Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl₂ for 4 h (immediate activation after injection; IAI), or cultured in vitro for 2~3 h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation 2~3 h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of 36.6%(15/41), 39.5% (17/43) and 46.3% (25/54), respectively. However, in the ABI group, only one embryo (1.8%, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage (4.9%, 2/41). However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.

INTRODUCTION   MATERIALS AND METHODS   RESULTS   DISCUSSION   REFERENCES

• Sangho Roh(Department of Craniomaxillofacial Reconstructive Science, School of Dentistry and Dental Research Institute, Seoul National University)