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Transgenic Lines (transgenic + line)
Selected AbstractsEnhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of MinosDEVELOPMENTAL DYNAMICS, Issue 1 2008Yasunori Sasakura Abstract Germline transgenesis with a Tc1/mariner superfamily Minos transposon was achieved in the ascidian Ciona intestinalis. Transgenic lines that express transposases in germ cells are very useful for remobilizing transposon copies. In the present study, we created transposase-expressing lines of Minos in Ciona. A Ciona gene encoding protamine (Ci - prm) is expressed in the testes and sperm. Transgenic lines expressing Minos transposase in the testes and sperm were created with a cis -element of Ci - prm, and used for enhancer detection. Double-transgenic animals between transposase lines and a transgenic line with an enhancer detection vector passed on several independent enhancer detection events to subsequent progeny. This technique allowed us to isolate transgenic lines that express GFP in restricted tissues. This system provides an easy and efficient method for large-scale enhancer detection in Ciona intestinalis. Developmental Dynamics 237:39,50, 2008. © 2007 Wiley-Liss, Inc. [source] Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficientINSECT MOLECULAR BIOLOGY, Issue 4 2002O. P. Perera Abstract Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac -mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species. [source] Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimerasPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2006Juan Pablo Sánchez Summary The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465,1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1. [source] A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plantsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2009M. Amine Badri Abstract We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens -mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2,h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production. [source] Progressive neurogenesis defines lateralis somatotopyDEVELOPMENTAL DYNAMICS, Issue 7 2010Jesús Pujol-Martí Abstract Fishes and amphibians localize hydromechanical variations along their bodies using the lateral-line sensory system. This is possible because the spatial distribution of neuromasts is represented in the hindbrain by a somatotopic organization of the lateralis afferent neurons' central projections. The mechanisms that establish lateralis somatotopy are not known. Using BAPTI and neuronal tracing in the zebrafish, we demonstrate growth anisotropy of the posterior lateralis ganglion. We characterized a new transgenic line for in vivo imaging to show that although peripheral growth-cone structure adumbrates somatotopy, the order of neurogenesis represents a more accurate predictor of the position of a neuron's central axon along the somatotopic axis in the hindbrain. We conclude that progressive neurogenesis defines lateralis somatotopy. Developmental Dynamics 239:1919,1930, 2010. © 2010 Wiley-Liss, Inc. [source] Efficient transposition of a single Minos transposon copy in the genome of the ascidian Ciona intestinalis with a transgenic line expressing transposase in eggsDEVELOPMENTAL DYNAMICS, Issue 4 2010Akiko Hozumi Abstract Transgenesis with transposons is an important technique for studying genetic functions. In the ascidian Ciona intestinalis, methods for germline transformation with the Tc1/mariner transposon Minos have been established. A system to remobilize a single Minos copy in the genome is needed to refine this transgenic technique. In this study, such an experimental system was established with a transgenic line expressing Minos transposase in eggs. In the eggs of a double transgenic animal from a cross between the egg transposase line and a transgenic line having a single Minos insertion, the transposon was transposed into new positions of the Ciona genome, thus creating new insertions. Some of the new insertions caused enhancer detection. The majority of the new insertion sites were mapped on different chromosomes from that of the transposon donor. This characteristic of Minos is in contrast to that of the Sleeping Beauty transposon, which causes frequent intrachromosomal transposition. Developmental Dynamics 239:1076,1088, 2010. © 2010 Wiley-Liss, Inc. [source] Overexpression of the transcription factor Msx1 is insufficient to drive complete regeneration of refractory stage Xenopus laevis hindlimbsDEVELOPMENTAL DYNAMICS, Issue 6 2009Donna M. Barker Abstract Xenopus laevis tadpoles are capable of hindlimb regeneration, although this ability declines with age. Bmp signaling is one pathway known to be necessary for successful regeneration to occur. Using an inducible transgenic line containing an activated version of the Bmp target Msx1, we assessed the ability of this transcription factor to enhance regeneration in older limbs. Despite considerable evidence correlating msx1 expression with regenerative success in vertebrate regeneration models, we show that induction of msx1 during hindlimb regeneration fails to induce complete regeneration. However, we did observe some improvement in regenerative outcome, linked to morphological changes in the early