Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs

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.