Flp and Cre expressed from Flp-2A-Cre and Flp-IRES-Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange
Flp and Cre expressed from Flp-2A-Cre and Flp-IRES-Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange
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DOI：
10.1093/nar/gks027
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发表时间：
2012-04-01
影响因子：
14.9
通讯作者：
Voziyanov, Yuri
中科院分区：
文献类型：
--
作者：
Anderson, Rachelle P.;Voziyanova, Eugenia;Voziyanov, Yuri
酶介导的盒交换（RMCE）是将感兴趣的DNA片段单向整合到预定基因组区域中的有力工具。在这份报告中，我们研究了Flp和Cre催化的双重RMCE的效率如何取决于表达重组酶的转录单位的性质。分析了以下重组酶转录单位：（i）Flp和Cre基因表达为位于不同载体上的单独转录单位，（ii）Flp和Cre基因表达为位于相同载体上的单独转录单位，（iii）Flp和Cre基因由单个启动子表达并由内部核糖体进入序列分开，以及（iv）Flp和Cre编码序列被2A肽分开并作为单个基因表达。我们发现，当Flp和Cre重组酶表达为Flp-2A-Cre和Flp-IRES-Cre转录单位时，可以实现最高水平的双重RMCE（转染细胞的35-45%）。相反，当Flp和Cre作为单独的转录单位表达时，达到最低水平的双重RMCE（类似于转染细胞的1%）。分析表明，这是相对的Flp到Cre的比例，关键影响双RMCE的效率。我们的研究结果将有助于最大限度地提高双RMCE的效率，旨在工程和重组基因组。
Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35-45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp-2A-Cre and Flp-IRES-Cre transcription units. In contrast, the lowest level of dual RMCE (similar to 1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp-to-Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes.