Tada N, Sato M, Kasai K, Ogawa S
Laboratory Animal Center, Hoechst Japan Limited, Saitama.
Transgenic Res. 1995 May;4(3):208-13. doi: 10.1007/BF01968786.
Vitrification is a technique for cryopreserving cells without crystallization due to elevation of the viscosity during the cooling process. We have developed a rapid and convenient mean of cryopreserving mouse preimplantation embryos by vitrification using a solution (hereafter named DPS) consisting of 2.75 M dimethylsulfoxide, 2.75 M propylene glycol and 1.0 M sucrose. In vitro fertilized pronucleate stage eggs were used because a large number of stage-matched eggs can be obtained at once. Only successfully fertilized eggs were collected and vitrified in DPS. After warming, two DNA constructs were injected into a total of 257 cryopreserved eggs, of which 175 (68%) survived the injection and were transferred into six recipients. All recipients became pregnant and gave birth to a total of 20 pups. When these DNA constructs were concomitantly injected into fresh eggs, 18% of eggs that were transferred developed into live pups, which was the same as the 18% figure for the cryopreserved eggs. With respect to transgenesis, 40% of the pups (8/20) developed from vitrified eggs were transgenic. In terms of the injected eggs that had been transferred, 4.5% of the 213 fresh eggs and 3.1% of the 112 vitrified eggs developed into transgenic mice. These results indicate that the efficiency of production of transgenic mice from vitrified eggs is comparable to that from fresh eggs.
玻璃化是一种在冷却过程中由于粘度升高而使细胞在不结晶的情况下进行冷冻保存的技术。我们已经开发出一种快速简便的方法，使用由2.75M二甲基亚砜、2.75M丙二醇和1.0M蔗糖组成的溶液（以下简称DPS）通过玻璃化来冷冻保存小鼠植入前胚胎。使用体外受精的原核期卵，因为可以一次性获得大量阶段匹配的卵。仅收集成功受精的卵并在DPS中进行玻璃化。解冻后，将两种DNA构建体注射到总共257个冷冻保存的卵中，其中175个（68%）在注射后存活并转移到6只受体动物体内。所有受体动物均怀孕并总共产下20只幼崽。当将这些DNA构建体同时注射到新鲜卵中时，转移的卵中有18%发育成活幼崽，这与冷冻保存卵的18%的比例相同。关于转基因，从玻璃化卵发育而来的幼崽中有40%（8/20）是转基因的。就已转移的注射卵而言，213个新鲜卵中有4.5%以及112个玻璃化卵中有3.1%发育成转基因小鼠。这些结果表明，从玻璃化卵生产转基因小鼠的效率与从新鲜卵生产的效率相当。
