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PubMedDate not supplied

Detection of genetically modified organisms in foods by protein- and DNA-based techniques: bridging the methods.

According to European Commission (EC) Regulation 1139/98, foods and food ingredients that are to be delivered to the final consumer in which either protein or DNA resulting from genetic modification is present, shall be subject to additional specific labeling requirements. Since 1994, genetically altered tomatoes, squash, potatoes, canola, cotton, and soy have been on the market. Recently, insect-resistant and herbicide-tolerant maize varieties have been introduced. Soy and maize are 2 of the most important vegetable crops in the world. During the past 4 years, both protein- and DNA-based methods have been developed and applied for detection of transgenic soy and maize, and their derivatives. For protein-based detection, specific monoclonal and polyclonal antibodies have been developed; for immunochemical detection, Western blot analysis and enzyme-linked immunosorbent assays are the most prominent examples. For detection of genetically modified organisms (GMOs) at the level of DNA, polymerase chain reaction-based methods are mainly used. For these reactions, highly specific primer sets are needed. This study compares the principally different methods. Specificity of methods and the possible risks of false-positive or false-negative results are considered in relation to sampling, matrix effects, and food processing procedures. In addition, quantitative aspects of protein- and DNA-based GM detection methods are presented and discussed. This is especially relevant as EC regulation 49/2000, which defines a threshold for an unintentional comingling of 1%, came into force on April 10, 2000.

DNA, Plant↗
PubMed2002

[A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)].

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.

DNA Primers↗
PubMed2006

A detection method of CryIAc protein for identifying genetically modified rice using the lateral flow strip assay.

We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.

Bacillus thuringiensis Toxins↗
PubMed2003

Weeds in fields with contrasting conventional and genetically modified herbicide-tolerant crops. II. Effects on individual species.

We compared the effects of the management of genetically modified herbicide-tolerant (GMHT) and conventional beet, maize and spring oilseed rape on 12 weed species. We sampled the seedbank before and after cropping. During the season we counted plants and measured seed rain and biomass. Ratios of densities were used to calculate emergence, survival, reproduction and seedbank change. Treatments significantly affected the biomass of six species in beet, eight in maize and five in spring oilseed rape. The effects were generally consistent, with biomass lower in GMHT beet and spring oilseed rape and higher in GMHT maize. With few exceptions, emergence was higher in GMHT crops. Subsequent survival was significantly lowered for eight species in beet and six in spring oilseed rape in the GMHT treatments. It was increased for five species in maize and one in spring oilseed rape. Significant effects on seedbank change were found for four species. However, for many species in beet and spring oilseed rape (19 out of 24 cases), seed densities were lower in the seedbank after GMHT cropping. These differences compounded over time would result in large decreases in population densities of arable weeds. In maize, populations may increase.

Agriculture↗
PubMed2006

A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

Animals↗
PubMed1998

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction.

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.

Anti-Bacterial Agents↗
PubMed2002

An alternative focus in strategic research on disease vectors: the potential of genetically modified non-biting mosquitoes.

We examine the constraints and the feasibility of field experiments involving the release of genetically modified (GM) pathogen-resistant mosquitoes, and whether there are alternatives to the research line based on the production of refractory strains. The production of a GM mosquito strain characterized instead by obligate primiparous and parous autogeny and by disrupted host seeking and biting behaviour could make the release more acceptable by the general public. Genetic transformation should act in this case to reverse some of the essential steps of the evolutionary process that gave rise to hematophagy. The replacement strategy could be based on the mass release of both sexes in a well defined ecological niche made temporarily empty of the natural population, thus avoiding the problems related to the need of sexual competitiveness of the released material. This option is encouraged by the growing evidence that competitive exclusion mechanisms influence the pattern of distribution of different taxa within Anopheles gambiae s.s. and by the fact that the plesiomorphic characteristics of vitellogenesis without a blood meal (autogeny), which exploits fat body reserve accumulated during larval life and food other than blood in adult life, persist as genetic variants in various hematophagous insect groups, and it has been found secondarily fixed in others showing stable reversions to primiparous and parous autogeny. If this has been the result of natural selection, then the artificial production of non-biting mosquito strains, by selection and/or transgenesis, should be feasible.

