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

1. Qi Zhou等（2003）首次报道了将M期供体细胞核卵浆注射，然后以一步方式去除母体有核板，随后用丁内酯-I活化处理，导致体细胞衍生的克隆大鼠的出生。在本研究中，齐周的方案生产克隆大鼠的重复性进行了研究。166枚胚胎移植到3只受体雌鼠体内后，均未着床并发育至足月.为了使用GO/G1期卵丘细胞成功生产克隆小鼠，将核注射到去核卵母细胞中，随后发生过早染色体凝聚（PCC）和用锶进行孤雌激活（和歌山等人，1998），在我们的实验室中被证明是一种可重复的和方便的技术。在本研究中，采用和歌山的方案， ...更多信息 被用来生产经过微小修改的克隆老鼠。用离子霉素+ 6-DMAP +放线菌酮联合激活后24 h，只有17%（67/394）的重构卵分裂。将188个克隆合子转移到9个受体中，未产生后代.大鼠卵母细胞一旦暴露于体外条件下就会自发激活。重构的大鼠卵母细胞中PCC的发生率受到由于卵母细胞自发激活而导致的促成熟因子（MPF ; p34激酶）活性快速丧失的干扰<cdc2>。此外，去核本身导致卵母细胞完全丧失支持供体核材料PCC的潜力。因此，在本研究中，使用与通过磷酸化或泛素化级联调节细胞周期相关的化学物质探索抑制大鼠卵母细胞自发激活的方案。用MG-132（一种蛋白酶体抑制剂）处理导致接受卵丘细胞核的去核卵母细胞中PCC发生率增加。少

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