Production of transgenic rats using cryopreserved pronuclear-stage zygotes

We investigated the application of cryopreserved pronuclear-stage zygotes for the production of transgenic rats. Most of the pronuclear-stage zygotes cryopreserved by conventional two-step freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7-60.2%) was higher than that of vitrified zygotes (5.5-22.1%). When the frozen-thawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0 and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclear-stage rat zygotes can be successfully cryopreserved by conventional two-step freezing for production of transgenic rats.