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PubMed2001

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

DNA Primers↗

PubMed2005

Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity.

BACKGROUND: We have performed a double-blind, placebo-controlled injection immunotherapy study with genetically modified derivatives of the major birch pollen allergen, Bet v 1 (Bet v 1-trimer, Bet v 1-fragments). OBJECTIVE: To investigate whether vaccination with genetically modified allergens induces allergen-specific antibodies in nasal secretions and to study whether these antibodies affect nasal allergen sensitivity. METHODS: A randomly picked subgroup of patients (n = 23; placebo, n = 10; trimer, n = 10; fragments, n = 3) was subjected to an extensive analysis of serum samples and nasal lavage fluids and to nasal provocation testing. Bet v 1-specific IgG(1-4) and IgA antibodies were determined in serum samples obtained before and after vaccination, after the birch pollen season, and 1 year after start of vaccination as well as in nasal lavage fluids obtained after the birch pollen season and 1 year after start of vaccination by ELISA. Nasal sensitivity to natural, birch pollen-derived Bet v 1 was determined by active anterior rhinomanometry after the birch pollen season and 1 year after start of vaccination. RESULTS: Vaccination with genetically modified Bet v 1 derivatives, but not with placebo, induced Bet v 1-specific IgG1, IgG2, and IgG4, and low IgA antibodies in serum, which also appeared in nasal secretions, but no IgG3 antibodies. The levels of therapy-induced Bet v 1-specific IgG4 antibodies in nasal secretions were significantly (P < .05) associated with reduced nasal sensitivity to natural, birch pollen-derived Bet v 1 as objectively determined by controlled nasal provocation experiments. CONCLUSION: Our data demonstrate that vaccination with genetically modified allergens induces IgG antibody responses against the corresponding natural allergen not only in serum but also in mucosal fluids, where they may protect against allergen-induced inflammation.

Adult↗

PubMed1995

Systemic delivery of a recombinant protein by genetically modified mesothelial cells reseeded on the parietal peritoneal surface.

To evaluate the ability of genetically modified peritoneal mesothelial cells to deliver recombinant proteins to the systemic circulation, we used our previously described mesothelial cell-based ex vivo gene therapy strategy. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transfected (using strontium phosphate DNA co-precipitation) with the plasmid pSVTKgh to express a secreted reporter gene product, human growth hormone (hgh). Such hgh-secreting mesothelial cells were reseeded on the denuded peritoneal surface of syngeneic recipients and delivery of the reporter gene product to the systemic circulation was monitored by analysis of serum samples for the presence of hgh at various times after mesothelial cell implantation. Polymerase chain reaction (PCR) analysis demonstrated that the hgh-transfected mesothelial cells repopulated the denuded areas and remained attached there for at least 12 weeks. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo and secreted hgh in sufficient quantity to be detected in the systemic circulation (ie statistically significant amounts of hgh could be measured in the serum of cyclosporine A-treated rats for at least 2 months; Mann-Whitney test, P < 0.05). Our results demonstrate the successful, sustained, systemic delivery of a recombinant protein by genetically modified peritoneal mesothelial cells following their reattachment to the peritoneal surface, and suggest the potential of ex vivo mesothelial cell-mediated gene therapy for the treatment of inherited or acquired disorders requiring delivery of therapeutic proteins to the circulation.

Animals↗

PubMed2000

Gene transfer from genetically modified food.

The current debate about the safety of genetically modified food includes some important scientific issues where more scientific data would aid the robustness of safety evaluation. One example is the possibility of gene transfer, especially from genetically modified plant material.

Bacteria↗

PubMed2002

Improvement of growth and nutritive value in chicks with non-genetically modified phytase product from Aspergillus niger.

