Cryopreservation for broader production of transgenic mice by DNA injection into zygotes.

Mutant mice are indispensable to biological and medical research, and transgenic mouse production by DNA injection into zygotes has been an important method for producing these mice which are used to examine the over-expression of genes and to analyze gene transcriptional regulatory sequences. Recently, cryopreservation of zygotes by a simple vitrification method has become popular, saving the labor of isolating zygotes following each injection. However, the DNA injection technique requires training in the use of special equipment, and the injection cannot be accomplished routinely in every laboratory. The exchange of live mice also risks the propagation of common murine pathogens and possible escape of the animals; therefore the transfer of mice in frozen zygotes or embryos is recommended. Here we propose injecting DNA into frozen and thawed zygotes, refreezing them before transportation to any destination, where they can be thawed and developed into pups. The rate of transgenic mouse production using this method does not decrease significantly, even if zygotes are frozen and thawed before and after their DNA injection, and will make transgenic studies more popular on a worldwide scale.