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PubMed1990

Immobilization of genetically engineered cells: a new strategy for higher stability.

The r-DNA clones improve the bioprocess and provide better economics, if and when properly developed. In recent times, many approaches were made to improve the stability of recombinants in a reactor which includes both genetic and environmental methods, but many of them were proved to be unsuccessful in the scale-up process. The immobilization technique, exploited recently for the cultivation of recombinants, in many cases gave high cell concentrations, better expression of cloned gene products and also maintained plasmid stability for longer periods in a host under continuous operation in comparison to a free cell system. Many plasmids and hosts were tested for improved stabilities. So far, no explanation was provided for higher stability in the immobilized system. However, it was observed to reduce the competition between the plasmid harboring and plasmid free cells in a matrix. The stability of recombinant strains under immobilization technique is affected by various factors, and these are important parameters for the commercial process. Thus, the immobilization system is promising for the successful cultivation and scale-up of genetically engineered cells.

Cloning, Molecular↗
PubMed2000

Comparison of phytase from genetically engineered Aspergillus and canola in weanling pig diets.

Ninety-six crossbred pigs with an average weight of 9.0 kg were used in a 5-wk trial to compare the efficacy of genetically engineered Aspergillus ficuum phytase, expressed in Aspergillus niger (Natuphos) or in canola seed (Phytaseed), for enhancing the utilization of phytate P in corn-soybean meal-based diets fed to young pigs and to evaluate the safety of Phytaseed phytase. Three levels of the two sources of phytase (250, 500, or 2,500 U/kg of diet) were added to a corn-soybean meal basal diet containing .35% total P, .09% available P, and .50% Ca. There were six pens per treatment (one barrow and one gilt/pen), except that the diet without added phytase was fed to 12 pens of pigs. Pen feed consumption and BW were recorded weekly. During wk 5, pen fecal samples were collected for determination of apparent digestibilities of DM, Ca, and P. At the end of wk 5, all barrows were killed, and the 10th rib on both sides was removed for determination of shear force and energy. Thirty pigs (six from the diet without added phytase and the diets with 500 and 2,500 U/kg phytase from both sources) were randomly selected for gross necropsy and histologic evaluation of liver, kidney, and bone tissues. Both sources of phytase were equally effective in increasing (P < .05) daily gain, gain:feed, apparent digestibilities of DM, P, and Ca, and 10th rib measurements. Fecal P excretion was reduced with phytase addition. Feed intake was increased by phytase levels during wk 4 to 5. No significant abnormalities were seen in any of the 30 pigs necropsied. The fit of a nonlinear function revealed that most measurements were reaching a plateau at 2,500 U/kg phytase. In summary, based on performance, bone measurements, and digestibilities of P, Ca, and DM of young pigs, the efficiency of Phytaseed was similar to that of Natuphos for enhancing the utilization of phytate P in corn-soybean meal-based diets. General necropsy and histologic examination of tissues indicated no toxic effect of phytase.

6-Phytase↗
PubMed1993

Survival differences among freeze-dried genetically engineered and wild-type bacteria.

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed.

Air↗
PubMed1993

Effects of pH and acetic acid on glucose and xylose metabolism by a genetically engineered ethanologenic Escherichia coli.

Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. The patented, genetically engineered, ethanologen Escherichia coli B (pLOI297) exhibits high-performance characteristics with respect to both yield and productivity in xylose-rich lab media. In addition to producing monomer sugar residues, thermochemical processing of biomass is known to produce substances that are inhibitory to both yeast and bacteria. During prehydrolysis, acetic acid is formed as a consequence of the deacetylation of the acetylated pentosan. Our investigations have shown that the acetic acid content of hemicellulose hydrolysates from a variety of biomass/waste materials was in the range 2-10 g/L (33-166 mM). Increasing the reducing sugar concentration by evaporation did not alter the acetic acid concentration. Acetic acid toxicity is pH dependent. By virtue of its ability to traverse the cell membrane freely, the undissociated (protonated) form of acetic acid (HAc) acts as a membrane protonophore and causes its inhibitory effect by bringing about the acidification of the cytoplasm. With recombinant E. coli B, the pH range for optimal growth with glucose and xylose was 6.4-6.8. With glucose, the pH optimum for ethanol yield and volumetric productivity was 6.5, and for xylose it was 6.0 and 6.5, respectively. However, the decrease in growth and fermentation efficiency at pH 7 is not significant. At pH 7, only 0.56% of acetic acid is undissociated, and at 10 g/L, neither the ethanol yield nor the maximum volumetric productivity, with glucose or xylose, is significantly decreased. The "uncoupling" effect of HAc is more pronounced with xylose and the potency of HAc is potentiated in a minimal salts medium. Controlling the pH at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance of the recombinant biocatalyst.

Acetates↗
PubMed2004

Prospects of genetic engineering of plants for phytoremediation of toxic metals.

Bioremediation is gaining a lot of importance in recent times as an alternate technology for removal of elemental pollutants in soil and water, which require effective methods of decontamination. Phytoremediation--the use of green plants to remove, contain or render harmless environmental pollutants--may offer an effective, environmentally nondestructive and cheap remediation method. The use of genetic engineering to modify plants for metal uptake, transport and sequestration may open up new avenues for enhancing efficiency of phytoremediation. Metal chelator, metal transporter, metallothionein (MT), and phytochelatin (PC) genes have been transferred to plants for improved metal uptake and sequestration. Transgenic plants, which detoxify/accumulate cadmium, lead, mercury, arsenic and selenium have been developed. A better understanding of the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits. As more genes related to metal metabolism are discovered, facilitated by the genome sequencing projects, new vistas will be opened up for development of efficient transgenic plants for phytoremediation.

Biodegradation, Environmental↗
PubMed2001

Dermal fibroblasts genetically engineered to release nerve growth factor.

Current surgical strategies for repair of critical nerves involves the transfer of normal donor nerve from an uninjured body location. One possible alternative to autogenous tissue replacement is the development of engineered constructs to replace those elements necessary for axonal proliferation. Delivery of growth factors is one strategy to enhance synthetic nerve constructs. Thus, this study focused on the delivery of nerve growth factor (NGF) by genetic engineering to begin approaching the microenvironment dictated, in part, by Schwann's cells. Rat dermal fibroblasts (DFBs) were modified genetically to release rat NGF. The reporter gene LacZ was used to assess the optimum nonviral transfection method commercially available before NGF transfection. FuGENE6 provided the optimum transfection efficiency (24% maximum, 20.1 +/- 1.9% 5-day average) as measured by beta-galactosidase catalytic activity. NGF release from transfected DFBs was assessed over a 3-day period. Compared with control (no transfection) DFBs and DFBs transfected with vector alone, DFBs transfected with an expression vector encoding rat beta-NGF demonstrated significantly (p < 0.05) higher levels of NGF, with a 3-day maximum of 111 pg NGF per milliliter. When normalized to cell number, NGF-transfected DFBs released 1.2 pg NGF per milliliter/10(3) cells. The NGF-transfected DFBs demonstrated a maximal NGF release rate at day 1 (1.2 ng NGF/10(6) cells per day), followed by a markedly lower, sustained release rate at days 2 and 3 (0.44 ng NGF/10(6) cells per day and 0.48 ng NGF/10(6) cells per day respectively). The release rate curves for control and vector-transfected DFBs also exhibited a maximal NGF release rate at day 1, but were followed by a decreasing release rate, potentially representing in vitro degradation of NGF present in fetal bovine serum. Although not first with the development of growth factor delivery through fibroblasts, these findings suggest that rat DFBs can be modified genetically to act like Schwann's cells to deliver NGF.

Animals↗
PubMed1992

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Animals↗
PubMed1996

Negative-strand RNA viruses: genetic engineering and applications.

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.

Animals↗
PubMed2000

Genetically engineered mouse models of mammary intraepithelial neoplasia.

