Freezing of spermatozoa and unfertilized oocytes is a useful tool for the conservation of mouse genetic resources. However, the proportion of frozen-thawed oocytes fertilized with spermatozoa in vitro is low because spermatozoa, especially those frozen-thawed, can not penetrate into oocytes because of hardening of the zona pellucida following premature release of cortical granules. To produce offspring efficiently from cryopreserved transgenic mouse gametes, we fertilized frozen-thawed gametes by using intracytoplasmic sperm injection (ICSI) and assessed pre- and postimplantation development of embryos. Compared with fresh unfertilized oocytes, frozen-thawed unfertilized oocytes were highly tolerant to damage by injection, as the survival rates after injection of frozen spermatozoa were 51 and 78%, respectively. Frozen-thawed oocytes that survived after sperm injection developed normally to the blastocyst stage and gave rise to offspring. Moreover, offspring with transgenes also were obtained from frozen gametes fertilized by ICSI. These results demonstrate that ICSI is an efficient technique for producing offspring from transgenic spermatozoa showing low fertility and that use of frozen-thawed oocytes leads to conservation of genetic resources because suboptimally preserved gametes are not wasted.