A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the Ct- and Tm values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product

High glucose disturbs different developmental parameters of the mouse blastocyst in vitro

Diabetic embryopathy is the leading cause of neonatal death and I or congenital malformations in diabetic mothers’infants. In response to the difficulties with pregnancy outcome, there has been much effort to extend understanding of the disruptive mechanisms involved in diabetic embryopathy. Most of our knowledge on the deleterious influence of maternal diabetes on embryo development comes from experimental studies on rodent embryos undergoing organogenesis. These studies have demonstrated the embryotoxicity of diabetic serum and the implication of glucose as a direct teratogen. Much less studies have been performed during the pré-implantation period of embryo development. However, there is increasing recognition that poorly controlled maternal diabetes before the time of implantation may be an important risk factor for fetal complications in the human population. One of the most determinant events that occur during the pré-implantation period is the first cell differentiation with the emergence of two stem cell lineages: the embryoblast or Inner Cell Mass which will form the fetus and the trophectoderm which will form the placenta. The changes induced in the uterine environment by maternal diabetes may disrupt at this stage of development, the differentiation and proliferation of both cell lineages that could have severe consequences on further embryo development. This study will investigate the influence of high glucose concentrations on the in vitro development of the mouse pré-implantation embryo and particularly the differential impact of glucose on the two cell lineages composing the blastocyst stage embryo. The first part of this work will present different developmental aspects of the pré-implantation embryo and particularly of the blastocyst stage embryo. We will also summarize observations reported in vivo and in vitro by previous studies of the deleterious impact of diabetes on rodent pré-implantation embryos. Finally, some concepts proposed to explain the diabetic embryopathy in the post-implantation rodent embryos would be presented. Personal contribution in this area of research will follow with three manuscripts investigating the impact of high glucose concentrations on mouse blastocyst cell death and differentiation. The main results and perpectives will be discussed in the final part of the thesis.Thèse de doctorat en sciences biomédicales (biologie du développement) (SBIM 3) --UCL, 200

Control of trophectoderm differentiation by inner cell mass-derived fibroblast growth factor-4 in mouse blastocysts and corrective effect of FGF-4 on high glucose-induced trophoblast disruption.

Previous studies have suggested that fibroblast growth factor-4 (FGF-4) may be a paracrine signal used by inner cell mass (ICM) cells to maintain adjacent trophectoderm (TE) cells in an undifferentiated state. In the present work, immunocytochemical analysis of mouse blastocysts confirmed that FGF-4 was predominantly detected in the ICM before and after spreading over a fibronectin-coated culture substrate. Addition of human recombinant FGF-4 did not influence morphological progression, cell allocation and proliferation in ICM and TE lineages or mitosis and karyorhexis frequencies during blastocyst expansion. Addition of FGF-4 to outgrowing blastocysts, in contrast, induced a significant decrease in the surface of the trophoblast outgrowths formed by the TE cells and in the proportion of giant trophoblasts per outgrowth. The fact that blastocysts display excessive trophoblast expansion and spreading over their culture substrate upon pre-exposure to high concentrations of glucose in vitro was used to further assess the regulatory effect of FGF-4. Addition of FGF-4 was indeed found to fully neutralize the disruptive impact of high glucose on trophoblast outgrowths. Altogether, our data indicate that ICM-derived FGF-4 participates actively in the regulation of trophoblast development

SYBR Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products

Identification of crops present in food and/or feed matrices represents an important step in the screening strategies targeting genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their presence in an integrated screening approach, a set of SYBRGreen real-time polymerase chain reaction (qPCR) methods has been developed which can be used under the same assay conditions and at similar efficiency for each of the abovementioned crops. Each qPCR method is shown to meet the performance criteria (i.e. specificity, limit of detection and PCR efficiency) set by the European Network of GMO Laboratories (ENGL). When combined with the equivalent qPCR methods targeting GMO elements, these crop-specific SYBRGreen qPCR methods can aid the development of an efficient tool for determining GMO presence in food and/or feed products.JRC.I.4-Molecular biology and genomic

Four new SYBRGreen qPCR screening methods for the detection of Roundup Ready, LibertyLink, and CryIAb traits in genetically modified products

SYBR®Green qPCR methods for the detection of the Roundup Ready® Cp4-EPSPS, LibertyLink® “PAT” and “BAR” and the Bacillus thuringiensis CryIAb/Ac traits as present in genetically modified organisms (GMO) were developed. The specificity, sensitivity, dynamic range and PCR method efficiency was determined for each of them. All methods comply with the performance criteria set by the European Network of GMO Laboratories (ENGL), ISO norms and Codex guidelines. The developed methods are specific and generate amplicons of 108, 73, 109 and 69 bp respectively for “Cp4-EPSPS”, “CryIAb/Ac”, “PAT” and “BAR” targets. Single-target Sybricon plasmids comprising the corresponding PCR amplicons have been constructed and used as controls in PCR analysis. These four SYBR®Green qPCR methods for GMO analysis complete the set of qPCR methods applied in the so-called combinatory SYBR®Green qPCR screening (abbreviated as "CoSYPS''; Van den Bulcke et al, 2010, Anal Bioanal Chem, 396(6):2113-23). Due to their trait-specific nature, these methods allow discriminating better between the different GMO. Compared to GMO screening applying only common targets such as the 35S promoter or the NOS terminator, screening with trait-specific methods reduces the number of possible GMO candidates present in a sample, reducing the cost of the analysis. The application of these methods in CoSYPS analysis is demonstrated using two GEMMA proficiency test samples and reference material from the genetically modified rapeseed event GT73. This set of SYBR®Green qPCR trait-specific methods represents a very interesting novel set of GMO analysis methods allowing cost-effective identification of GM materials in products.JRC.I.3-Molecular Biology and Genomic