A randomized library approach to identifying functional lox site domains for the Cre recombinase.
A randomized library approach to identifying functional lox site domains for the Cre recombinase.
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DOI：
10.1093/nar/gkm604
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发表时间：
2007
影响因子：
14.9
通讯作者：
Leinwand LA
中科院分区：
文献类型：
--
作者：
Sheren J;Langer SJ;Leinwand LA
噬菌体P1 Cre/loxP位点特异性重组系统在许多基因工程过程中是有用的工具。Cre重组酶已显示作用于与其噬菌体识别序列loxP的DNA序列显著不同的DNA序列。然而，鲜为人知的是，功能性lox样序列的序列要求。在这项研究中，我们已经实现了一个随机库的方法来确定功能lox网站域的序列特征。我们创建了一个随机间隔区文库和一个随机臂文库，然后在体内和体外测试它们的重组。从间隔区库的结果表明，虽然有很大的可塑性，间隔区对之间的身份是最重要的因素影响的功能，特别是在体外反应。在功能性loxP重组反应中存在一个完全随机化的臂揭示了只有三个野生型loxP臂是在表达Cre的细菌中成功重组所必需的，并且对于最有效的重组序列，在随机化臂的前三个和后三个位置处存在核苷酸偏好。最后，我们发现，在体外Cre重组反应是更严格的评估哪些序列可以支持有效的重组相比，294-CRE系统。
The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.
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