Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice
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DOI：
10.3791/60808
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发表时间：
2020-04-01
影响因子：
1.2
通讯作者：
Yasuda, Ryohei
中科院分区：
文献类型：
--
作者：
Nishizono, Hirofumi;Darwish, Mohamed;Yasuda, Ryohei
转基因小鼠的使用已经成为了解基因功能和破译人类疾病潜在机制的关键。CRISPR/Cas9系统允许研究人员以前所未有的效率、保真度和简单性修改基因组。利用这项技术，研究人员正在寻找一种快速、高效和简单的方法来生产转基因小鼠。在此，我们介绍了一种改进的单细胞胚胎冷冻保存方法，该方法可以提高冻融胚胎的发育率。通过将其与优化的电穿孔条件相结合，该方案允许在短时间内以高效率和低马赛克率产生敲除和敲入小鼠。此外，我们展示了我们的优化方案的一步一步的解释，包括CRISPR试剂的准备，体外受精，单细胞胚胎的冷冻和解冻，CRISPR试剂的电穿孔，小鼠的生成，以及创始人的基因分型。使用这种方案，研究人员应该能够以无与伦比的轻松、快速和高效的方式准备转基因小鼠。
The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.