Animals↗
PubMed1995

[L-DOPA-producing primary fibroblasts genetically modified with a retrovirus vector system].

Although intracerebral grafting has become a new strategy for the treatment of Parkinson's disease, many problems related to the grafts remain. We focused on primary skin fibroblasts as grafts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing the cDNA of human tyrosine hydroxylase (TH) (pLTHSNL) or cytomegalovirus promoter (pCTHSNL) as a foreign promoter, and catecholamine production and release by these genetically modified fibroblasts, were analyzed in vitro immunocytochemically and by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The cells were supplemented with biopterin (BH4; (6R)-L-erythro-tetrahydrobiopterin), a cofactor required for TH activity, and they produced and released L-DOPA into the culture medium. When exposed to the combination of a foreign promoter and BH4, L-DOPA production increased in a time-dependent manner, and was unaffected by the number of cell-passages or the duration of liquid-nitrogen freezing. This suggests that the foreign gene (THcDNA)-containing retrovirus vector had integrated into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with THcDNA using this retrovirus vector system appear to be a promising graft for transplantation and gene therapy of Parkinson's disease in the future.

Animals↗
PubMedDate not supplied

Regulation of L-DOPA production by genetically modified primary fibroblasts transfected with retrovirus vector system.

Although intracerebral grafting has become established as a new strategy for the treatment of Parkinson's disease, there are many problems regarding such grafts. We focused on the grafting of primary skin fibroblasts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing cDNA of human tyrosine hydroxylase (TH) type 1 (LTHSNL) or of cytomegalovirus promoter (CTHSNL) as a foreign promoter. In these genetically modified fibroblasts, L-DOPA production and release were analyzed in vitro by immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Being supplemented with the biopterin (BH4:(6R)-L-erythro-tetrahydrobiopterin) cofactors required for TH activity, these cells produced and released L-DOPA into the culture medium. When CTHSNL and BH4 were combined, L-DOPA production increased with time, regardless of the number of cell passages, or the duration of liquid nitrogen freezing. This suggests that the foreign gene (THcDNA) containing retrovirus vector integrates into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with a retrovirus vector system containing the TH cDNA may be promising grafts for transplantation and gene therapy in Parkinson's disease.

Animals↗
PubMed2005

Genetically modified plants and food hypersensitivity diseases: usage and implications of experimental models for risk assessment.

The recent advances in biotechnology in the plant industry have led to increasing crop production and yield that in turn has increased the usage of genetically modified (GM) food in the human food chain. The usage of GM foods for human consumption has raised a number of fundamental questions including the ability of GM foods to elicit potentially harmful immunological responses, including allergic hypersensitivity. To assess the safety of foods derived from GM plants including allergenic potential, the US FDA, Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), and the EU have developed approaches for evaluation assessment. One assessment approach that has been a very active area of research and debate is the development and usage of animal models to assess the potential allergenicity of GM foods. A number of specific animal models employing rodents, pigs, and dogs have been developed for allergenicity assessment. However, validation of these models is needed and consideration of the criteria for an appropriate animal model for the assessment of allergenicity in GM plants is required. We have recently employed a BALB/c mouse model to assess the potential allergenicity of GM plants. We have been able to demonstrate that this model is able to detect differences in antigenicity and identify aspects of protein post-translational modifications that can alter antigenicity. Furthermore, this model has also enabled us to examine the usage of GM plants as a therapeutic approach for the treatment of allergic diseases. This review discusses the current approaches to assess the allergenic potential of GM food and particularly focusing on the usage of animal models to determine the potential allergenicity of GM foods and gives an overview of our recent findings and implications of these studies.

Animals↗
PubMed2001

Are genetically modified mice useful for the understanding of acute pancreatitis?

