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Results for “Genetic Engineering”

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PubMed1992

Construction of a genetically engineered strain producing propionylspiramycin.

Our work has demonstrated the cloning of propionyl acylase gene and the expression of S. mycarofaciens mutant in S. lividans TK54. In this paper, we report the transformation of pIJM9 recombinant plasmid containing the propionyl acylase gene into spiramycin producer S. spiramyceticus. The results of colony hybridization and Southern hybridization showed that No. 61 transformant harbored the pIJM9 recombinant plasmid. TLC and bioautography showed that the Rf value of one component of the fermentation products of No. 61 transformant was similar to that of propionylspiramycin. The HPLC retention time of the components of the fermentation products of No. 61 transformant and that of propionylspiramycin were also similar. Mass spectrum analysis showed that there was propionylspiramycin II in the fermentation products of No. 61 transformant. According to these results, No. 61 transformant is shown to be a genetic engineered strain producing propionylspiramycin.

Acyltransferases↗

PubMed2000

Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease.

We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.

Animals↗

PubMed1989

Growth of genetically engineered Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere.

The effect of the addition of a recombinant plasmid containing the pglA gene encoding an alpha-1,4-endopolygalacturonase from Pseudomonas solanacearum on the growth of Pseudomonas aeruginosa and Pseudomonas putida in soil and rhizosphere was determined. Despite a high level of polygalacturonase production by genetically engineered P. putida and P. aeruginosa, the results suggest that polygalacturonase production had little effect on the growth of these strains in soil or rhizosphere.

Genetic Engineering↗

PubMed2006

Use of genetically engineered bone-marrow stem cells to treat femoral defects: an experimental study.

BACKGROUND: Treatment of osteonecrosis continues to be a challenging problem. The replacement of necrotic bone with graft materials that promote osteogenesis and angiogenesis may provide better outcomes for early stage disease. In this study, genetically engineered bone-marrow stem cells were used to enhance repair of a defect in the distal aspect of the femur. METHODS: Cloned bone-marrow stem cells were transfected with traceable genes. Osteoblastic and angiogenic properties of the cells were analyzed. A defect was created bilaterally in the distal portion of the femur of twenty-four mice to mimic a core decompression procedure. The cloned cells were transplanted into each defect of the right femur while the left femur served as control. Bone formation was evaluated radiographically and histomorphometrically. In addition, in twenty-four additional mice, the cells were injected into subcutaneous sites, muscles, and into the renal capsule (eight mice in each group) to evaluate ectopic osteogenesis. RESULTS: Radiopaque tissue appeared two weeks after the cells were transplanted into bone defects and at ectopic sites. Histologic analysis demonstrated that these tissues consisted of newly formed bone from transplanted cells that expressed traceable genes. Four of six bone defects that received cell transplantation were filled with new bone at four weeks, and all of the defects (n = 6) demonstrated complete healing at six weeks. On the control side, complete repair was seen in only two of six bone defects at four weeks and in three of six defects at six weeks. Histomorphometric analysis showed that transplantation of marrow stem cells into bone defects produced more bone at an earlier time-point than occurred in the controls. CONCLUSIONS: This study demonstrated that cloned bone-marrow stem cells can directly form bone after transplantation into bone defects and at ectopic sites, indicating that the in vitro expanded bone-marrow stem cells can serve as a graft material to enhance bone repair and to treat osteonecrosis. CLINICAL RELEVANCE: As an alternative graft material, bone-marrow stem cells may provide new and as yet technologically unachievable solutions to many clinical problems in the areas of musculoskeletal reconstruction and tissue regeneration.

Animals↗

PubMed1996

Anticipating public reaction to the use of genetic engineering in infant nutrition.

Public acceptance of new food products will be the major determinant of their commercial success. I present selected findings from two national social science research projects aimed at gauging consumer knowledge of and understanding consumer attitudes about biotechnology, as well as about the specific use of genetic engineering in milk production. People are more willing to accept products if they understand them and are assured by credible sources that such products are safe and effective. Perceived health concerns and ethical issues will be important to many people and are an important challenge to acceptance. Women tend to be more skeptical about these new products than are men. I make recommendations for more-effective educational efforts, based on social science research, that could increase acceptance by key consumer groups and, ultimately, the commercial success of bioengineered food products.

Biotechnology↗

PubMed2004

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma.

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.

CD3 Complex↗

PubMed2002

Comparison of mixed cell culture containing genetically engineered BGMK and CaCo-2 cells (Super E-Mix) with RT-PCR and conventional cell culture for the diagnosis of enterovirus meningitis.

