Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain
Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain
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DOI：
10.1016/j.jneumeth.2019.01.010
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发表时间：
2019-04-01
影响因子：
3
通讯作者：
Takao, Keizo
中科院分区：
文献类型：
--
作者：
Darwish, Mohamed;Nishizono, Hirofumi;Takao, Keizo
背景：CRISPR/Cas9技术已经经历了许多修改，以减少工作量并缩短高效制造突变小鼠所需的时间。使用新鲜胚胎在每次实验前的卵母细胞准备和受精过程中耗费时间和精力，而冻融胚胎克服了这一限制。然而，1-细胞胚胎的冷冻保存是具有挑战性的。新方法：我们介绍了一种改进的1-细胞C57BL/6J胚胎冷冻保存方法和优化的电穿孔条件，用于在解冻后1小时将CRISPR试剂输送到胚胎中。结果：与新鲜胚胎相比，冷冻-解冻的1-细胞胚胎显示出相似的存活率和惊人的高发育率。利用该方法，我们在4周内获得了几个突变小鼠系：非同源末端连接敲除小鼠(NHEJ)和同源定向修复敲入小鼠(HDR)，突变率分别为100%和75%，嵌合率较低。与已有的方法(S)相比，本方法将近交系冻融胚胎与电穿孔技术相结合，实验室人员可以在较短的时间内进行胚胎操作的基础训练，从而在较短的时间内产生突变小鼠。结论：我们建立了一种简单、经济、健壮的转基因小鼠的产生方法，避免了回交，具有效率高、马赛克率低的特点。它使制备人类疾病的小鼠模型成为一项简单的任务，具有前所未有的轻松、速度和效率。
Background: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging.New method: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing.Results: Freeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks.Comparison with existing method (s): Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods.Conclusion: We developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.