The effect of a novel cryoprotective agent, carboxylated ε-poly-L-lysine, on the developmental ability of re-vitrified mouse embryos at the pronuclear stage.

Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new cryoprotective agent (CPA), carboxylated ε-poly-l-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2±3.1%) than those vitrified with DMSO and EG at three times (44.2±2.7%) which was equivalent to that of fresh embryos (70.0±3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos.