New methods for the safety testing of transgenic food

This result addressed the question whether in vitro experiments combined with new transcriptomics technologies would be useful for the safety testing of transgenic foods. For this feasibility study, the in vitro effect of GNA lectin, PHA-E lectin and Bt toxin on gene expression in intestinal epithelial cell lines from human (Caco-2) and rat (IEC-6) was assessed using prior knowledge obtained from the biodegradation and cytotoxicity experiments. Finally, the gene expression patterns obtained in vitro with PHA-E were compared with those found in vivo (28-day and 90-day feeding studies). IEC-6 cells and undifferentiated/differentiated Caco-2 cells were exposed to non-cytotoxic concentrations of the lectins, Bt toxin, and peptic-tryptic (PT) digests. Total RNA was isolated and hybridised to microarrays containing either 3136 rat-specific or 2352 human-specific cDNAs. Also RNA from untreated cells (control) and cells exposed to a pepsin-trypsin mixture (digest control) was isolated and hybridised to the cDNA microarrays. Many genes were found to be differentially regulated upon exposure of the cells to the three proteins or corresponding PT digests. The most striking effect was seen in IEC-6 cells and differentiated Caco-2 cells exposed to PHA-E. From the three proteins, PHA-E resulted in the largest number of differentially expressed genes. Furthermore, the number of genes differentially expressed upon exposure to the PT digest of PHA-E was approximately ten-fold lower. When the results from the human-derived Caco-2 cells and rat IEC-6 cells were compared, only little correlation could be found. To assess the validity of the gene expression results obtained in vitro, gene expression profiling was performed on jejunal scrapings sampled from two groups of male and female rats in the PHA-E 28-day study. The rats from one group were fed on a purified diet with 60% rice, the rats from the other group were fed on the same diet but supplemented with 0.08% PHA-E. Total RNAs from the scrapings were isolated and hybridised to the same rat-specific microarrays as used for the in vitro experiments with IEC-6. When the data obtained with the male and female PHA-fed rats were compared, the majority of differentially expressed genes, including some immunologically relevant ones, were differentially expressed in a gender-specific fashion. Furthermore, comparison of the data from the PHA-E 28-day and Caco-2/IEC-6 studies showed poor correlation between in vivo and in vitro. Two possible explanations for this lack of correlation were envisaged: IEC-6 and Caco-2 cells are not appropriate models to mimic the effects on intestinal epithelial cells of the jejunum; or intestinal scrapings consist of so many different cell types that the effects on the target cell type (epithelial cells) are masked. In order to examine the latter possibility, in the subsequent PHA-E 90-day study epithelial cells were differentially isolated from the jejunal segments of female rats using a procedure that has been proven to produce a sequential isolation of epithelial cells from villus to crypt. Rats were from the control group (purified diet with non-transgenic rice), from the group fed with purified diet containing PHA rice, and from the group fed with purified diet containing PHA rice and 0.1% purified PHA. Total RNA was isolated from samples containing mainly either crypt or villus cells. The most striking results were found in villus cells from rats fed with the diet containing purified PHA-E. Interestingly, immunologically relevant genes which were differentially regulated in these latter cells were also found to be regulated in a similar fashion in scrapings from female rats fed with PHA-E in the 28-day study. Gender-specific effects of PHA-E on the intestine were also observed upon macroscopic examination of the intestine of rats in the acute and 28-day study. Although, in the 90-day study microarray hybridisations were performed with RNAs from distinct fractions of cells, it did not improve the in vitro-in vivo correlation. Therefore, the combined results suggest that Caco-2 and IEC-6 cells are in vitro culture systems that are too simple to screen for effects of PHA-E on the intestine. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

This result comprised a number of 90-day feeding and toxicity studies in rats, which are the core studies of the project. The specific objectives of this task were to: - Design an animal study with a selection of biomarkers, which will render the 90-day studies sensitive and specific to possible adverse effects of the tested transgenic rice strains. - Test high amounts of the transgenic rice implemented in a purified diet to mimic and challenge the potential human exposure situation. - Use pure recombinant protein spiked to the transgenic rice as the parallel positive controls. Three 90-day studies addressing the safety of transgenic rice lines expressing PHA-E-lectin, GNA-lectin and Cry1Ab-protein were carried out. All transgenic rice lines were tested against the respective parental lines. In the study with PHA-E-lectin, an additional dose group given PHA-E rice spiked with pure recombinant PHA-E was included to increase the specificity of the study. The amounts of recombinant Cry1Ab-protein and GNA-lectin available were not sufficient to be used as spiking material and therefore no positive control