Laboratory of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, University of Lausanne, Lausanne, Switzerland.
Nucleic Acids Res. 2011 Aug;39(15):e104. doi: 10.1093/nar/gkr436. Epub 2011 Jun 7.
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.
真核生物中的基因转移和表达常常受到一些稳定维持的基因拷贝数和表观遗传沉默效应的限制。沉默可能受到表观遗传调节序列如基质附着区（MAR）的限制。在这里，我们显示 MAR 包含载体的连续转染允许转基因表达的协同增加。这种发现部分可以通过增加细胞进入细胞核和 DNA 的基因组整合来解释，这种效应既需要 MAR 元件又需要迭代转染。荧光原位杂交分析经常显示单个整合事件，表明在连续转染中引入的 DNA 可以重组。高表达也与细胞分裂周期有关，因此当同源重组最活跃时，DNA 的核转运发生。利用非同源末端连接或同源重组缺陷的细胞表明，有效的整合和表达可能需要基于同源重组的 MAR 包含质粒的基因组整合，以及与串联基因拷贝相关的表观遗传沉默事件的缺失。我们得出结论，MAR 元件可能促进同源重组，并且可以对细胞和载体进行工程改造，以利用这种特性来介导高效的基因转移和表达。
