Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos.
Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos.
复制标题
使用 δC31 整合酶介导非洲爪蟾胚胎中 DNA 的插入。
DOI：
10.1007/978-1-61779-992-1_13
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Weeks,DanielL
中科院分区：
文献类型：
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作者：
Li,YouE;Allen,BryanG;Weeks,DanielL
用于产生转基因Xenopus胚胎的两种最常见的方法，限制性内切酶介导的插入和I-SceI大范围核酸酶利用精子，卵子或早期胚胎基因组中相对常见但空间不可预测的双链断裂。这些方法还倾向于插入转基因的多聚体拷贝。另一种方法是使用噬菌体或转座子衍生的整合酶或重组酶来介导转基因的更多位点特异性插入。phiC 31整合酶的使用需要用于插入的限定序列，并且与转基因的单拷贝的插入相容。我们描述了我们用来促进phiC 31整合酶转基因插入的方案，包括使用绝缘子序列来减少转基因活性的位置效应破坏。
The two most common methods used to generate transgenicXenopusembryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These methods also tend to insert multimeric copies of the transgene. An alternative is to use bacteriophage- or transposon-derived integrase or recombinase to mediate more site-specific insertion of the transgene. The use of phiC31 integrase requires a defined sequence for insertion and is compatible with insertion of a single copy of the transgene. We describe the protocol we use to facilitate phiC31 integrase transgene insertion including the use of insulator sequences to reduce position effect disruption of transgene activity.