Production of transgenic mice by microinjection of DNA into vitrified pronucleate stage eggs
- 67 Downloads
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 2.75m dimethylsulfoxide, 2.75m propylene glycol and 1.0m 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.
Keywordstransgenic mice microinjection vitrification pronucleate stage eggs
Unable to display preview. Download preview PDF.
- Hogan, B., Costantini, F. and Lacy, E. (1986) Manipulating the Mouse Embyo. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press.Google Scholar
- Kasai, M., Komi, J.H., Takakamo, A., Tsudera, H., Sakurai, T. and Machida, T. (1990) A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability.J. Reprod. Fert. 89, 91–97.Google Scholar
- Nakagata, N. (1989) Survival of two-cell mouse embryos derived from fertilizationin vitro after ultrarapid freezing and thawing.Japan. J. Fert. Steril. 34, 470–3 (In Japanese).Google Scholar
- Scheffen, B., Van Der Zwalmen, P. and Massip, A. (1986) A simple and efficient procedure for preservation of mouse embryos by vitrification.Cryo-Letters 7, 260–9.Google Scholar
- Tada, N., Sato, M., Amann, E. and Ogawa, S. (1993b) Stage-dependent viability of mouse preimplantation embryos vitrified with sugar-containing solutions.J. Reprod. Dev. 39, 139–44.Google Scholar
- Tada, N., Sato, M., Kasai, K. and Ogawa, S. (1994) Successfulin vitro andin vivo development of cryopreserved mouse oocytes fertilized by cryopreserved mouse epididymal spermatozoa.J. Reprod. Dev. 40, 65–70.Google Scholar
- Toyoda, Y., Yokoyama, M. and Hoshi, T. (1971) Studies on the fertilization of mouse eggsin vitro. IIn vitro fertilization of eggs by fresh epididymal sperm.Japan. J. Anim. Reprod. 16, 147–51 (In Japanese).Google Scholar
- Whittingham, D.G. (1971b) Culture of mouse ova.J. Reprod. Fert. (Suppl)14, 7–21.Google Scholar