Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.

Immunotherapy of cancer can lead to the selection of antigen loss variants, which provides strong rationale to target oncogenes that are essential for tumor growth or viability. To investigate this concept, we tagged the HER2/neu oncogene with epitopes from ovalbumin to confer recognition by T-cell receptor transgenic CD8 + (OT-I) and CD4 + (OT-II) T cells. Transgenic mice expressing neu OT-I/OT-II developed mammary adenocarcinomas at 6 to 10 months of age. Adoptively transferred naive OT-I cells (with or without OT-II cells) proliferated vigorously on encountering neu OT-I/OT-II -expressing tumors. This was followed by the complete regression of 37% of tumors, whereas others showed partial/stable responses (40%) or progressive disease (23%). Those tumors undergoing complete regression never recurred. In mice with multiple primary tumors, simultaneous regressions and nonregressions were often seen, indicating that immune evasion occurred at a local rather than systemic level. The majority of nonregressing tumors expressed Neu OT-I/OT-II and MHC class I, and many avoided rejection through a profound block to T-cell infiltration. Thus, T cells directed against an essential oncogene can permanently eradicate a subset of spontaneous, established mammary tumors. However, in other tumors, local barriers severely limit the therapeutic response. To maximize the efficacy of immunotherapy against spontaneous cancers, predictive strategies that take into account the heterogeneity of the tumor microenvironment will be required. [Cancer Res 2007;67(13):6442-50]

Purpose: In vitro studies suggest that ovarian cancer evades immune rejection by fostering an immunosuppressive environment within the peritoneum; however, the functional responses of ovarian cancer^specific T cells have not been directly investigated in vivo. Therefore, we developed a new murine model to enable tracking of tumor-specific CD8 + T-cell responses to advanced ovarian tumors. Experimental Design: The ovarian tumor cell line ID8 was transfected to stably express an epitope-tagged version of HER-2/neu (designated Neu OT-I/OT-II

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