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PubMed1983

Applications of genetic engineering to the pharmaceutical industry.

A frequent motive for genetic engineering is to transfer the gene for the desired protein from a cell that is difficult or impossible to grow into a cell which is easy and inexpensive to handle. In the pharmaceutical field this usually means isolation of a gene coding for a protein secreted by human cells, the protein often being glycosylated. While bacterial cells are an inexpensive host in which to express genes, they do not produce proteins which are glycosylated, nor necessarily properly processed. Thus while there are examples of successful bacterial systems, there is also an intense interest in expression in animal cells. The situation is often complicated by our lack of knowledge of the function, or even the necessity, of glycosylation. Examples of successes and problems in production of insulin and interferons in several hosts have been presented.

Animals↗
PubMed1995

Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells.

V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP have been shown to serve as analytical tools for studying metabolism related problems in toxicology and pharmacology. Here, the application of rat and human CYP1A1 and CYP1A2 is demonstrated for comparative studies on the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Live cells were cultivated for 2 days in the presence of these chemicals. Thereafter, the supernatant medium was checked for metabolites by gas chromatography and mass spectrometry. Marked cytochromes P450 and species dependent differences in the metabolite profiles were observed. Most important was the finding, that human cytochrome P450 1A1 almost exclusively oxidized benzo[a]pyrene in the 7,8,9,10-position, yielding the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene whereas the rat cytochrome P450 1A1 oxidized benzo[]pyrene in the 4,5-position and 7,8,9,10-position. The importance of this finding is underlined by results from cytotoxicity studies. Benzo[a]pyrene was twice as cytotoxic in the human cytochrome P450 1A1 than in the rat cytochrome P450 1A1 expressing V79 cells. Species and cytochrome P450 specific metabolite profiles were also observed for phenanthrene and benz[a]anthracene.

Animals↗
PubMed1995

[Detection of genetically engineered plants by polymerase chain reaction (PCR) using the FLAVR SAVR tomato as an example].

In this study, a practical approach for the detection of a genetically engineered tomato is demonstrated by polymerase chain reaction (PCR) assays. The Flavr Savr tomato available on the market which was used as an example, contains a resistance gene for the antibiotic kanamycin (kanr) and a gene construct for the inhibition of fruit ripening and softening (antisense polygalacturonase). The presence of these DNA's could only be detected in the genetically engineered tomato.

Base Sequence↗
PubMed1986

[Actinomycetes--the test-organisms of genetic engineering].

The paper contains a short review of the data on using the methods of genetic engineering in studies of genetics and molecular biology in Streptomyces. The techniques of DNA introduction into actinomycetes and wide-spread vectors are briefly described. The origin of the actinomycete plasmids as chromosomal segments capable of autonomous replication is discussed. In this view, it is suggested that genetic instability in actinomycetes is connected with excision of specific DNA sequences from the chromosome at frequencies characteristic of recombination events. Also, amplification of short DNA segments within the chromosome resulting in tandem repeats is a consequence of unequal crossing over between direct repeats flanking the amplifying DNA and, possibly, of induction of replication of this DNA. The data on molecular cloning of actinomycete genes for primary metabolism and those for resistance to and biosynthesis of antibiotics, on using actinomycetes as the hosts for foreign genes to be expressed, as well as on analysis of nucleotide sequences of actinomycete DNA, are presented.

Actinomycetaceae↗
PubMed1995

Applying genetic engineering to the structural analysis of proteins.

Genetic engineering offers many techniques that can be applied to the structural analysis of proteins. These techniques aid in the characterization of the protein but also can be applied to the generation of completely new diagnostic and therapeutic agents.

Amino Acid Sequence↗
PubMed2003

Tumor targeting properties of indium-111 labeled genetically engineered Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment (chTNT)-3 antibody.

Genetic engineering techniques have allowed the construction of Fab' and F(ab')2 constructs of chimeric tumor necrosis treatment antibody (chTNT-3), a chimeric monoclonal antibody (MAb) that targets necrotic regions of solid tumors. The purpose of this study is to evaluate the in vitro and in vivo properties of Fab' and F(ab')2 constructs radiolabeled with indium-111 (111In) using diethylentriamine pentaacetic acid (DTPA) conjugation to develop a clinically useful imaging agent for the detection of necrosis in solid tumors. Optimization of the MAb-to-DTPA ratio showed that a 1:2 ratio gave the best immunoreactivity while providing good radiolabeling efficiency and high specific activity for all three DPTA conjugates. In addition, 111In-labeled Fab' and F(ab')2 conjugates were found to have faster whole body clearance times and better biodistribution profiles compared to parental 111In-labeled chTNT-3 in tumor-bearing mice. Although radiolabeled Fab' and F(ab')2 constructs showed lower tumor uptake than radiolabeled chTNT-3, biodistribution results showed that these constructs had significantly lower uptake in liver, spleen, and other normal organs (except the kidney), and therefore had higher tumor-to-organ ratios. In addition, a comparison of all derivatives showed that the F(ab')2 reagent gave the best results in tumor imaging studies. These results demonstrate that stable, a genetically engineered F(ab')2 construct can be successfully radiolabeled with 111In to produce potential imaging reagents for the imaging and monitoring of tumor necrosis.

