Helmholtz Zentrum München, Technische Universität München, Institut für Entwicklungsgenetik, Ingolstädter Landstrasse 1, 85764 München, Neuherberg, Germany.
Nucleic Acids Res. 2010 May;38(9):e106. doi: 10.1093/nar/gkq044. Epub 2010 Feb 5.
Recombinase-mediated cassette exchange (RMCE) exploits the possibility to unidirectionally exchange any genetic material flanked by heterotypic recombinase recognition sites (RRS) with target sites in the genome. Due to a limited number of available pre-fabricated target sites, RMCE in mouse embryonic stem (ES) cells has not been tapped to its full potential to date. Here, we introduce a universal system, which allows the targeted insertion of any given transcriptional unit into 85 742 previously annotated retroviral conditional gene trap insertions, representing 7013 independent genes in mouse ES cells, by RMCE. This system can be used to express any given cDNA under the control of endogenous trapped promoters in vivo, as well as for the generation of transposon 'launch pads' for chromosomal region-specific 'Sleeping Beauty' insertional mutagenesis. Moreover, transcription of the gene-of-interest is only activated upon Cre-recombinase activity, a feature that adds conditionality to this expression system, which is demonstrated in vivo. The use of the RMCE system presented in this work requires one single-cloning step followed by one overnight gateway clonase reaction and subsequent cassette exchange in ES cells with efficiencies of 40% in average.
重组酶介导的盒式交换（RMCE）利用了这样一种可能性，即可以将任何侧翼带有异型重组酶识别位点（RRS）的遗传物质单向交换到基因组中的靶位点。由于可用的预制靶位点数量有限，迄今为止，RMCE 在小鼠胚胎干细胞（ES 细胞）中的应用尚未充分发挥其潜力。在这里，我们引入了一种通用系统，该系统允许通过 RMCE 将任何给定的转录单元靶向插入到 85742 个先前注释的逆转录病毒条件性基因陷阱插入中，这些插入代表了小鼠 ES 细胞中 7013 个独立的基因。该系统可用于在体内表达任何给定的 cDNA，受内源性捕获启动子的控制，以及用于生成转座子“启动子”，以便进行染色体区域特异性“Sleeping Beauty”插入诱变。此外，只有在 Cre 重组酶活性存在的情况下，目标基因的转录才会被激活，这一特性为该表达系统增加了条件性，这在体内得到了证明。在这项工作中使用的 RMCE 系统只需要一个单克隆化步骤，然后进行一个过夜的 gateway clonase 反应，随后在 ES 细胞中进行盒式交换，平均效率为 40%。
