Efficient generation of conditional knockout mice via sequential introduction of lox sites.
Efficient generation of conditional knockout mice via sequential introduction of lox sites.
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DOI：
10.1038/s41598-017-08496-8
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发表时间：
2017-08-11
影响因子：
4.6
通讯作者：
Hatada I
中科院分区：
文献类型：
--
作者：
Horii T;Morita S;Kimura M;Terawaki N;Shibutani M;Hatada I
Cre/lox条件敲除技术是基因功能分析的基础。CRISPR/Cas与两组指导RNA和单链寡核苷酸的组合使得能够同时插入两个lox序列。然而，该方法在同一染色体上的两个位点处诱导双链断裂，这导致不期望的染色体缺失并降低侧翼lox（flox）率。为了解决这个问题，我们研究了分别在1细胞和2细胞胚胎阶段顺序引入每个lox序列的方法。顺序方法适用于显微注射和电穿孔系统。与普通的同时显微注射相比，顺序电穿孔提高了flox效率，导致具有floxed等位基因的后代的高产率。最后，我们利用Cre合子通过连续电穿孔直接制备了同时含有Cre转基因和floxed等位基因的Cre/lox小鼠，与普通方法相比，这加快了条件性基因敲除小鼠的产生。
Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.
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影响因子：
64.5
作者：
Yang H;Wang H;Shivalila CS;Cheng AW;Shi L;Jaenisch R
通讯作者：
Jaenisch R
通过核酸酶 Zygote 电穿孔进行高效 CRISPR/Cas9 介导的小鼠基因组编辑。
影响因子：
3.3
作者：
Qin W;Dion SL;Kutny PM;Zhang Y;Cheng AW;Jillette NL;Malhotra A;Geurts AM;Chen YG;Wang H
通讯作者：
Wang H
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Charpentier, Emmanuelle
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46.9
作者：
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通讯作者：
Kim, Jin-Soo
影响因子：
3.7
作者：
Kaneko T;Mashimo T
通讯作者：
Mashimo T