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At least 667 records · Page 37Linked to original sources
PubMed2005

Genetically engineered dopamine beta-hydroxylase gene promoters with better PHOX2-binding sites drive significantly enhanced transgene expression in a noradrenergic cell-specific manner.

A continuously growing body of evidence suggests that dysregulation of noradrenergic (NA) neurons is implicated in the etiology and pathophysiology of various human diseases such as depression, drug addiction, and autonomic dysfunction. An efficient NA neuron-specific promoter is potentially valuable to investigate the precise role of NA neurons in normal as well as in diseased brain and to treat the associated disorders by gene therapy. In this study, we tested a novel strategy to modify genetically the promoter of the human dopamine beta-hydroxylase (hDBH) gene to overcome its inherent weakness while maintaining its cell-type specificity. We optimized the nucleotide sequence motifs of PHOX2-binding sites (PRS2 and PRS3) residing within the hDBH promoter. Optimization of both PRS2 and PRS3 motifs significantly increased their binding affinities to PHOX2A, leading to a dramatic increase in the promoter strength (>20-fold). More importantly, these modifications do not alter the level of transgene expression in non-NA cells either in vitro or in vivo, demonstrating tight cell-type specificity. This work shows that a cellular gene promoter can be genetically modified to strengthen its promoter activity without losing cell-type specificity by optimizing critical cis-regulatory elements. Our genetically engineered promoter may be useful for cell-type-specific gene targeting as well as for generating in vivo animal models with altered gene expression in a specific cell type.

Animals↗
PubMed1996

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.

Animals↗
PubMed1991

Cellular replacement therapy for neurologic disorders: potential of genetically engineered cells.

Neural transplantation, a mode of cellular replacement, has been used as a therapeutic trial for Parkinson's disease. Studies indicate that tonic release of the metabolites from the graft that can be utilized by the host brain, is likely to be the major mechanism responsible for the therapeutic effect. The use of fetal tissue is complicated by ethical controversy and immunological incompatibility. Autografting adult tissue has not been successful mainly due to poor survival. Genetically engineered cells are promising alternative sources of donor cells. We have investigated the potential of primary skin fibroblasts as donor cells for intracerebral grafting. Primary skin fibroblasts survive in the brain and remain in situ. A number of genes (nerve growth factor, tyrosine hydroxylase, glutamic acid decarboxylase, and choline acetyltransferase) have been successfully introduced and expressed in the primary fibroblasts. The L-dopa-secreting primary fibroblasts exhibited a behavioral effect in a rat model of Parkinson's disease up to 8 weeks after being grafted into denervated striatum. Factors that can maximize gene transfer, transgene expression, and fibroblast survival in the brain make up the future direction of investigation.

Animals↗
PubMed2000

Antitumour effects of genetically engineered Salmonella in combination with radiation.

The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with X-rays in mice bearing B16F10 or Cloudman S91 melanomas. Each treatment alone slowed tumour growth and prolonged survival, and the combined treatments produced supra-additive antitumour effects. That is, in dose-response studies with single doses of Salmonella and increasing doses of radiation, the two agents together caused suppression of tumour growth that was greater than that calculated for additivity. The results suggest that the combination of these genetically engineered Salmonella with radiotherapy could be a new and beneficial treatment for solid tumours.

Animals↗
PubMedDate not supplied

Genetically engineered fluorescent cell marker for labeling CD34+ hematopoietic stem cells.

To address the challenge of labeling and tracking stem cells in vivo, we have engineered a fluorescent cell marker CD34EGFP by utilizing the mechanism of the cell-specific activity of CD34 promoter in CD34(+) stem cells. A retroviral vector derived from a murine stem cell virus was constructed to integrate the CD34EGFP gene into the genome of the cells for labeling. Our experiment demonstrates that the 454 bp segment upstream of the murine CD34 cDNA sequence has full function of promoter activity and can serve as a cell-specific promoter for driving the expression of EGFP in CD34(+) hematopoietic stem cells (HSC), providing a living color for labeling stem cells. The CD34EGFP marker was tested in various types of cells, including terminally differentiated cells, CD34(+) mouse myeloid leukemia progenitor cells, CD34(-) hematopoietic cells, and CD34(+) HSCs. We show that the engineered CD34EGFP cell marker is expressed in the CD34(+) stem or progenitor cells but not in CD34(-) or terminally differentiated cells. RT-PCR assay indicates that the transcription level of the CD34EGFP gene from CD34 promoter is almost the same as that from CMV promoter in CD34(+) progenitor cells. The approach we present here offers a framework for genetic engineering of fluorescent cell markers for labeling and tracking stem cells in vivo. We anticipate that a variety of cell markers could be generated by coupling variants of fluorescent proteins with various cell-specific promoters.

Animals↗
PubMed2004

Genetically engineered negative signaling molecules in the immunomodulation of allergic diseases.

