STUDIES ON DEVELOPMENT OF PRACTICAL TECHNIQUE FOR PRODUCING CLONE COWS BY NUCLEAR TRANSFER

核移植生产克隆牛实用技术开发研究

• 批准号: 05556050
• 负责人: NAKAHARA Tatsuo
• 金额: $ 10.82万
• 依托单位: TOKYO UNIVERSITY OF AGRICULTURE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05556050/
• 关键词: NUCLEAR TRANSFER; CLONE CATTLE; SERIAL NUCLEAR TRANSFER; PARTHENOGENETIC ACTIVATION; CO-CULTURE; EMBRYO TRANSFER; 細胞周期

The present project was conducted to improve techniques for producing clone cattle by nuclear transfer and to apply for practical use it. The goal of the project was to produce 26 clone cattle from 85 identical embryos, in which the pregnancy rate after transfer to recipient cows was estimated as 30%. Through the project term, all investigators have achieved their aim and the technique for embryo cloning by nuclear transfer was significantly improved. The results are summarized as follows :1) We showed that about 30 (maximum 43) identical embryos were produced by the serial nuclear transfer system, and live young were obtained from such embryos after transfer to recipient cows.2) It was showed that live young can be produced from cryopreserved unclear transferred embryos, and that cryopreserved unfertilized eggs can be used as recipient cytoplasm.3) In co-culture system, mouse fetus fibroblast cells showed high sustaining ability for development of nuclear transferred embryos.4) Effective activation system that consisted of repetitive electric pulses was established, by which the developmental ability of nuclear transferred eggs was improved.5) Aphidicolin, DNA polymerase I inhibitor, which was used for synchronizing cell cycle of donor nuclei, was effective to improve developmental ability of nuclear transferred eggs.6) The present project has achieved the aim that is establishment of an effective cloning technique by nuclear transfer, and total of 40 live young were produced by nuclear transfer.

本项目旨在改进核移植生产克隆牛的技术，并将其应用于实际生产中，目标是用85枚完全相同的胚胎生产26头克隆牛，移植到受体母牛后的妊娠率估计为30%。通过项目的实施，所有研究者都达到了预期的目的，核移植胚胎克隆技术得到了显著的提高。结果总结如下：1）我们发现，大约30结果表明：（1）在连续培养体系中，小鼠胚胎成纤维细胞对核移植胚胎的发育具有很强的维持能力;（2）建立了有效的重复电脉冲激活体系，提高了核移植胚胎的发育能力;（3）建立了一种新的核移植胚胎激活体系，使核移植胚胎的发育能力得到了提高;（4）建立了一种新的核移植胚胎激活体系，使核移植胚胎的发育能力得到了提高;（5）Aphidicolin，应用DNA聚合酶I抑制剂使供体细胞核的细胞周期同步化，可有效提高核移植卵的发育能力。6）本研究实现了建立一种有效的核移植克隆技术的目的，共获得40只活仔。

项目成果

Takano, H.., Shimizu, S., Koyama, K., Kozai, C., Kato, Y.and Tsunoda, Y.: "Production of offspring by nuclear transferred bovine embryos produced in vitro." J.Reprod. Dev.40. 167-170 (1994)

Takano, H..、Shimizu, S.、Koyama, K.、Kozai, C.、Kato, Y. 和 Tsunoda, Y.：“通过体外产生的核移植牛胚胎生产后代。”

Takano, H.: "Effect of cell cycle stage of douor nuclei on the development of boviue nuclear transferred embryos" J. Reprod. Dev.(In press).

Takano, H.：“双核细胞周期阶段对牛核移植胚胎发育的影响”J. Reprod。

Kato,T.: "Effect of the culture density of mouse zygotes on the development in vitrc and vivo" Theriogenology. 41. 1315-1322 (1994)

Kato,T.：“小鼠受精卵培养密度对体外和体内发育的影响”动物发生学。

Kato, Y., Odagaki, S.and Tsunoda, Y.: "Effects of leukemia inhibitory factor (LIF) on the developmental ability of mouse 8-cell embryos in vitro and in vitro." J.Mamm. Ova. Res.10. 194-199 (1993)

Kato, Y.、Odagaki, S. 和 Tsunoda, Y.：“白血病抑制因子 (LIF) 对小鼠 8 细胞胚胎体外和体外发育能力的影响。”

Kato, Y.and Tsunoda, Y.: "Effect of the culture density of mouse zygotes on the development in vitro and in vivo." Theriogenology. 41. 1315-1322 (1994)

Kato, Y. 和 Tsunoda, Y.：“小鼠受精卵培养密度对体外和体内发育的影响。”