Improved protocol for the vitrification and warming of rat zygotes by optimizing the warming solution and oocyte...

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.