High-level transgene expression by homologous recombination-mediated gene transfer

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expressio

Evaluation of the potential of functionalised calcium carbonate as carrier for essential oils with regard to antimicrobial packaging applications

Functionalised calcium carbonates (FCCs) are inorganic mineral-based particles with a high porosity and extended surface area consisting of hydroxyapatite and calcium carbonate crystal structures. Therefore, FCCs have a high potential to be used as a carrier for active substances such as essential oils (EOs), which are well known for their antimicrobial activities, and control their release in antimicrobial packaging applications. In this study, different EOs were loaded on FCCs, and their antimicrobial activities were studied against Listeria innocua in in vitro tests and in food tests using sliced cooked chicken breast. FCCs loaded with thyme or oregano EO (10 wt%) showed the highest reduction in microbial load in in vitro tests at 37°C (≥8.6 log cfu/filter) as well as at 7°C after 6 days (≥7.0 log cfu/filter for thyme EO and 6.5 log cfu/filter for oregano EO). However, in food tests, FCC loaded with either EO did not show any significant antimicrobial activity. FCCs loaded with cinnamon or rosemary EO (10 wt%) did not show any significant antimicrobial activity in in vitro tests. On the other hand, they showed a significant reduction in microbial load (1.7 log cfu/g for cinnamon and 2 log cfu/g for rosemary) in food tests. Differences in antimicrobial activities in in vitro and food tests are probably due to the interaction of the components of the EOs and the components of the food such as fat and proteins

High-level transgene expression by homologous recombination-mediated gene transfer

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression