Ultrarapid freezing of DNA-injected and nuclear-transplanted mouse embryos = 외래유전자 주입 및 핵치환된 생쥐 수정란의 초급속 동결

Abstract

We determined whether the ultrarapid freezing method is applicable to micromanipulated mouse embryos. One-cell mouse embryos were microinjected with MThGH gene. Nuclei from one-cell embryos of Fi(C57BL x CBA) mice were transplanted into enucleated one-cell embryos of ICR mice. The injected and nucleated embryos that developed to 2-cell stage were cryopreserved by ultrarapid freezing. The embryos equilibrated in freezing medium(3 M DMSO + 0.25 M sucrose + 2% FBS in PBS) were directly immersed into liquid nitrogen and then thawed in 37°C water. Developmental rates of the microinjected and nuclear-transplanted embryos to blastocyst stage after ultrarapidly freezing and thawing were 31% and 55%, respectively. The frozen-thawed embryos were transferred to pseudopregnant recipients, which then gave birth to 17 offsprings. Twelve(14% of the transferred embryos) and five(20%) offsprings were derived from microinjected and nuclear-transplanted embryos, respectively. The results indicate that the DNA injected and nuclear-transplanted mouse embryos are cryopreservable at 2-cell stage by ultrarapid freezing method.