the control and to the administration of unrelated peptides. As far as is known this is the first report documenting a biological effect of enod40 at the cellular level in non-legume plants.

Arabidopsis↗