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PubMed1993

Kinetics of the persistence of chromosomal DNA from genetically engineered Escherichia coli introduced into soil.

Investigations to quantify bacterial survival and DNA persistence of a genetically engineered population of Escherichia coli introduced into soil microcosms were carried out. The survival of E. coli was monitored by plate counting and immunofluorescence methods, whereas the persistence of the DNA was evaluated by using a most-probable-number-polymerase chain reaction method. Whereas the E. coli population density declined below the plate-counting-technique detection threshold (10(2) CFU.g-1) after 15 days, 10(3) extracellular and 5 x 10(5) total DNA target sequences were still detected after 40 days. Additionally, the E. coli cell counts fell below the detection limit of the immunofluorescence method (10(5) cells.g-1) before the end of the experiment. Colony hybridizations did not reveal gene transfer to the indigenous microflora. These results confirm the persistence of residual E. coli target sequences that could not be detected by the classical cell counting method and offer promising applications for the environmental detection of microorganisms, either engineered, pathogenic, or released for beneficial effects.

Base Sequence↗

PubMedDate not supplied

Gene therapy to develop a genetically engineered cardiac pacemaker.

While cardiac pacemakers are frequently used for the treatment of bradydysrhythmias (from diseases of the cardiac conduction system), their use is still limited by complications that can be life-threatening and expensive. Genetic engineering approaches offer an opportunity to modulate cellular automaticity in a manner that could have significant therapeutic potential. It is well known that ventricular myocytes exhibit a more negative diastolic potential than do pacemaker cells, in large part because of the inward rectifying potassium current/K1 (which pacemaker cells lack). Taking advantage of these intrinsic electrophysiological differences, a biological pacemaker has recently been developed by Miake et al (Nature 2002; 419:132-133) using adenoviral gene transfer approaches. By isolating the gene responsible for/K1 (the Kir2.1 gene), mutating it to make it a dysfunctional channel (a dominant-negative), inserting the mutated gene into an adenoviral vector, and delivering the virus to the hearts of guinea pigs, the investigators were able to successfully convert some ventricular myocytes to pacemaker cells. While issues of safety and long-term efficacy need to be further established, the results of these experiments provide proof of principle that gene transfer offers great promise for treatment of electrophysiological disorders including conduction system disease.

Action Potentials↗

PubMed1990

Genetically engineered V79 Chinese hamster cells metabolically activate the cytostatic drugs cyclophosphamide and ifosfamide.

V79 cells, genetically engineered to express active cytochromes P450IIB1 and P450IA1, were used to study the cytotoxicity and mutagenicity of cyclophosphamide and ifosfamide. Cyclophosphamide, tested up to a concentration of 2 mM, was not cytotoxic in V79 nor in the P450IA1-expressing V79-derived cell line XEM2. Pronounced cytotoxicity was, however, observed in the P450IIB1-expressing V79-derived cell line SD1. Induction of gene mutations (acquisition of 6-thioguanine resistance) was observed in SD1 cells as well, but the effects were weak. Ifosfamide was inactive in V79 cells, but was cytotoxic in SD1 cells. Ifosfamide mustard, an active metabolite of ifosfamide, was equally cytotoxic and showed similar mutagenic effects in SD1 and parental V79 cells. The results indicate that cyclophosphamide and ifosfamide are metabolically activated by cytochrome P450IIB1. In contrast, cytochrome P450IA1 was not capable of activating cyclophosphamide. Thus, V79-derived cell lines defined for their expression of a specific form of cytochrome P-450 can be used as diagnostic tools to identify the cytochrome P-450 that is responsible for the metabolic activation of drugs.

Animals↗

PubMed1995

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs.

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Base Sequence↗

PubMed2001

A review and rationale for the use of genetically engineered animals in the study of traumatic brain injury.

The mechanisms underlying secondary cell death after traumatic brain injury (TBI) are poorly understood. Animal models of TBI recapitulate many clinical and pathologic aspects of human head injury, and the development of genetically engineered animals has offered the opportunity to investigate the specific molecular and cellular mechanisms associated with cell dysfunction and death after TBI, allowing for the evaluation of specific cause-effect relations and mechanistic hypotheses. This article represents a compendium of the current literature using genetically engineered mice in studies designed to better understand the posttraumatic inflammatory response, the mechanisms underlying DNA damage, repair, and cell death, and the link between TBI and neurodegenerative diseases.

