Cre-loxP biochemistry
Cre-loxP biochemistry
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DOI：
10.1016/s1046-2023(02)00244-x
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发表时间：
2002-11-01
期刊：
影响因子：
4.8
通讯作者：
Van Duyne, GD
中科院分区：
文献类型：
--
作者：
Ghosh, K;Van Duyne, GD
Cre重组酶现在被广泛用于在体外和体内进行DNA分子的复杂操作。对于体外实验，显然需要用作对照或提供改变的活性或特异性的Cre和Cre突变体的高纯度制剂。利用Cre变体的体内实验通常也需要体外表征，并且一些应用涉及纯化酶的转染以实现细胞中的瞬时活性。这篇综述概述了一个详细的协议，用于纯化天然Cre和描述简单的测定，可用于测试重组活性在体外。还介绍了用于生物物理研究的捕获Cre-loxP位点特异性重组中间体的实验设计。所描述的方法应该是有用的任何研究者与纯化的Cre重组酶的需要，并应广泛适用于相关的位点特异性重组系统。(C)2002 Elsevier Science（美国）。All rights reserved.
Cre recombinase is now widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. For in vitro experiments, there is a clear need for highly pure preparations of Cre and of Cre mutants that serve as controls or supply an altered activity or specificity. In vivo experiments utilizing Cre variants also often require in vitro characterization and some applications involve transfection of purified enzyme to achieve transient activity in the cell. This review outlines a detailed protocol for purification of native Cre and describes straightforward assays that can be used to test for recombination activity in vitro. The design of experiments to trap the intermediates of Cre-loxP site-specific recombination for biophysical studies is also presented. The methods described should be useful to any investigator with a need for purified Cre recombinase and should be broadly applicable to related site-specific recombination systems. (C) 2002 Elsevier Science (USA). All rights reserved.