Nakagata Naomi, Nakao Satohiro, Mikoda Nobuyuki, Yamaga Katsuma, Nakagawa Yoshiko, Sakuma Tetsushi, Yamamoto Takashi, Takeo Toru
Division of Reproductive Biotechnology and Innovation, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
Division of Reproductive Engineering, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
PLoS One. 2025 Sep 8;20(9):e0328718. doi: 10.1371/journal.pone.0328718. eCollection 2025.
Zygotes are used to create genetically modified animals by electroporation using the CRISPR-Cas9 system. Such zygotes in rats are obtained from superovulated female rats after mating. Recently, we reported that in vivo-fertilized zygotes had higher cryotolerance and developmental ability than in vitro-fertilized zygotes in Sprague Dawley (SD) and Fischer 344 rats. To apply the in vitro-fertilized zygotes in creating genetically modified rats, we need to address their low cryotolerance and developmental ability. Hence, we evaluated the effects of warming solutions containing different sucrose concentrations (0-0.3 M) and the oocyte donor's age (3-7-week-old SD rats) on the viability of vitrified-warmed zygotes after in vitro fertilization and on developmental ability by embryo transfer in SD rats. A warming solution containing 0.1 M sucrose enhanced the survival rate of vitrified-warmed zygotes and their rate of development to two-cell embryos. Additionally, zygotes derived from 6- and 7-week-old female rats had higher cryotolerance and developmental ability than those from 3-week-old ones. Next, vitrified-warmed rat zygotes produced using the optimized protocol underwent genome editing by electroporation with Cas9 ribonucleoprotein and gRNA introduced to disrupt the Tyr gene. We then found that 86.5% of the pups derived from zygotes demonstrated mutation of the targeted gene. Therefore, the improved protocol for vitrifying and warming rat zygotes is useful for preserving and producing genetically modified rats.
通过使用CRISPR-Cas9系统进行电穿孔，合子被用于创建转基因动物。大鼠中的此类合子是在交配后从超排卵的雌性大鼠获得的。最近，我们报道在斯普拉格-道利（SD）大鼠和费希尔344大鼠中，体内受精的合子比体外受精的合子具有更高的耐冻性和发育能力。为了将体外受精的合子应用于创建转基因大鼠，我们需要解决它们较低的耐冻性和发育能力问题。因此，我们评估了含有不同蔗糖浓度（0 - 0.3M）的解冻溶液以及卵母细胞供体年龄（3 - 7周龄的SD大鼠）对体外受精后玻璃化冷冻-解冻合子活力以及通过胚胎移植在SD大鼠中的发育能力的影响。含有0.1M蔗糖的解冻溶液提高了玻璃化冷冻-解冻合子的存活率及其发育到二细胞胚胎的比率。此外，来自6周龄和7周龄雌性大鼠的合子比来自3周龄雌性大鼠的合子具有更高的耐冻性和发育能力。接下来，使用优化方案产生的玻璃化冷冻-解冻大鼠合子通过电穿孔用Cas9核糖核蛋白和引入的gRNA进行基因组编辑以破坏Tyr基因。然后我们发现，来自合子的幼崽中有86.5%表现出靶向基因的突变。因此，改进的大鼠合子玻璃化冷冻和解冻方案对于保存和生产转基因大鼠是有用的。
