An Accumulative Site-Specific Gene Integration System Using Cre Recombinase-Mediated Cassette Exchange
An Accumulative Site-Specific Gene Integration System Using Cre Recombinase-Mediated Cassette Exchange
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DOI：
10.1002/bit.22619
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发表时间：
2010-04-15
影响因子：
3.8
通讯作者：
Kamihira, Masamichi
中科院分区：
文献类型：
--
作者：
Kameyama, Yujiro;Kawabe, Yoshinori;Kamihira, Masamichi
Cre-loxP系统经常用于动物细胞中的位点特异性重组。重组反应的平衡和特异性可以使用突变的loxP来控制。在本研究中，我们设计了一个累积的位点特异性基因整合系统，使用Cre重组酶和突变的loxP，其中Cre介导的盒交换反应是无限重复的目标基因整合到loxP目标位点。为了评估该系统的可行性和有用性，使用Cre重组酶蛋白和质粒在体外重复并确认了一系列整合反应。还对中国仓鼠卵巢（CHO）细胞的基因组进行了累积基因整合。结果表明，该系统适用于多个基因重复整合到质粒和CHO细胞基因组上的靶位点。这种基因整合系统提供了一种新的策略，基因扩增和基因功能的生物学分析，通过遗传修饰的细胞和生物体。Biotechnol. Bioeng. 2010; 105：1106-1114. (C)2009 Wiley Periodicals，Inc.
The Cre-loxP system is frequently used for site-specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms. Biotechnol. Bioeng. 2010; 105: 1106-1114. (C) 2009 Wiley Periodicals, Inc.