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At least 559 records · Page 31Linked to original sources
PubMed2000

Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene.

The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L. x Populus tremuloides Michx.). We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes. The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA. In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression. Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA. Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants. This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth.

Blotting, Northern↗
PubMed2006

An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants.

Proteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After confirming the importance of trypsin- and chymotrypsin-like activities in crude protein extracts of potato (Solanum tuberosum L.) leaves, transgenic lines of potato expressing either tomato cathepsin D inhibitor (CDI) or bovine aprotinin, both active against trypsin and chymotrypsin, were generated by Agrobacterium tumefaciens-mediated genetic transformation. Leaf crude protein extracts from CDI-expressing lines, showing decreased levels of cathepsin D-like and ribulose 1,5-bisphosphate carboxylase/oxygenase hydrolysing activities in vitro, conducted decreased turnover rates of the selection marker protein neomycin phosphotransferase II (NPTII) relative to the turnover rates measured for transgenic lines expressing only the marker protein. A similar stabilizing effect on NPTII was observed in leaf protein extracts from plant lines coexpressing bovine aprotinin, confirming the ability of ectopically expressed broad-spectrum serine proteinase inhibitors to reproduce the protein-stabilizing effect of low-molecular-weight proteinase inhibitors generally added to protein extraction media.

Agrobacterium tumefaciens↗
PubMed2006

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system.

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.

Animals↗
PubMed2002

Comparison of a new pmp22 transgenic mouse line with other mouse models and human patients with CMT1A.

Charcot-Marie-Tooth disease type 1A is a dominantly inherited demyelinating disorder of the peripheral nervous system. It is most frequently caused by overexpression of peripheral myelin protein 22 (PMP22), but is also caused by point mutations in the PMP22 gene. We describe a new transgenic mouse model (My41) carrying the mouse, rather than the human, pmp22 gene. The My41 strain has a severe phenotype consisting of unstable gait and weakness of the hind limbs that becomes obvious during the first 3 weeks of life. My41 mice have a shortened life span and breed poorly. Pathologically, My41 mice have a demyelinating peripheral neuropathy in which 75% of axons do not have a measurable amount of myelin. We compare the peripheral nerve pathology seen in My41 mice, which carry the mouse pmp22 gene, with previously described transgenic mice over-expressing the human PMP22 protein and Trembler-J (TrJ) mice which have a P16L substitution. We also look at the differences between CMT1A duplication patients, patients with the P16L mutation and their appropriate mouse models.

Animals↗
PubMed2000

A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing.

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

Base Sequence↗
PubMed2005

DNA hypomethylation reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei of Arabidopsis thaliana.

Fluorescent chromatin tagging makes possible tracking of specific loci in vivo and in situ. Loci tagged by the lac operator (lacO)/GFP-LacI/Nuclear Localization Signal (NLS) system show rapid motility and constrained chromatin dynamics in somatic nuclei of a transgenic line, designated EL702C, in Arabidopsis thaliana. The tagged loci associated with each other significantly more often than expected at random, due to homologous pairing of the lacO tandem repeat arrays. Furthermore, these arrays associated significantly more often than average euchromatic regions with heterochromatic chromocenters (CCs). We show now that the inserted lacO array in this transgenic line became strongly methylated at CG sites in the T3 generation, which can be reversed upon transfer into the mutant backgrounds of decrease in DNA methylation 1 (ddm1) and methyltransferase 1 (met1). Concomitantly, the tagged loci showed lower association frequencies as compared with the transgenics in wild-type background, which is correlated with a significant decrease in allelic and ectopic pairing of the lacO repeat arrays as visualized by fluorescence in situ hybridization. In contrast, the preferential association of the lacO arrays with heterochromatin, locus mobility in somatic nuclei and transcription of neighboring transgenes were not altered by reduced DNA methylation in ddm1 and met1 backgrounds. Our results show that repeat arrays can activate hypermethylation of the inserted locus that correlates with high frequencies of homologous pairing in somatic cells. In contrast, the preferential association of these inserted arrays with CCs in plant cells occurs through another mechanism.

Arabidopsis↗
PubMed2001

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities.

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.

Animals↗
PubMed2000

A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing.

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R(2) plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R(1) plants were resistant and resistance was not observed in R(2) plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

Artificial Gene Fusion↗
PubMed2000

Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis.

Urokinase-type plasminogen activator (uPA) is a potent catalyst of extracellular proteolysis, which also binds to a high-affinity plasma membrane receptor (uPAR). Binding of uPA may influence pericellular proteolysis and/or activate intracellular signal transduction. Transgenic mice overexpressing either uPA or uPAR in basal epidermis and hair follicles had no detectable cutaneous alterations. In contrast, bi-transgenic mice overexpressing both uPA and uPAR, obtained by crossing the two transgenic lines, developed extensive alopecia induced by involution of hair follicles, epidermal thickening and sub-epidermal blisters. The phenotype was due to uPA catalytic activity since combined overexpression of uPAR and uPAR-binding but catalytically inactive uPA in the same tissue was not detrimental in another bi-transgenic line. It was accompanied by increased plasmin-generating capacity, up-regulation and activation of matrix metalloproteinases type-2 and -9, and cleavage of uPAR. Thus, combined overexpression of uPA and uPAR acts in synergy to promote pathogenic extracellular proteolysis.

Animals↗
PubMed2007

Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

Alcohol Oxidoreductases↗
PubMed2004

Targeted mutagenesis of the Sap47 gene of Drosophila: flies lacking the synapse associated protein of 47 kDa are viable and fertile.

BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila. RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels. CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila.

Alternative Splicing↗
PubMed2000

The role of homodimers in surfactant protein B function in vivo.

Surfactant protein B (SP-B) is detected in the airways as a sulfhydryl-dependent dimer (M(r) approximately 16,000). To test the hypothesis that formation of homodimers is critical for SP-B function, the cysteine residue reported to be involved in SP-B dimerization was mutated to serine (Cys(248) --> Ser) and the mutated protein was targeted to the distal respiratory epithelium of transgenic mice. Transgenic lines which demonstrated appropriate processing, sorting, and secretion of human SP-B monomer were crossed with SP-B +/- mice to achieve expression of human monomer in the absence of endogenous SP-B dimer (hSP-B(mon), mSP-B-/-). In two of three transgenic lines, hSP-B(mon), mSP-B-/- mice had normal lung structure, complete processing of SP-C proprotein, well formed lamellar bodies, and normal longevity. Pulmonary function studies revealed an altered hysteresis curve for hSP-B(mon), mSP-B-/- mice relative to wild type mice. Large aggregate surfactant fractions from hSP-B(mon), mSP-B-/- mice resulted in higher minimum surface tension in vitro compared with surfactant from wild type mice. Surfactant lipids supplemented with 2% hSP-B monomer resulted in slower adsorption and higher surface tension than surfactant with 2% hSP-B dimer. Taken together, these data indicate a role for SP-B dimer in surface tension reduction in the alveolus.

Animals↗
PubMed2005

Production of CETD transgenic mouse line allowing ablation of any type of specific cell population.

Diphtheria toxin A-chain (DT-A) is a potent inhibitor of protein synthesis. As little as a single molecule of DT-A can result in cell death. DT-A gene driven by a tissue-specific promoter is used to achieve genetic ablation of a particular cell lineage. However, this transgenic approach often results in aberrant depletion of unrelated cells. To avoid this, we established a method for specific depletion of a cell population by controlled expression of the DT-A gene via the Cre-loxP system. We produced five transgenic mice carrying CETD construct containing loxP-flanked enhanced green fluorescent protein (EGFP) cDNA and the DT-A gene. Transfection of primary cultured cells derived from CETD transgenic fetus with Cre expression plasmid resulted in extensive cell loss, as expected. Bigenic (double transgenic) offspring obtained by crossbreeding between CETD and MNCE transgenic mice in which Cre expression is controlled by the myelin basic protein (MBP) promoter exhibited embryonic lethality, suggesting expression of Cre at embryonic stages. Intravenous injection of Cre expression vector to CETD mice led to generation of glomerular lesions, probably due to predominant depletion of glomerular epithelial cells. This Cre-loxP-based cell ablation technology is powerful and convenient method of generating mice lacking any chosen cell population.

Animals↗
PubMed2000

Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice.

The short persistence of cecropin B peptide in plants, due to post-translational degradation, is a serious impediment in its effective utilization for developing bacterial resistance transgenic plants. Two DNA constructs encoding the full-length precursor of cecropin B peptide and the mature sequence of cecropin B peptide preceded by a signal peptide derived from rice chitinase gene were transformed in rice. The differences in the transcriptional levels in independent transgenic lines showed moderate to high expression of cecropin B gene that correlated well with the differences in cecropin B accumulation observed by Western blot analysis. The development of lesions resulting from infection by Xanthomonas oryzae pv. oryzae was significantly confined in the infected leaflet of transgenic lines, when compared with the control plants.

