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PubMed1999

The development of and perspectives for genetic engineering of malaria parasites.

The genetic manipulation of malaria parasites is a rapidly emerging technology that offers great promise for the investigation of many aspects of infection. Currently it is possible to transform avian, rodent, primate as well as human parasites, the latter three on a stable, drug selectable basis. This review focuses on the history of the development of the technology, current abilities and future perspectives.

Animals↗

PubMed1999

Encapsulated, genetically engineered cells, secreting glucagon-like peptide-1 for the treatment of non-insulin-dependent diabetes mellitus.

Non-insulin-dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose-induced insulin secretion by pancreatic beta cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon-like peptide-1 (GLP-1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP-1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP-1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP-1 which has a longer half-life in vivo but which is as potent as the wild-type peptide. The genetically engineered cells are then encapsulated in semi-permeable hollow fibers for implantation in diabetic hosts for constant, long-term, in situ delivery of the peptide. This approach may be a novel therapy for type II diabetes.

Animals↗

PubMed1999

Mammalian spermatogenesis investigated by genetic engineering.

Genes involved in mammal spermatogenesis can now be identified through mutants created by genetic engineering. Information has been obtained on male meiosis, but also on the factors regulating the proliferation, maintenance and differentiation of male germ cells. Its has also increased our knowledge of the germ cell phenotype emerging from an altered germ cell genotype. This review is focused on data from genes expressed in male germ cells and on the question of how germ cells and Sertoli cells cope with the molecular lesions induced. The conservation of a wild-type phenotype of male germ cells in mutant mice is discussed, and how the mouse genetic background can lead to different germ cell phenotypes for a given gene mutation.

Animals↗

PubMedDate not supplied

Rescue of motoneurons from axotomy-induced cell death by polymer encapsulated cells genetically engineered to release CNTF.

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) results from the progressive loss of motoneurons, leading to death in a few years. Ciliary neurotrophic factor (CNTF), which decreases naturally occurring and axotomy-induced cell death, may result in slowing of motoneuron loss and has been evaluated as a treatment for ALS. Effective administration of this protein to motoneurons may be hampered by the exceedingly short half-life of CNTF, and the inability to deliver effective concentration into the central nervous system after systemic administration in vivo. The constitutive release of CNTF from genetically engineered cells may represent a solution to this delivery problem. In this work, baby hamster kidney (BHK) cells stably tranfected with a chimeric plasmid construct containing the gene for human or mouse CNTF were encapsulated in polymer fibers, which prevents immune rejection and allow long-term survival of the transplanted cells. In vitro bioassays show that the encapsulated transfected cells release bioactive CNTF. In vivo, systemic delivery of human and mouse CNTF from encapsulated cells was observed to rescue 26 and 27% more facial motoneurons, respectively, as compared to capsules containing parent BHK cells 1 wk postaxotomy in neonatal rats. With local application of CNTF on the nerve stump and by systemic delivery through repeated subcutaneous injections, 15 and 13% more rescue effects were observed. These data illustrate the potential of using encapsulated genetically engineered cells to continuously release CNTF to slow down motoneuron degeneration following axotomy and suggest that encapsulated cell delivery of neurotrophic factors may provide a general method for effective administration of therapeutic proteins for the treatment of neurodegenerative diseases.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Safety assessment of genetically engineered food: detection and monitoring of glyphosate-tolerant soybeans.

A detection technique for the genetically engineered food, glyphosate-tolerant soybean (GTS), was designed. Commercial soybeans imported from North America were cultured in pots and genomic DNA was isolated from their leaves. To detect the genes, promoter and terminator, involved in the expression of glyphosate tolerance, PCR was done using the genomic DNA and chemically synthesized primers specific to the genes. DNAs with predicted sizes were amplified and confirmed by DNA sequencing to be the genes responsible for the expression of glyphosate tolerance. Glyphosate-tolerant soybeans were found to form approximately 1.1% of the commercial soybeans, when commercially available soybeans were cultivated and number of soybeans resistant to glyphosate was found. This level is somewhat lower than an estimated value announced officially on the basis of the cultivation area of the glyphosate-tolerant soybeans.

Base Sequence↗

PubMed1995

Genetic elements of plant viruses as tools for genetic engineering.

Viruses have developed successful strategies for propagation at the expense of their host cells. Efficient gene expression, genome multiplication, and invasion of the host are enabled by virus-encoded genetic elements, many of which are well characterized. Sequences derived from plant DNA and RNA viruses can be used to control expression of other genes in vivo. The main groups of plant virus genetic elements useful in genetic engineering are reviewed, including the signals for DNA-dependent and RNA-dependent RNA synthesis, sequences on the virus mRNAs that enable translational control, and sequences that control processing and intracellular sorting of virus proteins. Use of plant viruses as extrachromosomal expression vectors is also discussed, along with the issue of their stability.

DNA Replication↗

PubMedDate not supplied

Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor.