group was included in either of these studies. When assessing the safety of genetically modified crops the most common approach is to test whether the GM-crop is substantial equivalent to the conventional parental crop. In the present SAFOTEST project, it was the idea to include in the test design of the animal studies a third group where the rats should be given a diet based on the transgenic rice and spiked with either Bt toxin, GNA lectin or PHA-E lectin. The levels of the recombinant proteins added to the diets should correspond to the lowest observed adverse effect level, which should be identified in 28-day studies and in vitro studies. As mentioned earlier this concept was only possible to conduct for PHA-E rice, due to lack of Bt toxin and GNA lectin. The 90-day studies with Bt rice GNA rice were therefore conducted s conventional safety studies where the transgenic rice was tested against its parental line. In the study with Bt rice very few significant findings were observed. The most striking findings were a significant increase in testis and uterus weight of the rats fed Bt rice. When it comes to uterus weight, the female rats being in different stages of the estrous cycle could explain these differences. This is in accordance with observations of estrous related hyperemia and edema of the uterine wall of more animals in the group fed on Bt rice. The differences in testis weight could be explained by histopathological findings of degeneration of the seminiferous tubules. In the study with GNA rice the most striking effect observed was increased water intake of both male and female rats fed GNA rice. Although not an adverse effect it self, it could indicate some kind of disturbance in the organisms of the rats resulting from a change in the chemical composition of the transgenic line. However, the compositional analysis of the GNA rice and the parental line did not reveal any changes that could be directly linked to an increase in water consumption. The study with PHA-E rice following the original SAFOTEST concept with an additional group fed the PHA-E rice spiked with 0.1% pure recombinant PHA-E lectin was performed with only female rats, which in preliminary studies had shown to be the most sensitive sex towards the lectins. In the group given PHA-E rice spiked with PHA-E lectin, a large number of parameters (body weight, food and water consumption, blood biochemistry, microbiology and organ weights) were found to be significantly affected compared to the group given parental rice. For some of the parameters significant differences were also seen between the group fed PHA-E rice and the group fed parental rice. The outcome of the 90-day study with PHA-E rice showed that we by using the SAFOTEST concept could perform scientific meaningfully studies. The use of an additional group given the transgenic rice spiked with the recombinant protein, the so-called positive control, did not increase the sensitivity of the study. However, it enables us to demonstrate the sensitivity and specificity of the study and thereby to increase the confidence to the animal model used. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

This result comprised a number of tightly linked studies which were addressing aspects such as the in vitro biodegradability and cytotoxicity of the recombinant lectins and Bt toxin. The information and material generated in these studies were used for subsequent experiments to study the effect of these proteins on gene expression in intestinal epithelial cell lines derived from human (Caco-2 cells) and rat (IEC-6 cells). The biodegradability, i.e. proteolytic degradation, of PHA-E, GNA and Bt toxin was analysed by incubation in simulated gastric fluid (SGF, containing pepsin, pH2.0) and subsequent incubation in simulated intestinal fluid (SIF, containing trypsin, pH7.0). Two protocols were used which differed in the ratio of protein to digestion enzyme (low in the one, high in the other). It could be concluded from these experiments that in SGF the Bt toxin was degraded more rapidly than PHA-E and GNA, that further treatment in SIF resulted in more degradation only in the case of PHA, and that degradation was increased at the low ratio of protein to enzyme. The cytotoxicity of the lectins, Bt toxin, and peptic-tryptic (PT) digests of these proteins has been determined in order to define the appropriate protein/peptide concentration to be applied for gene expression profiling. Dose-response experiments were performed using Caco-2 cells (both differentiated and undifferentiated) and IEC-6 cells. LDH leakage and conversion of MTT were the cytological parameters examined. Only digests generated according to the high ratio of protein to enzyme method were found to be suitable for further experimentation. The high concentration of digestion enzymes in the other method appeared to be detrimental for the cells. It could be concluded from these experiments that, irrespective of the type of protein or digest under investigation, 100microgram/ml was the non-cytotoxic concentration to be used for gene expression profiling experiments in Caco-2. With respect to IEC-6, it was concluded that 50microgram/ml of GNA /Cry1Ab protein or PT digest, was the concentration to be used for gene expression studies. Compared to these latter proteins, PHA-E appeared to be more toxic for IEC-6 cells and 20microgram/ml was chosen as the concentration for the gene expression profiling experiments. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The objective was to produce adequate quantities of seed from the transgenic rice lines for the other partners involved in SAFOTEST. After establishment of the lines expressing the proteins of interest, seed from the primary transformants were multiplied in environmentally controlled rooms at 30C with a 16 hour photoperiod; seeds were subsequently sent to Zhejiang University, China for bulking up in the field. Between 200-250 kg of each rice line was harvested and despatched to be used by the other SAFOTEST partners. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The aim was to produce and verify constructs for expression of snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) in microorganisms to produce gram quantities of purified recombinant protein for feeding studies. The Pichia pastoris strain X33 was transformed with pPICZB expression vector containing the structural gene for PHA-E, including the sequence encoding the signal peptide. After transformation, transformants were selected for zeocin resistance. Similarly, the Pichia pastoris strain X33 was transformed with pPICZB expression vector containing the structural gene for GNA, including the sequence encoding the signal peptide. After transformation, transformants were selected for zeocin resistance. The DNA construct (pOS4301) for expression of Cry1Ab was obtained from the Bacillus Genetic Stock Centre (BGSC) at the Ohio State University, USA (BGSC No. ECE54). The construct was assembled by Ge et al. (1989). Briefly, a wild-type full-length Bacillus thuringiensis Cry1Ab cDNA was cloned into vector pKK223-3 (Pharmacia), which contains the inducible tac promoter (Ptac) and transformed into Escherichia coli strain JM103. Gram quantities of the recombinant proteins were produced by large-scale fermentation. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The objective was to compare the chemical compositions of transgenic and parental rice grains used in the feeding studies. Two different methodologies were evaluated as tools for an analytical comparison: - Targeted analysis of (anti-) nutrients and toxicants, and - Non-targeted analysis of low molecular weight compounds by means of metabolite profiling. - Targeted analysis Comprehensive analytical characterization of rice material was performed by application of validated standard protocols. Analysis of the chemical compositions included proximate analysis (water, ash, fibre, starch, sugars, fat, protein), analysis of lipids (fatty acid distribution, phospholipids), amino acids, vitamins (B1, B6, niacin, pantothenic acid, folic acid) and minerals (sodium, potassium, magnesium, calcium, manganese, iron, copper, zinc, molybdenum, phosphorous, selenium). Differences between the lines were assessed for statistical significance and evaluated in the light of natural variations described in literature. In order to check the microbiological quality, fungal and bacterial counts were determined and the material was tested for mycotoxins. As regards contaminants, the material was analysed for heavy metals and pesticides. Conventional methods for the targeted analysis of minor food constituents usually require laborious sample preparation steps in order to isolate analytes from the food matrix prior to analysis. On-line coupled liquid chromatographygas chromatography (on-line LC-GC) was evaluated as a technique for the rapid analysis of minor grain constituents preventing time-consuming sample preparation steps. A method based on on-line LC-GC was developed for the analysis of phytosteryl ferulates, a group of minor rice constituents exhibiting antioxidative and cholesterol-lowering activities. On-line LC-GC proved to be a powerful analytical tool for the targeted analysis of minor rice constituents without laborious sample preparation steps. The suitability of Near Infrared Spectroscopy (NIRS) for non-destructive determination of minor compounds was investigated by using phytic acid, an antinutritional rice grain constituent, as example. Applicability of the methodology for rapid screening of rice samples from different origins regarding low, medium, and high phytic acid contents was demonstrated. Results obtained by NIRS for GM and parental rice lines were in agreement with those determined by the traditional method. Sample preparation is reduced to a minimum by the non-destructive approach. - Non-targeted analysis A gas chromatographic metabolite profiling method for unbiased screening of a broad spectrum of compounds was elaborated using rice as model crop. It is based on a fractionation approach which eventually allows non-targeted investigation of major and minor rice grain constituents. General applicability of the technique for analysis of cereals with metabolite compositions similar to rice was demonstrated. Reliability of the profiling approach was proven by investigation of rice, maize and barley samples with known and expected differences on the metabolite level. Software tools for comparative analysis of metabolite profiling data were developed. They include methods for manual and automated correction of retention times and responses by means of corresponding standards. This allows transfer of normalised datasets into databases and eventually automated unbiased chromatogram comparison. Application of the metabolite profiling methodology to the analysis of GM and conventional rice revealed the suitability of a non-targeted approach for detection of statistically significant differences between metabolite levels in different genotypes. The usefulness of databases established through investigation of a broad spectrum of conventional rice cultivars for evaluation of differences in metabolite profiling data was demonstrated. The unbiased screening of a broad spectrum of compounds combined with powerful methods for data analysis may help to increase the chances to detect unintended effects due to the application of recombinant DNA techniques. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