Treatment of patients with acute pancreatitis has greatly improved due to a better understanding of the pathophysiology of the disease. This pathophysiology includes the activation and release of pancreatic enzymes in the interstitium, the autodigestion of the pancreas, and a multiple organ dysfunction after their release into the systemic circulation. Moreover, significant evidence exists that synthesis and release of proinflammatory cytokines and chemokines are also responsible for the local injury and systemic dispersion of the inflammation. The use of knockout mice devoid of active pro- or anti-inflammatory mediators allows examination of the effects of a specific cytokine without any drawbacks induced by pharmacological manipulations. The results obtained from these genetically modified mice show that numerous mediators have a major role in the pathophysiology of acute pancreatitis. They also clearly demonstrate that a single genetic deletion cannot completely prevent the occurrence of pancreatic or distant organ injury. However, the fact that the immune system is characterized by redundancies of ligands and receptors complicates the full understanding of each report. The utility of such experimental models might have limitations, and a full extrapolation of experimental data from genetically modified mice to humans must be done with caution.

Acute Disease↗
PubMed2001

Variable phenotypic presentation of iron overload in H63D homozygotes: are genetic modifiers the cause?

BACKGROUND: First considered as a polymorphism of the HFE gene, the H63D mutation is now widely recognised as a haemochromatosis associated allele. But few H63D homozygotes with clinical manifestations of hereditary haemochromatosis (HH) have been reported. Concurrently, an increasing number of genes have been shown to interact with HFE in iron metabolism. AIMS: To describe the clinical expression of iron overload (IO) associated with H63D homozygosity, and search for potential genetic modifiers (within the HFE or other genes) that could explain the variability of the phenotypes. PATIENTS AND METHODS: We retrospectively analysed the clinical phenotype of 56 H63D homozygotes referred for a personal or family history of IO. For each subject we examined intragenic HFE haplotypes and transferrin receptor (TfR) gene polymorphisms and searched for the Y250X mutation on the TFR2 gene. Additionally, we sequenced the HFE gene of H63D homozygotes with HH. RESULTS: Fifty of 56 subjects had biological and/or clinical abnormalities of iron metabolism. Up to two thirds of patients (n=34) had no acquired cause of IO. Among these, 12 had a phenotypic diagnosis of HH. In the iron loaded group there was a strong prevalence of male patients. No correlation was found between the potential genetic modifiers and phenotypes. No additional mutation of HFE was identified. CONCLUSION: The variable phenotypes associated with H63D homozygosity do not appear to be linked to other HFE mutations, to the TFR2 Y250X mutation, or to HFE or TfR gene intragenic polymorphisms. The exact role of H63D homozygosity in IO and HH needs to be further investigated in unselected populations.

Adult↗
PubMed2005

Genetically modified Plasmodium highlights the potential of whole parasite vaccine strategies.

A genetically modified malaria sporozoite might breathe new life into the traditional approach to vaccine development, that of using whole organisms. Mueller and colleagues recently knocked out a gene, UIS3, from the rodent parasite, Plasmodium berghei, and demonstrated that the sporozoite forms could not develop beyond the stage of the life cycle in the liver (thus not giving rise to clinical disease, which is associated with blood infection) but could induce protection against subsequent challenge with genetically intact sporozoites. UIS3(-) sporozoites or irradiated sporozoites might find success where subunit approaches are struggling.

Animals↗
PubMed2003

Assessment of allergenic potential of genetically modified foods: an agenda for future research.

Speakers and participants in the workshop "Assessment of the Allergenic Potential of Genetically Modified Foods" met in breakout groups to discuss a number of issues including needs for future research. These groups agreed that research should progress quickly in the area of hazard identification and that a need exists for more basic research to understand the mechanisms underlying food allergy. A list of research needs was developed.

Animals↗
PubMed2003

Behavioral correction of Parkinsonian rats following the transplantation of immortalized fibroblasts genetically modified with TH and GCH genes.