wound epithelium and a corresponding increase in proliferation in the underlying distal mesenchyme, neither of which are maintained later. Additionally, we show that Msx1 is not able to rescue limb regeneration in a Bmp signalling-deficient background, indicating that additional Bmp targets are required for regeneration in anuran limbs. Developmental Dynamics 238:1366,1378, 2009. © 2009 Wiley-Liss, Inc. [source] Enhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of MinosDEVELOPMENTAL DYNAMICS, Issue 1 2008Yasunori Sasakura Abstract Germline transgenesis with a Tc1/mariner superfamily Minos transposon was achieved in the ascidian Ciona intestinalis. Transgenic lines that express transposases in germ cells are very useful for remobilizing transposon copies. In the present study, we created transposase-expressing lines of Minos in Ciona. A Ciona gene encoding protamine (Ci - prm) is expressed in the testes and sperm. Transgenic lines expressing Minos transposase in the testes and sperm were created with a cis -element of Ci - prm, and used for enhancer detection. Double-transgenic animals between transposase lines and a transgenic line with an enhancer detection vector passed on several independent enhancer detection events to subsequent progeny. This technique allowed us to isolate transgenic lines that express GFP in restricted tissues. This system provides an easy and efficient method for large-scale enhancer detection in Ciona intestinalis. Developmental Dynamics 237:39,50, 2008. © 2007 Wiley-Liss, Inc. [source] Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retinaDEVELOPMENTAL NEUROBIOLOGY, Issue 5 2008Ann C. Morris Abstract Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Disease-related epitope spread in a humanized T cell receptor transgenic model of multiple sclerosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2004Stephan Ellmerich Abstract While EAE has been an invaluable model for the immunopathogenesis of multiple sclerosis, it has sometimes been difficult to bridge the gap between findings and therapies in the rodent models and the cellular and molecular interactions that can be studied in the human disease. Humanized transgenic models offer a means of achieving this, through the expression of disease-implicated HLA class II molecules, co-expressed with a cognate HLA-class II-restricted, myelin-specific TCR derived from a human T cell clone implicated in disease. We have generated such a transgenic line, called line 8, that co-expresses a high level of HLA-DR15 and a human TCR specific for HLA-DR15/MBP 85,99. T cells from the transgenic line are skewed to the CD4 single-positive compartment and produce IFN-, in response to peptide from mylein basic protein. Mice develop a spontaneous disease phenotype, showing poverty of movement, although this rarely develops into paralysis except following immunization with peptide. On induction of paralysis by immunization with peptide, disease correlates with epitope spread to a number of additional, HLA-DR15-restricted myelin epitopes. This model should be valuable for analyzing epitope spread in a humanized immunogenetic environment and for the testing of specific immunotherapies. [source] Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eyeEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007Thorsten Belz Abstract During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness. [source] Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regenerationGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 8 2010April DeLaurier Adult transgenic zebrafish expressing eGFP under the control of the zinc finger transcription factor Sp7 gene, which is expressed in osteoblasts but not chondrocytes. In this line, eGFP expression recapitulates the endogenous gene pattern of expression in the otic placode, otic vesicle and developing skeletal structures. GFP-positive cells are also observed in adult skeletal structures and in regenerating fins. This transgenic line will be a very useful tool for studying otic development and the development and regeneration of the skeleton. See the article by DeLaurier et al. in this issue. [source] Construction and characterization of a doxycycline-inducible transgenic system in Msx2 expressing cellsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2009Congxing Lin Abstract Homeobox gene Msx2 is widely expressed during both embryogenesis and postnatal development and plays important roles during organogenesis. We developed an Msx2 -rtTA BAC transgenic line which can activate TetO-Cre expression in Msx2 -expressing cells upon doxycycline (Dox) treatment. Using the Rosa26-LacZ (R26R) reporter line, we show that rtTA is activated in Msx2 -expressing organs including the limb, heart, external genitalia, urogenital system, hair follicles and craniofacial regions. Moreover, we show that in body appendages, the transgene can be activated in different domains depending on the timing of Dox treatment. In addition, the transgene can also be effectively activated in adult tissues such as the hair follicle and the urogenital system. Taken together, this Msx2 -rtTA;TetO-Cre system is a valuable tool for studying gene function in the development of the aforementioned organs in a temporal and spatially-restricted manner, as well