Protocol development optimal for cell-cycle stage of somatic donor cells in production of cloned rats

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1. Qi Zhou et al. (2003) have first reported that ooplasmic injection of M-phase donor cell nucleus, followed by removal of maternal karyoplate in a one-step manner and the subsequent activation treatment with buthylactone-I, resulted in the birth of somatic cell-derived cloned rats. In the present study, the reproducibility of the Qi Zhou's protocol for production of cloned rats was investigated. None of 166 embryos reconstructed from fetal fibroblasts arrested at the metaphase stage (DA origin) and ovulated oocytes (SD origin) implanted and developed to full-term after transfer into 3 recipient females.2. For the successful production of cloned mice using GO/G1-phase cumulus cells, nuclear injection into enucleated oocytes, followed by incidence of premature chromosome condensation (PCC) and parthenogenetic activation with strontium (Wakayama et al., 1998), was proved to be a reproducible and convenient technique in our laboratory. In the present study, the Wakayama's protocol was ap … More plied to produce cloned rats with minor modifications. Only 17% of the reconstructed oocytes (67/394) cleaved 24 h after a combined treatment for activation with ionomycin + 6-DMAP + cycloheximide. Transfer of 188 presumptive cloned zygotes into 9 recipients failed to produce any offspring.3. Rat oocytes activate spontaneously once they are exposed to an in vitro condition. The incidence of PCC in the reconstructed rat oocytes was interfered with a rapid loss of maturation-promoting factor (MPF ; p34^<cdc2> kinase) activity due to the spontaneous oocyte activation. In addition, enucleation per se caused a complete loss of oocyte potential to support PCC of the donor nuclear materials. Therefore in the present study, regimens to inhibit spontaneous activation of rat oocytes were explored using chemicals associated with regulation of cell cycle via phosphorylation or cascades for ubichitination. Treatment with MG-132, a proteasome inhibitor, contributed to an increase of PCC incidence in enucleated oocytes receiving cumulus cell nuclei. Less