BACKGROUND: Enteroviral meningitis has traditionally been diagnosed by cell culture. More recently, molecular techniques and shell vials with a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (BGMK cells) (Super E-Mix) have been described. OBJECTIVE: We compared the results of this new cell culture technique with two reverse transcriptase polymerase chain reaction (RT-PCR) techniques and conventional cell culture to assess the accuracy of these various methods. STUDY DESIGN: 2 ml of cerebrospinal fluid (CSF) was obtained from 72 patients with suspected viral meningitis. 600 microl was used for conventional cell culture and 200 microl was inoculated to each of two Super E-Mix vials. One vial was incubated for 24 h, then stained with pan-enterovirus antibody, and the other was examined for CPE daily for 5 days and stained when positive or at the end of incubation. The final aliquot (500 microl) was centrifuged at 25,000g, and the pellet in 140 microl of supernatant was used for ribonucleic acid (RNA) extraction and tested by commercial single-step (Chemicon, Temecula, CA) and two-step (Argene, Varilhes, France) RT-PCR methods. RESULTS: Conventional culture was 51% sensitive and 100% specific. The Super E-Mix was slightly higher (sensitivity 76%, specificity 100%). The Chemicon and Argene PCR methods had a sensitivity of 93% and 88% and specificity of 90% and 97%, respectively. CONCLUSION: The Super E-Mix procedure had greater sensitivity than conventional cell culture, but RT-PCR was the most sensitive technique with this type of specimen. The RT-PCR methods performed equally.

Adolescent↗

PubMed2000

Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor.

The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris. Restriction endonuclease sites located at both 5'- and 3'-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e. EcoRI and SmaI or AvrII). The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1. An OX26 scFv high producer clone [GS115 His+ Mut+ (pPIC-OX26 SacI)] was isolated and used for large-scale production and characterization. Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography. The identity of the OX26 scFv was confirmed by Western blot analyses with both anti c-myc and anti poly-His antibodies. Minor immunoreactive bands corresponding to hyperglycosylated and partially processed alpha-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7. The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.

Antibodies↗

PubMed2005

Biodegradation of azo dyes by genetically engineered azoreductase.

A azoreductase gene with 537 bp was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was efficiently expressed in Escherichia coli and its biodegradation characteristics for azo dyes were investigated. Furthermore, the reaction kinetics and mechanism of azo dyes catalyzed by the genetically engineered azoreductase were studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.

Azo Compounds↗

PubMed1989

[Risk assessment of the industrial application of genetically engineered microorganisms].

Factors affecting the persistence, dissemination of GEM's and their gene transfer to indigenous microorganisms are important in assessing potential risks posed by environmental release of GEM's. Risk assessment should essentially contain case studies of the extent and ways of release, GEM stability and gene transfer under laboratory and in situ-conditions. Some aspects of a risk assessment study for the industrial production of alpha-Amylase from a genetically engineered Bacillus subtilis strain are discussed.

Bacteria↗

PubMed2006

Microfluidic protein detection through genetically engineered bacterial cells.

Protein microarray technology, in which a large number of capture ligands are spatially arrayed at a high density, presents an attractive method for high-throughput proteomic analysis. Toward this end, we demonstrate the first cell-based protein detection in a microsystem, wherein Escherichia coli cells are genetically engineered to express the desired capture proteins on the membrane surface and are spatially arrayed as sensing elements in a microfluidic device. An E. coli clone expressing peptide ligands with high affinity and high specificity for target molecules was isolated a priori. Then these cells were electrokinetically immobilized on gold electrodes using dielectrophoresis, thus allowing each sensor element to be electrically addressable. Flow cytometry and subsequent fluorescence analysis verified the highly specific capture and detection of target molecules by the bacteria. Finally, through the coexpression of peptide-based capture ligands on the cell surface and fluorescent protein in the cytoplasm, we demonstrate an effective means of directly linking the fluorescence intensity to the density of capture ligands.

Escherichia coli↗

PubMed1990

Genetic engineering of plants for virus resistance.

Historically, control of plant virus disease has involved numerous strategies which have often been combined to provide effective durable resistance in the field. In recent years, the dramatic advances obtained in plant molecular virology have enhanced our understanding of viral genome organizations and gene functions. Moreover, genetic engineering of plants for virus resistance has recently provided promising additional strategies for control of virus disease. At present, the most promising of these has been the expression of coat-protein coding sequences in plants transformed with a coat protein gene. Other potential methods include the expression of anti-sense viral transcripts in transgenic plants, the application of artificial anti-sense mediated gene regulation to viral systems, and the expression of viral satellite RNAs, RNAs with endoribonuclease activity, antiviral antibody genes, or human interferon genes in plants.

Base Sequence↗

PubMed2003

Genetically engineered probiotics.

Probiotic micro-organisms have been used for many years. Originating as food supplements, they are now most often administered orally and offer an attractive alternative for treating of intestinal disorders. A better understanding of the mechanisms by which these micro-organisms act has now opened up possibilities for designing new probiotic strains. Through genetic engineering, it is possible not only to strengthen the effects of existing strains, but also to create completely new probiotics. These need not necessarily be composed only of bacterial products but can also include elements of regulatory systems or enzymes derived from a foreign-human-source. If designed carefully and with absolute attention to biological safety in its broadest sense, the development of genetically modified probiotics has the potential to revolutionize alimentary health.

