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Genetic Manipulation (genetic + manipulation)
Selected AbstractsMOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 5 2006Nicole Poulsen Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non-selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co-transformation allowing for the simultaneous introduction of a non-selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana -specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms. [source] Current Opinions on the Functions of Tocopherol Based on the Genetic Manipulation of Tocopherol Biosynthesis in PlantsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2008Yin Li Abstract As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactive oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed. [source] Genetic Manipulation of Rubisco: Chromatium vinosum rbcL is expressed in Nicotiana tabacum but does not form a functional proteinANNALS OF APPLIED BIOLOGY, Issue 1 2002P J MADGWICK Summary N. tabacum lines that lacked functional Rubisco were transformed with plasmids encoding a chloroplast transit peptide in frame with C. vinosum rbcL and stable transformants generated. However, the transgene was transcribed at a low level and no Rubisco activity or C. vinosum large subunits were detectable in any line. [source] Genetic manipulation, whole-cell recordings and functional imaging of the sensorimotor cortex of behaving miceACTA PHYSIOLOGICA, Issue 1 2009C. C. H. Petersen Abstract Sensory processing, sensorimotor integration and motor control are amongst the most basic functions of the brain and yet our understanding of how the underlying neuronal networks operate and contribute to behaviour is very limited. The relative simplicity of the mouse whisker sensorimotor system is helpful for detailed quantitative analyses of motor control and perception during active sensory processing. Recent technical advances now allow the measurement of membrane potential in awake-behaving mice, using whole-cell recordings and voltage-sensitive dye imaging. With these recording techniques, it is possible to directly correlate neuronal activity with behaviour. However, in order to obtain causal evidence for the specific contributions of different neuronal networks to behaviour, it is critical to manipulate the system in a highly controlled manner. Advances in molecular neurobiology, gene delivery and mouse genetics provide techniques capable of layer, column and cell-type specific control of gene expression in the mouse neocortex. Over the next years, we anticipate considerable advances in our understanding of brain function through measuring and manipulating neuronal activity with unprecedented precision to probe the molecular and synaptic mechanisms underlying simple forms of active sensory perception and associative learning. [source] Transgenic Animals in Cardiovascular Disease ResearchEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Michael Bader Worldwide, the highest morbidity and mortality results from such cardiovascular diseases as hypertension, myocardial infarction, cardiac and renal failure, as well as stroke. Since the cardiovascular system and its regulation is quite complex, study of these disorders has been grossly limited to whole organism models. As a result, in recent years, transgenic technology has played a significant role in the discovery of specific gene products for cardiovascular regulation and disease aetiology. Genetic manipulation in rats and mice has altered the expression of numerous genes. In this review, some of the important new genetically modified animals (i.e. transgenic models) with alterations in hormone and second messenger systems involved in cardiovascular regulation are summarized. [source] Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastorisFEMS YEAST RESEARCH, Issue 6 2009Tamara Wriessnegger Abstract Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris, which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae. Using P. pastoris psd1, and psd2, mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP),ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris, which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes. [source] Antibacterial Coatings: Genetically Engineered Phage Fibers and Coatings for Antibacterial Applications (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 2 2010Mater. Genetic manipulation of viruses can be used to fabricate antibacterial fibers and coatings comprising crosslinked M13 bacteriophages, which are modified to bind silver ions. On page 209, Krystyn Van Vliet and co-workers demonstrate the bactericidal effects of such silverized phage fibers against several types of bacterial challenges including those potentially arising from use as wound dressings or antibacterial textiles. [source] Targeted Expression of SHH Affects Chondrocyte Differentiation, Growth Plate Organization, and Sox9 Expression,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2004Sara Tavella Abstract The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation. Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates. Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization. Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter. Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones. [source] Regulation of Osteogenesis-Angiogenesis Coupling by HIFs and VEGF,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2009Ernestina Schipani Abstract Bone is a highly vascularized tissue, but the function of angiogenesis in bone modeling and remodeling is still poorly defined, and the molecular mechanisms that regulate angiogenesis in bone are only partially elucidated. Genetic manipulations in mice have recently highlighted the critical role of the hypoxia-inducible-factor/vascular endothelial growth factor pathway in coupling angiogenesis and osteogenesis. In this brief perspective, we review the current understanding of the mechanisms responsible for this coupling. Elucidation of such mechanisms will expand our knowledge of bone development and homeostasis, and it may aid in the design of new therapies for accelerating bone regeneration and repair. [source] Fragile X syndrome, the Fragile X related proteins, and animal modelsMICROSCOPY RESEARCH AND TECHNIQUE, Issue 3 2002André T. Hoogeveen Abstract The Fragile X syndrome (FraX), which is characterized among other physical and neurologic impairments by mental retardation, is caused by the absence of the product of the FMR1 gene. The Fragile X Mental Retardation Protein (FMRP) is a member of a novel family of RNA-binding proteins. The latter includes two other proteins highly homologous with FMRP: the fragile X related proteins 1 and 2 (FXRP1 and FXRP2). Characterization of FXRPs, including their interaction with FMRP, will provide critical information about the mechanisms of action of FMRP and the role of this group of proteins in FMRP-deficient conditions such as FraX. Genetic manipulations of FMRP and the FXRPs should also provide valuable tools for investigating pathophysiology and gene therapies in FraX. The present review summarizes the strategies used for identifying the FXRPs, their chromosomal localization, molecular structure, and tissue distribution. It also reviews interactions between different members of this family of RNA-binding proteins. Animal models, both knockout and transgenic, of FMRP and the FXRPs are discussed. Phenotypic features of the FMR1 knockout mouse, the FMR1 transgenic rescue mouse, and other novel strategies for manipulating and delivering FMRP and FXRPs to the brain and other tissues are described. Microsc. Res. Tech. 57:148,155, 2002. © 2002 Wiley-Liss, Inc. [source] Transgenic mice for studies of the renin,angiotensin system in hypertensionACTA PHYSIOLOGICA, Issue 4 2004J. L. Lavoie Abstract Hypertension is a polygenic and multi-factorial disorder that is extremely prevalent in western societies, and thus has received a great deal of attention by the research community. The renin,angiotensin system has a strong impact on the control of blood pressure both in the short- and long-term, making it one of the most extensively studied physiological systems. Nevertheless, despite decades of research, the specific mechanisms implicated in its action on blood pressure and electrolyte balance, as well as its integration with other cardiovascular pathways remains incomplete. The production of transgenic models either over-expressing or knocking-out specific components of the renin,angiotensin system has given us a better understanding of its role in the pathogenesis of hypertension. Moreover, our attention has recently been refocused on local tissue renin,angiotensin systems and their physiological effect on blood pressure and end-organ damage. Herein, we will review studies using genetic manipulation of animals to determine the role of the endocrine and tissue renin,angiotensin system in hypertension. We will also discuss some untraditional approaches to target the renin,angiotensin system in the kidney. [source] Enhancement of the NAD(P)(H) Pool in Escherichia coli for BiotransformationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007F. Heuser Abstract In pyridine nucleotide-dependent, reductive whole cell biotransformation with resting cells of Escherichia coli, the availability of intracellular NAD(P)(H) is a pivotal point for an efficient and highly productive substrate conversion. The question whether an increase of the intracellular NAD(P)(H) concentration could increase the productivity was discussed controversially in the past. This is the first report on an E. coli strain with an increased NAD(P)(H) pool which was tested in a reductive biotransformation system for an increased productivity. Biotransformation was performed with a strain overexpressing a gene encoding an (R)-specific alcohol dehydrogenase for the stereospecific, NADPH-dependent reduction of methyl acetoacetate (MAA) to (R)-methyl-3-hydroxybutanoate (MHB). Cofactor regeneration was implemented via glucose oxidation by coexpression of a gene encoding glucose dehydrogenase. The specific MHB productivity (mmol mg,1 cell dry weight,1h,1) enabled a comparison between the E. coli,BL21(DE3) wild-type and a genetically modified strain. The enhancement of the NAD(P)(H) pool was achieved by genetic manipulation of the NAD(H) biosynthetic pathways. After simultaneous overexpression of the pncB and nadE genes, encoding nicotinic acid phosphoribosyltransferase and NAD synthetase, measurements of the total NAD(P)(H) pool, sizes showed a 7-fold and 2-fold increased intracellular concentration of NAD(H) and NADP(H), respectively. However, the implementation of an E.,coli strain carrying a genomically integrated pncB gene with an upstream T7,promoter for biotransformation did not result in reproducible increased specific cell productivity. [source] Natural transformation of Vibrio fischeri requires tfoX and tfoYENVIRONMENTAL MICROBIOLOGY, Issue 8 2010Amber Pollack-Berti Summary Recent evidence has indicated that natural genetic transformation occurs in Vibrio cholerae, and that it requires both induction by chitin oligosaccharides, like chitohexaose, and expression of a putative regulatory gene designated tfoX. Using sequence and phylogenetic analyses we have found two tfoX paralogues in all sequenced genomes of the genus Vibrio. Like V. cholerae, when grown in chitohexaose, cells of V. fischeri are able to take up and incorporate exogenous DNA. Chitohexaose-independent transformation by V. fischeri was observed when tfoX was present in multicopy. The second tfoX paralogue, designated tfoY, is also required for efficient transformation in V. fischeri, but is not functionally identical to tfoX. Natural transformation of V. fischeri facilitates rapid transfer of mutations across strains, and provides a highly useful tool for experimental genetic manipulation in this species. The presence of chitin-induced competence in several vibrios highlights the potential for a conserved mechanism of genetic exchange across this family of environmentally important marine bacteria. [source] Toward detoxifying mercury-polluted aquatic sediments with rice genetically engineered for mercury resistanceENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2003Andrew C. P. Heaton Abstract Mercury contamination of soil and water is a serious problem at many sites in the United States and throughout the world. Plant species expressing the bacterial mercuric reductase gene, merA, convert ionic mercury, Hg(II), from growth substrates to the less toxic metallic mercury, Hg(0). This activity confers mercury resistance to plants and removes mercury from the plant and substrates through volatilization. Our goal is to develop plants that intercept and remove Hg(II) from polluted aquatic systems before it can undergo bacterially mediated methylation to the neurotoxic methylmercury. Therefore, the merA gene under the control of a monocot promoter was introduced into Oryza sativa L. (rice) by particle gun bombardment. This is the first monocot and first wetland-adapted species to express the gene. The merA -expressing rice germinated and grew on semisolid growth medium spiked with sufficient Hg(II) to kill the nonengineered (wild-type) controls. To confirm that the resistance mechanism was the conversion of Hg(II) to Hg(0), seedlings of merA -expressing O. sativa were grown in Hg(II)-spiked liquid medium or water-saturated soil media and were shown to volatilize significantly more Hg(0) than wild-type counterparts. Further genetic manipulation could yield plants with increased efficiency to extract soil Hg(II) and volatilize it as Hg(0) or with the novel ability to directly convert methylmercury to Hg(0). [source] Reduced plasticity of cortical whisker representation in adult tenascin-C-deficient mice after vibrissectomyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2004Anita Cybulska-Klosowicz Abstract The effect of the extracellular matrix recognition molecule tenascin-C on cerebral plasticity induced by vibrissectomy was investigated with 2-deoxyglucose (2DG) brain mapping in tenascin-C-deficient mice. Unilateral vibrissectomy sparing row C of vibrissae was performed in young adult mice. Two months later, cortical representations of spared row C vibrissae and control row C on the other side of the snout were visualized by [14C]2DG autoradiography. In both wild-type and tenascin-C-deficient mice, cortical representation of the spared row was expanded in all layers of the barrel cortex. However, the effect was significantly more extensive in wild-type animals than in the mutant. Elimination of tenascin-C by genetic manipulation thus reduces the effect of vibrissectomy observed in the somatosensory cortex. No increase in number of fibres in the vibrissal nerve of spared vibrissae was seen, and occurrence of additional nerve to the spared follicle was very rare. Thus, in tenascin-C-deficient