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.

Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis.

Periodicity, the cyclical rise and fall in microfilaria (mff) numbers in the peripheral blood over time, is observed in many filarial infections. It is correlated with the necessity for these larval stages to be ingested by the blood feeding vector before they can be transmitted to a new vertebrate host. Microfilariae of the dog heartworm Dirofilaria immitis have been described to show periodicity, but the circadian pattern does not seem to be consistent. Most publications describe the lowest mff-concentrations in the peripheral blood in the early morning, while the highest counts occurred either in the afternoon, in the late evening or shortly after midnight. Sixteen dogs were experimentally infected with D. immitis isolates originating from Italy (one isolate, 14 dogs), and the USA (two isolates, one dog each). The dogs were housed indoors with a natural light source (windows) and heating that prevented temperature-drops below 20°C during winter. When patency was reached, blood samples were collected at weekly and monthly intervals over a period of up to 3 years, and at given hours of the day (morning, noon, evening) for the duration of one year in order to determine seasonal, as well as daily variations of microfilaremia. Despite the fact that the dogs were kept indoors, there was an apparent seasonality of the D. immitis-microfilaremia, with peaks in summer and 5-49-times lower counts in winter. This difference was statistically significant and the ratio remained constant over the years, regardless of the fact that the mff-counts increased from the first to the second year of patency. Since the temperature was kept constantly in a range between 20 to 26°C (with some single outliners in both directions) the climatic conditions may not explain this observation. Therefore, day length may be the most obvious reason for the seasonality in the given study set-up. Interestingly, the Italian D. immitis-isolate lost seasonality after three passages of experimental infections in dogs. The circadian cycle of mff in the peripheral blood varied considerably between dogs and season. There was no consistent or apparent pattern, which led to the conclusion that many individual factors seem to influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.