Animals↗

PubMed2006

Genetically engineered poly-gamma-glutamate producer from Bacillus subtilis ISW1214.

The pgsBCA-gene disruptant from Bacillus subtilis ISW1214, i.e., MA41, does not produce poly-gamma-glutamate (PGA). We newly constructed an MA41 recombinant bearing the plasmid-borne PGA synthetic system, in which PGA production was strictly controlled by the use of xylose. Unlike the parent strain, ISW1214, the genetically engineered strain produced abundant PGA in both L-glutamate-rich and D-glutamate-rich media.

Bacillus subtilis↗

PubMed2006

Genetically engineered human cancer models utilizing mammalian transgene expression.

Cancer models are vital to cancer biology research, and multiple cancer models are currently available that utilize either murine or human cells, each with particular strengths and weaknesses. The ability to transform primary human cells into tumors through the expression of specific transgenes offers many advantages as a cancer model, including genetic malleability and the ability to transform specific cell types. Until recently, the conversion of primary human cells into tumors through transgene expression required the use of viral genetic elements, which unfortunately adds uncertainty regarding which cancer pathways are affected and how they are affected. In recent years multiple reports have described the transformation of primary human cells into tumors using only mammalian transgenes. This review focuses on these five cancer models, comparing the different cell types which were transformed into tumors and which transgenes were expressed, as well as the cancer pathways affected in the disparate models. These genetically-engineered human cancer models offer a valuable tool to complement existing cancer models and further cancer research.

Animals↗

PubMed1993

Crystallization of a genetically engineered water-soluble primary penicillin target enzyme. The high molecular mass PBP2x of Streptococcus pneumoniae.

A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals have been grown at 15 degrees C, from solutions containing 8% polyethylene glycol 10,000 at pH values ranging from 3.9 to 6.0. These crystals diffract to a resolution of 3.5 A and have a space group P6(1)22 (or enantiomorph) with unit cell dimensions of a = b = 162.2 A, c = 171.8 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one molecule of enzyme per asymmetric unit. The breakdown of a chromogenic cephalosporin derivative diffused into a crystal reveals clearly that the enzyme is active in the crystalline state.

Bacterial Proteins↗

PubMed2005

Genetically engineered vaccines.

The application of recombinant DNA technology to allergen research has provided the sequence information and genetic material to produce new types of allergy vaccines. One general strategy has been to use the knowledge to produce synthetic peptides that represent selected T-cell or B-cell epitopes. The production of genetically engineered allergens provides an alternative strategy to construct hypoallergenic vaccines, which can provide a better and less selected representation of the epitopes. Many strategies have been used to produce such hypoallergens, and their ability to reduce allergenicity has been amply demonstrated by skin and nasal provocation tests. The retention of T cell-stimulating activity has also been demonstrated, and a consistent feature of the vaccines has been, despite the reduced immunoglobulin E (IgE)-binding reactivity, the ability to induce anti-allergen IgG antibody. The lead hypoallergens have been polypeptide fragments and trimeric constructs of the birch allergen Bet v 1. A clinical trial with these medicaments has shown the ability to modify IgE and IgG antibody production, skin test reactivity, and symptom scores. This is the first trial of a recombinant allergy vaccine, and it has set a benchmark for further studies. A new generation of hypoallergens is now being produced based on the detailed knowledge of the tertiary structures of the allergens and of the T-cell and B-cell epitopes. The modifications have been made to change the topography of the allergens while retaining a stable, folding structure. In the case of Bet v 1, tertiary structures of hypoallergens have been determined. Structurally modeled hypoallergens have been produced for pollen, venom, food, and latex allergens, with promising characteristics from preclinical studies.

Allergens↗

PubMed2004

Novel tracing paradigms--genetically engineered herpesviruses as tools for mapping functional circuits within the CNS: present status and future prospects.

The mammalian CNS is composed of an extremely complex meshwork of highly ordered interconnections among billions of neurons. To understand the diverse functions of this neuronal network we need to differentiate between functionally related and nonrelated elements. A powerful labeling method for defining intricate neural circuits is based on the utilization of neurotropic herpesviruses, including pseudorabies virus and herpes simplex virus type 1. The recent development of genetically engineered tracing viruses can open the way toward the conception of novel tract-tracing paradigms. These new-generation tracing viruses may facilitate the clarification of problems, which were inaccessible to earlier approaches. This article first presents a concise review of the general aspects of neuroanatomical tracing protocols. Subsequently, it discusses the molecular biology of alpha-herpesviruses, and the genetic manipulation and gene expression techniques that are utilized for the construction of virus-based tracers. Finally, it describes the current utilization of genetically modified herpesviruses for circuit analysis, and the future directions in their potential applications.

Animals↗