In order to provide sufficient recombinant protein for food safety assessments, methods were developed for the high level expression and large-scale purification of recombinant protein in Pichia pastoris and Eshericia coli. Methods were developed for the production of two lectins, GNA and PHA-E and a Bt protein cryIA(b). Gram quantities of the proteins were produced and their authentic biologicalm activity was confirmed. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The results obtained in SAFOTEST as well as the overall test design was evaluated at the final project workshop in May 2004, where also recommendations for the future testing procedures to be applied to GM plant foods for the European market were discussed. The discussion and interest in the workshop Plenary early on centred on the use of the tiered approach (and not a base-set) and thereby excluded a mandatory request for the 90-day safety study, which is an inherent part of the SAFOTEST approach. The conclusions of the plenary at the SAFOTEST workshop, as it went, only acknowledged the tiered approach where the 90-day safety study becomes an option on a case-by-case basis. More specifically the plenary concluded that - The requests for molecular characterisation in the Guidance Document should be revised in the light of the discussions at this Work Shop. - Short term toxicity studies could be shorter than 28-days, even not necessary depending on the case. - Guidelines should also be revised concerning request for data regarding agricultural performance. - The use of nutritional adjustments of the overall diet in feeding trials should depend on the purpose of the test: Testing for toxicity assessment of the insert or for identification of unintended effects (it should be kept in mind that minor un-intended effects may not be discovered if obvious nutritional deficiencies in the GM-food group are not adjusted!). - Development of a guideline for the use of defined animal diets designed for the inclusion of high amounts of food material should be considered by OECD. - Spiking should not be used as a routine, but on indication. - If spiking are used, it still is a matter of discussion and judgment if there should be both a spiked control group and a spiked GM group. - Use of the Margin-Of-Safety approach, where intake of the GM-food in the animal test should be compared with the expected intake in man, should be considered in order to proceed in the risk characterisation. - In the end it was stressed that the scientific assessment should not loose sight of the data available and thrive at reaching a comprehensive view of the safety of the GM-food. Finally the Workshop Plenary felt that the segment of the SAFOTEST concept directed towards the spiking will need further experience and data in order to determine its future usefulness in the hazard characterisation of GM plants. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The aim was to produce and verify constructs for expression of snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) in the rice (Oryza sativa L.) cultivars ASD16, EYI105, and Xiushui 11 respectively. Two different methods of plant transformation were used to produce transgenic rice plants, namely particle bombardment (GNA or PHA-E expressing rice) and Agrobacterium mediated transformation (Bt). For generation of GNA expressing rice, both the gna coding sequence (from plasmid pLECGNA2) and the promoter region were used to produce the ubi-gna plasmid for subsequent transformation. Similarly, for generation of PHA-E expressing rice, both the pha-e coding sequence and the promoter region were used to produce the ubi-pha-e plasmid. Plants containing the gene construct for expression of either GNA or PHA-E were generated by particle gun bombardment of mature embryos. In addition to the expression pasmid, the pWRG1515 plasmid (containing both the hygromicineresistance gene, hpt, and the reporter gene gusA) was also used. Following co-transformation, calli were transferred to a proliferation medium to allow plantlet formation and transformants selected on the basis of hygromycin resistance. Putative transformants were screened for the presence of the transgene by PCR. Bt (cry1Ab) expressing rice was generated by Agrobacterium mediated transformation. The T-DNA region of the binary vector pKUB was used for transformation. In addition to the cry1Ab synthetic insecticidal protein gene, the transformation cassette also included a hygromycin phosphotransferase gene, a neomycin phosphotransferase gene, the termination signal of nopaline synthase, the maize ubiquitin promoter, and the coding region for the gus gene. After selection of putative transformants (based upon hygromycin resistance and/or gus assay) transgene expression in the resulting plants was confirmed by immuno-assay using Western blot analysis. Immunodotblot assay was additionally employed to quantify transgene expression in rice tissues, including seeds. Bt rice showed the lowest levels of transgene expression in seeds when compared to the other rice tissues. In contrast, GNA and PHA-E rice plants exhibited the highest level of expression in the seeds, compared to other tissues. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

