Studies on bovine nuclear transfer(cloning)and related technologies

牛核移植（克隆）及相关技术研究

• 批准号: 10556058
• 负责人: TAKAHASHI Yoshiyuki
• 金额: $ 8.38万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10556058/
• 关键词: Cattle; Nuclear transfer; Cloning

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

目前的研究旨在建立以胚胎卵裂球和体细胞为核供体的可靠牛核移植（NT）程序，并开发卵母细胞/卵泡冷冻保存和腔前卵泡体外培养等相关技术。我们开发了一种新的卵母细胞去核技术，利用 NT 移植后获得的预激活卵母细胞，获得高效的克隆牛。在使用体细胞作为核供体的克隆牛生产实验中，我们研究了影响NT胚胎发育的几个因素，结果表明细胞周期之间存在协调性。受体细胞质和核供体是最关键的，当体细胞用作G0/G1期的核供体和M期受体细胞质的组合时，可以产生具有高发育能力的胚胎。用子宫上皮细胞、初乳来源的乳腺上皮细胞和耳来源的成纤维细胞重建的NT胚胎移植后获得了核供体，但NT胚胎移植后的流产和克隆牛的异常现象时有发生。为了制备大量受体卵母细胞，我们研究开发了用于冷冻保存卵母细胞的窦前卵母细胞培养系统和冷冻保存方案我们证实，来自早期腔前卵泡的牛卵母细胞在成熟培养前培养超过 8 天即可获得发育能力。使用小鼠卵母细胞进行多次研究后，我们成功地冷冻保存了小鼠腔前卵泡，并证实了乙二醇和棉子糖。来自玻璃化培养的腔前卵泡的卵母细胞可以发育到囊胚阶段。

项目成果

Dela Pena EC: "エチレングリコールとラフイノースを用いたマウス卵子のガラス化における前処理の効果."北海道牛受精卵移植研究会会報. 19. 31-34 (2000)

Dela Pena EC：“使用乙二醇和棉子糖预处理对小鼠卵母细胞玻璃化的影响。”北海道牛受精卵移植研究组通报。19. 31-34 (2000)

Sawai T, Moriyasu S, Kageyama S, Yamamoto Y, Ashino M, Kitano N, Matsuzaki S, Yamashina H, Okamoto H, Hujikawa A, Minamihashi A: "Birth of a clone calf after the transfer of nuclear transfer embryos reconstructed with uterine epithelial cells."Bull Soc Ho

Sawai T、Moriyasu S、Kageyama S、Yamamoto Y、Ashino M、Kitano N、Matsuzaki S、Yamashina H、Okamoto H、Hujikawa A、Minamihashi A：“子宫上皮重建核移植胚胎移植后克隆牛的诞生

笹本良彦: "ウシの超音波誘導経膣採卵において卵胞吸引時の吸引針の回転と吸引針の違いが卵子回収率に及ぼす影響."北海道牛受精卵移植研究会報. 18. 19-22 (1999)

Yoshihiko Sasamoto：“卵泡抽吸过程中抽吸针的旋转以及抽吸针的差异对超声引导下经阴道牛卵母细胞回收率的影响。”北海道牛受精卵移植研究组通报18。 19-22（1999）

Kishi M, Itagaki Y, Takakura R, Sudo T, Yoshinarai M, Kashima N: "Effects of polythylenegycol and DMSO on the fusion of oocytes and their subsequent in vitro development of nuclear transplanted bovine embryos reconstructed with colostrum-derived mammary g

Kishi M、Itagaki Y、Takakura R、Sudo T、Yoshinarai M、Kashima N：“聚乙二醇和 DMSO 对卵母细胞融合及其随后用初乳来源的乳腺 g 重建的核移植牛胚胎的体外发育的影响”

Mohamed Nour MS: "Preparation of youn preactivated oocytes with high enucleation efficiency for bovinenuclear transfer"Theriogenology. 51・3. 661-666 (1999)

Mohamed Nour MS：“为牛核移植制备具有高去核效率的预激活卵母细胞”51・3 (1999)。