German Research Center for Biotechnology (GBF), RDIF/Epigenetic Regulation, Mascheroder Weg 1, D-38124, Braunschweig, Germany.
Cytotechnology. 2006 Mar;50(1-3):93-108. doi: 10.1007/s10616-006-6550-0. Epub 2006 Jun 14.
The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like PhiC31, which recombine attP and attB target sites in a one-way reaction - at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and PhiC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is 'clean' in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.
位点特异性重组酶的可用性彻底改变了具有可预测特性的细胞系的合理构建。早期的努力是为具有单个重组酶靶位点的预先表征的基因组基因座提供地址，该靶位点作为携带兼容标签的载体整合的地址。这种高效的程序必须等待像 PhiC31 这样的重组酶，该重组酶在单向反应中重组 attP 和 attB 靶位点 - 至少在高等真核细胞的细胞环境中。尽管如此，这些程序还是导致了已知会引起表观遗传沉默的原核载体序列的共同引入。本文综述了为位点特异性重组酶 (SR) 的主要成员 Flp、Cre 和 PhiC31 开发的更先进的重组酶介导的盒交换 (RMCE) 技术的实际现状。在 RMCE 中，基因组地址由一组异源重组酶靶 (RT-) 位点组成，允许将中间序列交换为感兴趣的基因 (GOI)，作为类似盒的一部分。该过程将 GOI 锁定到位，并且它是“干净的”，因为它不会共同引入原核载体部分，也不会留下选择标记。