The specific objectives were to produce transgenic rice plants expressing snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) constitutively expressed throughout the rice tissue and therefore provide the necessary transgenic plant material for the programme. Quantitative immunological techniques were employed to assay levels of accumulation of transgene products in the transgenic rice plants, and to generate lines of progeny plants containing defined levels of transgene products in the harvested seeds. Primary transformants of rice plants containing the gene constructs for expression of the selected foreign proteins (GNA, PHA-E) were produced by particle gun bombardment of mature, seed-derived callus. Calli were bombarded with gold particles coated with DNA of the plasmids to be co-transformed together with the plasmid containing the selectable marker gene. After bombardment, calli were transferred to proliferation medium to allow plantlet formation. Plantlets were separated, transferred to rooting medium and after root induction, to soil. Plantlets were screened for transgene presence by PCR while still in tissue culture (when approx. 5cm high), using primers directed against the appropriate transgene coding sequence. Plantlets negative for the transgene were discarded at this stage. Rooted plantlets growing on in soil were screened for the presence and accumulation of the transgene product by immunological assay (Western blotting after analysis of total protein by SDS-PAGE). The remaining plants were grown on to maturity in growth rooms under high-light conditions, and allowed to set seed by selfing. Immuno-dot blot assays were used to quantify levels of accumulation of transgene products in selected primary transformant rice plants; quantification was carried out by densitometry of X-ray films exposed to the blots, using Bio Rad Molecular Analyst software. Based on these assays, positive and negative expressors for the transgenes were selected. These plants were used as the basis for transgenic rice lines and controls. Seed from subsequent generations were assayed to establish defined levels of transgene product accumulation in the selected lines. PHA-E expression at the mature stage in the selected lines was highest in seeds (approximately 0.78% total soluble protein), and lowest in the root (Approximately 0.051% total soluble protein). Stability of transgene expression was also assessed employing quantitative immunoassays (dot-blot) in progeny plants from the selected primary transformants. The expression levels in seeds from three successive generations (T1-T3) was 0.75, 0.74, and 0.77% of the total soluble protein respectively, indicating no significant differences over time. At T4 (the latest generation) seeds still showed some degree of heterozygocity. GNA expression levels were highest in seeds (0.98%) and lowest in leaves (0.25%). As with PHA-E, GNA expression in the seeds was relatively constant over three consequetive generations (T1-T3, 1.01, 1.4, 1.2% respectively). Seeds from the final generation (T6) were all homozygous after screening by immunoassay. Bt toxin expression levels were lowest in seeds (0.018%) and highest in the leaf sheath (0.28%). The level of expression in seeds over three successive generations (T9-T11) was 0.017, 0.016, and 0.015% respectively, showing no significant differences over time. The final generation of the seeds (T11) were homozygous. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

This result comprised the 28-day feeding and toxicity studies, which formed the basis for the 90-day animal studies in the project. The specific objectives of this task were to: -Optimise the composition of the purified diet to include the maximum concentration of rice. - Identify sensitive target organs and specific biomarkers for the identification of adverse effects of the PHA-E lectin in the rat model. - Establish the lowest-observed-adverse-effect-levels (LOAEL) for the lectin, which are needed for the later 90-day study (Task VI). In order to fulfil the objectives of the project, PHA-E lectin seemed to be the most useful model compounds. Consequently, the project partners decided that all efforts in making the recombinant proteins should concentrate on PHA-E lectin on behalf of the GNA lectin and Bt toxin. The increased production of the PHA-E lectin resulted in a yield that was sufficient to perform all scheduled studies for this compound. In the preliminary small-scale acute rat study the purpose was to reveal the toxic potential of the PHA-E lectin expressed in P. pastoris. Based on findings such as oedema and thickening of the epithelium of the small intestine and sign of proliferation in the gut lympoid tissue it was concluded that 10 mg/rat/day (corresponding to about 0.1% in the feed) would generate adverse effects in the animals. In order to avoid mortality and serious adverse effects that could compromise the evaluation