Animals↗

PubMedDate not supplied

Antibodies and genetically engineered related molecules: production and purification.

Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification.

Animals↗

PubMedDate not supplied

Reflectance confocal microscopy for characterization of mammary ductal structures and development of neoplasia in genetically engineered mouse models of breast cancer.

The earliest steps of breast cancer begin with aberrations in mammary ductal structure. Techniques that enable an investigator to image in situ and then analyze the same tissue using biochemical tools facilitates identification of genetic networks and signaling pathways active in the imaged structure. Cellular confocal microscopy (VivaCell-TiBa, Rochester, New York) is used to image mammary ductal structures and surrounding vasculature in situ in intact wild-type and genetically engineered mice that develop ER alpha-initiated ductal carcinoma in situ (DCIS) and ER alpha-driven invasive mammary cancer. In wild-type mice, normal mammary ductal structures that appear from puberty through lactation are visualized and serially sectioned optically, and a developmental atlas is created. Altering tissue preparation enabled visualization of the vasculature surrounding the ductal structures. In the genetically engineered mice, aberrant mammary ductal structures and cancers are imaged and compared to corresponding normal structures. Different preparation techniques are able to preserve tissue for routine histological analyses and RNA isolation. Comparative studies demonstrate that reflectance confocal imaging provides more cellular detail than carmine-alum-stained mammary gland whole mounts and equivalent detail with hematoxylin and eosin stained tissue sections. In summary, reflectance confocal microscopy is a tool that can be used to rapidly and accurately analyze mammary gland structure.

Adenocarcinoma↗

PubMedDate not supplied

A total delivery system of genetically engineered drugs or cells for diseased vessels. Concept, materials, and fabricated prototype device.

The development of a percutaneous procedure using a catheterized system for diseased vessels has been increasingly in demand in conjunction with gene therapy using genetically engineered drugs (antisense) and cells. The authors' strategic concept realizes revascularization at narrowed, diseased sites and delivery of drugs or cells into the diseased tissues or targeted cells. An inflatable, drug-releasing double balloon is installed at the tip of a catheter. The outer balloon, fabricated with micropores (diameters of 20 and 30 mm) by an excimer laser ablation technique, releases a viscous solution containing a photoreactive polymer and drug or cells on inflation of the inner balloon. A photoresponsive water-soluble polymer, molecularly designed for its ability to achieve prolonged local residency of antisense DNA at the tissue level and enhanced transmembrane transport at the cellular level, is premixed with antisense oligonucleotide drug. On light irradiation, the nonionic polymer is reversibly converted to a positively charged polymer that can be complexed with highly negatively charged antisense DNA (c-myb), which may enhance the transmembrane delivery of antisense. On cessation of irradiation, the complex slowly dissociates to function intracellularly as an antisense drug, resulting in inhibition of cell proliferation. Thus, our integrated, dual-function balloon system may contribute to mechanical dilatation gene therapies at diseased vessels.

Angioplasty, Balloon, Coronary↗

PubMed1997

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa.

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Enzyme Activation↗

PubMedDate not supplied

Genetically engineered HSV in the treatment of glioma: a review.

Central nervous system malignancies, particularly glioblastoma multiforme, pose significant problems for the development of novel therapeutics. In the absence of advances with standard surgical and chemotherapeutic approaches, the utilisation of genetically engineered viruses, both as direct oncolytic agents as well as for the delivery of foreign proteins, represents a significant advance in the experimental approach to management of patients with these incurable tumours. Among other viruses, HSV offers an opportunity to directly influence the replication of tumour cells within the central nervous system. Because of its propensity to replicate in neuronal tissue as well as its large coding capacity, it provides an experimental model for the development of novel therapeutics. The status of these experimental approaches will be summarised in this review.

Animals↗

PubMedDate not supplied

[Achievements and problems of genetic engineering of Crucifereceae plants].

Plants of the Brassicaceae family are important oil, vegetable and feed crops. The review is devoted to the latest achievements in genetic engineering of plants from this family. Results concerning development of effective methods both of Agrobacteium-mediated transformation and of direct gene uptake are considered. Particularly, possibilities of plant genetic modification with the aim to improve agronomically and commercially important traits are stressed. Problems of biologically safe introduction of transgenic plants into agricultural production are discussed.

