Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Iwate, 020-8551, Japan.
Department of Chemistry and Biological Sciences, Faculty of Science and Engineering, Iwate University, Iwate, 020-8551, Japan; Division of Fundamental and Applied Sciences, Graduate School of Science and Engineering, Iwate University, Iwate, 020-8551, Japan.
Cryobiology. 2019 Oct;90:71-74. doi: 10.1016/j.cryobiol.2019.08.004. Epub 2019 Aug 22.
Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.
精子保存是保存有价值动物品系的一种有用技术。大鼠精子可以在简单的 Tris-EDTA 溶液（TE 缓冲液）中冷冻或冻干，通过胞质内精子注射（ICSI）受精的卵母细胞可以发育成后代。利用成簇规律间隔短回文重复序列（CRISPR）和 CRISPR 相关蛋白 9（Cas9）系统进行基因组编辑，可以快速生产基因修饰大鼠。最近的创新方法称为 TAKE 方法，可以通过将内切酶电穿孔到胚胎中轻松产生基因组编辑大鼠。尽管已经应用了各种大鼠品系进行基因组编辑，但使用作为精子保存的品系进行基因组编辑需要更长的时间，因为它需要在从精子再生的动物成熟后收集胚胎。为了缩短生产周期，我们直接将 Cas9 蛋白和 gRNA 电穿孔到用 TE 缓冲液中的冷冻或冻干精子注射的卵母细胞中。在来自冷冻或冻干精子的 ICSI 胚胎的发育过程中，直到 30V 至电穿孔都没有显示出效果。此外，后代中的基因组编辑率很高（冷冻精子为 56%，冻干精子为 50%）。这些结果表明，ICSI 和 TAKE 方法的结合可用于从作为遗传资源保存的精子快速生产基因组编辑动物。