Animals↗
PubMed2000

Therapeutic anti-tumor response induced with epitope-pulsed fibroblasts genetically engineered for B7.1 expression and IFN-gamma secretion.

Mouse fibroblasts (H-2(b)) were genetically engineered to express a co-stimulatory B7.1 and an IFN-gamma (Fb/IFN-gamma/B7.1). The Fb/IFN-gamma/B7.1 cells were then pulsed with an ovalbumin epitope (amino acids 257-264, SIINFEKL, H-2K(b)-restricted) as a model antigen (Fb/IFN-gamma/B7.1/OVA) and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2(b)). Genetically engineered fibroblasts lacking either IFN-gamma or B7.1 were constructed and used as controls. Immunization with the Fb/IFN-gamma/B7.1/OVA cells induced strong cytotoxic activity against OVA-expressing EL4 (EG7) tumor cells but not against other H-2(b) tumor cells, such as EL4, C1498, and B16F1. The magnitude of the cytotoxic response in mice with the Fb/IFN-gamma/B7.1/OVA cells was significantly higher than that in mice immunized with any other cell construct. CD8(+) T cells with OVA-specific cytotoxic activity were predominant in mice immunized with Fb/IFN-gamma/B7.1/OVA cells. Furthermore, treatment with Fb/IFN-gamma/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. Anti-tumor CTL immunity by the Fb/IFN-gamma/B7.1/OVA cells could be induced without the help of host antigen-presenting cells, CD4(+) T cells, or NK1.1(+) cells. Our results suggest that fibroblasts can be genetically modified into efficient antigen-presenting cells for the induction of antigen-specific CTL response in cancer immunotherapy.

Animals↗
PubMed1996

Competition in a spatially heterogeneous environment: modelling the risk of spread of a genetically engineered population.

In recent years regulations have been developed to address the risks of releasing genetically engineered organisms into the natural environment. These risks are generally considered to be proportional to the exposure multiplied by the hazard. Exposure is, in part, determined by the spatial spread of the organisms, a component of risk suited to mathematical analysis. In this paper we exampine a mathematical model describing the spread of organisms introduced into a hetereogeneous environment, focusing on the risk of spread and plausibility of containment strategies. Two competing populations are assumed, one the natural species and the other an engineered species or strain, both of which move randomly in a spatially heterogenous environment consisting of alternating favourable and unfavourable patches. The classical Lotka-Volterra competition model with diffusion is used. Analyses of the possible spread and invasion of engineered organisms are thus reduced to finding periodic travelling wave solutions to the model equations. We focus on whether a very small number of engineered organisms can spatially invade a natural population. Initially we investigate the problem for spatially periodic diffusion coefficients and demonstrate that, under the right circumstances and a large enough unfavourable patch, invasion does not succeed. However, if spatially periodic carrying capacities are assumed along with spatially varying diffusion rates, the situation is far more complex. In this case containment of the engineered species is no longer only a simple function of the unfavourable patch length. By using perturbation solutions to the nonuniform steady states, approximate invasion conditions are obtained.

DNA, Recombinant↗
PubMed1989

Novel method for monitoring genetically engineered microorganisms in the environment.

A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of DNA) and has an advantage over the plate count technique, which can detect only culturable microorganisms. The method may be useful for monitoring GEMs in complex environments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.

Colony Count, Microbial↗
PubMed1981

A genetic engineering manifesto for the genus Bacillus.

The genus Bacillus is widely studied in industry and academia because of the secretion of enzymes and antibiotics. Genetic manipulation is available in several species and genetic engineering capabilities are presently developed or under construction. Bacteriophage cloning vehicles are available which facilitate isolation and selection of individual genes and plasmid cloning vehicles are available for amplification of genes and gene products. The application of these techniques to industrially important genes is just beginning in Bacillus and continued genetic engineering in this genus promises to enhance production of numerous products in the near future.

Anti-Bacterial Agents↗
PubMed1992

Natural genetic engineering in evolution.

