Lauth Matthias, Spreafico Fabio, Dethleffsen Kathrin, Meyer Michael
Division of Molecular Genetics, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.
Nucleic Acids Res. 2002 Nov 1;30(21):e115. doi: 10.1093/nar/gnf114.
Work of the last decade has proven the 'one gene- one product-one function' hypothesis an oversimplification. To further unravel the emerging 'one gene-multiple products-even more functions' concept, new methods (such as subtle knock-in and tightly regulated conditional mutations) for the analysis of gene function in health and disease are required. Another class of improvements (such as tetraploid fusion and cassette exchange) addresses the efficiency with which targeted mutant strains can be generated. Recombinase-mediated cassette exchange (RMCE), which in theory is well suited for the rapid generation of multiple alleles of a given locus, is hampered by its low efficiency in the absence of selection and, especially in vivo, by the promiscuity of the participating recombinase recognition sites. Here we present a novel approach which circumvents this problem by the use of two independent recombinase systems. The strategy, which uses loxP on one and FRT on the other side of the cassette together with a Cre/Flpe expression vector, prevents excisive events and results in higher rates of cassette integration without selection than previously described. This method has a huge potential for the generation of allelic series in embryonic stem cells and, importantly, in pre-implantation embryos in vivo.
过去十年的研究已证明“一个基因-一种产物-一种功能”假说是一种过度简化的说法。为了进一步阐明新出现的“一个基因-多种产物-更多功能”概念，需要新的方法（如精细的基因敲入和严格调控的条件性突变）来分析健康和疾病状态下的基因功能。另一类改进方法（如四倍体融合和盒式交换）解决了产生靶向突变株的效率问题。理论上，重组酶介导的盒式交换（RMCE）非常适合快速产生给定基因座的多个等位基因，但在缺乏选择的情况下其效率较低，尤其是在体内，由于参与的重组酶识别位点的混杂性，其应用受到阻碍。在此，我们提出一种新方法，通过使用两个独立的重组酶系统来规避这一问题。该策略在盒式结构的一侧使用loxP，另一侧使用FRT，并结合Cre/Flpe表达载体，可防止切除事件的发生，且在无选择的情况下，盒式整合率高于先前报道。这种方法在胚胎干细胞中，更重要的是在体内植入前胚胎中产生等位基因系列方面具有巨大潜力。