of the study, a level of 0.08% PHA-E lectin in the diet was therefore chosen as the highest dose level in the 28-day study with PHA-E lectin. Before initiating the animal studies one very important item was to test whether the rats liked the taste of raw brown rice and whether they could tolerate high inclusion levels of rice in the diet. The results from the tolerance study showed that Wistar rats readily eat and tolerate up to 60% of raw brown rice in a purified diet for 28 days. In the first 28-day study with PHA-E lectin focussing on general toxicity, no dose-related effects were seen on clinical observations, body weight, body weight gain, and food consumption. For clinically parameters as blood chemistry, haematology and microbiology, a few numbers of significant clinical signs were observed in the study period, but none of them are considered to be treatment-related. At the necropsy, animals given PHA-E lectin had a tendency to slightly thicker jejunum compared to animal from the control group. Despite these macroscopic observations, the findings could not be confirmed by histologic examinations. In general no dose-related findings in organ weights, macroscopic pathology and histopathology were observed. The second 28-day study with PHA-E lectin focussed on immunotoxicity of the PHA-E lectin and especially designed to reveal this. Other end-points addressing general toxicity were included as well. In the study a statistically significant increase in absolute and relative weight of the small intestine of female rats and relative weight for male rats was observed in the highest dose group. In addition, the absolute and relative pancreas weight was significantly increased in nearly all female groups given PHA-E lectin compared to the control group. To conclude on the two studies with PHA-E lectin, the toxicity of PHA-E lectin expressed in P. pastoris was lower than expected. Accordingly, a dose-response relationship for PHA-E could not be established based on these studies. An effect level of 0.08% PHA-E has been established based on the increased weight of the small intestine. As a consequence, the small intestine and related parameters and partly the pancreas will be the target organs for the future 90-day study with PHA-E. As the PHA-E level of 0.08% in the feed only caused mild effects, it was decided to increase the level of spiked PHA-E-lectin in the 90-day study with PHA-E rice to 0.1%. Based on the findings in the acute study we had expected an effect of the PHA-E lectin at a lower dose level than 0.08%. However, the rats seemed to in some way have been adapted to the effects of the PHA-E lectin indicating that the effects were acute and reversible of nature. Nevertheless, agglutination assays indicated that PHA-E lectin had very similar biological activity to authentic plant standards. The glycosylation of recombinant PHA-E was typical of that previously seen for Pichia pastoris produced proteins. The special formulated purified diet with 60% of rice tested in the preliminary tolerance study as well in the two above-mentioned 28-day studies proved to sustain growth and health and yield reproducible experimental results. This type of diet was therefore with only minor corrections the diet that was used in the 90-day studies. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Molecular characterisation of transgenic materials by Southern blot analysis was conducted. Genomic DNA was isolated from leaves of transgenic or parental plants. Approximately 10 µg aliquots of genomic DNA were subjected to digestion with appropriate restriction enzymes, separated on agarose gel, blotted and fixed onto nylon membranes. DNA was hybridised with 32P or non-radioactively labelled DNA probes. For PHA-E expressing rice, Southern hybridisation of transformants demonstrated the presence of three insertions in the selected transgenic line. This result was obtained using both PHA-E coding DNA and the ubiquitin promoter as DNA probes. Southern blot analysis of the genomic DNA from GNA expressing plants indicated that there was only one insertion event of the transgene in the rice genome. A genomic library was constructed, using a Lambda vector, to identify the site of insertion. Screening of the library with radioactively labelled gna coding DNA revealed that the transgene was integrated into a non protein coding region of the rice genome. For Bt expressing rice, an internal fragment of the gus (uidA) gene and a cry1Ab fragment generated by PCR were used as probes for hybridisation. Results from Southern analyses clearly showed that the transgene was inserted at a single locus. Inverse PCR was also employed in an attempt to characterise the site of insertion of the transformation cassette. Bt expressing rice was transformed via Agrobacterium T-DNA, thus IPCR primers were designed based on the sequences of the left and right borders of the vector. The generated PCR product of approx 1.2 KB was cloned into a sequencing vector. After sequencing, Similarity studies using BLASTX or BLASTN indicated that transposition of the transgene occurred within a retrotransposable element-coding region in the rice genome. Furthermore, the results indicate that both single and multiple insertion events may occur when particle bombardment is used to transform plants. More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

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