Escherichia coli cells engineered to express an Hg2+ transport system and metallothionein accumulated Hg2+ effectively over a concentration range of 0.2-4 mg/L in batch systems. Bioaccumulation was selective against other metal ions and resistant to changes in ambient conditions such as pH, ionic strength, and the presence of common metal chelators or complexing agents (Chen, S.-L.; Wilson, D. B. Appl. Environ. Microbiol. 1997, 63, 2442-2445; Biodegradation 1997, 8, 97-103). Here we report the characterization of the bioaccumulation system based on its kinetics and an isotherm. Bioaccumulation was rapid and followed Michaelis-Menten kinetics. A hollow fiber bioreactor was constructed to retain the genetically engineered cells. The bioreactor was capable of removing and recovering Hg2+ effectively at low concentrations, reducing a 2 mg/L solution to about 5 microgram/L. A mathematical equation that quantitatively described Hg2+ removal by the bioreactor provides a basis for the optimization and extrapolation of the bioreactor. The genetically engineered E. colicells and the bioreactor system have excellent properties for bioremediation of Hg2+-contaminated environments.

Bioreactors↗

PubMed2003

Site-specific selfish genes as tools for the control and genetic engineering of natural populations.

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Animals↗

PubMed1998

Apoptosis-mediated regulation of recombinant human granulocyte colony-stimulating factor production by genetically engineered fibroblasts.

We investigated the feasibility of an inducible apoptosis system to regulate cells genetically engineered for ectopic cytokine production. In a previous study, cDNA encoding the ligand-binding domain of the rat estrogen receptor was fused to the sequence for murine Fas transmembrane and cytoplasmic regions, and expression of the fusion protein (MfasER) in L929 fibroblasts resulted in estrogen-dependent apoptosis. We applied this MfasER/estrogen strategy to apoptosis-mediated regulation of cytokine production, using the human granulocyte colony-stimulating factor (G-CSF) as a model. Upon estrogen treatment, the G-CSF producers expressing MfasER showed an apoptotic phenotype and died in several hours, with termination of G-CSF production. This estrogen-induced apoptosis was not influenced by whether the target cells were proliferating or resting, unlike a conventional suicide system involving the herpes simplex virus 1 thymidine kinase (HSVtk). That is, estrogen induced prompt and extensive apoptosis in the resting cells which expressed MfasER, while ganciclovir treatment induced only partial reduction of the resting cells which expressed HSVtk. These results imply the feasibility of apoptosis-mediated regulation of cytokine production by genetically modified cells for supplement gene therapy.

Animals↗

PubMed2006

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon.

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30 degrees C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10(5)-10(7) CFU ml(-1)), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30 degrees C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg(-1) MP and 25 mg kg(-1) carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.

Biodegradation, Environmental↗

PubMed1994

High density cultivation of genetically-engineered CHO cell lines with microcarrier culture systems.

Genetically-engineered CHO cell lines, r beta-13 and CLF-8B2, were cultivated with the MC-1 microcarrier culture system. The cell density could be enhanced by increasing the concentration of microcarrier. At a microcarrier concentration of 10 mg/ml, the cell density could reach 4 to 5 x 10(6) cells/ml. It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers. We were thus able to scale up cultivation using a simple method, i.e. by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume. Using a perfusion culture system, we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0.5 to 3 working volumes. Prourokinase was stably secreted.

Animals↗

PubMedDate not supplied

Impact of genetic engineering on the understanding of spermatogenesis.

To date, about 100 genes have been found, by genetic engineering, to be implicated in spermatogenesis. Primordial germ cells, spermatogonia, spermatocytes I and elongating spermatids are particularly sensitive. Transgenic and knockout mice permit an approach to be made to the question of genetic factors involved in DNA damage repair, thermal injury, sperm chromatin compaction and sex-specific recombination. Knockout mice reveal unexpected functional redundancies of testis-specific genes. This review considers how functional divergences can exist among homologous genes from different species, and to what extent the phenotypes of knockout mice can be similar to those from spontaneous mutations. Additional anomalies in reproductive function have frequently been found in these mice, as were found factors leading to tumour susceptibility and/or various diseases. Finally, knockout mice remind us that, in nearly all cases, hemizygous individuals retain a fertility and a wild-type sperm phenotype, although half of the spermatozoa share a genetic defect. The findings strongly emphasize the importance of understanding epidemiology in male infertility, to identify hereditary forms of impaired spermatogenesis, and to create DNA and pathological germ cell banks.

Animals↗

PubMed1992

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.

Biodegradation, Environmental↗

PubMed1995

Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN-gamma, IL-4, IL-6, or TNF-alpha.

Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.

3T3 Cells↗

PubMed1989

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants.

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].

Base Sequence↗

PubMed1996

Genetically engineered smooth muscle cells as linings to improve the biocompatibility of cardiovascular prostheses.

