Seki Shinsuke, Kawabe Toshiaki, Matsumura Kazuaki, Higashiya Misako, Oikawa Takanori, Fujii Yuriko, Yano Megumi, Yamazaki Wataru, Eto Tomoo
Experimental Animal Division, Bioscience Education and Research Support Center, Akita University, 1-1-1 Hondo, Akita, Akita, 010-8543, Japan.
ARK Resource Co., Ltd., 456 Osazu, Misato-machi, Shimomashiki-gun, Kumamoto, 861-4401, Japan.
Cryobiology. 2025 Sep;120:105260. doi: 10.1016/j.cryobiol.2025.105260. Epub 2025 Jun 9.
Genome-edited animals can be created by introducing CRISPR/Cas9 systems into one-cell stage embryos, even in non-mice embryos. We developed a vitrification method for rat one-cell embryos of the F344 inbred, Long-Evans, and SD strains. Successful cryopreservation requires the avoidance of intracellular ice formation (IIF). We hypothesized that a new cryopreservation method could be developed by rapid warming to avoid IIF during warming, via cryopreservation in conventional cryotubes using small-volume vitrification and rapid warming. When cryopreserved one-cell embryos in a cryotube with 15 μL cryopreservation solution (mixture of 5 μL 5 % propylene glycol and 10 μL PEPeS) were warmed by adding 1 ml of 0.3 M sucrose solution at 50 °C, they developed to term at 67.2 % (F344), 56.3 % (Long-Evans), and 65.0 % (SD), which were comparable with those (60.3 %, 58.3 %, 67.0 %, respectively) of non-cryopreserved control embryos. Thus, rat one-cell embryos from several strains can be cryopreserved even with cryotubes via rapid warming.
通过将CRISPR/Cas9系统导入单细胞期胚胎，甚至是在非小鼠胚胎中，都可以培育出基因编辑动物。我们开发了一种用于F344近交系、Long-Evans和SD品系大鼠单细胞胚胎的玻璃化方法。成功的冷冻保存需要避免细胞内冰晶形成（IIF）。我们推测，可以通过快速升温来开发一种新的冷冻保存方法，即在传统冷冻管中使用小体积玻璃化和快速升温的方法，以避免升温过程中的IIF。当将保存在含有15 μL冷冻保存溶液（5 μL 5%丙二醇和10 μL PEPeS的混合物）的冷冻管中的单细胞胚胎在50°C下加入1 ml 0.3 M蔗糖溶液进行快速升温时，它们的发育成功率在F344品系中为67.2%，在Long-Evans品系中为56.3%，在SD品系中为65.0%，这与未冷冻保存的对照胚胎（分别为60.3%、58.3%、67.0%）相当。因此，即使使用冷冻管，通过快速升温也可以冷冻保存来自多个品系的大鼠单细胞胚胎。
