Studies on bovine nuclear transfer(cloning)and related technologies

本研究以牛胚胎卵裂球和体细胞为核供体，建立了可靠的牛核移植程序，并发展了卵母细胞/卵泡的冷冻保存和腔前卵泡的体外培养等相关技术。将卵裂球重组的NT胚胎移植后，获得了克隆小牛，并证实了其一致的生长和受精能力。在利用体细胞作为核供体生产克隆小牛的实验中，我们研究了影响NT胚胎发育的几个因素。结果表明，供核细胞和受体胞质之间的细胞周期协调是最关键的，供体处于G0/G1期和受体胞质处于M期的组合可产生高发育…的胚胎当体细胞被用作核供体时，有更多的细胞能力。用子宫上皮细胞、初乳来源的乳腺上皮细胞和耳源性成纤维细胞构建的NT胚胎移植后获得克隆小牛，但移植后的胚胎经常出现流产和畸形。为了制备大量的受体卵母细胞，我们研究了以牛、小鼠卵母细胞和腔前卵泡为材料的卵泡/卵母细胞冷冻保存体系和冷冻保存方案。我们证实了来源于腔前卵泡的牛卵母细胞在成熟培养前培养8天以上即可获得发育能力。在用小鼠卵母细胞进行了多次玻璃化冷冻试验后，研制出了一种新型玻璃化冷冻液(VS)。利用这种新的VS，我们成功地冷冻了小鼠腔前卵泡，证实了来自玻璃化培养的腔前卵泡的卵母细胞可以发育到囊胚期。较少

Present studies were conducted to establish reliable bovine nuclear transfer(NT)procedure using embryonic blastomeres and somatic cells as nuclear donors, and to develop related technologies such as cryopreservation of oocytes/follicles and in vitro culture of preantral follicles.In experiments for NT using embryonic blastomeres, we have developed a new oocyte-enucleation technique with a high efficiency by using pre-activated oocytes. Clone calves were obtained after the transfer of NT embryos reconstituted with blastomeres, and we confirmed their consistent growth and fertility. In experiments for the production of clone calves using somatic cells as nuclear doner, we have investigated the several factors affecting the development of NT embryos. Results indicate that cell-cycle coordination between recipient cytoplast and nuclear donors is most critical, and that the combination of nuclear donor in the G0/G1 phase and recipient cytoplast in the M phase produces embryos with high deve … More lopmental capacity when somatic cells are used as nuclear donors. Clone calves were obtained after the transfer of NT embryos reconstructed with uterine epithelial cells, colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells ; however, abortion after the transfer of NT embryos and abnormalities in the clone calves were frequently observed.To prepare a large number of recipient oocytes, we have studied to develop a pre-antral follicle culture system and cryopreservation protocol for the cryopreservation of follicles/oocytes using cattle and mouse oocytes and preantral follicles. We confirmed that bovine oocytes derived from early preantral follicles could acquire developmental capacity if they were cultured for more than 8 days before maturation culture. A new vitrification solution(VS), a mixture of ethylene glycol and raffinose was developed after the several investigation using mouse oocytes. Using this new VS, we have succeeded to cryopreserve mouse preantral follicles, and confirmed that the oocytes derived from vitrified-cultured preantral follicles could develop to the blastocyst stage. Less