Quantifying the risks of invasion by genetically engineered organisms.
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Gene Frequency↗
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Results for “Genetic Engineering”
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The ethics of genetic engineering.
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Agriculture↗
Don't ban genetic engineering.
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Advisory Committees↗
The case against perfection: what's wrong with designer children, bionic athletes, and genetic engineering.
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Biomedical Enhancement↗
Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics.
Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems.
Calcium↗
The use of a genetically engineered Pseudomonas species (Shk1) as a bioluminescent reporter for heavy metal toxicity screening in wastewater treatment plant influent.
Heavy metals are known to be inhibitory and toxic to the activated-sludge microbial community in biological wastewater treatment plants. Toxicity screening of aqueous mixtures of these heavy metal ions in plant influent could use both chemical and biological methods. As a biological method, luminescent bacterial bioreporters offer the advantages of a simple test procedure and rapid response. Current biologically based methods for screening aqueous streams for toxicity are labor-intensive, inaccurate, or difficult to use in continuous monitoring applications. In the present study, a system was developed that is simple and easily automated. This system is based on the bacterium Shk1, a genetically engineered bioluminescent Pseudomonad whose parent strain was originally isolated from activated sludge. Compared with other bioluminescence-based systems (specifically, the Microtox assay), the system of the present study more accurately reflects the effects of the toxicity of common metal ions on activated-sludge respirometry without being overly sensitive to typical constituents of wastewater. The use of Shk1 as a bioluminescent reporter for heavy metal toxicity testing for the application of wastewater treatment influent toxicity screening is presented in this study.
Biological Assay↗
Production of 'hybrid' antibiotics by genetic engineering.
The recent development of molecular cloning systems in Streptomyces has made possible the isolation of biosynthetic genes for some of the many antibiotics produced by members of this important genus of bacteria. Such clones can now be used to test the idea that novel antibiotics could arise through the transfer of biosynthetic genes between streptomycetes producing different antibiotics. The likelihood of a 'hybrid' compound being produced must depend on the substrate specificities of the biosynthetic enzymes, about which little is known. In attempts to demonstrate hybrid antibiotic production, we therefore began with strains producing different members of the same chemical class of compounds in order to maximize the chance of success. Here we report the production of novel compounds by gene transfer between strains producing the isochromanequinone antibiotics actinorhodin, granaticin and medermycin. These experiments were made possible by the recent cloning of the whole set of genes for the biosynthetic pathway of actinorhodin from Streptomyces coelicolor A3(2) (ref. 8). We believe that this represents the first report of the production of hybrid antibiotics by genetic engineering.
Anthraquinones↗
Genetically engineered plants, endangered species, and risk: a temporal and spatial exposure assessment for Karner blue butterfly larvae and Bt maize pollen.
Genetically engineered maize (Zea mays) containing insecticidal endotoxin proteins from Bacillus thuringiensis (Bt) delta-endotoxin proteins has been adopted widely in the Midwestern United States. The proteins are toxic to several lepidopteran species and because a variety of maize tissues, including pollen, may express the endotoxins, the probability of exposure to nontarget species, including endangered species, needs to be understood. The objective of this study was to assess the potential temporal and spatial exposure of endangered Karner blue butterfly larvae (Lycaeides melissa samuelis) to Bt maize pollen in Wisconsin using probabilistic exposure techniques and geographic information systems analysis. Based on degree-day modeling of butterfly phenology and maize pollen shed, there is some potential for temporal exposure of larvae to maize pollen. However, in the majority of years and locations, maize pollen shed most likely will occur after the majority of larval feeding on wild lupine (Lupinus perennis). The spatial analysis indicates that some Karner blue butterfly populations occur in close proximity to maize fields, but in the vast majority of cases the butterfly's host plant and maize fields are separated by more than 500 m. A small number of potential or existing Karner blue butterfly sites are located near maize fields, including sites in two of the four counties where temporal overlap is most likely. The exposure assessment indicates that these two counties should receive the highest priority to determine if Karner blue butterfly larvae are actually at risk and then, if needed, to reduce or prevent exposure.
Animals↗
Noninterference of cytochrome P4501A2 in the cytotoxicity of tacrine using genetically engineered V79 Chinese hamster cells for stable expression of the human or rat isoform and two human hepatocyte cell lines.