1. Non-genetically modified (non-GM) phytase product derived from Aspergillus niger possesses various side active enzymes including alpha-amylase, protease, cellulase and hemicellulase. In contrast, the product of genetically modified (GM) phytase product has much less side active enzyme since the capacity of phytase production is reinforced by gene modification. In the present study we have tried to determine whether the difference of side enzyme activity of phytase product affects growth performances and nutritive value in chicks; in addition we tried to characterise the physiological change induced by the difference of side active enzymes. 2. Single Comb White Leghorn male chicks at 7 d of age were fed on experimental barley-based diets for 10 d. The feeding trial was of a factorial design (3 x 2 x 2), having three types of dietary phytase products (control, non-GM or GM phytase products derived from A. niger at 1000 U/kg diet), two levels of dietary available P supplement (0 or 6 g/kg diet) and two levels of dietary protein (CP 180 or 120 g/kg). 3. The non-GM phytase product caused a 6% increase in final body weight and feed efficiency compared with the control and the GM phytase product without interacting with dietary protein and available P level. However, in birds given available P-free diet, both non-GM and GM phytase products induced a 20% increase in plasma P concentration, suggesting no difference in phytase activity between the non-GM and GM phytase products. 4. The balance study showed that the metabolisable energy of the non-GM phytase product (15.6 +/- 0.05 kJ/g diet) was significantly higher among the treatments (control, 15.1 +/- 0.05; GM phytase product 15.3 +/- 0.07). The non-GM phytase product also increased the rate of food passage through the crop, and caused a drastic reduction in intestinal weight, perhaps as a consequence of digestion of non-starch polysaccharides. 5. We conclude that the side active enzymes in non-GM phytase product improve growth performance and nutritive value of the diet in chicks. However, the efficacy of phytase activity should not be different between non-GM and GM phytase products.

6-Phytase↗

PubMed2004

[Identification for genetically modified maize T14/T25 with real time fluorescent PCR method].

To identify genetically modified (GM) maize T14/T25 lines, a real-time fluorescent PCR (RTF PCR) assay was performed in this study. Primers and Taqman probes specific for inserted genes in the T14/T25 were used to conduct the real-time fluorescent (RTF) PCR and PCR assays. The RTF PCR method was established to detect and identify GM maize lines. The results show that the TaqMan probe could identify T14/T25 maize used, while other GM and NO-GM maize didn't be detected. The RTF PCR could be a new method for detecting other genetically modified organism.

DNA Primers↗

PubMed2005

Science and norms in policies for sustainable development: assessing and managing risks of chemical substances and genetically modified organisms in the European Union.

Use of chemical substances and genetically modified organisms cause complex problems characterised by scientific uncertainty and controversies. Aiming at sustainable development, policies for assessment, and management of risks in the two areas are under development in the European Union. The article points out that both science and norms play a central role in risk assessment as well as risk management and suggests that the precautionary principle, the principle of public participation, and the polluter pays principle, all adopted in the European Union, offer a way to operationalise the concept of sustainable development. It is shown, however, that a number of steps ought to be taken to better implement the principles through different policy measures. In doing so, and by recognising the role of both science and norms, the decision-making on risks related to the use of chemicals or genetically modified organisms can be improved to better promote sustainable development.

Chemical Industry↗

PubMed2005

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination.

Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies.

Adoptive Transfer↗

PubMed2005

Ethical, legal and social issues of genetically modifying insect vectors for public health.

The use of genetically modified (GM) insects for control of human disease can be consistent with common ethical norms of international society to reduce human suffering. This paper considers a range of ethical issues including animal rights, informed consent, community consensus and environmental viewpoints. Each community needs to decide its own priorities for methodology of disease policy guidance for ethical genetic engineering, and to negotiate with neighbouring countries. The approach to genetically modify insects raises few intrinsic ethical issues; however, important environmental and human health concerns need to be assessed before release of any GM insects. The policy that each community adopts should be the product of open dialogue involving all sectors of society. It can be expected that this process will take years and not all communities will endorse genetic control approaches to insect vectors.

Animal Rights↗

PubMed2001

Allergenicity test of genetically modified soybean in Sprague Dawley rats.