mice functional plasticity seems to be impaired within the CNS. [source] Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2002Sangmi Chung Abstract Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1, promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic l -amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate. [source] Therapeutic angiogenesis and vasculogenesis for tissue regenerationEXPERIMENTAL PHYSIOLOGY, Issue 3 2005Paolo Madeddu Therapeutic angiogenesis/vasculogenesis holds promise for the cure of ischaemic disease. The approach postulates the manipulation of spontaneous healing response by supplementation of growth factors or transplantation of vascular progenitor cells. These supplements are intended to foster the formation of arterial collaterals and promote the regeneration of damaged tissues. Angiogenic factors are generally delivered in the form of recombinant proteins or by gene transfer using viral vectors. In addition, new non-viral methods are gaining importance for their safer profile. The association of growth factors with different biological activity might offer distinct advantages in terms of efficacy, yet combined approaches require further optimization. Alternatively, substances with pleiotropic activity might be considered, by virtue of their ability to target multiple mechanisms. For instance, some angiogenic factors not only stimulate the growth of arterioles and capillaries, but also inhibit vascular destabilization triggered by metabolic and oxidative stress. Transplantation of endothelial progenitor cells was recently proposed for the treatment of peripheral and myocardial ischaemia. Progenitor cells can be transplanted either without any preliminary conditioning or after ex vivo genetic manipulation. Delivery of genetically modified progenitor cells eliminates the drawback of immune response against viral vectors and makes feasible repeating the therapeutic procedure in case of injury recurrence. It is envisioned that these new approaches of regenerative medicine will open unprecedented opportunities for the care of life-threatening diseases. [source] A new molecular tool for transgenic diatomsFEBS JOURNAL, Issue 13 2005Control of mRNA, protein biosynthesis by an inducible promoter, terminator cassette Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen-free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms. [source] Histone modifications and chromatin dynamics: a focus on filamentous fungiFEMS MICROBIOLOGY REVIEWS, Issue 3 2008Gerald Brosch Abstract The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems. Filamentous fungi show a wide range of morphologies and have developed a complex network of genes that enables them to use a great variety of substrates. This fact, together with the possibility of simple and quick genetic manipulation, highlights these organisms as model systems for the investigation of gene regulation. However, little is still known about regulation at the chromatin level in filamentous fungi. Understanding the role of chromatin in transcriptional regulation would be of utmost importance with respect to the impact of filamentous fungi in human diseases and agriculture. The synthesis of compounds (antibiotics, immunosuppressants, toxins, and compounds with adverse effects) is also likely to be regulated at the chromatin level. [source] Tools for the genetic manipulation of Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 8 2007Lenka Pribylova Abstract A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP,kanMX,loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase. [source] Transgenic and knock-out mouse pups: the growing need for behavioral analysisGENES, BRAIN AND BEHAVIOR, Issue 3 2002I. Branchi Few laboratories working with transgenic and knock-out mice analyze the neurobehavioral consequences of genetic manipulation in early ontogeny. However, the study of behavioral endpoints during the early postnatal period in genetically modified mice is important not only to assess possible developmental abnormalities, but also to better understand and disentangle the effects of genetic manipulations in adulthood. We propose that the assessment of neurobehavioral development represents an appropriate strategy to identify possible compensatory and/or unexpected effects. Nowadays, a large number of experimental protocols that take into account the practical constraints imposed by the peculiar physiological and behavioral responses of an immature subject are available to assess the neurobehavioral profile of developing mice. While this knowledge should be applied to the field of transgenic and knock-out mice in general, it should be recommended, in particular, for the study of mouse models of neurodevelopmental disorders. [source] Modulating tone: the overture of S1P receptor immunotherapeuticsIMMUNOLOGICAL REVIEWS, Issue 1 2008Hugh Rosen Summary: Modulation of complex functions within the immune system has proven to be surprisingly sensitive