Foci of atypical mammary epithelium have been associated with breast cancer in many species including mouse and man. The advent of targeted genomics has led to the creation of numerous genetically engineered mice (GEM) which display focal atypical lesions associated with mammary cancer. Some early lesions in GEM have a remarkable morphological similarity to pre-cancers in humans. While the malignant potential of atypical foci have been thoroughly documented in the non-GEM by tissue transplantation, a review of the literature reveals that precursor lesions in GEM remain incompletely described and only partially documented. Their validation as appropriate models of human breast preneoplasia awaits classical transplantation studies. Here, we review the literature characterizing early lesions of GEM models of mammary cancer, discuss the principles of the Focality, Atypia, and Association and present an introduction of mammary transplantation for model Validation.

Animals↗
PubMed1991

Targeting of tumor necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibody-tumor necrosis factor hybrid molecule.

The construction, synthesis and secretion of a genetically engineered antibody-cytokine fusion molecule is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to produce a Fab-like antibody-TNF conjugate. At the gene level, the heavy chain gene of an antitransferrin receptor antibody was linked to a synthetic TNF gene encoding human TNF. Transfection of the heavy chain-TNF gene into a myeloma derived cell line which was producing the light chain of the same antibody, allowed the isolation of a cell line secreting a fusion protein of the expected molecular weight and composition. The culture supernatant of the cell line contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells. Cytotoxicity towards the human cancer cells was inhibited by an excess of the original antitransferrin receptor antibody, indicating that the antibody-TNF molecules are targeted to the transferrin receptor rich tumor cells. Since the antibody genes used are chimeric (i.e. composed of mouse variable and human constant regions) and since DNA encoding human TNF was used, the hybrid protein is an example of a humanized immunotoxin-like molecule. These results illustrate the possibilities of antibody engineering technology to create and produce improved agents for cancer therapy. Furthermore, they demonstrate for the first time the ability of myeloma cells to secrete an antibody-cytokine chimeric molecule.

Amino Acid Sequence↗
PubMed2002

Genetic engineering of fibrous proteins: spider dragline silk and collagen.

Various strategies have been employed to genetically engineer fibrous proteins. Two examples, the subject of this review, include spider dragline silk from Nephila clavipes and collagen. These proteins are highlighted because of their unique mechanical and biological properties related to controlled release, biomaterials and tissue engineering. Cloning and expression of native genes and synthetic artificial variants of the consensus sequence repeats from the native genes has been accomplished. Expression of recombinant silk and collagen proteins has been reported in a variety of host systems, including bacteria, yeast, insect cells, plants and mammalian cells. Future utility for these proteins for biomedical materials is expected to increase as needs expand for designer materials with tailored mechanical properties and biological interactions to elicit specific responses in vitro and in vivo.

Amino Acid Sequence↗
PubMed2000

Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation.

Enzymatic phosphorylation and dephosphorylation reactions were used to modify a genetically engineered variant of spider dragline silk. The approximately 25 kDa protein was phosphorylated with cyclic AMP-dependent kinase and dephosphorylated with calf intestinal alkaline phosphatase. Phosphorylation inhibited beta-sheet assembly of the protein and enhanced solubility to about 5 mg/mL in water, compared to about 20% of this level upon enzymatic dephosphorylation. The cyclability of the phosphorylation-dephosphorylation system was confirmed by MALDI with a model peptide. Kinetic studies conducted with [gamma-(32)P]ATP illustrate that the phosphorylation reaction proceeds over 6 h. Secondary structure of the phosphorylated and dephosphorylated proteins was determined by CD and FTIR. The results illustrate that an enzymatic phosphorylation event can be used to control the solution structure of a protein like silk, which has a tendency to prematurely precipitate due to the formation of beta-sheets.

Amino Acid Sequence↗
PubMed1997

A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.

A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed. Recombinant B. thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430. For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B. thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis. The resulting strain had a broader activity spectrum than that of the parental strain. It contained only B. thuringiensis DNA and was free of antibiotic resistance genes. This should facilitate regulatory approval for its development as a commercial biopesticide.

