Cre重组酶
重组酶
Cre-Lox重组
体外
生物
计算生物学
DNA
合成生物学
转染
体内
体外重组
细胞生物学
重组
遗传学
转基因
细胞培养
基因
转基因小鼠
分子克隆
基因表达
作者
Kaushik Ghosh,Gregory D. Van Duyne
出处
期刊:Methods
[Elsevier]
日期:2002-11-01
卷期号:28 (3): 374-383
被引量:89
标识
DOI:10.1016/s1046-2023(02)00244-x
摘要
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.
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