as for tissue lineage tracing of Msx2 -expressing cells. When induced postnatally, this system can also be used to study gene function in adult tissues without compromising normal development and patterning. genesis 47:352,359, 2009. © 2009 Wiley-Liss, Inc. [source] Gain of function of Tbx1 affects pharyngeal and heart development in the mouseGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 3 2009Francesca Vitelli Abstract Mammalian development is highly sensitive to Tbx1 gene dosage reduction. Gene function insights can also be learned from increased or ectopic expression. The authors generated a novel mouse transgenic line, named COET, which expresses Tbx1 upon Cre-mediated recombination. The authors crossed this transgenic line with Tbx1Cre animals to activate expression in the Tbx1 -expression domain. Compound mutant COET;Tbx1Cre/+ animals died after birth and showed heart enlargement. At E18.5, compound mutants showed ventricular septal defects and thymic abnormalities. The authors crossed compound mutants into a Tbx1 null background to understand whether this phenotype is caused by gene overdosage. Results showed that gene dosage reduction at the endogenous locus could not rescue heart and thymic defects, although the transgene rescued the loss of function phenotype. Thus, the transgenic phenotype appears to be due to gain of function. Resultant data demonstrate that Tbx1 expression must be tightly regulated to be compatible with normal embryonic development. genesis 47:188,195, 2009. © 2009 Wiley-Liss, Inc. [source] Cre-mediated recombination in pituitary somatotropesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2009Igor O. Nasonkin Abstract We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick ,-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in. Cre activity is detectable in the developing pituitary after initiation of Gh transcription and persists through adulthood with high penetrance in Gh expressing cells and lower penetrance in lactotropes, a cell type that shares a common origin with somatotropes. This Gh-cre transgenic line is suitable for efficient, cell-specific deletion of floxed regions of genomic DNA in differentiated somatotropes and a subset of lactotrope cells of the anterior pituitary gland. genesis 47:55,60, 2009. © 2008 Wiley-Liss, Inc. [source] Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2007Teresa Gallardo Abstract Cell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoter-driven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at ,e15, and was >95% efficient in male and female germ cells by birth. Although there was a potent maternal effect with some animals showing more widespread recombination, there was no ectopic activity in most adults. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of gametogenesis and as a general deletor line. genesis 45:413,417, 2007. Published 2007 Wiley-Liss, Inc. [source] Nas transgenic mouse line allows visualization of Notch pathway activity in vivoGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2006Céline Souilhol Abstract The Notch signaling pathway plays multiple and important roles in mammals. However, several aspects of its action, in particular, the precise mapping of its sites of activity, remain unclear. To address this issue, we generated a transgenic line carrying a construct consisting of a nls-lacZ reporter gene under the control of a minimal promoter and multiple RBP-J, binding sites. Here we show that this transgenic line, which we termed NAS (for Notch Activity Sensor), displays an expression profile that is consistent with current knowledge on Notch activity sites in mice, even though it may not report on all these sites. Moreover, we observe that NAS transgene expression is abolished in a RBP-J,-deficient background, indicating that it indeed requires Notch/RBP-J, signaling pathway activity. Thus, the NAS transgenic line constitutes a valuable and versatile tool to gain further insights into the complex and various functions of the Notch signaling pathway. genesis 44: 277,286, 2006. © 2006 Wiley-Liss, Inc. [source] Preservation of a transgenic strain of the sawfly, Athalia rosae (Hymenoptera) by artificial fertilization using cryopreserved spermINSECT MOLECULAR BIOLOGY, Issue 1 2005M. Hatakeyama Abstract Germline transformation using a piggyBac -derived vector is feasible in the sawfly, Athalia rosae. A previously generated transgenic line carrying green fluorescence protein (GFP) genes as reporters was successfully maintained and preserved without consecutive rearing. Sperm taken from males that were frozen directly in liquid nitrogen and stored at ,80 °C for a year were microinjected into mature unfertilized eggs dissected from female ovaries. A fraction of the sperm-injected eggs was fertilized and developed into diploid females, and all of them expressed GFP. Haploid male progeny from these females segregated into GFP-positive and GFP-negative individuals in a ratio of 1 : 1 indicating heterozygosity of the parental females. The GFP genes were stably inherited staying at the location where they were originally integrated. [source] Correlated changes in fertility and fitness traits in lines of oMt1a-oGH transgenic mice selected for increased 8-week body weight,JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 