Factors affecting electrofusion of 2-cell mouse embryos.

Parameters of electrofusion of 2-cell mouse embryos were optimized for application as a model for nuclear transplantation. There was considerable lysis of embryos with M2 as the medium for fusion; however, 100% fusion (n = 58) was obtained with a single 0.31-kv / cm, 1280-μsec pulse. With mannitol and sucrose solutions as the medium, a wide range of field strengths (0.31–1.41 kv / cm for 0.26 M sucrose solution and 0.31 to 2.04 kv / cm for 0.3 M mannitol solution) and durations of the electrical pulse (10–1280 μsec) resulted in high rates of fusion (often 100%). Likewise, osmolarity of sucrose and mannitol solutions did not affect the rate of fusion using a 0.47-kv / cm pulse. With a field strength of 2.04 kv / cm, the proportion of embryos that fused in mannitol solution increased (P<0.05) and the proportion that were lysed decreased (P<0.05) as osmolarity increased. Both fused (162642, 25%) and control embryos (3272, 44%) continued to develop in culture for 48 h, after which they began to compact. Fused embryos were only at the 4-cell stage by this time, while control embryos were at the 8-cell stage. Optimal pulse durations are plotted for field strengths between 0.31 and 1.41 kv / cm with 0.26 M sucrose as fusion medium.