Animals↗
PubMed2000

Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes.

The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA. Four lines of the transgenic plants were self-crossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization. Two lines, D1232 and D1653, showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leafy head. Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage, and the cuttings from head leaves had more roots than rosette leaves at heading stage. It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin. These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant organs and for the breeding of new variety with rapid growth trait.

Brassica↗
PubMed2026

Mammalian growth factors enhance regeneration in transgenic tomato lines.

Genome editing technologies are now available for many crop species, greatly enhancing our ability to investigate gene function and transforming the field of plant transgenesis. However, the capacity to regenerate whole plants from cell culture remains a major limiting factor in many crops. Even in species with regeneration potential, certain genotypes remain recalcitrant. The physiological state of plant cells plays a central role in growth and development and is closely associated with kinase-mediated signaling networks. Notably, several defense-related genes activated during cellular repair processes following transgenesis share significant homology with mammalian defense genes. In this study, we evaluated whether supplementation with three mammalian growth factors could enhance regeneration efficiency in tomato. We selected two cytokines and a pro-inflamatory factor showing homology with plant kinase genes. We compared the percentage of transgenic plants generated through CRISPR-Cas9-mediated mutagenesis of four genes involved in sugar and organic acid metabolism across six tomato lines exhibiting varying regeneration capacities. Over three years of transformation experiments, we demonstrated that the addition of mammalian growth factors during transgenesis significantly improved regeneration frequency, particularly in recalcitrant tomato genotypes. Furthermore, growth factor supplementation not only enhanced transformation efficiency in difficult-to-transform lines but also increased the production of stable secondary lines.

Solanum lycopersicum↗
PubMed2006

Cloning of two individual cDNAS encoding 9-cis-epoxycarotenoid dioxygenase from Gentiana lutea, their tissue-specific expression and physiological effect in transgenic tobacco.

Two 9-cis-epoxycarotenoid dioxygenase (NCED) cDNAs have been cloned from a petal library of Gentiana lutea. Both cDNAs carry a putative transit sequence for chloroplast import and differ mainly in their length and the 5'-flanking regions. GlNCED1 was evolutionary closely related to Arabidopsis thaliana NCED6 whereas GlNCED2 showed highest homology to tomato NCED1 and A. thaliana NCED3. The amounts of GlNCED2 transcript were below Northern detection in G. lutea. In contrast, GlNCED1 was specifically expressed at higher levels in developing flowers when petals start appearing. By genetic engineering of tobacco with coding regions of either gene under a constitutive promoter, their function was further analyzed. Although mRNA of both genes was detectable in the corresponding transgenic plants, a physiological effect was only found for GlNCED1 but not for GlNCED2. In germination experiments of GlNCED1 transgenic lines, delayed radicle formation and cotyledon appearance were observed. However, the transformants exhibited no improved tolerance against desiccation stress. In contrast to other plants with over-expressed NCEDs, prolonged delay of seed germination is the only abscisic-acid-related phenotypic effect in the GlNCED1 transgenic lines.

Adaptation, Physiological↗
PubMed2002

Overexpression of the wheat FK506-binding protein 73 (FKBP73) and the heat-induced wheat FKBP77 in transgenic wheat reveals different functions of the two isoforms.

The FK506-binding proteins (FKBPs) belong to the peptidyl prolyl cis-trans isomerase (PPIase) family, and catalyse the rotation of the peptide bond preceding a proline. They are conserved in organisms from bacteria to man. In order to understand the function of plant FKBP isoforms, we have produced transgenic wheat plants overexpressing each of the two wheat FKBPs: wFKBP73 (which is expressed in young vegetative and reproductive tissues under normal growth conditions) and wFKBP77 (which is induced by heat stress). Transgenic lines overexpressing wFKBP77 at 25 degrees C showed major morphological abnormalities, specifically relating to height, leaf shape, spike morphology and sterility. In these plants, the levels of hsp90 mRNA were over two fold higher than in controls, indicating a common regulatory pathway shared between wFKBP77 and Hsp90. Transgenic lines overexpressing wFKBP73 showed normal vegetative morphology, but the grain weight and composition was altered, corresponding to changes in amylase activity during seed development.

Conserved Sequence↗