Regulation of Cre recombinase by ligand-induced complementation of inactive fragments
Regulation of Cre recombinase by ligand-induced complementation of inactive fragments
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DOI：
10.1093/nar/gng131
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发表时间：
2003-11-01
影响因子：
14.9
通讯作者：
Herman, JP
中科院分区：
文献类型：
--
作者：
Jullien, N;Sampieri, F;Herman, JP
Cre重组酶广泛用于实验动物基因组的改造。然而，由于对其活动缺乏有效的时间控制，其用途仍然有限。为了克服这一点，我们开发了DiCre，Cre的可调控片段互补系统。该酶被分成两个部分，分别与FKBP 12（FK 506结合蛋白）和FRB（FKBP 12-雷帕霉素相关蛋白的结合结构域）融合。这些可以被雷帕霉素有效地异源二聚化。基于在不同位点分裂Cre并使用不同接头肽的几种变体在指示细胞系中进行了测试。分离的融合蛋白没有重组酶活性。基于在Asn 59和Asn 60之间分裂Cre共表达互补片段的稳定转化体显示影响0.05-0.4%细胞的低背景活性。雷帕霉素诱导快速重组，48-72 h达到100%，EC 50为0.02 nM。因此，配体诱导的二聚化可以有效地调节Cre，并且应该有助于实现其活性的严格时间控制，例如在产生条件性敲除动物的情况下。
Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.