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首页> 外文期刊>Journal of Molecular Biology >Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology.
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Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology.

机译：复用RMCE：Flp重组酶介导的盒式交换技术的多功能扩展。

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摘要

There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRTxFRT), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F'xF). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting ("multiplexing") reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell).
机译：有充分的迹象表明，但尚无证据表明，扩展的48 bp Flp重组酶靶标（FRT）代表了所研究的所有真核基因组中的独特靶标，并且重组酶介导的盒式交换不受基因组假位点的影响。这鼓励了本研究，在该研究中我们探索了在给定细胞中交换两个不同的基因组锚定的盒的可行性，每个盒的侧翼是一组独特的两个异源FRT位点。突变FRT必须满足成功进行重组酶介导的盒交换的两个主要先决条件：（i）与一对FRT野生型位点（FRTxFRT）相当的自我识别能力，以及（ii）如果一部分是交叉相互作用，则可以忽略不计一组异源位点（F'xF）。我们应用两步策略来探索各种新创建的FRT间隔突变体的这些特性。作为我们筛选步骤的结果，我们确定了成功应用于平行Flp介导的基因组靶向（“多重”）反应（即在给定细胞中同时交换两个单独的靶标盒）的位点组合。

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