The results of molecular genetics have frequently been difficult to explain by conventional evolutionary theory. New findings about the genetic conservation of protein structure and function across very broad taxonomic boundaries, the mosaic structure of genomes and genetic loci, and the molecular mechanisms of genetic change all point to a view of evolution as involving the rearrangement of basic genetic motifs. A more detailed examination of how living cells restructure their genomes reveals a wide variety of sophisticated biochemical systems responsive to elaborate regulatory networks. In some cases, we know that cells are able to accomplish extensive genome reorganization within one or a few cell generations. The emergence of bacterial antibiotic resistance is a contemporary example of evolutionary change; molecular analysis of this phenomenon has shown that it occurs by the addition rearrangement of resistance determinants and genetic mobility systems rather than by gradual modification of pre-existing cellular genomes. In addition, bacteria and other organisms have intricate repair systems to prevent genetic change by sporadic physicochemical damage or errors of the replication machinery. In their ensemble, these results show that living cells have (and use) the biochemical apparatus to evolve by a genetic engineering process. Future research will reveal how well the regulatory systems integrate genomic change into basic life processes during evolution.

Animals↗
PubMed1984

A genetically engineered mutant of alpha 1-antitrypsin protects connective tissue from neutrophil damage and may be useful in lung disease.

The effectiveness of a genetically engineered mutant of human alpha 1-antitrypsin (358 Met----Val) as an inhibitor of connective tissue breakdown was tested in a model of inflammation. The degradation of basement membrane collagen by stimulated neutrophils was efficiently inhibited by a tenfold lower concentration (0.2 mg/ml) of the mutant inhibitor than of the normal alpha 1-antitrypsin (2.4 mg/ml). Effective inhibition by normal alpha 1-antitrypsin occurred at much lower concentrations when azide or catalase was added, or when normal neutrophils were replaced by those from a donor with chronic granulomatous disease. These results confirm that neutrophils augment tissue proteolysis by the oxidative inactivation of the methionine at the reactive centre of alpha 1-antitrypsin. The replacement of this methionine by valine gives an effective inhibitor that is not inactivated by neutrophil oxidants. The availability of this genetically engineered mutant suggests the possibility of prophylaxis of lung dysplasias, notably emphysema, and of the shock syndromes associated with massive neutrophil activation.

Basement Membrane↗
PubMed2003

Improving plant genetic engineering by manipulating the host.

Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to "improve" transformation by altering the bacterium, future successes might come from manipulation of the plant. Recent studies that identified several plant genes involved in Agrobacterium-mediated transformation, and their over-expression in currently transformable species, suggest that this approach holds great promise for improving the transformation of recalcitrant, but agronomically important, crops.

DNA, Bacterial↗
PubMed2005

The promise of genetically engineered mice for cancer prevention studies.

Sophisticated genetic technologies have led to the development of mouse models of human cancers that recapitulate important features of human oncogenesis. Many of these genetically engineered mouse models promise to be very relevant and relatively rapid systems for determining the efficacy of chemopreventive agents and their mechanisms of action. The validation of such models for chemoprevention will help the selection of appropriate agents for large-scale clinical trials and allow the testing of combination therapies.

Animals↗
PubMed2002

Increased production of nutriments by genetically engineered crops.

Plants are the basis of human nutrition and have been selected and improved to assure this purpose. Nowadays, new technologies such as genetic engineering and genomics approaches allow further improvement of plants. We describe here three examples for which these techniques have been employed. We introduced the first enzyme involved in fructan synthesis, the sucrose sucrose fructosyltransferase (isolated from Jerusalem artichoke), into sugar beet. The transgenic sugar beet showed a dramatic change in the nature of the accumulated sugar, 90% of the sucrose being converted into fructan. The use of transgenic sugar beet for the production and isolation of fructans will result in a more efficient plant production system of fructans and should promote their use in human food. The second example shows how the over-expression of the key enzyme of flavonoid biosynthesis could increase anti-oxidant levels in tomato. Introduction of a highly expressed chalcone isomerase led to a seventyfold increase of the amount of quercetin glucoside, which is a strong anti-oxidant in tomato. We were also able to modify the essential amino acid content of potato in order to increase its nutritional value. The introduction of a feedback insensitive bacterial gene involved in biosynthesis of aspartate family amino acids led to a sixfold increase of the lysine content. Because the use of a bacterial gene could appear to be controversial, we also introduced a mutated form of the plant key enzyme of lysine biosynthesis (dihydrodipicolinate synthase) in potato. This modification led to a 15 times increase of the lysine content of potato. This increase of the essential amino acid lysine influences the nutritional value of potato, which normally has low levels of several essential amino acids. These three examples show how the metabolism of primary constituents of the plant cell such as sugar or amino acids, but also of secondary metabolites such as flavonoids, can be modified by genetic engineering. Producing fructan, a soluble fiber, increasing the level of flavonoids, an antioxidant, in tomato or increasing the level of essential amino acids in potato are all clear examples of plant genetic modifications with possible positive effects on human nutrition.

Crops, Agricultural↗