Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation
Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation
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DOI：
10.3390/cells9051088
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发表时间：
2020-05-01
期刊：
影响因子：
6
通讯作者：
Hatada, Izuho
中科院分区：
文献类型：
--
作者：
Horii, Takuro;Kobayashi, Ryosuke;Hatada, Izuho
使用Cre-loxP系统产生条件性敲除小鼠有利于基因的功能分析。侧翼有两个loxP位点（floxed）的小鼠可以通过CRISPR/Cas9基因组编辑系统直接从受精卵中获得。我们以前报道过，在1-和2-细胞胚胎阶段通过电穿孔（EP）连续敲入（KI）每个loxP位点与同时KI相比，增加了具有floxed等位基因的小鼠数量。然而，EP在2-细胞期经常诱导卵裂球融合。这些融合的胚胎不能发育到足月，因为它们是四倍化的。在这项研究中，我们研究了以下三种条件，以抑制卵裂球融合的EP在2-细胞阶段：（1）高渗处理，（2）钙（Ca 2+）-无处理，和（3）肌动蛋白聚合抑制。2-细胞期胚胎的高渗处理阻止了卵裂球融合并促进了囊胚发育;然而，KI效率降低。无钙处理和细胞松弛素B（CB）抑制肌动蛋白聚合降低融合率，并没有对发育和KI效率产生负面影响。这些结果表明，在2-细胞阶段的无Ca 2+和CB处理与顺序EP方法组合可有效产生floxed小鼠。
The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.