Eukaryotic plasmid vectors encoding the tyrosine hydroxylase (TH) gene and GTP cyclohydrolase-1 (GCH) gene were constructed and introduced into immortalized fibroblasts obtained from SV40 large antigen (LT(AG)) transformed rat primary fibroblasts. TH and GCH positive clones were selected and identified by immunohistochemistry and RT-PCR, respectively. Hemi-parkinsonian rats created using 6-hydroxydopamine (6-OHDA) were used to assess the therapeutic effect created by the co-implantation of immortalized fibroblasts genetically modified by TH or GCH genes. Animal behavior was significantly improved two weeks following implantation and behavioral correction was maintained for over 14 weeks. Behavioral improvement was paralleled by exogenous TH gene expression, identified by TH immunohistochemistry and RT-PCR analyses. The transplanted cells survived for at least 38 weeks as demonstrated by fibronectin immunohistochemical staining. Tumor formation or host reaction was not seen, although TH expression was negative for 20 weeks after the implantation. This work demonstrates that the co-transplantation of immortalized fibroblasts genetically modified by TH and GCH genes may be developed as a valuable approach to the treatment of Parkinson's disease.

Animals↗
PubMed1994

Detoxification of the plant toxin fluoroacetate by a genetically modified rumen bacterium.

We isolated the fluoroacetate dehalogenase gene (H1), from Moraxella species strain B, and placed it under the transcriptional control of a 154 bp fragment of the erm gene promoter. The promoter/gene construct was attached to the Butyrivibrio fibrisolvens shuttle vector pBHerm, and the resulting dehalogenase expression plasmid (pBHf) was transferred to B. fibrisolvens OB156 by electroporation. The erm gene promoter directed expression of dehalogenase activity in both E. coli and B. fibrisolvens OB156. Cell-free lysates of the genetically modified OB156 defluorinated 10.6 nmol fluoroacetate/min/mg protein. Growing cultures of OB156 were able to detoxify fluoroacetate in the culture medium, at the rate of 9.9 nmol/min/mg. Plasmid pBHf was retained by 100% of OB156 cells after 500 generations of non-selective culture. The restriction pattern of pBHf remained unchanged after extensive non-selective growth and host bacteria continued to produce active dehalogenase. The construction of rumen bacteria that are able to detoxify an important natural poison supports the feasibility of using genetically modified rumen bacteria to aid animal production.

Animals↗
PubMed2000

Safety evaluation of genetically modified foods.

The concept of substantial equivalence has been accepted as the cornerstone of the health hazard assessment of genetically modified (GM) foods (OECD 1993). Substantial equivalence is the most practical approach to address the safety of foods or food components derived from GM crops and is based on comparison of the phenotypic and compositional characteristics of the parent crop and the GM crop. Basically, three categories of GM crops can be considered (FAO/WHO 1996; EU 1997): (a) GM crops which have the same composition as the parent crop, (b) GM crops which have the same composition as the parent crop with the exception of a well-defined trait, and (c) GM crops which are different from the parent crop. For the safety assessment of the first category of GM foods only a molecular characterisation of the genetic insert is sufficient, whereas for the second category a safety assessment of the expressed protein(s) is also required. For the last category an extensive evaluation including bioavailability and wholesomeness studies are required, beside the molecular characterisation and safety assessment of the expressed protein(s) and their products. By molecular characterisation is meant the position, nature, stability and number of copies of the inserted DNA. Substantial equivalence is established by the determination of the phenotypic characteristics (e.g. resistance against diseases, agronomic properties) and the complete chemical composition of the plant including nutrients, toxicants, antinutrients, and allergens. The toxicity of the expressed protein(s) is assessed by their homology with known protein toxins, degradation in the gastro-intestinal tract, stability to food processing and acute toxicity in rodents. The possible allergenicity of the expressed proteins is evaluated by comparison of their amino acid sequence with that of known allergens and determination of their stability to digestion and food processing. If the source of the genetic insert is allergenic then the use of solid-state immunoassays, skin prick tests and even food challenge tests can be considered.

Animals↗
PubMed2006

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability.

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.

Arabidopsis↗