BACKGROUND: The seeding of the blood-contacting surfaces of cardiovascular prostheses with autologous endothelial cells to improve their biocompatibility has had little success. In most instances, cells have sloughed off under flow conditions. The performance of left ventricular assist devices (LVADs) designed to stabilize patients awaiting donor hearts for transplantation has been remarkably good. After prolonged implantation, pump surfaces become covered with a pannus of smooth muscle-like cells (myofibroblasts). Occasional islands of endothelial cells have been identified on top of such cell layers. Therefore, in an attempt to accelerate the beneficial conditioning and improve biomaterial-blood compatibility of LVAD internal surfaces, their seeding with autologous, genetically engineered smooth muscle cells (SMCs) was investigated. METHODS AND RESULTS: Since routine testing of the Thermocardiosystems HeartMate LVAD is carried out in calves, SMCs were isolated from calves, propagated in culture, and transduced with NO synthase genes to yield stable production of NO. Previous studies had demonstrated that SMCs attached strongly to the biomaterials that compose the internal surfaces of LVADs. Transduction of NO synthase gene expression in the SMCs was achieved by electroporation and antibiotic (G418) selection. Inhibition of smooth muscle cell proliferation by NO has been documented, and the same molecule has been shown to inhibit platelet adhesion to cell surfaces. Cells transduced with NO synthase expressed enzyme protein at consistently high levels for several passages in culture; however, NO production was dependent on the supplementation of culture medium with a source of tetrahydrobiopterin (sepiapterin). Under such conditions, transduced cells were growth-inhibited compared with mock-transfected controls. Induction of GTP cyclohydrolase (the rate-limiting enzyme for the production of tetrahydrobiopterin) expression also resulted in NO production by NO synthase-transduced cells. CONCLUSIONS: Preliminary studies have shown that SMCs form strong attachments to the surface materials of LVADs and that their proliferation rates could be controlled after transformation with NO synthase under conditions that support production of NO. Therefore, genetically engineered SMCs may provide an improved blood biomaterial interface for cardiovascular prostheses.

Animals↗

PubMed2002

Role of human cytochrome P450 (CYP) in the metabolic activation of nitrosamine derivatives: application of genetically engineered Salmonella expressing human CYP.

The role of human cytochrome P450 (CYP) in the metabolic activation of tobacco-related N-nitrosamines was examined by Salmonella mutation test using a series of genetically engineered Salmonella typhimurium YG7108 strains each co-expressing a form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) together with human NADPH-cytochrome P450 reductase. Seven tobacco-related N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosonornicotine, N-nitrosoanabasine, and N-nitrosoanatabine were used. The CYP2A6 was found to be responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined. On the basis of the evidence, genetic polymorphism of the CYP2A6 gene appeared to be one of the factors determining cancer susceptibility caused by smoking. Previously, we found the whole deletion of the CYP2A6 gene (CYP2A6*4C) as a type of genetic polymorphism in Japanese. We hypothesized that individuals possessing the gene homozygous for CYP2A6*4C were incapable of activating tobacco-related N-nitrosamines and showed lower susceptibility to lung cancer induced by tobacco smoke. Thus, the relationship between the CYP2A6*4C and the susceptibility to the lung cancer was evaluated. The frequency of the CYP2A6*4C was significantly lower in the lung cancer patients than healthy volunteers, suggesting that the subjects carrying the CYP2A6*4C alleles are resistant to carcinogenesis caused by N-nitrosamines because of the poor metabolic activation capacity. Taking these results into account, CYP2A6 is an enzyme enhancing lung cancer risk.

Aryl Hydrocarbon Hydroxylases↗

PubMed2002

Insights into the regulation of gastric acid secretion through analysis of genetically engineered mice.

The regulation of acid secretion in the stomach involves a complex network of factors that stimulate secretion in response to the ingestion of a meal and maintain homeostasis of gastric pH. Genetically engineered mouse models have provided a new opportunity to investigate the importance and function of specific molecules and pathways involved in the regulation of acid secretion. Mouse mutants with disruptions in the three major stimulatory pathways for acid secretion in parietal cells, gastrin, histamine, and acetylcholine, have been generated. Disruption of the gastrin pathway results in a major impairment in both basal and induced acid secretion. Histamine and acetylcholine pathway mutants also have significant alterations in acid secretion, although the impairment does not appear to be as severe as in gastrin pathway mutants, perhaps due in part to the hypergastrinemia that occurs. Mice with a disruption in the somatostatin pathway have increased gastric acid secretion, which confirms an important negative regulatory role for this factor. This review discusses these genetically engineered mouse models, as well as others, that provide insight into the complex regulation of in vivo gastric acid secretion. The regulation of growth and cellular morphology of the stomach in these mouse models is also presented. In addition, transgene promoters that are expressed in the gastric epithelium are discussed because these promoters will be important tools to alter cellular physiology in new mouse models in the future.

Acetylcholine↗