A real-time quantitative PCR detection method specific to widestrike transgenic cotton (event 281-24-236/3006-210-23).

In compliance with global regulations on transgenic crops, a real-time quantitative PCR method specific to Widestrike transgenic cotton (event 281-24-236/3006-210-23, OECD Unique Identifier DAS-24236-5/DAS-21023-5) was established on the basis of the DNA sequences in the junction between the transgene insert and cotton genome. The optimized method consists of a DNA extraction method for cotton seeds and three PCR systems corresponding to a cotton-specific endogenous reference DNA sequence SAH7 (Sinapis Arabidopsis Homolog 7) and specific detection of event 281-24-236 and event 3006-210-23. The method performance including specificity, sensitivity, accuracy, and precision was determined at a dynamic range of Widestrike DNA levels from 0.04% to 5.0%. The limits of detection (LOD) and quantification (LOQ) were < or =0.04% and < or =0.09%, respectively, at 100 ng of DNA sample per reaction. The quantification results using either the event 281-24-236 or 3006-210-23 system were consistent, and the relative deviation from the expected (true) value was in the range of +/-25%. The robustness of the method was demonstrated by a series of tests with deviations from the optimized assay parameters such as annealing temperature, extension time, PCR instrument, interlaboratory transferability, etc. All the measurements from these tests met the criteria set by EU JRC-CRL (European Commission Joint Research Centre-Community Reference Lab). This real-time quantitative PCR method is accurate and robust, and is recommended as a global benchmark method for the detection and quantification of Widestrike cotton. The method including description, protocol, and performance results is available on the JRC-CRL website (http://gmo-crl.jrc.it/statusofdoss.htm).

The value of short amino acid sequence matches for prediction of protein allergenicity.

Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens.

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.

Compositional equivalency of Cry1F corn event TC6275 and conventional corn (Zea mays L.).

Maize (Zea mays L.) plants have been transformed to express a Cry1F insecticidal crystal protein originally isolated from Bacillus thuringiensis Berliner. This protein controls lepidopteran pests of maize, including the European corn borer, Ostrinia nubilalis (Hübner). As part of the safety assessment for crops containing transgenes, a compositional analysis of the food and feed is conducted. This analysis is designed to detect unintended changes in the nutrient and antinutrient content of the raw commodities produced by the crop due to the insertion of the genes into the genomic DNA of the plant (pleotropic effects). Samples of transgenic and nontransgenic maize forage and grain were collected from six field sites located in the U.S. and Canada. Forage samples were analyzed for proximates and minerals, and grain was further analyzed for fatty acids, amino acids, vitamins, secondary metabolites, and antinutrients. Results demonstrated that maize expressing the Cry1F protein was equivalent to nontransgenic maize with respect to these important components. Comparison of the variability within the nontransgenic and transgenic hybrid, as compared to composition values reported in the literature, suggest that factors other than transgenes may contribute more substantially to the composition of crops.

Functional analysis of two matrix attachment region (MAR) elements in transgenic maize plants.

Matrix attachment regions (MARs) are binding sites for nuclear scaffold proteins in vitro, and are proposed to mediate the attachment of chromatin to the nuclear scaffold in vivo. Previous reports suggest that MAR elements may stabilize transgene expression. Here, we tested the effects of two maize MAR elements (P-MAR from the P1-rr gene, and Adh1-MAR from the adh1 gene) on the expression of a gusA reporter gene driven by three different promoters: the maize p1 gene promoter, a wheat peroxidase (WP) gene promoter, or a synthetic promoter (Rsyn7). The inclusion of P-MAR or Adh1-MAR on P::GUS transgene constructs did not reduce variation in the levels of GUS activity among independent transformation events, nor among the progeny derived from each event. The Adh1-MAR element did not affect GUS expression driven by the WP promoter, but did modify the spatial pattern of expression of the Rsyn7::GUS transgene. These results indicate that, in transgenic maize plants, the effects of MAR elements can vary significantly depending upon the promoter used to drive the transgene.

High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system.

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation. Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation. A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.