Allergenicity of genetically-modified (GM) soybean was evaluated in male Sprague Dawley rats. To confirm the GM soybean used in this study, the polymerase chain reaction (PCR) was performed using the chromosomal DNA of soybeans. The PCR result provided the clear discrimination of genetically-modified (GM) soybeans. To evaluate the allergenicity of GM soybean and non-GM control one, the soybean homogenate was sensitized subcutaneously 3 times a week for 3 weeks. The doses of soybean were 0, 2 and 20 mg/kg in the protein basis. A week after the last sensitization, antisera were recovered from individual animals. When the sera were injected intradermally on the clipped back of unsensitized rats with various dilutions, followed by a challenge with 20 mg/kg of soybean homogenate containing 1% Evans blue, no sign of passive cutaneous anaphylaxis reaction was detected. In addition, when the sera were treated in the cultures of peritoneal mast cells, the increase of histamine release by anti-(GM soybean) sera was not observed when compared to that by anti-(non-GM soybean) sera. The present results indicate that the GM soybean might not act as a strong allergen in male Sprague Dawley rats.

Animals↗

PubMed2006

Effects of feeding calves genetically modified corn bt11: a clinico-biochemical study.

Genetically modified corn Bt11 is insect-resistant and expresses Cry1Ab toxin, an insecticidal protein, in kernels. Although Bt11 corn is considered safe based on animal performance, there are no reports available on the clinico-biochemical effects of feeding it to cattle. In this study, we evaluated the effects of feeding Bt11 to calves, using blood and ruminal clinico-biochemical parameters. Our three-month-long feeding experiment demonstrated that calves (n=6), fed with a ration containing 43.3% of Bt11 corn kernels as dry matter, did not develop any discernible clinical, hematological, biochemical, or ruminal abnormalities as compared with control calves (n=6) fed non-Bt11 corn. The results suggest that the transgenic Bt11 has no negative clinico-biochemical effects on calves.

Alanine Transaminase↗

PubMed2001

How to make foods safer--genetically modified foods.

It is the responsibility of companies developing genetically modified foods, and of regulatory authorities that approve their marketing, to ensure that they are at least as safe as the traditional foods they are intended to replace in the diet. This requires that any novel material introduced into the food material should not be allergenic. If the novel gene has come from an allergenic source, e.g. nuts, it is necessary to demonstrate using immunological procedures applied to the IgE fractions of pooled sera from individuals with confirmed allergies that the novel protein is non-allergenic. When the novel gene is from a non-allergenic source then it is necessary to demonstrate lack of significant amino acid sequence homology to known allergens together with sensitivity to food manufacturing and digestive processes. Consumer confidence in genetically modified foods would be significantly improved if hypoallergenic varieties of crops and food products that are currently allergenic could be developed. Techniques such as antisense technology and single site amino acid substitution have been shown to have such potential.

Animals↗

PubMed2006

Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization.

BACKGROUND: With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Advances in biotechnology have resulted in an increasing number of genetically engineered foods, and among these soybean is one of the most widespread. OBJECTIVE: To develop and characterize a murine model of IgE-mediated soybean sensitization induced by intragastric immunization, in the presence of Cholera Toxin, with wild-type soybean extract (wt-SE) or with genetically modified soybean extract (gm-SE). METHODS: Balb/c mice born in our animal facilities, from females fed on soy-free food, were fed with the same soy-free food and used in all the experiments. Mice were sensitized by gavages with soybean extracts, and allergen-specific IgE and IgG responses were studied by direct ELISA and ELISA inhibition. Antigen-specific cell proliferation and cytokine production were evaluated in spleen cell cultures. Results Sensitization with both soybean extracts induced high levels of antigen-specific IgE and IgG1 and low levels of specific IgG2a. Both wt-SE and gm-SE were able to inhibit the binding of specific IgE from mice immunized with gm-SE to the same antigen used for the ELISA coating. A comparable proliferative response was obtained with the homologous as well as with the heterologous extracts. CONCLUSION: In sensitized mice, we observed a predominantly T-helper type 2 (Th2)-type immune response, with increased soybean-specific IgE and IgG1 antibodies and a concomitant increase of IL-4 and IL-5 production. RESULTS: obtained by specific IgE ELISA inhibition and by antigen-specific T cell proliferation demonstrated that wt-SE and gm-SE shared B and T epitopes. The present murine model of soybean sensitization established by the oral route should provide valuable information about risk assessment for food allergy from new proteins of genetically modified foods.

