BackgroundRNA interference (RNAi) is a regulatory cellular process that controls post-transcriptional gene silencing. During RNAi double-stranded RNA (dsRNA) induces sequence-specific degradation of homologous mRNA via the generation of smaller dsRNA oligomers of length between 21-23nt (siRNAs). siRNAs are then loaded onto the RNA-Induced Silencing multiprotein Complex (RISC), which uses the siRNA antisense strand to specifically recognize mRNA species which exhibit a complementary sequence. Once the siRNA loaded-RISC binds the target mRNA, the mRNA is cleaved and degraded, and the siRNA loaded-RISC can degrade additional mRNA molecules. Despite the widespread use of siRNAs for gene silencing, and the importance of dosage for its efficiency and to avoid off target effects, none of the numerous mathematical models proposed in literature was validated to quantitatively capture the effects of RNAi on the target mRNA degradation for different concentrations of siRNAs. Here, we address this pressing open problem performing in vitro experiments of RNAi in mammalian cells and testing and comparing different mathematical models fitting experimental data to in-silico generated data. We performed in vitro experiments in human and hamster cell lines constitutively expressing respectively EGFP protein or tTA protein, measuring both mRNA levels, by quantitative Real-Time PCR, and protein levels, by FACS analysis, for a large range of concentrations of siRNA oligomers.ResultsWe tested and validated four different mathematical models of RNA interference by quantitatively fitting models' parameters to best capture the in vitro experimental data. We show that a simple Hill kinetic model is the most efficient way to model RNA interference. Our experimental and modeling findings clearly show that the RNAi-mediated degradation of mRNA is subject to saturation effects.ConclusionsOur model has a simple mathematical form, amenable to analytical investigations and a small set of parameters with an intuitive physical meaning, that makes it a unique and reliable mathematical tool. The findings here presented will be a useful instrument for better understanding RNAi biology and as modelling tool in Systems and Synthetic Biology.

Research on the environmental risks of gene flow from genetically modified (GM) crops to wild relatives has traditionally emphasized recipients yielding most hybrids. For GM rapeseed (Brassica napus), interest has centred on the 'frequently hybridizing' Brassica rapa over relatives such as Brassica oleracea, where spontaneous hybrids are unreported in the wild. In two sites, where rapeseed and wild B. oleracea grow together, we used flow cytometry and crop-specific microsatellite markers to identify one triploid F 1 hybrid, together with nine diploid and two near triploid introgressants. Given the newly discovered capacity for spontaneous introgression into B. oleracea, we then surveyed associated flora and fauna to evaluate the capacity of both recipients to harm cohabitant species with acknowledged conservational importance. Only B. oleracea occupies rich communities containing species afforded legislative protection; these include one rare micromoth species that feeds on B. oleracea and warrants further assessment. We conclude that increased attention should now focus on B. oleracea and similar species that yield few crophybrids, but possess scope to affect rare or endangered associates.

Summary• Biocontainment methods for genetically modified crops closest to commercial reality (chloroplast transformation, male sterility) would be compromised (in absolute terms) by seed-mediated gene flow leading to chloroplast capture. Even in these circumstances, however, it can be argued that biocontainment still represses transgene movement, with the efficacy depending on the relative frequency of seed-and pollen-mediated gene flow.• In this study, we screened for crop-specific chloroplast markers from rapeseed (Brassica napus) amongst sympatric and allopatric populations of wild B. oleracea in natural cliff-top populations and B. rapa in riverside and weedy populations.• We found only modest crop chloroplast presence in wild B. oleracea and in weedy B. rapa, but a surprisingly high incidence in sympatric (but not in allopatric) riverside B. rapa populations. Chloroplast inheritance models indicate that elevated crop chloroplast acquisition is best explained if crop cytoplasm confers selective advantage in riverside B. rapa populations.• Our results therefore imply that chloroplast transformation may slow transgene recruitment in two settings, but actually accelerate transgene spread in a third. This finding suggests that the appropriateness of chloroplast transformation for biocontainment policy depends on both context and geographical location.

In the era of post-genomic research two new disciplines, Systems and Synthetic biology, act in a complementary way to shed light on the ever-increasing amount of data produced by novel high-throughput techniques. Systems biology aims at developing a formal understanding of biological processes through the development of quantitative mathematical models (bottom-up approach) and of 'reverse engineering' (top-down approach), whose aim is to infer the interactions among genes and proteins from experimental observations (gene regulatory networks). Synthetic biology on the other hand uses mathematical models to design novel biological 'circuits' (synthetic networks) able to perform specific tasks (for example, periodic expression of a gene of interest), or able to change the behavior of a biological process in a desired way (for example, modify metabolism to produce a specific compound of interest). The use of a pioneering approach that combines biology and engineering, to describe and/or invent new behaviors, could represent a valuable resource for studying complex diseases and design novel therapies. The identification of regulatory networks will help in identifying hundreds of genes that are responsible for most genetic diseases and that could serve as a starting point for therapeutic intervention. Here we present some of the main genetics and medical applications of these two emerging fields.

We constructed and modeled a novel synthetic network which may be able to exhibit bistable expression of a reporter gene in mammalian cells. This network is based on an aptamer-fused short-hairpin RNA (shRNA) directed against a single mRNA encoding both a EGFP reporter gene and the repressor tTR-KRAB, which, in turn, represses transcription of the shRNA. The activity of the shRNA can be controlled by an inducer molecule (theophylline) which prevents the aptamer-fused shRNA to be properly processed. Repression of the tTR-KRAB can be relieved by treatment with doxycyline. This reciprocal negative feed-back loop can exhibit a bistable response, as shown through the mathematical analysis performed here. Specifically, the network can be controlled to induce sustained expression of a shRNA, or the reporter gene, with a transient input of two different inducer molecules.