Alive Mice Birth After Electrofusing Cumulus Cell With Enucleated Oocyte

Up to now, most of the somatic cell nuclear transfer (SCNT) mice were produced by injection method except for one case by electrofusing fibroblast cells with enucleated oocytes, and the cloning procedure was still far away from the routine. The present study examined the feasibility and effect of electrofusion technology to the production of SCNT mice. The fresh cumulus cells were transferred into the perivitelline space of the enucleated Mâ…¡oocytes, electrofusion was performed in the 0.3M mannitol fusion medium without Ca²⁺, the stimulation was one 2.4kv/cm, 20usec electrical pulse. The reconstructed embryos were cultured for 1 hour in vitro and then activated by 6 hours treatment of 10mM Sr²⁺. Some of the activated reconstructed embryos were cultured in vitro for 3.5 days, and then transferred into the uterus of the 2.5 days pseudopregnant mice, the others were cultured to the 2-cell stage in vitro, and transferred into the oviduct of 0.5 day pseudopregnant foster. At 19.5 days, the somatic cloned mice were delivered by Caesarean secession. Cloned embryos derived from injection method (Wakayama et al, Nature 1998) were used as control. Results showed the activation rates of SCNT embryos derived from electrofusion and injection method were 90.3% and 87.3% respectively (P>0.05). However, there was significant difference at cleavage (82.4% VS 92.9, P<0.05) and blastocyst development (47.3% VS 57.2%, P<0.05) between electrofusion and injection groups. We also studied the factor that can effect on the electrofusion efficiency, including aging of the oocyte, alteration of the electrofusion parameter, different strain mice, different donor cell and so on. The results suggested that the electrofusion parameter and condition were reasonable and feasible. The present experiment indicated that SCNT in mouse could succeed by the two NT methods. Since electrofusion were already widely used in nuclear transfer research, it should be easier to be applied in mouse SCNT research in the point of view of technique.