PURPOSE OF REVIEW: This review summarizes current knowledge regarding the control of human mast cell and basophil signaling and recent developments using a new therapeutic platform consisting of a human bifunctional gamma and epsilon heavy chain (Fc gamma-Fc epsilon) protein to inhibit allergic reactivity. RECENT FINDINGS: Crosslinking of Fc gamma RIIb to Fc epsilon RI on human mast cells and basophils by a genetically engineered Fc gamma-Fc epsilon protein (GE2) leads to the inhibition of mediator release upon Fc epsilon RI challenge. GE2 protein was shown to inhibit cord blood-derived mast cell and peripheral blood basophil mediator release in vitro in a dose-dependent fashion, including inhibition of human IgE reactivity to cat. IgE-driven mediator release from lung tissue was also inhibited by GE2. The mechanism of inhibition in mast cells included alterations in IgE-mediated Ca mobilization, spleen tyrosine kinase phosphorylation and the formation of downstream of kinase-growth factor receptor-bound protein 2-SH2 domain-containing inositol 5-phosphatase (dok-grb2-SHIP) complexes. Proallergic effects of Langerhan's like dendritic cells and B-cell IgE switching were also inhibited by GE2. In vivo, GE2 was shown to block passive cutaneous anaphylaxis driven by human IgE in mice expressing the human Fc epsilon RI and inhibit skin test reactivity to dust mite antigen in a dose-dependent manner in rhesus monkeys. SUMMARY: The balance between positive and negative signaling controls mast cell and basophil reactivity, which is critical in the expression of human allergic diseases. This approach using a human Fc gamma-Fc epsilon fusion protein to co-aggregate Fc epsilon RI with the Fc gamma RII holds promise as a new therapeutic platform for the immunomodulation of allergic diseases and potentially other mast cell/basophil-dependent disease states.

Animals↗
PubMed1994

Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.

BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.

Escherichia coli↗
PubMed1996

Improved production of phenomycin by a genetically engineered Escherichia coli.

We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.

Anti-Bacterial Agents↗
PubMed1993

Genetically engineered microorganisms to rescue plants from frost injury.

Ice nucleation active bacteria belonging to genera Pseudomonas, Xanthomonas and Erwinia contribute to frost damage to plants by initiating the formation of ice in plants that would otherwise supercool and avoid the damaging ice formation. The biological control of frost injury can be achieved by the application of non-ice nucleation active bacteria to the plant surfaces before they become colonized by Ice+ species. ice genes have been cloned from Pseudomonas and isogenic Ice- derivatives constructed via genetic manipulations. These genetically engineered microorganisms (GEMs) have been released into the environment to control the frost damage. The incidence of frost injury to the plants has, thereby, been reduced by 50-85% during natural frosts. These GEMs do not survive in soil and show no aerial dispersal in the environment.

Amino Acid Sequence↗
PubMed2006

The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation.

PsEND1 is a pea anther-specific gene that displays very early expression in the anther primordium cells. Later on, PsEND1 expression becomes restricted to the epidermis, connective, endothecium and middle layer, but it is never observed in tapetal cells or microsporocytes. We fused the PsEND1 promoter region to the cytotoxic barnase gene to induce specific ablation of the cell layers where the PsEND1 is expressed and consequently to produce male-sterile plants. Expression of the chimaeric PsEND1::barnase gene in two Solanaceae (Nicotiana tabacum and Solanum lycopersicon) and two Brassicaceae (Arabidopsis thaliana and Brassica napus) species, impairs anther development from very early stages and produces complete male-sterile plants. The PsEND1::barnase gene is quite different to other chimaeric genes previously used in similar approaches to obtain male-sterile plants. The novelty resides in the use of the PsEND1 promoter, instead of a tapetum-specific promoter, to produce the ablation of specific cell lines during the first steps of the anther development. This chimaeric construct arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. This strategy represents an excellent alternative to generate genetically engineered male-sterile plants, which have proved useful in breeding programmes for the production of hybrid seeds. The PsEND1 promoter also has high potential to prevent undesirable horizontal gene flow in many plant species.

Arabidopsis↗
PubMed2007

Functionality of the beta/six site-specific recombination system in tobacco and Arabidopsis: a novel tool for genetic engineering of plant genomes.

The beta recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the beta recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-beta recombinase fusion which can localise to the cell nucleus. Beta recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, beta recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, beta recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional / recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the beta/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.

Amino Acid Sequence↗
PubMed2000

Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance.

Metabolic acclimation via the accumulation of compatible solutes is regarded as a basic strategy for the protection and survival of plants in extreme environments. Certain plants accumulate significant amounts of glycinebetaine (betaine), a compatible quaternary amine, in response to high salinity, cold and drought. It is likely that betaine is involved in the protection of macrocomponents of plant cells, such as protein complexes and membranes, under stress conditions. Genetic engineering of the biosynthesis of betaine from choline has been the focus of considerable attention as a potential strategy for increasing stress tolerance in stress-sensitive plants that are incapable of synthesizing this compatible/protective solute. Three distinct pathways for the synthesis of betaine have been identified in spinach, Escherichia coli and Arthrobacter globiformis, and various genes and cDNAs for the proteins involved are available. Moreover, each of the pathways has been exploited to a greater or lesser extent in efforts to convert betaine-deficient plants to betaine accumulators. In this review, the potential of several recent examples of transgenic approaches to the enhancement of stress tolerance in plants is summarized and discussed.

Adaptation, Physiological↗