Tacrine (THA) is the only drug currently approved for the treatment of Alzheimer's disease. A common side effect of this drug in humans is major hepatotoxicity. THA-induced toxicity may be related to a metabolic pathway implicating cytochrome P450 1A2 (CYP1A2). The purpose of this study was to clarify the role of the metabolic conversion of THA by CYP1A2 in the cytotoxicity of THA. The cytotoxicity of THA was evaluated in two human hepatocyte cell lines, HepG2 and Chang liver, and on the V79 Chinese hamster cell line, which does not express cytochrome P450 activity, and its variants, genetically engineered for expression of human or rat CYP1A2. Cells expressing human CYP1A2 metabolized THA to form its 1-OH derivative (Vmax = 9.36 +/- 0.57 pmol min(-1) mg(-1) total protein), whereas no metabolism was observed with the nonexpressing parental cells. In all cell lines, THA induced a marked decrease in cell viability and a strong inhibition of RNA and protein synthesis. However, these cytotoxic effects did not differ in parental V79 cells and variant cells expressing human or rat CYP1A2. The IC50 were tenfold higher for cell viability than for RNA and protein inhibition after 3 hr of incubation but were similar after 24 hr (P < 0.0001), indicating that this early inhibition was not a transient effect and could lead to cell death. These results strongly suggest that THA-induced cytotoxicity is not mediated by CYP1A2.
Animals↗
Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.
The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.
Aryl Hydrocarbon Hydroxylases↗
Transplant of polymer-encapsulated cells genetically engineered to release nerve growth factor allows a normal functional development of the visual cortex in dark-reared rats.
Visual experience is necessary for the normal development of the visual system. Dark-reared mammals show abnormal vision when reintroduced into a normal environment. The absence of visual experience during the critical period results in reduced and/or inappropriate neural responses in visual cortical neurons. The change in electrical activity induced by dark rearing is probably reflected by the modulation of specific unknown molecules. Neurotrophins are present in the developing visual cortex and their production depends on visually driven electrical activity. Recent findings support the possibility that an important link between electrical activity in the visual pathway and correct development of visual properties is represented by neurotrophins. We advance the hypothesis that the visual abnormalities present in dark-reared animals could be due to a decreased production of a neurotrophin secondary to the lack of visual stimulation. We report that some properties of visual cortical response such as receptive field size, orientation selectivity, adaptation to repeated stimulation, response latency and visual acuity are virtually normal in dark-reared rats transplanted with polymer-encapsulated baby hamster kidney cells genetically engineered to release nerve growth factor.
Adaptation, Ocular↗
Production and characterization of genetically engineered antibody molecules.
Expression of antibody heavy- and light-chain genes by transfection permits the production of monoclonal antibodies with improved biological and antigen-binding properties. The immunoglobulin genes are placed in vectors containing a gene for encoding a protein that provides a biochemically selectable function in eukaryotic cells; these vectors are transfected into myeloma and hybridoma cells. Selection of drug-resistant cells permits the efficient isolation of the rare cells that express the transfected DNA. By placing heavy and light chains on plasmids with different selectable markers, one can deliver heavy- and light-chain genes simultaneously to the same cell. The transfected immunoglobulin genes are efficiently expressed and the proteins produced are a faithful mirror of the genes that were introduced. Using the standard techniques of genetic engineering and gene transfection, we can now produce antibodies of widely varying structures, including chimeric antibodies with segments derived from different species. These antibodies provide useful reagents to study structure-function relationships within the antibody molecule. Ultimately it will be possible to produce a new generation of antibody molecules with improved antigen-binding properties and effector functions.
Animals↗
Genetically engineered plant allergens with reduced anaphylactic activity.
Allergy immunotherapy is based on the administration of increasing amounts of the disease-eliciting allergens in order to yield allergen-specific non-responsiveness. Success of this therapy is associated with modulation of the immune response to allergenic molecules at the level of T-helper cells and the induction of blocking antibodies. The extracts used for immunotherapy are highly heterogenous preparations from natural sources and contain additional components, mostly proteins which are not well defined. Recombinant DNA technology offers novel tools for production of pure and well-characterised allergens for specific immunotherapy. However, high IgE reactivity of pure recombinant allergens is associated with an increased risk of potentially life-threatening anaphylactic reactions. A major improvement in allergen-specific immunotherapy may be achieved by using genetically engineered recombinant allergens with reduced anaphylactic activity. Recently the site- directed mutagenesis technique has been applied successfully to produce variants of major grass, birch and oilseed rape allergens with reduced IgE reactivity but retained T-cell reactivity. These modified allergens with reduced anaphylactic potential are novel candidates for safer and more effective allergen-specific immunotherapy.