2 2000F. Siewerdt Correlated responses in fitness and fertility traits were compared in transgenic and nontransgenic lines of mice selected for increased 8-week body weight. Two replicates of lines which either carried or did not carry the sheep metallothionein-1a sheep growth hormone transgene (oMt1a-oGH) were established. Host lines had been previously selected for rapid growth or selected randomly. Within-litter selection was carried out for 13 generations, and a randomly selected control line was kept for each set of replicate lines. Mice were genotyped every generation for the presence of the transgene, but this information was not used in selection decisions. The oMt1a-oGH construct was activated by adding 25 mm ZnSO4 to the drinking water from 3 weeks (weaning) until 8 weeks of age. Zinc stimulation of the transgene was not carried out during mating, gestation and lactation. Correlated responses in fitness traits were measured by regression of least-squares means (as deviations from the control lines) on generation number. Two fitness indexes were defined to combine the information on individual fitness traits. The proportion of infertile matings was higher in generations 7 to 13 than in generations 0 to 6. Correlated responses to selection showed an increase in the cohabitation to littering interval in nontransgenic lines and an increase in litter sizes in lines from the selected background. Preweaning pup survival did not change over generations. Overall fitness increased in the transgenic line from the selection background whereas no changes were observed in the transgenic line from the control background. The initial frequency of 0.5 of the transgene was reduced to less than 0.10 in the selected background, but increased to an average of 0.62 in the control lines. The comparison of specific mating groups involving transgenic and nontransgenic mates revealed that the only consistent disadvantage in having a transgenic parent was the increase in the length of the cohabitation to littering interval. Major fitness problems were not associated with the oMt1a-oGH transgene, which makes this construct a potential choice for use in livestock breeding programmes. Zusammenfassung Korrelierte Reaktionen der Merkmale Fertilität und Fitness wurden bei transgenen und nicht trans-genen Mäuselinien verglichen. Es wurden zwei Nachzüchtungen von Linien erstellt, die entweder das ovine Metallothionein-1a Wachstumshormon transgen tragen bzw. es nicht tragen. Zuvor waren Wirts-linien entweder auf schnelles Wachstum selektiert oder zufällig ausgesucht worden. Die Selektion erfolgte innerhalb der Würfe auf erhöhtes 8-Wochengewicht für 13 Generationen, und eine zufällig selektierte Kontroll-Linie wurde für jede der replizierten Linien gehalten. In jeder Generation wurden die Mäuse auf die Anwesenheit des Transgens genotypisiert, aber diese Information wurde nicht für Selektionsentscheidungen herangezogen. Das oMt1a-oGH Konstrukt wurde durch Zugabe von 25 mm ZnSO4 zum Trinkwasser ab der dritten Woche (Absetzen) bis zum Alter von acht Wochen aktiviert. Während der Paarung, der Trächtigkeit und der Laktation wurde keine Zink-Stimulation der transgenen Tiere durchgeführt. Korrelationen zwischen Fitnessmerkmalen und der Generationsnummer wurden durch Regression der kleinsten Quadrate (als Abweichung von den Kontroll-Linien) erhalten. Es wurden zwei Fitness-Indizes definiert, um die Information individueller Fitness-Merkmale zu kombinieren. Der Anteil an unfruchtbaren Paarungen war von der 7. bis zur 13. Generation höher als bei der 0. bis zur 6. Generation. Korrelationen bezüglich Selektion zeigten ein Anstieg der Kohabitation mit dem Wurfintervall bei nicht transgenen Tieren und einen Anstieg der Wurfgrößen bei den Linien der selektierten Gruppe. Das Überleben von Jungen vor dem Absetzten veränderte sich nicht über die Generationen. Allgemeine Fitness stieg bei der transgenen Linie der selektierten Gruppe an, während bei der transgenen Linie der Kontrollgruppe keine Veränderungen beobachtet wurden. Ein Vergleich von spezifischen Paarungsgruppen, die transgene und nicht transgene Paarungen einbezog, ergab, daß der einzige Nachteil eines transgenen Elternteils darin besteht, daß die Länge der Kohabitation mit dem Wurfintervall ansteigt. Mit dem oMt1a-oGH Transgen waren keine schweren Fitness-Probleme verbunden, so daß dieses Konstrukt eine potentielle Wahl für die Verwendung bei Nutztier-Zuchtprogrammen sein könnte. [source] Low levels of mutant ubiquitin are degraded by the proteasome in vivoJOURNAL OF NEUROSCIENCE RESEARCH, Issue 11 2010Paula van Tijn Abstract The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB+1, a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB+1 properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB+1. In these mice, UBB+1 protein is scarcely detectable in the neuronal cell population. Accumulation of UBB+1 commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB+1 protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB+1 is continuously degraded and disease-related UBB+1 accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB+1 and its role in neurodegenerative disease. © 2010 Wiley-Liss, Inc. [source] Increased Susceptibility of Rice Following Insertion of Amylopullulanase Gene, to Brown Spot Caused by Bipolaris oryzaeJOURNAL OF PHYTOPATHOLOGY, Issue 9 2008M.-Y. Ting Abstract Transgenic rice expressing an amylopullulanase (APU) from the bacterium Thermoanaerobacter ethanolicus 39E produces grains which are less expensive to process for production of sugar syrup and protein-enriched flour. During risk assessment of the transgenic line in a field test, brown spot disease caused by Bipolaris oryzae was found more severe on the transgenic line APU than on its parental line TNG67. When lines APU and TNG67 were inoculated at seedling, tillering or heading stage with B. oryzae isolated from line TNG67, the disease was more severe on line APU than on line TNG67 at heading stage, but not at the seedling or tillering stage. However, when B. oryzae isolated from line APU was used in the inoculation tests, the disease was more severe on line APU than on line TNG67 at seedling stage, but not at the tillering or heading stage. To our knowledge, this is the first report of an unintended change in a transgenic plant to become more susceptible to a disease than the non-transgenic plant. [source] Probing Pineal-specific Gene Expression with Transgenic Zebrafish,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Daisuke Kojima The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis -acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P20 -rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P20 -rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N -ethyl- N -nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P20 -rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland. [source] Expression of Cre Recombinase in Pigment CellsPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2002Laurence Guyonneau Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination. [source] A metabolomic study of substantial equivalence of field-grown genetically modified wheatPLANT BIOTECHNOLOGY JOURNAL, Issue 4 2006John M. Baker Summary The ,substantial equivalence' of three transgenic wheats expressing additional high-molecular-weight subunit genes and the corresponding parental lines (two lines plus a null transformant) was examined using metabolite profiling of samples grown in replicate field trials on two UK sites (Rothamsted, Hertfordshire and Long Ashton, near Bristol) for 3 years. Multivariate comparison of the proton nuclear magnetic resonance spectra of polar metabolites extracted with deuterated methanol,water showed a stronger influence of site and year than of genotype. Nevertheless, some separation between the transgenic and parental lines was observed, notably between the transgenic line B73-6-1 (which had the highest level of transgene expression) and its parental line L88-6. Comparison of the spectra showed that this separation resulted from increased levels of maltose and/or sucrose in this transgenic line, and that differences in free amino acids were also apparent. More detailed studies of the amino acid composition of material grown in 2000 were carried out using gas chromatography-mass spectrometry. The most noticeable difference was that the samples grown at Rothamsted consistently contained larger amounts of acidic amino acids (glutamic, aspartic) and their amides (glutamine, asparagine). In addition, the related lines, L88-6 and B73-6-1, both contained larger amounts of proline and ,-aminobutyric acid when grown at Long Ashton than at Rothamsted. The results clearly demonstrate that the environment affects the metabolome and that any differences between the control and transgenic lines are generally within the same range as the differences observed between the control lines grown on different sites and in different years. [source] Bioengineered ,golden' indica rice cultivars with ,-carotene metabolism in the endosperm with hygromycin and mannose selection systemsPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2003Karabi Datta Summary Vitamin-A deficiency (VAD) is a major malnutrition problem in South Asia, where indica rice is the staple food. Indica-type rice varieties feed more than 2 billion people. Hence, we introduced a combination of transgenes using the biolistic system of transformation enabling biosynthesis of provitamin A in the endosperm of several indica rice cultivars adapted to diverse ecosystems of different countries. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytoene synthase (psy), while lycopene ,-cyclase (lcy) and phytoene desaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35S P). Transgenic plants were recovered through selection with either CaMV35S P driven hph (hygromycin phosphotransferase) gene or cestrum yellow leaf curling virus promoter (CMP) driven pmi (phophomannose isomerase) gene. Molecular and biochemical analyses demonstrated stable integration and expression of the transgenes. The yellow colour of the polished rice grain evidenced the carotenoid accumulation in the endosperm. The colour intensity correlated with the estimated carotenoid content by spectrophotometric and HPLC analysis. Carotenoid level in cooked polished seeds was comparable (with minor loss of xanthophylls) to that in non-cooked seeds of the same transgenic line. The variable segregation pattern in T1 selfing generation indicated single to multiple loci