Animals↗
PubMed2005

Genetic engineering of terpenoid metabolism attracts bodyguards to Arabidopsis.

Herbivore-damaged plants release complex mixtures of volatiles that attract natural enemies of the herbivore. To study the relevance of individual components of these mixtures for predator attraction, we manipulated herbivory-induced volatiles through genetic engineering. Metabolic engineering of terpenoids, which dominate the composition of many induced plant volatile bouquets, holds particular promise. By switching the subcellular localization of the introduced sesquiterpene synthase to the mitochondria, we obtained transgenic Arabidopsis thaliana plants emitting two new isoprenoids. These altered plants attracted carnivorous predatory mites (Phytoseiulus persimilis) that aid the plants' defense mechanisms.

Animals↗
PubMed2006

A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus.

A 28-d experiment was conducted using 126 crossbred barrows to evaluate the addition of a genetically engineered Escherichia coli phytase to diets that were 0.15% deficient in available P. Growth performance, bone strength, ash weight, and the apparent absorption of P, Ca, Mg, N, energy, DM, Zn, Fe, and Cu were the response criteria. The pigs (2 pigs/pen) averaged 7.61 kg of BW and 30 d of age initially. The low-P basal diet was supplemented with 0, 100, 500, 2,500, or 12,500 units (U) of E. coli phytase/kg of diet, or 500 U of Peniophora lycii phytase/kg of diet. The positive control (PC) diet was adequate in available P. Pigs were fed the diets in meal form. Fecal samples were collected from each pig from d 22 to 27 of the experiment. There were linear and quadratic increases (P < 0.001) in 28-d growth performance (ADFI, ADG, and G:F), bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg (P < or = 0.01 for quadratic) with increasing concentrations of E. coli phytase. Pigs fed the low-P diets containing 2,500 or 12,500 U/kg of E. coli phytase had greater (P < or = 0.01 or P < 0.001, respectively) values for growth performance, bone breaking strength and ash weight, and the apparent absorption (g/d and %) of P, Ca, and Mg than pigs fed the PC diet. The addition of E. coli phytase did not increase the apparent percentage absorption of N, GE, DM, Zn, Fe, or Cu. There were no differences in the efficacy of the E. coli or P. lycii phytase enzymes at 500 U/kg of low-P diet for any criterion measured. In conclusion, there were linear increases in growth performance, bone breaking strength and ash weight, and the apparent absorption of P, Ca, and Mg with increasing addition of E. coli phytase up to 12,500 U/kg of diet. Also, all of these criteria were greater for pigs fed the low-P diets containing 2,500 or 12,500 U of E. coli phytase/kg than for pigs fed the PC diet. The addition of 500, 2,500, or 12,500 U of E. coli phytase/kg of low-P diet reduced P excretion (g/d) in manure by 35, 42, and 61%, respectively, compared with pigs fed the PC diet.

6-Phytase↗
PubMed1989

Genetically engineered (chimeric) antibodies.

The diversity, specificity, and biologic activity of antibodies make them potentially ideal reagents in the clinic and the laboratory, but conventional antibodies do not always fill the bill. Naturally occurring antibodies are heterogeneous, and human hybridoma antibodies are hard to produce. Genetic engineering techniques offer antibodies with just the properties desired.

Animals↗
PubMed2004

Genetically engineered phytoremediation: one man's trash is another man's transgene.

Plant-based environmental remediation, or phytoremediation, has been widely pursued in recent years as a favorable clean-up technology and is an area of intensive scientific investigation. For the vast majority of field applications, vegetative 'phyto-crops' are selected specifically for their capacity for site decontamination and not for additional concurrent or post-remediation utility. By contrast, a recent publication by Ellis and colleagues highlights potential anti-carcinogenic uses for plants genetically engineered primarily for detoxification of selenium-polluted soils and sediments.

Anticarcinogenic Agents↗