Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells
Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cre-mediated cassette exchange in ES cells
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DOI：
10.1093/jb/mvj208
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发表时间：
2006-12-01
影响因子：
2.7
通讯作者：
Yamamura, Ken-ichi
中科院分区：
文献类型：
--
作者：
Araki, Kimi;Araki, Masatake;Yamamura, Ken-ichi
Cre-lox系统是胚胎干细胞遗传操作的重要工具。我们先前报道了使用突变体lox 66/71和lox 2272组合的盒交换策略显示出高重组效率和稳定性。然而，效率受到染色体靶lox位点的位置的强烈影响。为了富集成功的盒交换事件，甚至在显示较低重组效率的克隆中，我们改进了交换载体。将白喉毒素A片段基因置于非交换区用于阴性选择，并将嘌呤霉素N-乙酰转移酶基因代替新霉素磷酸转移酶基因用于阳性选择。通过减少随机整合，成功的盒交换的频率增加至2-4倍。此外，通过增加第三个lox位点诱导分子内重组，提高了盒式交换本身的重组效率，频率最高可提高5倍，其中交换克隆的比例达到50- 70%。这种策略对于其他重组酶介导的盒交换应该是有用的。
The Cre-lox system is an important tool for genetic manipulation in embryonic stem cells. We previously reported that the cassette exchange strategy using the mutant lox66/71 and lox2272 combination showed high recombination efficiency and stability. However, the efficiency was strongly affected by the position of chromosomal target lox sites. To enrich successful cassette exchange events, even in clones showing lower recombination efficiency, we have improved exchange vector. The Diphtheria toxin A fragment gene was placed in the un-exchanged region for negative selection and the puromycin N-acetyl-transferase gene, instead of the neomycin phosphotransferase gene, was used for positive selection. By reducing random integration, the frequency of successful cassette exchange increased up to 2-4 fold. Furthermore, by adding the third lox site to induce intrarmolecular recombination, the recombination efficiency of cassette exchange itself was improved, and the frequency increased to maximum 5 fold, in which the percentage of exchanged clones reached to 50-70%. This strategy should be useful for other recombinase-mediated cassette exchanges.