insertion of the transgenes in the genome. This is the first report of using nonantibiotic pmi driven by a novel promoter in generating transgenic indica rice for possible future use in human nutrition. [source] Systemic Potato virus YNTN infection and levels of salicylic and gentisic acids in different potato genotypesPLANT PATHOLOGY, Issue 4 2005-Stres, H. Kre Endogenous levels of free and conjugated salicylic (SA) and gentisic (GA) acids, both putative signal molecules in plant defence, were analysed in order to investigate their involvement in the resistance of four potato (Solanum tuberosum) genotypes with different susceptibilities to Potato virus YNTN (PVYNTN) infection: the highly susceptible cv. Igor and its extremely resistant transgenic line, the extremely resistant cv. Sante and the tolerant cv. Pentland Squire. The lowest levels of free and conjugated SA were observed in the extremely resistant cv. Sante, while free GA, which was detected in all the other varieties, was absent. The extremely resistant transgenic cv. Igor contained the highest basal total SA level and the lowest level of total GA of all four cultivars. In susceptible cv. Igor, but not in resistant transgenic cv. Igor, a systemic increase of free SA was measured 1 day postinfection (dpi). Even more significant increases of free and conjugated SA and GA were detected 11 dpi when systemic symptoms appeared. In inoculated but not in upper noninoculated leaves of resistant transgenic cv. Igor, significant increase of SA conjugates occurred, but not before 11 dpi. The increase of SA and GA in susceptible cv. Igor could contribute to the general elevated levels of phenolic compounds as a response to stress caused by virus infection. It appears that basal levels of SA and GA do not correlate with resistance to PVYNTN in potato plants. [source] Comparative proteomic and transcriptional profiling of a bread wheat cultivar and its derived transgenic line overexpressing a low molecular weight glutenin subunit gene in the endospermPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Federico Scossa Abstract We carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that overexpresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding nontransformed genotype. Proteomic analyses showed that, during seed development, several classes of endosperm proteins were differentially accumulated in the transformed endosperm. As a result of the strong increase in the amount of the transgenic protein, the endogenous glutenin subunit, all subclasses of gliadins, and metabolic as well as chloroform/methanol soluble proteins were diminished in the transgenic genotype. The differential accumulation detected by proteomic analyses, both in mature and developing seeds, was paralleled by the corresponding changes in transcript levels detected by microarray experiments. Our results suggest that the most evident effect of the strong overexpression of the transgenic glutenin gene consists in a global compensatory response involving a significant decrease in the amounts of polypeptides belonging to the prolamin superfamily. It is likely that such compensation is a consequence of the diversion of amino acid reserves and translation machinery to the synthesis of the transgenic glutenin subunit. [source] The Cytoskeletal Regulator Zyxin is Required for Viability in Drosophila melanogasterTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 9 2010Patricia J. Renfranz Abstract The zyxin family of proteins function as cytoskeletal regulators in adhesion, actin assembly, and cell motility. Though fibroblasts derived from zyxin-null mice show striking defects in motility and response to mechanical stimuli, the mice are viable and fertile. In Drosophila melanogaster, the family is represented by a single homologue, Zyx102. To study the role of zyxin during development, we generated a zyx102 RNA-interference transgenic line that allows for the conditional knockdown of Zyx102. When UAST-zyx102-dsRNAi expression is driven broadly by Actin5C-GAL4, loss of Zyx102 results in lethality during the pharate adult stage, a narrow developmental window during which the fly must molt, resorb molting fluid, fill adult trachea with air, and execute a behavioral program to eclose. Zyx102 knockdown animals attempt to emerge, but their adult trachea do not fill with air. If dissected from the pupal case, knockdown individuals appear morphologically normal, but remain inviable. Anat Rec 293:1455,1469, 2010. © 2010 Wiley-Liss, Inc. [source] Characterization of Arabidopsis mur3 mutations that result in constitutive activation of defence in petioles, but not leavesTHE PLANT JOURNAL, Issue 5 2008Jennifer D. Tedman-Jones Summary A screen was established for mutants in which the plant defence response is de-repressed. The pathogen-inducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M2 seedlings screened for constitutive ICS1:luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1 -mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes. [source] DNA hypomethylation reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei of Arabidopsis thalianaTHE PLANT JOURNAL, Issue 4 2005Koichi Watanabe Summary 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. [source] | |||||||||||||||||||||||||||||||||||||