Biotechnology↗

PubMed2003

Phytoremediation of organomercurial compounds via chloroplast genetic engineering.

Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 micro M PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 micro M PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3'-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.

Biodegradation, Environmental↗

PubMed2001

[Genetically engineered drugs and their application with the example of erythropoietin].

Over the past two decades, many genetically engineered drugs have been developed and approved for the treatment of patients. Typically, these drugs are characterized by a high and specific activity in the presence of optimal safety. They include hormones, enzymes, growth and coagulation factors, antibodies as well as vaccines. All these proteins are generated using recombinant DNA technology. An expression vector with the gene encoding for the protein of interest is introduced into an appropriate microorganism or cell line. The biochemical machinery of the host cell then translates the genetic information into the corresponding protein. Large scale production of the recombinant drugs uses biotechnological processes. The genetically modified organisms are grown in bioreactors from which the desired protein is finally isolated and purified. This review focuses on the production and clinical application of recombinant erythropoietin in the areas of nephrology, hemato-oncology and elective surgery.

Biotechnology↗

PubMed2006

Genetically engineered avidins and streptavidins.

Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, K(d) approximately 10(-14)-10(-16) M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology.

Amino Acid Sequence↗

PubMed1994

An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems.

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20-30 days after injection was proven by PCR analysis.

Base Sequence↗

PubMed2001

Genetically engineered human herpes simplex virus in the treatment of brain tumours.

Central nervous system malignancies--particularly glioblastoma multiforme--pose significant problems for the development of novel therapeutics. In the absence of advances with standard surgical and chemotherapeutic approaches, the utilization of genetically engineered viruses--both as direct oncolytic agents (virus therapy) and for the delivery of foreign proteins (gene therapy)--represents a significant advance in the experimental approach to the management of patients with incurable tumours. Among other viruses, herpes simplex virus (HSV) offers an opportunity to influence the replication of tumour cells directly within the central nervous system. The propensity for HSV to replicate in tumour cells, and its large coding capacity, provide an experimental model for the development of novel therapeutics. The status of these experimental approaches and Phase I studies are summarized.

Animals↗

PubMedDate not supplied

Use of genetically engineered mice in drug discovery and development: wielding Occam's razor to prune the product portfolio.

Genetically engineered mice (GEMs) that either overexpress (transgenic) or lack (gene-targeted, or "knock-out") genes are used increasingly in industry to investigate molecular mechanisms of disease, to evaluate innovative therapeutic targets, and to screen agents for efficacy and/or toxicity. High throughput GEM construction in drug discovery and development (DDD) serves two main purposes: to test whether a given gene participates in a disease condition, or to determine the function(s) of a protein that is encoded by an expressed sequence tag (EST, an mRNA fragment for a previously uncharacterized protein). In some instances, phenotypes induced by such novel GEMs also may yield clues regarding potential target organs and toxic effects of potential therapeutic molecules. The battery of tests used in phenotypic analysis of GEMs varies between companies, but the goal is to define one or more easily measured endpoints that can be used to monitor the disease course--especially during in vivo treatment with novel drug candidates. In many DDD projects, overt phenotypes are subtle or absent even in GEMs in which high-level expression or total ablation of an engineered gene can be confirmed. This outcome presents a major quandary for biotechnology and pharmaceutical firms: given the significant expense and labor required to generate GEMs, what should be done with "negative" constructs? The 14th century philosophical principle known as Occam's razor-that the simplest explanation for a phenomenon is likely the truth-provides a reasonable basis for pruning potential therapeutic molecules and targets. In the context of DDD, Occam's razor may be construed to mean that correctly engineered GEMs lacking obvious functional or structural phenotypes have none because the affected gene is not uniquely essential to normal homeostasis or disease progression. Thus, a "negative" GEM construct suggests that the gene under investigation encodes a ligand or target molecule without significant therapeutic potential. This interpretation indicates that, at least in a market-driven industrial setting, such "negative" projects should be pruned aggressively so that resources may be redirected to more promising DDD ventures.

Animals↗

PubMed2001

What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function.

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.

Bacteriophage lambda↗