Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells
Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells
复制标题
DOI：
10.1186/1472-6750-10-29
复制
发表时间：
2010-03-31
影响因子：
3.5
通讯作者：
Yamamura, Ken-ichi
中科院分区：
文献类型：
--
作者：
Araki, Kimi;Okada, Yuka;Yamamura, Ken-ichi
背景：cre介导的小鼠胚胎干（ES）细胞的位点特异性整合重组是基因组工程的有用工具，可以实现精确和重复的位点特异性整合。为了促进整合反应，以前已经开发了一种左元件/右元件（LE/RE）突变策略，利用一对lox位点在lox序列的LE或RE中发生突变。LE和RE突变体lox之间的重组产生了一个野生型loxP位点和一个LE+RE双突变体lox位点，该位点两侧都有突变，对Cre的亲和力较低，从而实现了稳定的整合。我们之前在胚胎干细胞中成功地展示了lox71 （LE突变体）和lox66 （RE突变体）的整合重组。最近，其他LE/RE突变位点在大肠杆菌中显示出更高的重组效率。然而，它们在哺乳动物细胞中的重组效率仍有待分析。结果：利用胚胎干细胞，我们比较了6个RE突变体lox位点，重点研究了它们与lox71的重组效率。所有RE突变体的lox位点表现出相似的重组效率。然后，我们分析了重组产物，即LE+RE双突变位点在ES细胞中连续和强Cre活性下的稳定性。两个RE突变体loxJTZ17和loxKR3比lox66/71双突变体产生更稳定的LE+RE双突变体lox。结论：两个突变位点loxJTZ17和loxKR3比lox66更适合于cre介导的ES细胞整合或转化。
Background: Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type loxP site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.Results: Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71. All of the RE mutant lox sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant lox site, under continuous and strong Cre activity in ES cells. Two RE mutants, loxJTZ17 and loxKR3, produced more stable LE+RE double mutant lox than did the lox66/71 double mutant.Conclusion: The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.