Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.

中文翻译：

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通过电穿孔引入胚胎的核酸酶可视化生产基因组编辑小鼠


基因组编辑技术有助于基因工程动物的快速高效生产。这些动物有助于阐明人类疾病的机制。最近，开发了一种新的电穿孔技术（TAKE：电穿孔动物敲除系统技术），通过使用电穿孔而不是显微注射方法将核酸酶引入完整胚胎中来生产基因组编辑动物。本研究的目的是提高使用 TAKE 方法生产基因组编辑动物的效率。在传统方案中，很难确认核酸酶在胚胎中的引入以及操作过程中的通电。仅使用引入核酸酶的胚胎进行胚胎移植，将提高基因组编辑动物的生产效率。本研究使用 ATTO-550 荧光标记的核酸酶检查了将核酸酶引入胚胎的可视化情况。通过 TAKE 方法，用 Cas9 蛋白和荧光标记的双向导 RNA（与 crRNA 和 tracrRNA 与 ATTO-550 混合）靶向酪氨酸酶基因转染胚胎。电穿孔后存活的所有胚胎均显示出荧光。在这些带有荧光的胚胎中，43.7% 发育成形态正常的后代。此外，91.7%的后代受到酪氨酸酶基因的编辑。这项研究首次证明，通过 TAKE 方法将核酸酶引入胚胎可以使用荧光标记的核酸酶进行可视化。这种改进的 TAKE 方法可用于生产基因组编辑动物并确认操作过程中的通电情况。