Targeted integration of DNA using mutant lox sites in embryonic stem cells
Targeted integration of DNA using mutant lox sites in embryonic stem cells
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DOI：
10.1093/nar/25.4.868
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发表时间：
1997-02-15
影响因子：
14.9
通讯作者：
Yamamura, KI
中科院分区：
文献类型：
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作者：
Araki, K;Araki, M;Yamamura, KI
通过使用一对突变型lox位点，即右元件（RE）突变型lox位点和左元件（LE）突变型lox位点，已经实现了定点DNA整合[Albert等人（1995）Plant J.，7，649-659]，在小鼠胚胎干（ES）细胞中。我们建立了携带单拷贝的野生型loxP或LE突变体lox位点作为靶标的ES细胞系，并检查了由Cre瞬时表达诱导的携带loxP或RE突变体lox位点的靶向载体的位点特异性整合频率。由于我们的靶向载体含有完整的neo基因，随机整合体可以形成集落，如在基因靶向事件的情况下通过同源重组。利用我们的系统，通过突变体lox位点的位点特异性整合的频率达到最大值16%。相反，野生型loxP位点产生非常低频率（< 0.5%）的位点特异性整合事件。这种突变的lox系统将用于ES细胞中DNA的“敲入”整合。
Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albert et al. (1995) Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type loxP or LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression, Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (< 0.5%) of site-specific integration events, This mutated lox system will be useful for 'knock-in' integration of DNA in ES cells.