to alterations in the lysophospholipid sphingosine 1-phosphate (S1P) receptor-ligand rheostat. This has become increasingly evident from both chemical and genetic manipulation of the S1P system, with pharmacological effects upon lymphoid cells, dendritic cell function, as well as vascular interfaces. The integrated immune system, perhaps as a result of its relatively recent evolutionary ontogeny, has selected for a number of critical control points regulated by five distinct high affinity G-protein-coupled receptor subtypes with a shared ligand, with receptors distributed on lymphocytes, dendritic cells, and endothelium. All of these cellular components of the axis are capable of modulating immune responses in vivo, with the impact on the immune response being very different from classical immunosuppressants, by virtue of selective spatial and temporal sparing of humoral and myeloid elements of host defense. Pharmacological subversion of the S1P rheostat is proving to be clinically efficacious in multiple sclerosis, and both the scope and limitations of therapeutic modulation of the S1P axis in immunotherapy are becoming clearer as understanding of the integrated chemical physiology of the S1P system emerges. [source] In vivo analysis of gut function and disease changes in a zebrafish larvae model of inflammatory bowel disease: A feasibility studyINFLAMMATORY BOWEL DISEASES, Issue 7 2010Angeleen Fleming PhD Abstract Background: The aim of this study was to develop a model of inflammatory bowel disease (IBD) in zebrafish larvae, together with a method for the rapid assessment of gut morphology and function in vivo thereby enabling medium-throughput compound screening. Methods: Assays were performed using larval zebrafish from 3,8 days postfertilization (d.p.f.) in 96-well plates. Gut morphology and peristalsis were observed in vivo using fluorescent imaging following ingestion of fluorescent dyes. IBD was induced by addition of 2,4,6-trinitrobenzenesulfonic acid (TNBS) to the medium within the well. Pathology was assessed in vivo using fluorescent imaging and postmortem by histology, immunohistochemistry, and electron microscopy. Therapeutic compounds were evaluated by coadministration with TNBS. Results: A novel method of investigating gut architecture and peristalsis was devised using fluorescent imaging of live zebrafish larvae. Archetypal changes in gut architecture consistent with colitis were observed throughout the gut. Significant changes in goblet cell number and tumor necrosis factor alpha (TNF-,) antibody staining were used to quantify disease severity and rescue. Prednisolone and 5-amino salicylic acid treatment ameliorated the disease changes. Candidate therapeutic compounds (NOS inhibitors, thalidomide, and parthenolide) were assessed and a dissociation was observed between efficacy assessed using a single biochemical measure (TNF-, staining) versus an assessment of the entire disease state. Conclusions: Gut physiology and pathology relevant to human disease state can be rapidly modeled in zebrafish larvae. The model is suitable for medium-throughput chemical screens and is amenable to genetic manipulation, hence offers a powerful novel premammalian adjunct to the study of gastrointestinal disease. (Inflamm Bowel Dis 2010) [source] Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vectorINSECT MOLECULAR BIOLOGY, Issue 2 2002N. F. Lobo Mosquito-vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world-wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white-eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat-shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ-line transformation vector for Aedine mosquitoes. [source] Genetic Engineering and Autonomous AgencyJOURNAL OF APPLIED PHILOSOPHY, Issue 3 2003Linda Barclay abstract,In this paper I argue that the genetic manipulation of sexual orientation at the embryo stage could have a detrimental effect on the subsequent person's later capacity for autonomous agency. By focussing on an example of sexist oppression I show that the norms and expectations expressed with this type of genetic manipulation can threaten the development of autonomous agency and the kind of social environment that makes its exercise likely. [source] Strategies for Directing the Differentiation of Stem Cells Into the Osteogenic Lineage In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004Boon Chin Heng Abstract A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone-related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro. [source] Controlled and localized genetic manipulation in the brainJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2006Rachel Aronoff Abstract Brain structure and function are determined in part through experience and in part through our inherited genes. A powerful approach for unravelling the balance between activity-dependent neuronal plasticity and genetic programs is to directly manipulate the genome. Such molecular genetic studies have