Allergens↗
Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.
Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system (CNS). They develop from proliferating oligodendrocyte precursor cells (OPCs), which arise in germinal zones, migrate throughout the developing white matter and divide a limited number of times before they terminally differentiate. Thus far, it has been possible to purify OPCs only from the rat optic nerve, but the purified cells cannot be obtained in large enough numbers for conventional biochemical analyses. Moreover, the CNS stem cells that give rise to OPCs have not been purified, limiting one's ability to study the earliest stages of commitment to the oligodendrocyte lineage. Pluripotent, mouse embryonic stem (ES) cells can be propagated indefinitely in culture and induced to differentiate into various cell types. We have genetically engineered ES cells both to positively select neuroepithelial stem cells and to eliminate undifferentiated ES cells. We have then used combinations of known signal molecules to promote the development of OPCs from selected, ES-cell-derived, neuroepithelial cells. We show that the earliest stages of oligodendrocyte development follow an ordered sequence that is remarkably similar to that observed in vivo, suggesting that the ES-cell-derived neuroepithelial cells follow a normal developmental pathway to produce oligodendrocytes. These engineered ES cells thus provide a powerful system to study both the mechanisms that direct CNS stem cells down the oligodendrocyte pathway and those that influence subsequent oligodendrocyte differentiation. This strategy may also be useful for producing human cells for therapy and drug screening.
Aging↗
Genetically engineered cells with regulatable GABA production can affect afterdischarges and behavioral seizures after transplantation into the dentate gyrus.
Intractable seizures originating in the mesial temporal lobe can often be controlled by resection. An alternative to removing hippocampal tissue may be transplantation of GABA-producing cells. Neural cell transplantation has been performed in hundreds of patients, including some with temporal lobe epilepsy. This study evaluates the seizure-suppressing capabilities of engineered GABA-producing cells transplanted into the dentate gyrus. Immortalized neurons were engineered to produce GABA under the control of doxycycline. The cells were characterized for GABA production in vitro and for their ability to raise GABA concentrations in vivo. Cells were transplanted bilaterally into the dentate gyrus of rats and tested in two separate paradigms. Afterdischarge thresholds and durations were tested with granule cell stimulation, and the development of behavioral seizures, induced by daily electrical stimulation of the major excitatory input pathway into the dentate gyrus, was assessed in the presence, or the absence, of doxycycline. GABA production was under the tight control of doxycycline. Cells engineered to produce GABA raised tissue GABA concentrations in the hippocampus compared with non GABA-producing cells, and this was abolished when doxycycline was administered. GABA-producing cells raised the threshold, and shortened the duration of hippocampal afterdischarges elicited by granule cell stimulation. Lastly, the appearance of stage 5 seizures was slowed in the kindling paradigm, compared with a group that received non-GABA-producing cells, and compared with a group that received GABA-producing cells but was administered doxycycline. This study shows that targeted hippocampal implants of genetically engineered cells have the potential to raise GABA levels and to affect seizure development. The ability to suppress the production of GABA, and to modulate the physiological effects of the transplanted cells provides an important level of experimental control. These techniques, combined with stem cell technology, may advance cell-based therapies for epilepsy and other diseases of the CNS.
Amino Acids↗
Metabolism-mediated neurotoxicity: the significance of genetically engineered cell lines and new three-dimensional cell cultures.
Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.
Animal Testing Alternatives↗
Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T1 NMR relaxation.
Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence alpha 184-200 from the Torpedo californica receptor. This construct binds alpha-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control [Aronheim, A., Eshel, Y., Mosckovitz, R., & Gershoni, J. M. (1988) J. Biol. Chem. 263, 9933-9937]. In this study agonist binding to alpha 184-200 is demonstrated by monitoring the T1 relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with alpha-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified alpha 184-200 constructs, respectively. The KD values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while KD's with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain alpha 184-200 may comprise a major component of the cholinergic agonist site.
Acetylcholine↗
Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing.
The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at -5 and -12 degrees C. Plants colonized by the non-INA strain were undamaged at -5 degrees C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (-7, -9, and -12 degrees C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At -12 degrees C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.
Journal Article↗