Administration, Oral↗

PubMed2002

Detection of genetically modified organisms in foods.

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.

Blotting, Southern↗

PubMedDate not supplied

Impact of genetically modified crops on soil- and plant-associated microbial communities.

Transgenic or genetically modified plants possess novel genes that impart beneficial characteristics such as herbicide resistance. One of the least understood areas in the environmental risk assessment of genetically modified crops is their impact on soil- and plant-associated microbial communities. The potential for interaction between transgenic plants and plant residues and the soil microbial community is not well understood. The recognition that these interactions could change microbial biodiversity and affect ecosystem functioning has initiated a limited number of studies in the area. At this time, studies have shown the possibility that transgenes can be transferred to native soil microorganisms through horizontal gene transfer, although there is not evidence of this occurring in the soil. Furthermore, novel proteins have been shown to be released from transgenic plants into the soil ecosystem, and their presence can influence the biodiversity of the microbial community by selectively stimulating the growth of organisms that can use them. Microbial diversity can be altered when associated with transgenic plants; however, these effects are both variable and transient. Soil- and plant-associated microbial communities are influenced not only by plant species and transgene insertion but also by environmental factors such as field site and sampling date. Minor alterations in the diversity of the microbial community could affect soil health and ecosystem functioning, and therefore, the impact that plant variety may have on the dynamics of the rhizosphere microbial populations and in turn plant growth and health and ecosystem sustainability, requires further study.

Bacteria↗

PubMed2000

Persistent, antigen-specific, therapeutic antitumor immunity by dendritic cells genetically modified with an adenoviral vector to express a model tumor antigen.

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of cellular immune responses. Using a BALB/c syngeneic colon carcinoma cell line expressing a model tumor antigen beta-galactosidase (betagal), we previously reported (Song et al, J Exp Med 1997; 186: 1247-1256) that immunization of mice with a single injection of DCs genetically modified with an adenovirus vector expressing betagal confers potent protection against a lethal intravenous tumor challenge, as well as suppression of pre-established lung tumors, resulting in a significant survival advantage. In the present study, we have addressed the question: how long does the memory of tumor antigen- specific immunity persists after DC priming in vivo using this genetically modified DC-based cancer vaccination strategy? To accomplish this, two groups of mice were evaluated: (1) mice surviving >400 days following protection from an initial intravenous tumor challenge after immunization with DC genetically modified to express betagal; and (2) mice surviving >300 days that had previously demonstrated regression of pre-established lung tumors after treatment with DC immunization. By analyzing the antigen-specific cytotoxic T lymphocyte response and challenging these long-term survival mice with a second subcutaneous tumor administration, the data demonstrate that a single administration of DC genetically modified to express a model antigen induces long-lasting, antigen-specific antitumor immunity in both naive and tumor-bearing hosts, observations that have important implications in the development of genetically modified DC-based antitumor vaccination strategies. Gene Therapy (2000) 7, 2080-2086.

Adenoviridae↗

PubMed1998

Targeted expansion of genetically modified bone marrow cells.

The ability to specifically target a mitogenic signal to a population of genetically modified primary cells would have potential applications both for gene and cell therapy. Toward this end, a gene encoding a fusion protein containing the FK506-binding protein FKBP12, fused to the intracellular portion of the receptor for thrombopoietin (mpl), was introduced into primary murine bone marrow cells. Dimerization of this fusion protein through the addition of a dimeric form of the drug FK506, called FK1012, resulted in a marked proliferative expansion of marrow cells that was restricted to the genetically modified population. FK1012's proliferative effect was sustained and reversible. An apparent preference for differentiation along the megakaryocytic lineage was observed. This approach allows for the specific delivery of a mitogenic signal to a population of genetically modified primary cells and may have applications for studies in hematopoiesis and receptor biology, and for gene and cell therapy.

Animals↗

PubMed2006

Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti.

Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.

Aedes↗