been greatly aided by the remarkable progress of large-scale genome sequencing efforts. Sophisticated mouse genetic manipulations allow targeted point-mutations, deletions and additions to the mouse genome. These can be regulated through inducible promoters expressing in genetically specified neuronal cell types. However, despite significant progress it remains difficult to target specific brain regions through transgenesis alone. Recent work suggests that transduction vectors, like lentiviruses and adeno-associated viruses, may provide suitable additional tools for localized and controlled genetic manipulation. Furthermore, studies with such vectors may aid the development of human genetic therapies for brain diseases. [source] Some highlights of research on aging with invertebrates, 2006,2007AGING CELL, Issue 5 2007Linda Partridge Summary The invertebrate model organisms continue to be engines of discovery in aging research. Recent work with Drosophila stem cells has thrown light on their human equivalents, and on the role of stem cells and their niches in the decline in fecundity with age. Inspired by observations of aging in bacteria and yeast, a new theoretical study has revealed evolutionary forces that could favour asymmetry in the distribution of damaged cell constituents at division, and hence pave the way for the evolution of aging and selective maintenance of integrity of the germ line. Mechanisms of nutrient sensing and cell signalling in the response of lifespan to dietary restriction have been elucidated. Powerful invertebrate models of human aging-related disease have been produced, and used to start to understand how the aging process acts as a risk factor for disease. In the near future, studies of invertebrate aging are likely to move away from an exclusive reliance on genetic manipulation towards a more biochemical and physiological understanding of these systems. [source] Consumer response to functional foods produced by conventional, organic, or genetic manipulationAGRIBUSINESS : AN INTERNATIONAL JOURNAL, Issue 2 2004Bruno Larue The agro-food industry is developing a "second generation" of genetically modified (GM) foods that can offer functional health benefits to consumers. Many consumers, however, are turning to organic foods in order to avoid GM foods. This report attempts to differentiate consumer valuation of functional health properties in conventional, organic, and GM foods. A representative sample of 1,008 Canadian household food shoppers responded to twelve stated-choice experiments during a telephone survey. Because opinions about organic and GM foods varied greatly, random parameters logit models were used to analyze their choices. Results indicate that many Canadian consumers will avoid GM foods, regardless of the presence of functional health properties. For others, the introduction of GM functional plant foods should increase acceptance of GM production methods, but many consumers will likely avoid functional foods derived from GM animals. The organic food industry could also profit from the introduction of organic functional foods. [EconLit citations: I120; D120.] © 2004 Wiley Periodicals, Inc. Agribusiness 20: 155,166, 2004. [source] Molecular and Biochemical Evidence for Phenylpropanoid Synthesis and Presence of Wall-linked Phenolics in Cotton FibersJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 7 2009Ling Fan Abstract The mature cotton (Gossypium hirsutum L.) fiber is a single cell with a typically thickened secondary cell wall. The aim of this research was to use molecular, spectroscopic and chemical techniques to investigate the possible occurrence of previously overlooked accumulation of phenolics during secondary cell wall formation in cotton fibers. Relative quantitative reverse transcription-polymerase chain reaction analysis showed that GhCAD6 and GhCAD1 were predominantly expressed among seven gene homologs, only GhCAD6 was up-regulated during secondary wall formation in cotton fibers. Phylogenic analysis revealed that GhCAD6 belonged to Class I and was proposed to have a major role in monolignol biosynthesis, and GhCAD1 belonged to Class III and was proposed to have a compensatory mechanism for monolignol biosynthesis. Amino acid sequence comparison showed that the cofactor binding sites of GhCADs were highly conserved with high similarity and identity to bona fide cinnamyl alcohol dehydrogenases. The substrate binding site of GhCAD1 is different from GhCAD6. This difference was confirmed by the different catalytic activities observed with the enzymes. Cell wall auto-fluorescence, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and chemical analyses confirmed that phenolic compounds were bound to the cell walls of mature cotton fibers. Our findings may suggest a potential for genetic manipulation of cotton fiber properties, which are of central importance to agricultural, cotton processing and textile industries. 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